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DCDT+ User Manual - John Philo`s Software Home Page

1. ris I ToO RTRT i b f 4 4 ee ee es eet oe ee ee oe I if i i I oa ys i E 1 oe A gs i i i i 160E f d to m te o ft aar ae Por a ee ene oe oe ee i I I I i i i f ee bh ae i i i oa oa wee A 4 F PA FTP ata fat bandedJon t 4 AIF puttin ati ak as Us fl ml I I I I j I e i i I eet EE pos eee te As Clan eee E come Eee oO be 5 i i i i Si 1 ine i f i i Po wu tne le ae ae oe a oA EAT E E A T A E R E E E i i E sat i i i a i i i i i 3 ee e a hobo Tn T EE PEE EE AEEA AR E TS E AET E E EA TERA LA i i A b 2 see N ee 6 00B1 7 4 f 1 ppt nanaheeentnae I i i M i I I i i a e i oa 4008 T E E R R EE ft EN g I I i i i I I Te ee ee ee E NE A Mr Tr ea a aad i I i i i i i i i 0 00E 0 L 4 L L 4 L i L 4 4 4 L L i 00 03 06 00 12 15 18 2 24 27 30 33 36 30 42 45 42 Svedbergs This distribution shows one very broad peak with a maximum at 1 8 S Let us consider some sedimentation coefficient away from this maximum say at 1 2 S The distribution indicates a value of 1 2 AU S at this position Does this mean there is really this amount of material with a sedimentation coefficient of 1 2 S in this sample Possibly but not necessarily This distribution coul
2. Therefore although it is true that one can manually remove the effects of the region at the cell base by excluding the right end of the sedimentation coefficient range from the analysis that approach which is the only option is most DCDT implementations throws away a lot of good data Further that approach is dangerous because it may not be obvious from examination of the averaged data where the bad region begins This example also illustrates why it is a good idea to display and examine the dc dt data from each scan pair before averaging If you see dc dt curves data that look like this example then move back to the Set meniscus and data region page Section 3 2 2 drag the right hand green marker further to the left and then use the Next button to move forward and display all the individual dc dt curves to confirm that all the negative downward hooks at their right end are absent Why not simply set the default cutoff to a radius small enough to always avoid this problem The problem with that approach is that in many experiments you would be throwing away good data and this data may represent a significant fraction of the total potential range of sedimentation coefficients particularly if the cell is not completely filled 3 2 2 4 Set meniscus amp data region Jitter removal options Jitter removal options are displayed only in Advanced mode Section 1 3 and active only for interference data and pseudo absorbance data 98 DC
3. This brings up a dialog box showing the current version registration information and how to contact the author Registration This brings up a tabbed dialog box with information about why and how to register the program a page for entering and validating your program serial number and buttons to display the software license agreement or print an order form 73 DCDT User Manual DCDT User Manual aa J Registration Why and How Why Register How to Register License Agreement Activate Registration This software is distributed as shareware it is NOT public domain or freeware The essence of shareware is to provide you with software that you can ry before you buy while still rewarding the developer s time and effort to create and continue to support the program You may evaluate this software for a period of 60 days after which you must either purchase a license for its use or remove it from your computer You also are prohibited from publishing results or figures generated during the evaluation period until you purchasea license WHAT AM PAYING FOR 1 Development of the program code and Help file which required thousands of hours plus a considerable expense for programming languages and utilities 2 supportfor your questions and resolution of any problems that may arise 3 continued program development HOW DOES THE REGISTERED VERSION DIFFER FROM THE TRIAL VERSION There are no functional differ
4. If these parameters were not significantly correlated then the points would cluster along an ellipse that is oriented parallel to either the vertical or horizontal axis If the distribution was truly random the shape of the pattern would in principle be circular around the best fit values if the axes were scaled in units of standard deviations but in general the graph scaling will produce an ellipse rather than a circle 157 DCDT User Manual DCDT User Manual a This graph provides information that is quite similar to the cross correlation coefficients that can be viewed using the View parameter cross correlations button Section 3 2 10 3 However those values are based on a linear statistical model and assume the noise in the fitted data is random The cross correlation information obtained from these plots of bootstrap or Monte Carlo results is independent of such statistical assumptions Different pairs of parameters can be selected using the two drop down list boxes Obviously when the same parameter is chosen for both axes the result will be a diagonal line The size of the data points on this graph can be altered by clicking on any of them with the mouse the default size is automatically reduced as the number of data points increases to try to avoid too much overlap on the screen but this choice may not be optimum when the graph is exported or printed Other features of this dialog box The Copy button will place a copy of the cu
5. OD Svedberg pa FA Less Q Book Now A C If you are fitting D rather than M and wish to know what molecular mass is implied by the returned s and D values use the Mass diffusion calculator Section 3 1 1 4 1 menu command or push the calculator button on the Toolbar Section 3 1 2 The s and D values from the last fit will automatically be placed onto the form D If you are satisfied with the fit you will probably want to determine the true confidence intervals for the parameters and or print out a report both of which can be done from the Report fit results page Section 3 2 10 E It is highly recommended that you use the Save analysis as Section 3 1 1 1 3 command under the File Menu Section 3 1 1 1 or the equivalent Save button on the Toolbar Section 3 1 2 so you can quickly reload everything about this fit in the future and continue fitting with altered data or models if you choose to do so You are done 2 5 Tutorial Multi species fits Below is a general procedure for a multi species analysis in advanced mode Section 1 3 In many cases you would first do a single species analysis However for some samples it may be obvious from the shape of the g s distribution or known from other data that two or more species are present and thus starting with a multi 44 DCDT User Manual SR DCDT User Manual species fit may make the most scientific sense A Calculate the data to be fitted g s
6. These default values are used for the first analysis window opened each time the program is started When subsequent analysis windows are created the last values that were entered in any previous window are used which saves time when you are analyzing multiple samples in the same buffer or of the same protein dc dt options tab e Default meniscus clearance 0 015 cm This is the distance between the meniscus and the start of the data to be analyzed e Default data cutoff at cell base 7 15 cm Data beyond this radius will not be analyzed because that region is influenced by the accumulation of solutes at the base of the cell 68 DCDT User Manual SR DCDT User Manual Default cell region to test for and correct integer fringe shifts middle half see Set meniscus amp data region fringe jump removal options Section 3 2 2 5 se Philo 2000 algorithm to compute g s by default off see What is the difference between the broad and conventional g s algorithms Section 5 1 3 Fitting options tab Ge Confidence probability for parameter confidence limits 950 Fit converged when parameters change less than 005 Fit converged when variance change lt of statistical limit 10 _ Parameter cross correlation defined as high 97 Parameter cross correlation defined as very high 99 Maximum number of fit iterations before quitting 60 Ok cne Confidence probability for p
7. good data 7 DCDT User Manual DCDT User Manual aa Meniscus setting and data adjustments Meniscus at EEEERE cm good data begin 0 015 cm from meniscus good data end at radius 7 150 cm 18 16 14 Fringes oo T gt a More 1 When you first enter the the Set meniscus amp data region page Section 3 2 2 the program s meniscus wizard Section 6 6 will attempt to locate the meniscus position and will place the red vertical line at that position as shown above That position will be filled into the meniscus text box at the upper left and that text box is automatically given the focus e At this point the fringe jumps in these scans make it difficult to see the boundary movement Those will automatically be removed as soon as a meniscus position has been set e Note also that in this experiment the data acquisition region extended past the base of the cell producing lots of garbage data beyond 7 15 cm It is recommended that you start the acquisition a little to the left of the reference meniscus and end it a little to the left of the base of the cell 2 If you are satisfied with the wizard s choice of meniscus position then all you need to do is push the Enter key on the keyboard to accept that choice Otherwise you can click on the red meniscus marker and drag it to the position you want or simply type a value into the text box e To set the meniscus more accurately and that is im
8. 112 Set alter fitting parameters page 136 138 Should fit the offset 187 Show integrals over distribution 117 Simulate button 144 Simulation 57 60 93 94 Solvent density 133 134 Species plot 158 164 Standard and advanced modes 3 4 Standard mode 3 4 Statistics 154 Summary plot 158 164 Symbols used 191 Temperature equilibration 170 172 Temperature plot 100 101 Toolbar 74 75 Tools menu 65 True mass 132 133 True mean time 108 109 Tutorial Calculating the g s distribution 5 20 Tutorial How to select the scans to analyze 20 27 Tutorial Multi species fits 44 57 Tutorial Simulating experiments with the Claverie simulator 57 60 Tutorial Single species fit 31 44 Tutorial Using the g s overlay graph 27 31 Tutorials Calculating the g s distribution 5 20 Claverie simulation 57 60 Multi species fits 44 57 Single species fit 31 44 Tutorial Calculating the g s distribution 5 20 Tutorial How to select the scans to analyze 20 27 Tutorial Multi species fits 44 57 Tutorial Simulating experiments with the Claverie simulator 57 60 Tutorial Single species fit 31 44 Tutorial Using the g s overlay graph 27 31 Using the g s overlay graph 27 31 Use true mean time button 108 109 User interface elements 212 DCDT User Manual aa DCDT User Manual SR DCDT User Manual Analysis window 75 77 Clone analysis to new window 63 Derive g s distribution branch 85 86 Edit menu 63 65 F
9. s Svedbergs data fit residual 1 The relative vertical size of the overlay and residual plot portions can be altered using the mouse When the mouse hovers over the region in between these plots the cursor changes to a vertical arrow and the division point between the graphs can be moved by clicking and dragging up or down with the mouse 7 Residual plot This residual plot is functionally equivalent to but formatted differently than the one that is shown in the lower panel of the Summary plot 6 above and also on the Graphical fit monitor tab of the Perform least squares fit page C Users Public Documents Visual Studio Projects DCDT Help Source HelpStudio3 hs1240 html E Residual plot The value of the rms residual for this fit is displayed as the plot title k d Note that if you prefer alternate plotting methods such as plots with points connected by lines or stick plots the plotting method can be quickly changed by right clicking on the graph and using the Plotting Method sub menu on the pop up graph context menu 8 Fitted function This plot simply shows the best fit curve for the data region that was actually fitted 163 DCDT User Manual DCDT User Manual a Sesera vpe tomis i t ini 1 species fit to g s data 0 40 0 35 0 20 0 25 0 20 0 15 a s OD Svedberg 0 10 0 05 0 00 0 05 36 38 40 42 44 46 48 50 52 54 56 53 GO 62 64 66 68 70 s Svedbergs 9 Da
10. 150 DCDT User Manual SR DCDT User Manual calculations i Confidence Interval Options babae Confidence Probability Level P Standard values 68 3 1 00 sigma Input special value 90 1 65 sigma input as percentage 0 01 to 99 99 95 1 96 sigma 99 2 57 sigma Computation Method 3 Search error ellipse Johnson amp Faunt less rigorous faster Bootstrap select data subsets and re fit N times N 500 amp Monte Carlo add noise to simulated scans re fit N 500 rms noise in raw scans 0 003 OD Find confidence limits from bootstrap or M C from Q Computed standard deviationsof parameter distribution _ Observed parameter frequencies needs N gt 400 Canea R The confidence limits are often not symmetric around the best fit value and thus true confidence limits usually cannot be reported as a value Selecting a computation method Three radio buttons allow the user to select a preferred mathematical approach for computing the confidence intervals These are Search error ellipse This method moves each parameter away from its best fit value until the chi squared increases to a limit that is bases on F statistics it evaluates the shape of the error surface near the minimum The implementation is based on reference 2 Section 7 2 The search of parameter space is made along axes of a hyperellipse that represent eigenvectors of the covariance matrix The calculation of
11. Because the initial guesses were close this first step has already produced quite a good fit in this case 40 DCDT User Manual SR DCDT User Manual B v E F o a a Y exp Yt Sead Flies eet ee B Switching to the Fitting parameters monitor tab Section 3 2 9 6 by pushing the tab control located along the bottom allows you to follow the fit progress by monitoring the actual values of the parameters and the reductions of the sum of squared residuals or chi squared as the fit improves The image below shows these data during the pause after the first iteration 41 DCDT User Manual DCDT User Manual aa 177 82 6 365379E4 6 267662E 3 C Either continue using the 1 iteration button Section 3 2 9 2 until the fit converges or use the Do fit button Section 3 2 9 1 to iterate to convergence without further pauses When the fit converges the program will automatically advance to the Report fit results page Section 3 2 10 5 Review fit results The Report fit results page Section 3 2 10 below shows the final best fit parameters Section 3 2 10 1 the values for any parameters that were held fixed Section 3 2 10 2 and summarizes the fit statistics Section 3 2 10 6 This page is the same in either Advanced or Standard modes 42 DCDT User Manual SR DCDT User Manual Parameter or Constraint Value offset OD S 0 00000 es Ailes
12. Getting Started How to use this Help file 2 3 Program overview and organization 1 2 Standard and advanced modes 3 4 Good data end at radius 97 98 Good fit 189 Graph bootstrap results 155 158 Graph customization 172 175 206 DCDT User Manual SR DCDT User Manual Graph fit results page 158 164 Graph Monte Carlo results 155 158 Graphical fit monitor 141 143 Graphs g s overlay graph 86 87 Help menu 72 74 Help on Help 2 3 High cross correlation warning 154 155 How and why to equilibrate the rotor temperature 170 172 How should I cite this program 190 How to Avoid common mistakes 166 167 Customize graphs 172 175 Equilibrate the rotor temperature 170 172 Export graph images or data 175 176 Identify and remove rogue scans 177 183 Optimize data acquisition 167 170 Print graphs 172 Remove bad data points 176 177 Select scans to analyze 20 27 Zoom with the mouse 176 How to avoid common mistakes 166 167 How to customize graphs 172 175 How to export graph images or data to other programs 175 176 How to identify and remove rogue scans 177 183 How to optimize data acquisition for g s analysis 167 170 How to remove bad data points from scan files 176 177 How to use this Help file 2 3 How to zoom with the mouse 176 How to How and why to equilibrate the rotor temperature 170 172 How to avoid common mistakes 166 167 How to customize graphs 172 175 How to export graph images or data to othe
13. Magnification 100 6 eT The file name and full path of the restored fit and the program version that created the new Log are also entered into the Log these are not visible in the image above 3 1 3 4 User prompt area The User prompt area provides textual prompts for certain actions and displays a position read out when the user is specifying positions on graphs Those prompts will automatically be cleared after being displayed for 5 seconds 3 1 4 Navigation tree The Navigation Tree is a heirarchical tree similar to that used in Windows Explorer and other programs It 83 DCDT User Manual DCDT User Manual a provides a root node branch for each analysis window two major sub nodes for the major tasks Derive 9 s distribution branch Section 3 1 4 1 and Fit distribution as N species branch Section 3 1 4 2 and minor sub nodes for the individual steps within those tasks Navigation Tree x g s Overlay Graph a Analyses CAUsers ohn Philo Desktop Pt Derive g s distribution Load raw scans Set meniscus amp data region Select scans to analyze Calculate average de dt curve Calculate g s distribution B Fit distribution as N species Select fiting model Set initial guesses on graph Set alter fitting parameters Perform least squares fit Report fit results Graph fit results 5 C Users John Philo Desktop Philo D B Derive g s distribution Load raw scans Set meniscus amp data region Select
14. Noa In this case the initial guesses seem to give us a reasonable starting point If you see a theoretical curve that is grossly different than the experimental data you should probably go Back and modify the initial guesses and or the fitting model Fit the data use the Do fit Section 3 2 9 1 button or step through 1 iteration at a time using the 1 iteration Section 3 2 9 2 button Hopefully you will see the theoretical curve move to overlap the experimental data If it appears the fit is having trouble converging you may wish to switch to the Fitting parameters monitor tab Section 3 2 9 6 to follow how the parameter values are changing If you are trying to fit more species than are justified by the data it is likely that the concentration of one species may be dropping to zero or its sedimentation coefficient may be trying to go out of the range covered by the data If so halt the fit using the Cancel Section 3 2 9 4 button and go Back 3 steps to modify your fitting model When the fit converges it will automatically advance to the Report fit results page Section 3 2 10 G Examine whether this seems to be a reasonable fit 52 DCDT User Manual SR DCDT User Manual Co fringes 0 2445 s S 3 556 M kDa 58 11 Co fringes 0 0255 s S 5 447 Parameter or Constraint Value offset frnges S 0 0000 M constraint 1 M 2 2 x M 1 we AlLess Q Back Nex 29 The fit results in our example
15. R Note that s will also sometimes be written in this document as s 20 w to avoid problems in generating subscripts Convert to 5 20 w The Convert to S20 w buttons are present on the Set meniscus amp data region page Section 3 2 2 the Calculate average dc dt curve page Section 3 2 4 and the Calculate g s distribution page Section 3 2 5 When the data are in raw sedimentation coefficient unit pressing this button will bring up a dialog box shown below for entry of the data needed to perform the conversion discussed in more 121 DCDT User Manual DCDT User Manual aa detail below If this conversion has already been done as indicated by the button remaining in the down state then pressing the button will turn off the conversion and revert to raw s values Conversion to s 20 w Values for experimental conditions partial specific volume ml g 0 7198 solvent density g ml 1 003070 solvent viscosity cp 1 0185 Temperature C 20 0 Partial specific volume at 20 C 7 calculate temperature correction partial specific volume ml g 0 7198 ratio s 20 w s raw 1 02920 ratio D 20 w D raw 1 01647 ae It is generally desirable to present and report s values rather than raw uncorrected sedimentation coefficients Conversion from raw s to S compensates for the factors such as buffer viscosity which are relevant only to a particular experiment giving the sedimentatio
16. indicated by vertical green lines 2 set an initial guess for the sedimentation coefficient of each species vertical magenta lines 134 DCDT User Manual SR DCDT User Manual Fitting range M Species properties updating i 2 S Makenew concentration and peak width guesses based on new peak positions 0 5 toj 8 Keep previous peak position concentration and peak width values aot N normalized g s per Svedberg 9 o o D oo i pad 00 05 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 s Svedbergs How to set the fit range You may wish to exclude some data at low or high sedimentation coefficients either because those regions are noisy and unreliable or because the data in those regions are influenced by species that are too poorly defined to be included in the fit The actual setting of the sedimentation coefficient range is usually done by dragging the green vertical bars that mark the limits of data to include in the fit e During the range setting procedure the actual position of the limit bar is constantly displayed in the user prompt area at the bottom of the analysis window At the end of the drag operation the limit will be set to the nearest value for which there is an actual data point e Upon entry into this procedure if a range has not previously been set for these data the program defaults to the full data range When the upper or lower limit are at the limit of the dat
17. it will be very difficult for the fit to accurately evaluate its sedimentation and diffusion coefficients especially D Therefore if you want more accuracy for the amount and sedimentation coefficient of the dimer fraction and there is a good reason for believing this is a dimer such as evidence from non reducing gels or SEC MALLS it may be a good idea to constrain the D value of the dimer to be consistent with a mass twice that of monomer That is if s and D are the current values of s and D for the monomer and s and D are the current s and D values for the dimer then at every iteration of the fit the program can force the s D ratio to be exactly twice the s D ratio i e D S x D 2xS Similarly if the fraction of dimer is very small a few percent or the signal noise is poor it may also be appropriate to constrain the dimer sedimentation coefficients to be an appropriate multiple of the monomer s value If each monomer is a hard sphere then theory says that s 1 45 x s reference 10 Section 7 10 At the other extreme if two spherical monomers merged to form a spherical dimer then s 22 3 s 1 59 S Thus the difficulty with constraining sedimentation coefficients is that the ratios depend on conformation These constraints can be very useful for hypothesis testing If we see that a sample fits poorly as a single species and or the fit implies a molecular mass significantly less than a known value broader b
18. or fit file saved by version 1 After the file is loaded if the saved results include a completed fit the program will bring up the Report fit results page Section 3 2 10 to show the results from that fit Otherwise the program will display a graph of the 9 s or dc dt results depending on how many steps toward calculating g s had been completed The path to this restored fit will be added to the top of the recent files list at the bottom of the File menu Section 3 1 1 1 k d Tip 1 Once you have opened a saved analysis you may want to use the Clone analysis to new window command to create a second copy that can be altered while preserving the original analysis in an open window for reference 7 Tip 2 Since the dcdt file extension will be associated with this program you can also restore a fit by P double clicking on the file name from Windows Explorer or in the Recent Documents list on the Start Menu Note that when a saved analysis is loaded the program attempts to set the default folder for scan file loading to the folder from which the data were originally loaded If that folder is not currently available for example if the data were on a removable drive or a network drive to which you are not currently connected there is often a delay of several seconds while Windows tests for what drives and folders are currently available 3 1 1 1 2 Save analysis This command saves an updated version of an analysis that was prev
19. to indicate this automatic correction is active e The jitter wizard will be invoked as soon as you set a meniscus position either by using the Enter key to confirm the program s automatic determination of the meniscus location or by manually dragging the red meniscus marker i If the g s distribution is significantly negative not just the expected negative points due to random noise this is a sign that the jitter correction may be poor Try moving the correction region to another location To manually select the match region Two gray vertical bars are displayed on the graph to mark the inner and outer radii for the match region When the Manual button is pressed a prompt is given to drag those gray markers to a new position Whenever the markers are dragged away from the position set by the jitter wizard the Manual button will change to the depressed on state When you drag either gray bar both bars will move together maintaining the same separation between them If you drag the gray markers while holding the Shift key down the two gray bars can be moved independently Once the Manual button is pushed this operation must be completed by dragging a gray marker until it is completed other controls will be inactive Note that moving the right marker far to the right of the meniscus will affect the lowest sedimentation coefficient that is included in the dc dt curves and g s distribution Only data points which are to the right of
20. to wait for the diffusion pump to cool a bug in the instrument firmware If you need to release the vacuum hit Stop and then wait 5 minutes for the diffusion pump to cool before you push the Vacuum button Failure to cool the diffusion pump may get pump oil on your optics including the diffraction 171 DCDT User Manual DCDT User Manual a grating in the monochromator 20 000 to replace 4 3 How to work with graphs 4 3 1 How to print graphs All graphs can be quickly printed by right clicking on the graph to bring up this context menu and selecting the bottom Print Graph to Default Printer entry Viewing Style Border Style gt Font Size Legend Style Numeric Precision d Plotting Method gt Data Shadows gt Grid Options gt Mark Data Points Display Mouse Coordinates V Round Axis Scales After Zoom Maximize Customization Dialog Export Dialog Help Print Graph to Default Printer That choice will print a full page graph in landscape orientation to the current default printer the Windows default printer not the printer specified for analysis reports from the Options dialog Section 3 1 1 4 2 If you want to print to a specific printer print a graph of a specific size or control whether the graph is printed with color or in monochrome you can do this by selecting the Export Dialog menu entry 4 3 2 How to customize graphs Click and edit customizations The axe
21. 128 kDa so there should be no significant peak broadening of any species within this range of sedimentation coefficients Actually in this case the Auto adjust Section 3 2 3 1 was probably too cautious about avoiding peak broadening and it would probably be fine to push up the number of scans to 28 which would give somewhat higher signal noise However these automatic settings are giving perfectly usable results which is exactly what they are supposed to do As you gain more experience you will probably want to at least tweak the values set by Auto adjust Section 3 2 3 1 It is quick and easy to see what happens as you move the sliders and you will learn a lot from that too Now push the Next button Section 3 1 3 1 to move to the Calculate average dc dt curve page Section 3 2 4 Display the average dc dt curve adjust data range for 9 s if needed 12 DCDT User Manual SR DCDT User Manual L Setrangeforais 0 198 to sse S 0 00018 Scans 000351P1 to 0050 11 from C Usersvonn Philo Deskiop Philo Data Prot 0 00016 0 00014 0 00012 0 00010 0 00008 0 00006 de dt fringes sec 0 00004 0 00002 0 00000 0 00002 0 0 0 5 1 0 1 5 2 0 25 30 35 40 45 5 0 5 5 6 0 6 5 s Svedbergs esd i More 9 Back he E This page displays the average dc dt curve with experimental error bars on each data point gray vertical bars barely visible in this case Usually no adjustments are nee
22. 155 158 3 2 11 Graph fit results page 158 164 4 Howto 166 4 1 How to avoid common mistakes 166 167 4 2 How to optimize your experiments 167 4 2 1 How to optimize data acquisition for g s analysis 167 170 4 2 2 How and why to equilibrate the rotor temperature 170 172 4 3 How to work with graphs 172 4 3 1 How to print graphs 172 4 3 2 How to customize graphs 172 175 4 3 3 How to export graph images or data to other programs 175 176 4 3 4 How to zoom with the mouse 176 4 4 How to remove bad data points from scan files 176 177 DCDT User Manual SR DCDT User Manual 4 5 How to identify and remove rogue scans 177 183 5 Frequently asked questions 184 5 1 About g s distributions 184 5 1 1 What is the difference between s and s 184 185 5 1 2 What is the difference between g s and g s 185 5 1 3 What is the difference between the broad and conventional g s 185 187 algorithms 5 2 About fitting 187 5 2 1 Should fit the offset 187 5 2 2 Why do get different answers when fitting g s versus dc dt 188 189 5 2 3 What constitutes a good fit 189 5 2 4 What does high parameter cross correlation mean 189 190 5 3 How should I cite this program 190 6 Reference information 191 6 1 Symbols used 191 6 2 About using radial intensity scans 191 192 6 3 About this g s implementation 192 193 6 4 About the fitting functions 193 194 6 5 About the fitting algorithm 194 6 6 About the meniscus w
23. 48 39 Loaded 54 new scans from folder C XLAWIN XLIDATA antibody 00001 RA1 00002 RA1 00003 RA1 00004 RA1 00005 RA1 00006 RA1 00007 RA1 00008 RA1 00009 RA1 00010 RA1 00011 RA1 00012 RA1 00013 RA1 00014 RA1 00015 RA1 00016 RA1 00017 RA1 00018 RA1 00019 RA1 00020 RA1 B 00021 RA1 00022 RA1 00023 RA1 00024 RA1 00025 RA1 00026 RA1 00027 RA1 00028 RA1 00029 RA1 00030 RA1 00031 RA1 00032 RA1 00033 RA1 00034 RA1 00035 RA1 00036 RA1 00037 RA1 00038 RA1 00039 RA1 00040 RA1 00041 RA1 00042 RA1 00043 RA1 00044 RA1 00045 RA1 00046 RA1 00047 RA1 00048 RA1 00049 RA1 00050 RA1 00051 RA1 00052 RA1 00053 RA1 00054 RA1 Wi 4 26 2012 20 48 40 Scans despiked spike threshold 0 2 4 26 2012 20 48 42 Meniscus position set as 5 9521 cm as suggested by the Meniscus Wizard 4 26 2012 20 48 49 Maximum radius being fitted set to 7 070 cm 4 26 2012 20 48 54 Scans used for calculating dc dt were set by the Auto Adjust wizard to 12 total scans from NNN RA1 tn NNNR4 RA1 y Mogni caton e 10018 78 DCDT User Manual SR DCDT User Manual The example above shows the first portions of the Log generated during the analysis of some absorbance scans As you see each of the steps taken analyst inputs and options selected is documented If the 9 s distribution is fitted the full report from that fit including a summary graph for the fit is placed into the Log The full Log
24. C Shadow Line Line Line _ No Border No Border C No Border OK Cancel Apply Export The Export Dialog context menu item brings up a dialog that allows printing the graph exporting the image to a file or the Clipboard and even exporting the data that creates the graph Note however that if you want to export data this is usually done more easily using the Edit menu Section 3 1 1 2 For graphs that contain markers such as the vertical lines to indicate the meniscus position or regions of interest if you don t want those markers to appear in the exported graph first uncheck the Show Annotations menu item before you export the graph The Help menu item brings up a small graphing specific Help file that discusses some of these options in more detail The Print Graph to Default Printer item sends a full page graph in landscape orientation to the current default printer the Windows default printer not the printer specified for analysis reports from the Options dialog Section 3 1 1 4 2 If you want to print to a specific printer print a graph of a specific size or control whether the graph is printed with color or in monochrome you can do this by selecting the Export Dialog menu entry Most of these context menu customization features are implemented by the Gigasoft ProEssentials http gigasoft com graphing library used by this program 4 3 3 How to export graph images or data to other programs Rig
25. Manual aa When should this option not be used You should definitely not use multiple sections if your primary interest is in the g s distribution itself or calculating properties such as the weight average sedimentation coefficient from it It s use for those purposes would result in incorrect proportions for multiple components and sedimentation coefficients that are less accurate 3 2 4 Calculate average dc dt curve page This page shows the average dc dt curve corresponding to the scans selected via the Section 3 2 3 Select scans to analyze page Section 3 2 3 and provides options such as converting the sedimentation coefficients to S59 w Values or limiting the range of data to be used in calculating the 9 s distribution Each control on the image below is a clickable hotspot that will lead to further information or just use the numbered list below the image Display options X axis point spacing Manual adjustment Set range for g s Convert to Automatic 3 20 w Show error bars 0 005 S Automstic Reset zero Undo 0 25 to 8 045 S Scans 00120 1F1 10 001631P1 fom AWINXUDAT AlW snopaces 1 iP FU0425031110945 de dt fringes sec 00 as 1 0 15 20 25 3 0 35 40 45 50 55 60 65 70 75 60 85 5 20 w Svecberge To calculate the average the dc dt curve from each scan pair is first interpolated onto the uniform grid of X axis values Those interpolated curves are then averaged and the standard devia
26. N 2 F Desprke spike thfeshold 0 200 OD 00123 1P3 00124 IP3 00125 IP3 x Sample description Run Gaveta simulation RRR m A Celes Hoc _Next Controls on this page T Preselect data type and cell check box Section 3 2 1 1 2 Load new scan files button Section 3 2 1 2 Replace scans for this sample button Section 3 2 1 3 88 DCDT User Manual SR DCDT User Manual 4 Add more scans for this sample button Section 3 2 1 4 3 Delete selected scans button Section 3 2 1 5 6 Scans currently loaded in memory list box Section 3 2 1 6 Scans to be used in g s calculation list box Section 3 2 1 7 Data loading options Section 3 2 1 8 Run Claverie simulation to generate data button Section 3 2 1 10 10 Sample description Section 3 2 1 9 3 2 1 1 Load raw scans Preselect data type and cell check box MV Preselect data type and cell Data type Cell number Absorbance C Interference 1 C Fluoresence When this box is checked then the Data type radio buttons and Cell number up down spinner controls within this group will control the data type and cell number of the scans that are initially displayed in the File Open dialogs kd Using this option makes it easier to find the data you are seeking among the hundreds of scan files that may be present If you change your mind after you are already working within the File Open dialog you can still choose different
27. Pasted Species plot for entire data range area graph into log 80 DCDT User Manual SR DCDT User Manual Scans 00022 RA1 to 00033 RA1 from CAXLAWIN XLIDATA antibody 0 40 0 35 0 30 0 25 0 20 0 15 g s OD Svedberg 0 1 2 3 4 5 6 T 8 9 10 11 12 13 14 s Svedbergs MMM species1 data procedure by the authentication system is also added into the log The user name is added whenever a new analysis is started or when a previously saved analysis is opened Thus if user A does the initial analysis and later user B re opens the analysis file and re analyzes the data in a different manner the log will show who did what and when i If your IT system provides user authentication the user s identity is validated during a log in procedure then the analyst s name as given ab Buttons and other controls on the Log page The Print log button along the bottom of the log page allows the entire Log or a selected portion of it to be printed to the printer you select from a dialog box The Copy to clipboard button will place the entire Log or a selected portion of it onto the Clipboard in rich text RTF format so that it can be pasted into a word processor or electronic notebook software 81 DCDT User Manual DCDT User Manual aa e f you want to print or copy only a portion of the Log first highlight select the desired portion by dragging over it with the mouse or using the
28. Philo Data PKR K296R cell 00120001632 piae Ja CA APhBO Data PKR_K296R cel 00120 001631P1 olol j De amp lore x 5 cae a CA Philo Data PKR_X296R cell 00120 0016423 ep SST Se how negak over dsintubon Set integration regen Normatice Sonwent ec20 w Select scans to analyze Cakuate average doit curve Cakuate g s distribution Fi dstrbuton as N species weight average s 3 718 0 009 total c 0 275 0 000 baane C0120 152 mo ODIM IPA thom C UAE A CATA ancl Pe R_ DOR OOD I t 0 16 T T T T T T T T T T T T T Select fting modal 04 4 0 12 4 cy gt H _ owt J Navigation 2 g s z 9 z poe S 2 no 4 Tree ll sill 5 gig Analysis window Cakulate average doit curve b g 006 4 Cakuate g s distribution a 2 2 Fr dstrbuton as N spaces a 004 J Salect fting model a g a 002 4 p 00 Ci Users Jotn Phil Deaktop Phio Da p 02 4 Dative gia distribution Load raw scans Sat meniscus amp data ragion 004 10 158 20 26 30 36 40 45 60 Select scans to analyze 20w Sve el Cakuate average dot curve Cakuate gis dstrbuton Fe dstrbuton as N spaces r i Main menu amp toolbar f DCDT has a main program menu with the standard File View Window and other main headings you find in many Windows programs Analysis documents can be created opened and saved using the familiar New Open and Save commands under the File me
29. Q Bock Her Ei A Notice that by limiting the fitting range to the center of the main peak the molecular mass value has now risen to 153 kDa versus the 146 kDa obtained when the entire range including the aggregates was fitted as a single species see the easy way fit above By not fitting the data that are strongly influenced by aggregates data that obviously can t be properly fitted by a single species model we obtain a much better molecular mass estimate This value is also close to the 150 kDa expected for an antibody monomer but the mass calibration is probably uncertain by a few percent in this case because neither the density or partial specific volume are accurately known B You may want to push to the Next button to generate a species graph an overlay of the data and fitted curves or a residual plot to examine the quality of fit more closely The graph shown below where the theoretical fit is extended outside the actual range that was fitted helps to show that there is clearly a significant fraction of the sample sedimenting above 8 S that is not accounted for by this single species In this case the presence of those aggregates is also easily seen in the area type of species plot but low levels of minor components are often more easily seen in this line plot 43 DCDT User Manual DCDT User Manual a Scans 00034 RA1 to 00047_RA1 trom C iUsersionn Philio Desktop Pnilio Dataiantinodyi o 8 0 15 g s
30. Section 3 2 3 2 says that all species larger than only 1 kDa are being significantly broadened 23 DCDT User Manual DCDT User Manual aa Adjust scans used in computing de dt Scansused 30 of 60 lt Communal Position within run lt 4 Cam 00016 RA1 00031 RA1 00019 RA1 00034 RA1 00025 RA1 00040 RA1 meee SV ETSQE 2 The image above is the result of moving the top slider to the middle giving 30 scans used Now the average dc dt curve starts to resemble a peak but the curves from the 15 individual scan pairs are still fairly different and the peak broadening limit is still only 17 kDa 24 DCDT User Manual SR DCDT User Manual Adjust scans used in computing de dt Scansused 10 of 60 lt Troon Last scan 00035 RA1 209 Scans C0026 RA1 20 COOSS RA1 fom CXLAWINIXLUDAT Alsniibody T 8 a 2 3 a s Svedbergs 00030 RA1 00035 RA1 mm average 3 Above are the results after moving the slider further to the left dropping to 10 scans used Now we are in the right range the individual dc dt curves are close to the average some spread near the top of the peak is normal and the peak broadening limit is gt the mass of an antibody monomer Reducing the number of scans further down to 4 6 scans would increase the resolution for very high mass species but also would degrade the signal noise significantly Note that for absorbance scans it is
31. Set meniscus amp data region page Section 3 2 2 to graph the raw data Push the ID points button to display every data point as a clickable object Click on the bad data point and its x y coordinates and scan file name will be displayed OF oe oY Click on this same point a second time and you will be asked to confirm that you want to permanently remove that point and then asked whether you want to remove it in an irreversible or reversible manner explained below If you choose to make an irreversible deletion a back up copy of the un altered scan file will be created by adding an extra bak extension on the scan file name Although the easiest way to remove bad points is the one described above from the Load raw scans page Section 3 2 1 you can also right click on a scan file name and then automatically bring that scan into Notepad for editing If you do edit scan files using the right click pop up menu from the Load raw scans page Section 3 2 1 the program will automatically reload that edited scan file Alternatives for removing bad points from scan files Although it possible to simply delete the data row corresponding to a bad data point from scan files that procedure leaves no record that a bad data point was removed and does not permit easy restoration of the original data To address these concerns this program implements the University of Connecticut convention for marking bad data points in a manner where the origin
32. This should nearly always be true for absorbance data unless perhaps there are low mass absorbing species that are not well matched between sample and reference On the other hand if the g s data do not return to zero or go negative in regions away from the peak s then it will be impossible to obtain a good fit unless you include an offset With the dc dt data it is more difficult to judge the zero level since the data will not return to zero on the right side of peaks Be careful though because the presence of species sedimenting outside the range of sedimentation coefficients covered by your data can give the appearance of a zero offset problem With interference data and particularly for low concentration samples even the jitter removal Section 3 2 2 4 procedure may not get the zero level set correctly This issue is of course also related to whether or not you choose to manually adjust the zero levels of the data using either the Reset zero for average dc dt Section 3 2 4 4 or Reset zero for g s Section 3 2 5 7 procedures In principle if that manual adjustment is done correctly then the offset fitting parameter should be left at zero 187 DCDT User Manual DCDT User Manual aa 5 2 2 Why do I get different answers when fitting g s versus dc dt The short answer is that these two fitting approaches differ in the approximations involved and their sensitivity to certain errors Therefore the results should be somewhat diff
33. Tl 00092 IP3 6 8291 0 8463047 Fringes 57 58 59 60 61 62 63 64 65 66 67 68 69 70 7 1 72 73 74 Radius cm COOSONPS OO COOS1IFS COOS2IP3 A COOSZIFS VQOCE4IF3 COOESIFS M COOEEIFS COCETIF3 COOESIP3 COOESIFS COOSOIPS COOSTIPS COOSZIFS O COOSSIFS COOSLIF3 A COOSSIFS V COOSEIFS COOSTIPS m QOOSEIFS CCOSSIF3 A COICOIPS FOCIO1IPS COIODIPS CO1OSIPS COIOSIFS O OO10SIF3 COIDEIF3 COIOTIPS TOO10EIF3 001091P3 MCO11ONPS 001111IP3 amp 00112IP3 00113IP3 00114IP3 X CO1ISIPS O 001161P3 O 001171P3 00118IF3 A 001193 T O0I20IP3 00121IP3 M 00122IP3 00123IP3 001241IP3 COITSIPS 001251P3 6 COTZTIPS 001281P3 O 001293IP3 Click the same point again if you want to delete it x Less Back Next Once the rogue scan is identified use the Back button Section 3 1 3 1 to return to the Load raw scans page Section 3 2 1 Click on the file name of the rogue scan within the Scans currently loaded in memory Section 3 2 1 6 list box as shown below 182 DCDT User Manual SR DCDT User Manual ra Analysis1 IV Preselect data type and cell Scans currently loaded in memory 00080 IP3 Scans to be used ing s calculation 00080 IP3 Cell number Data type Absorbance 00081 IP3 00082 IP3 00083 IP3 00084 IP3 00085 IP3 00086 IP3 00087 IP3 00088 IP3 00089 IP3 00090 IP3 00091 IP3 00081 IP3 00082 IP3 00083 1IP3 00084 IP3
34. a good basic summary of the fit and whether it described the data well without actually plotting the contributions from each individual species 161 DCDT User Manual DCDT User Manual aa Select graph type from list Species plot for fitted range ine w Erin iCopy Addtotog Scans 00027 RA1 10 COOSE RA1 tom Cisedirdswisnmbody 0 4 on g s OD Svedberg 36 38 40 42 44 46 42 so s 54 s6 s 60 62 64 66 6s 70 s Svedbergs data species 1 The number of species and the type of data being fitted are indicated in the graph title line This plot is essentially the upper panel of the plot shown on the Graphical fit monitor tab of the Perform least squares fit page Section 3 2 9 6 Summary plot overlay residuals This plot creates a summary of the fit by combining an overlay plot 5 above with a residual plot 7 below into a single graph object with two sets of Y axes This plot is the same as the plot shown on the Graphical fit monitor tab of the Perform least squares fit page Section 3 2 9 and the one that is used in printed reports and automatically added to the Log page 25B418A9 EB1A 4DFC AB85 7555824F58A4 162 DCDT User Manual SR DCDT User Manual Select graph type from ist Summary overiay residuals w eprint copy Addto Leg 1 species fit to g s data 0G 0 DISvedber v xp vad 36 38 40 42 44 46 E so s2 54 s6 s 60 62 64 66 6 70
35. add many more scans in the analysis to improve the signal noise and see whether this peak is still present In such a test the peak for a real species would be broadened but it should not disappear so the qualitative result peak or no peak is enhanced even though this rule of thumb would be grossly violated For a more detailed analysis of how the accuracy of fitted parameters derived by the classic fitting methods is affected by the time span see Fig 6 in reference 8 Section 7 8 and the discussion therein Briefly simulations indicate that for M M max the errors in D relative to the value obtained with no peak broadening are 14 17 and those in M are 12 15 For smaller time spans these errors are reduced approximately linearly with the ratio of MMM max or equivalently approximately with the square of the number of scans 3 2 3 3 Convert to s 20 w This option will convert sedimentation coefficients for dc dt and g s curves as well as fitted values to the scale S20 w aa Note that s will also sometimes be written in this document as s 20 w to avoid problems in generating subscripts Convert to s 20 w The Convert to S20 w buttons are present on the Set meniscus amp data region page Section 3 2 2 the Calculate average dc dt curve page Section 3 2 4 and the Calculate g s distribution page Section 3 2 5 When the data are in raw sedimentation coefficient unit pressing this button will bring u
36. adjustments of the zero level Use with caution Undo will remove the manual adjustment 3 2 4 1 Calculate average dc dt X axis point spacing M X axis point spacing 0 005 s Automatic This group of controls is used to set 6s the increment of sedimentation coefficient between data points for both the average dc dt curve and the g s curve The default Automatic option which is on when the button appears depressed invokes a program wizard to set this value automatically The wizard will choose a value for ds that gives a total number of data points along the sedimentation coefficient axis between 1 and 2 times the number of data points in the corresponding region of the raw scans These automatic values are rounded to give intervals in a 1 2 2 5 3 4 5 10 sequence Alternatively any point spacing value can be entered manually into the text box to the left of the Automatic button which will toggle the Automatic button off If the Convert to s 20 w Section 3 2 3 3 option is on ds will be in s 20 w units If you manually set ds to a rounded value 02 05 and then turn the conversion on or off the interval will no longer be a rounded value When the Automatic option is on as the the conversion to s 20 w units is turned on or off the program will recalculate the interval to keep it as a rounded number If the ds value is altered after the average dc dt curve has already been calculated that calculation will b
37. always kept in raw units the conversion is only applied as graphs are generated and values are displayed and printed Generally this is transparent to the user but with regard to setting the ds intervals for the average dc dt and 9 s curves it is not possible to have conveniently rounded intervals between data points in both raw and S20 w scales Consequently switching between raw and S units will usually force a re calculation of the average dc dt curve and g s distribution because the X axis point spacing will change if that is being determined by the default Automatic option see Calculate average dc dt X axis point spacing Section 3 2 4 1 Thus the average dc dt curve and g s distribution will be slightly different in the different units the difference is not simply a multiplicative factor for the X axis However those differences will be negligible compared to the noise in the data How this conversion affects results from fitting When this conversion is used the sedimentation coefficients obtained by fitting will also be s values and the diffusion coefficients from fitting will be D values They will be marked as such on all program outputs and the values used to make the conversion will be printed along with the printed reports of fit results Switching between raw and s units will usually force a re calculation of the average dc dt curve and 9 s distribution because the X axis point spacing wi
38. and cubic octamer replace that table when the Show bigger oligomers button is pressed In this example species 2 is being constrained at the 1 45 ratio appropriate for a dimer of hard spheres These ratios for oligomers come from Garcia de la Torre J and V A Bloomfield 1981 reference 10 Section 7 10 k d In some cases it may be better to constrain the smaller species to be a sub multiple of a larger one For example if you had a sample where the native protein is a dimer but it contains a small fraction of a denatured or incompetent monomer it would be best to constrain the monomer mass to be half that of the dimer and perhaps set the monomer s value to be 0 69 that of the dimer i e the inverse of the 1 45 ratio shown in the table Other buttons When you are done setting constraints the Ok button will accept the current settings and return you to the Select fitting model page Section 3 2 6 If you want to undo any modifications and return to the settings present when this dialog box was opened use the Cancel button which will close the dialog and return you to the Select fitting model page Section 3 2 6 This group of 3 buttons provides quick mechanisms to clear out unwanted constraints The usage is obvious from the names of the buttons Note that if you reduce the number of species from the Select fitting model page Section 3 2 6 after constraints are active any constraints to or from species that no longer e
39. and partial specific volumes i solvent density 1 030000 gt sD Svedberg per Fick ajii i J Use the same partial specific buoyant molecular mass kDa volume for all species temperature must be known z k partial specific volume of Temperature C 20 0 HLE ee kDs eee oie 0 7320 temperature solvent density and a solute partial specific volume must a be known m A Less Q Back B Set the number of species to 1 it will probably already be at 1 C In the Data choice Section 3 2 6 3 section choose whether you are going to fit the 9 s or the dc dt data Fitting to dc dt is sometimes advantageous for very low molecular weight samples see reference 8 Section 7 8 D In the Fit diffusion or mass Section 3 2 6 4 section choose whether you prefer to use diffusion coefficient as a fitting parameter or M the ratio of s D instead E In the Function computation Section 3 2 6 2 section you will probably want to use the new improved method but you can choose the less accurate classic method if you wish F In the Weighting of data points section Section 3 2 6 5 choose whether you want to use a weighted fit data points with low standard deviation count more or a non weighted fit all points count equally The new theoretical weights option is recommended when you are fitting to the 9 s data G If you have chosen M as the fitting parameter be sure you have entered the correct partial
40. be indicated by a colored block with a caution symbol as shown below Be sure the rotor temperature is equilibrated before starting your run even the best software cannot overcome bad data Material that provides background information or details will be indicated by a colored block with the teacher symbol as shown below E The intellectually curious user will want to know that Such information may also be found within an expandable text block in some cases Send Comments link At the upper right corner of every topic is a link that allows you to send an e mail to the author if you find errors in the Help file if you have requests for new topics or tutorials or if you have other comments See Also links A See Also section like that below that leads you to related topics and further information will be found at the bottom of many topics The See Also link symbol at the upper left corner of the topic page takes you directly to the See Also list 1 3 Standard and advanced modes There are two program states or modes for each Section 3 1 3 analysis window 1 In Standard mode certain program options and steps are hidden to keep the user interface simple and keep features that may not be needed out of the way 2 In Advanced mode all program options and steps are shown and available for use Each Section 3 1 3 analysis window can be toggled between these modes using the and buttons the button text and graphic toggle
41. both the leftmost green marker and the rightmost gray marker will be used for data analysis Since the data in the match region are being forced to be zero it makes no sense to include them in the analysis To turn off jitter correction Jitter correction is off whenever both the Auto button and the Manual button are up off Simply press whichever button is down to turn jitter correction off 3 2 2 5 Set meniscus amp data region Fringe jump removal options 99 DCDT User Manual DCDT User Manual aa a Fringe jump removal options are displayed only in Advanced mode Section 1 3 and active only for interference data Fringe jump removal Auto Middle Manual This group of controls selects the method used to compensate for any fringe jumps that occur within a group of interference scans that are being analyzed The methods differ only in the region of the scan that is used in comparing successive scans for a possible fringe shift Four automatic methods are selectable via a drop down list box Those are 1 Entire scan compares the scans across the entire sample area to the right of the meniscus This will work well as long as the amount of sample movement between scans is not too large moderate concentrations and sedimentation rates which is generally true for scans appropriate for the dc dt method 2 Near meniscus confines the comparison to the part of the sample near the meniscus 60 data po
42. broad algorithm calculates a g s from each scan pair then averages these to get an average g s and then calculates 9 s from the average g s The broad algorithm also allows an alternate calculation of the average dc dt curve see below This broad algorithm has some advantages when the time span of the data is long and the peaks are being signficantly broadened hence the name broad algorithm When the time span is very short and the peaks are not being broadened the two are equivalent The actual g s and g s curves from the two algorithms are essentially identical what differs are the error bars on those values and therefore the uncertainties in derived fitted parameters weight average sedimentation coefficients etc The conventional algorithm tends to over estimate those error bars when the time span grows long and this is at least partially corrected by the broad algorithm Because the broad algorithm gives smaller error bars on 9 s it also tends produce lower uncertainties for the parameters derived by fitting to g s when the fit uses the classic method and is being weighted using the estimated error bars for each data point This improvement in precision is greatest for sedimentation coefficients and loading concentrations there is little improvement in the precision of D or M The graph below figure 3 from reference 8 Section 7 8 shows how the broad algorithm panel B reduces the error bars as more scans ar
43. constraints on sedimentation coefficients it is important to remember that the ratios for oligomers are sensitive to the shape and configuration of the oligomers and thus it is hard to predict the correct ratio For this reason for final results it may be desirable to relax such constraints However if you are trying to detect small amounts of one species and are more interested in knowing the amount of this species than its hydrodynamic properties you will obtain lower uncertainties for the concentrations and for the hydrodynamic properties of other species if the s ratio constraint is used In this example if we remove the constraint on the mass of the second species and re fit the data the fit does converge but in some cases the constraint s may be required for convergence Here we get the following results Species Parameter Best Value 95 0 Confidence Region Starting Valus View 0 242 0 237 0 243 cross correlations 3 549 3 533 3 553 59 15 58 16 61 53 Compute confidence intervals 0 028 0 027 0 033 5 395 5 269 5 419 Print 94 04 62 04 99 88 UIA Report and Fit statisties Sum of squared residuals 5 874445E3 Parameter or Constraint Value Degrees of freedom 1187 offset fringes S 0 0000 RMS residual 2 31911E 3 This un constrained fit is not too different from the earlier one although the best fit mass for species 2 has now dropped to be only 60 larger than species 2 Note however
44. data types or cells by making a different choice in the Files of type drop down list near the bottom of that dialog box Note that the Absorbance choice also includes radial intensity scans which can be converted to true absorbance or pseudo absorbance for when you are using two samples per cell See About using radial intensity scans Section 6 2 The cell selection spinner allows cell number 0 because SEDFIT creates cell 0 files when it saves simulated data It also allows cell 8 for those who replace the counterbalance with an additional cell The preselect mode is on by default when the program is installed You can turn off this default on the General Options tab of the Options dialog Section 3 1 1 4 2 3 2 1 2 Load raw scans Load new scan files button Load new scan files This button is used to initially load scan files into a new analysis window or to replace all the data that was previously loaded into that window It will bring up a standard Windows file open dialog which allows you to select and load multiple scan files you will want to load a minimum of 4 89 DCDT User Manual DCDT User Manual aa Tip 1 Generally you will want to load scans covering nearly the entire range of boundary movement i e all or nearly all the scans If you know your latest scans go long after the cell is empty there is no point in loading those and you also may want to skip the first 10 of the run where the meniscus re
45. distribution page Section 3 2 5 for each of these analysis windows and pushing the Normalize button Section 3 2 5 4 when we return to the 9 s overlay plot window we get the following much improved result 28 DCDT User Manual SR DCDT User Manual rin Overlay distributions o x 00 05 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 s Svedbergs C 042903 110946 cell3 00120 00164 IP3 O C 042903 110946 cell2 00120 00163 IP2 CA 042903 110946 cell1 00120 00163 IP1 Graph style Graph all Print Export Points C Lines Edit legend text c 1042903 1 10946 cell3 00120 00164 IP3 fi Points and lines Note that the graph updated automatically as we normalized the distributions From this graph it is apparent that at 1 1 mg ml the fraction of the shoulder at 5 5 S has increased significantly and the main peak near 3 5 S also shifts slightly to the right Those changes prove this protein exhibits reversible association On the other hand the fact that the shoulder changes little upon diluting from 0 32 to 0 11 mg ml suggests some of this material may be irreversible rather than reversible 47 For such comparisons it is best to be sure the scans chosen for analysis give nearly equal peak P broadening limits Section 3 2 3 2 for all the samples For samples within the same run that will usually be true if the same scan numbers are used for each sample Thus once you have analyze
46. error band on the distribution was as good or slightly better with 1 replicate Similarly the uncertainty in s and D values obtained by fitting the peak were as good and perhaps slightly better for the experiment using 1 replicate Furthermore the setup using 4 replicates requires gt 2 5 minutes per scan 8 minutes for 3 cells and such long scan times have drawbacks that may override signal noise considerations The consequence of having a low scan rate is that the total number of scan pairs in the analysis will usually be quite small Therefore moving the start of the analysis forward or back by a single scan will shift the range of sedimentation coefficients by a substantial amount and adding or subtracting even one scan pair may have a large effect on peak broadening and signal noise Overall the default velocity mode setting of continuous mode 0 003 cm spacing and 1 replicate is probably best in most situations In those rare situations where the random optical signal noise is dominant over the systematic noise very low or very high absorbance or when working at a wavelength with low light intensity and where the low signal noise means the sedimentation coefficients will not be highly accurate anyway the best setup should more closely follow the figure of merit criterion and it therefore will probably be best to use replicates 170 DCDT User Manual SR DCDT User Manual 4 2 2 How and why to equilibrate the rotor tempera
47. from the above analysis is contained in this drop down section as an example 79 4 26 2012 20 48 39 Loaded 54 new scans from folder C XLAWIN XLIDATA antibody 00001 RA1 00002 RA1 00003 RA1 00004 RA1 00005 RA1 00006 RA1 00007 RA1 00008 RA1 00009 RA1 00010 RA1 00011 RA1 00012 RA1 00013 RA1 00014 RA1 00015 RA1 00016 RA1 00017 RA1 00018 RA1 00019 RA1 00020 RA1 00021 RA1 00022 RA1 00023 RA1 00024 RA1 00025 RA1 00026 RA1 00027 RA1 00028 RA1 00029 RA1 00030 RA1 00031 RA1 00032 RA1 00033 RA1 00034 RA1 00035 RA1 00036 RA1 00037 RA1 00038 RA1 00039 RA1 00040 RA1 00041 RA1 00042 RA1 00043 RA1 00044 RA1 00045 RA1 00046 RA1 00047 RA1 00048 RA1 00049 RA1 00050 RA1 00051 RA1 00052 RA1 00053 RA1 00054 RA1 4 26 2012 20 48 40 Scans despiked spike threshold 0 2 4 26 2012 20 48 42 Meniscus position set as 5 9521 cm as suggested by the Meniscus Wizard 4 26 2012 20 48 49 Maximum radius being fitted set to 7 070 cm 4 26 2012 20 48 54 Scans used for calculating dc dt were set by the Auto Adjust wizard to 12 total scans from 00022 RA1 to 00034 RA1 4 26 2012 20 49 08 Lower sedimentation coefficient limit for g s computation was manually set to 0 3 S 4 26 2012 20 49 18 Current fitting model is 1 species fitting the g s data using the improved method The species molecular parameters are sedimentation coefficient and molecular mass in true mass units 4 26 2012 20 49 3
48. fs o ds s e s ads t s e s ds where n 1 0 1 2 correspond tos s Sz ands respectively z 1 The uncertainty in loading concentration c 0 is given by G fo ds 1 3 2 5 4 Calculate g s distribution Normalize option Normalize Normalization is used to re scale the g s distribution so the total area under the curve is exactly 1 100 For a normalized distribution the area of each peak gives the fraction of that species Normalization is particularly useful when you want to visually compare g s distributions from samples at different loading concentrations such as a dilution series on the g s overlay graph Section 3 1 5 Affects on fitting results When you fit individual species to a normalized distribution the fit returns the fraction of that species rather than its concentration in instrument units BE Note that the G s distribution the integral distribution is always normalized and thus technically the g s distribution should also be normalized since it is supposed to be the derivative of G s That is we should have g s 6 G s ds However the g s distributions created by Walter Stafford s original DCDT algorithm were not normalized so un normalized distributions become the usual convention for this method 3 2 5 5 Convert to s 20 w This option will convert sedimentation coefficients for dc dt and g s curves as well as fitted values to the S scale 20 w
49. gt ie wo oa 2 0 25 3 0 35 40 45 50 55 6 0 6 5 s 20 w Svedbergs When those values are correct push the Ok button and we now get the graph above in s 20 w units 18 DCDT User Manual SR DCDT User Manual Integration range Full Range 1 728 to 3 552 S Properties at time of analysis from g s Concentration 0 9370 0 0018 fringes Fraction of total 76 09 0 09 Number average s value 2 516 0 010S Weight average s value 2 601 0 008 S z average s value 2 685 0 011 S 2 1 average s value 2 764 l 0 021 S Properties extrapolated to t 0 from g s Loading concentration 1 0925 0 0021 fringes Fraction of total 76 01 0 08 Number average s value 2 529 0 009 S Weight average s value 2 614 0 008 S z average s value 2 697 0 013 S z 1 average s value 2 776 I 0 025 S Note stated uncertainties are 1 sigma and sedimentation coefficients are s 20 w values At this point if your data shows more than one peak you may wish to integrate that peak to compute the concentration of that species its fractional area and average sedimentation coefficients If you push the Set integration region Section 3 2 5 2 button and then click and drag with the mouse over the sedimentation coefficient region of interest those calculations will be done and displayed in this dialog box shown above these results are for the middle portion of the single peak e If you instead push the Sh
50. guess for species 1 It then positions the sedimentation coefficients for any additional species in positions appropriate for a dimer trimer tetramer hexamer series 1 If needed drag the magenta markers indicating the initial guesses for the sedimentation coefficients to the proper position o In this case the wizard has done all the work for us and these estimates are reasonable starting values for our monomer dimer mixture 2 If needed drag the green markers that define the left and right ends of the data to be fitted Your sample might for example contain aggregates or small fragments that you wish to exclude from the analysis by narrowing the range of sedimentation coefficients to be used for fitting 3 Push the Next button to go to the Set alter fitting parameters page Section 3 2 8 E Optionally alter the initial guesses or which parameters are being fitted 50 DCDT User Manual SR DCDT User Manual Fit this Parameter Value parameter Co 1 fringes 0 226 lV Yes s 1 S 3 455 lV Yes M 1 kDa 69 93 7 Yes Co 2 fringes 0 039 iW Yes s 2 S 5 010 lV Yes M 2 kDa 139 37 constrained offset fringes S 0 0000 J No Convergence control Convergence factor 10 Pammater racion 3 005 iG B Less Back 1 You may wish to alter the initial guesses for the sedimentation coefficient and D or M if you think the estimates made from the graph are poor The estimates for D or M m
51. is used within a modified Gauss Newton fitter to try to ensure that each iteration actually lowers the variance When the value falls below 1 that indicates the fitter is having difficulty finding improved parameters It is normal however for this value to fall just before the fit converges because further improvement is not possible 3 2 9 6 1 Increment reduction factor The increment reduction factor is a numerical multiplier used within a so called modified Gauss Newton fitter to improve the rate of convergence reference 2 Section 7 2 When this value falls below the nominal value of 1 this is an indication that the fitter either 1 is having some difficulty finding a good fit there are too many species or very poor initial guesses or 2 it is so near to convergence that it simply cannot find better parameter values How it works Let p1 p2 be the set of current fitting parameters Then at each iteration the Gauss Newton method calculates a set of increments to those parameters 5p1 5p2 that will define a new set of improved parameters p1 6p1 p2 dp2 which should give a lower variance In the modified method these increments are each multiplied by a scalar factor a the increment reduction factor and hence the new parameters will be p1 a x 5p1 p2 a x dp2 This modification is used because when the fit is far from the optimal parameters the Gauss Newton method tends to pick a reas
52. levels can be set within the Fitting Options tab of the Section 3 1 1 4 2 Options dialog Section 3 1 1 4 2 e These warnings will also appear in printed reports Section 3 2 10 5 The actual cross correlation matrix can be viewed by using the view parameter cross correlations button Section 3 2 10 3 on this page 3 2 10 8 Report fit results Graph bootstrap or Monte Carlo results Graph Monte Carlo results Graph bootstrap results These buttons become active whenever confidence limits have been calculated using either the Monte Carlo or bootstrap methods respectively the button name changes to match the method that was last used Pressing the button will bring up a dialog box that displays graphs of the parameter distributions using the table of parameter values from each round of bootstrap or Monte Carlo Initially this dialog box will display a histogram of the mass values for species 1 assuming that parameter was fitted like that shown below The vertical red line marks the best fit value The vertical green lines on the histogram show the 10 and 2o limits as calculated separately for the values above and below the mean The magenta curve shows the cumulative frequency the area under the distribution curve 155 DCDT User Manual DCDT User Manual a Graph Bootstrap Results esl Graph type Q Single parameter histogram Two parametercross correlation plot Parameter selection Para
53. may simply enter any desired values and the corresponding molecular mass will be calculated When numbers are manually entered in the molecular mass box then the corresponding D is calculated and the label of this D value changes to calculated D Species buttons After a fit has been completed a number of buttons at the top of the form will become active to correspond to 66 DCDT User Manual SR DCDT User Manual the number of species in that fit 2 in the case shown above Pressing these buttons will fill in the parameters from that fit The partial specific volume value associated with that species will also be used if that has been filled in on the Section 3 2 6 select fitting model page Copy button The Copy all values to Clipboard button will copy the labels and values for these entries as a small table to the clipboard with the labels and values separated by tab characters and a separate line for each type of entry About temperature The program automatically fills in the sample temperature based on the temperature recorded in the header of the last scan used in calculating the dc dt data but since the temperature may drift during the run you may wish to adjust that value manually The program internally converts the temperatures from Celsius to Kelvin scales Please note that changing the temperature on this form overrides the temperature recorded with the scans and also alters the value used elsewhere in
54. monomer mass as a fitting parameter See Constrain M or s ratios among species Section 3 2 6 6 Why this works The Svedberg equation shows that the s D ratio is directly related to molecular mass M RT M D 1 Ve where R is the gas constant V is the partial specific volume p is the solvent density and T is temperature Kelvin How this works As stated above internally the program always uses the value of D to determine the shape of the corresponding peak When you choose to use M as the fitting parameter during each iteration of the fitter the correct D value is calculated from the current values of s and M Since variations in the s value will also alter the apparent mass the s D ratio it is also necessary to adjust the derivatives of the fitting function to account for that effect 3 2 6 5 Select fitting model Weighting of data points 128 DCDT User Manual SR DCDT User Manual Weighting of data points Theoretical Experimental C None 5 This group of three radio buttons determines whether you wish to assign a statistical weight to each data B point or instead to weight all the data points equally the None option For weighted fits there are two choices for determining the weight to give each point 1 The Experimental weights option uses the standard deviation c assigned to each data point in the g s or dc dt data The weights are then given by 1 02 so the data points with the highest p
55. new data set is then fitted those parameters are stored and the process is repeated many times to build up a distribution of parameter values The confidence limits are then calculated from that parameter distribution based on options as described below The up down spinner next to this radio button sets the number of Monte Carlo rounds default 500 The rms noise level to add to the raw scans can be entered manually in the text box below this radio button If you are fitting simulated data you should enter the actual value used in the simulation When you first bring up this dialog after a new fit an estimate of the noise in the raw data is made and automatically entered into this text box If you later want to re calculate and re enter this estimate use the Recalc button See About the Monte Carlo method Section 6 9 for more details about how this estimate is made Finding the confidence limits from the bootstrap or Monte Carlo results Once a table of parameter values is built from the N rounds of bootstrap or Monte Carlo there are two choices for calculating the parameter confidence intervals Computed standard deviations of the parameter distribution With this choice first for each parameter the mean and standard deviation of the set of N values for that parameter is calculated and then the confidence limit is found assuming that distribution is random a Gaussian distribution This option is generally the best choice when the number
56. of conversion to the s20 w scale the Y axis must be contracted or expanded to keep the area unchanged Internally in the program the dc dt g s and g s data are always kept in raw units the conversion is only applied as graphs are generated and values are displayed and printed Generally this is transparent to the user but with regard to setting the ds intervals for the average dc dt and 9 s curves it is not possible to have conveniently rounded intervals between data points in both raw and s scales Consequently switching between raw and s _ units will usually force a re calculation of the average dc dt curve and 9 s distribution because the X axis point spacing will change if that is being determined by the default Automatic option see Calculate average dc dt X axis point spacing Section 3 2 4 1 Thus the average dc dt curve and 9 s distribution will be slightly different in the different units the difference is not simply a multiplicative factor for the X axis However those differences will be negligible compared to the noise in the data How this conversion affects results from fitting When this conversion is used the sedimentation coefficients obtained by fitting will also be s values and the diffusion coefficients from fitting will be D values They will be marked as such on all program outputs and the values used to make the conversion will be printed along with the printed reports of fit res
57. or dc dt See the procedure under Tutorial calculating the g s distribution Section 2 1 For this tutorial we are going to fit the data shown below from a sample that probably contains a small amount of dimer producing the shoulder on the right side of the main peak as shown below a Analysis1 00120 00162 P3 o G x Showintesraisoverdistrbuton poer la weight average s 3 691 0 008 total c 0 270 0 000 Scans 00120 0F3 to 001621P3 trom C UsersiJonn Philio DesinopPnilio DasaiPKR_KGS6R1 g s fringes Svedberg o o T 3 0 02 0 04 1 0 1 5 2 0 25 3 0 3 5 4 0 4 5 5 0 5 5 6 0 6 5 7 0 7 5 8 0 s Svedbergs wl R Less C Back Hex A B Set the fitting model to the desired number of species 45 DCDT User Manual 46 DCDT User Manual a Data choice fit a s data gt fit D for each species fit de dt data ft M D ratio instead of D Weighting of data points Theoretical Experimental None Constrain M or s ratios among species Set constraint values Molecular mass units Density and partial specific volumes solvent density g ml 1 001360 gt s D Svedberg per Fick z J Use the same partial specific T buoyant molecular mass kDa volume for all species temperature must be known eit of Temperature C 20 3 species 1 ml g molecular mass kDa temperature solvent density and solute p
58. percent high and M a few percent low For the classic method in many cases the accuracy of D and M is strongly affected by peak broadening for both g s and dc dt fits Peak broadening as the time span of the scans grows too large causes an overestimate of D and an underestimate of M See Peak broadening limit Section 3 2 3 2 about defining when the time span is too large When fitting to g s the peak broadening error tends to compensate for the opposing tendency to underestimate D so the apparent M may actually be closer to the expected or true value when fitting to 9 s than when fitting to dc dt but this will be true only over a certain range of broadening When the broadening is fairly strong the absolute errors are similar for both fitting approaches For slowly sedimenting species s lt 4 fitting to g s also underestimate the s value and therefore such results should probably not be used for shape analysis unless you manually correct for this effect for correction factors see reference 6 Section 7 6 This error is absent when you fit the dc dt data These errors in sedimentation coefficients when fitting 9 s can be significant even for larger species 6 S or greater particularly if they are run at low rotor speeds and also cause problems in trying to compare sedimentation coefficients from runs at different rotor speeds 188 DCDT User Manual SR DCDT User Manual On the other hand when species are poorly resol
59. plot is included at the end of the report Graph bootstrap or Monte Carlo results Section 3 2 10 8 This button is active whenever confidence intervals have been computed using either the bootstrap or Monte Carlo methods the name on the button changes to whichever method was last used It will open a dialog box that generates graphs of the parameter distributions either histograms of a single parameter or two parameter correlation plots 7 Fit statistics Section 3 2 10 6 This area displays some of the statistical parameters characterizing the fit 3 High cross correlation warning Section 3 2 10 7 This warning panel is only displayed when there are one or more pairs of parameters that are highly correlated there is 1 in this example This is a warning that some of the parameters may be poorly determined and you may want to consider fixing or constraining some of the parameters 3 2 10 1 Report fit results Fitted parameters 148 DCDT User Manual SR DCDT User Manual Fitted Parameters Species Parameter Best Value 95 0 Confidence Region Starting Value Co AU 0 9626 0 9579 0 9685 s S 6 196 6 191 6 204 M kDa 149 76 148 11 151 29 Co AU 0 0213 0 0144 0 0268 s S 8 968 8 535 9 430 M kDa 249 66 121 00 467 08 Co AU 0 0088 0 0058 0 0121 s S 11 440 10 671 12 261 Co AU 0 0058 0 0038 0 0086 s S 13 832 13 287 14 414 This area of the Report fit results page Section 3 2 10 disp
60. proceed without pausing after each iteration This is the normal way to complete a fit The fitting algorithm will iterate seeking parameters that give the lowest sum of squared residuals lowest x 2 for weighted fits The iterations will halt when the iterations no longer give improvement the fit has converged At that point the program will automatically move to the Report fit results page Section 3 2 10 to display the results Once fitting is started it can be aborted using the Cancel button Section 3 2 9 4 3 2 9 2 Perform fit 1 iteration button This button will allow the fit to proceed through only one iteration after which it will pause k d This procedure allows time for examination of trends in the parameter values and whether the initial guesses for the parameters are reasonable This may be a good choice for the initial stages of a difficult fit If the parameters are moving in a direction that makes no physical sense you can go back to the Set alter fitting parameters page Section 3 2 8 and perhaps try holding one or more parameters constant until the other parameters get into a better range or perhaps try using parameter constraints Section 3 2 6 6 At any point the fit may be continued without pausing by using the Do Fit button Section 3 2 9 1 If the requested single iteration produces convergence the program will automatically move to the Report fit results page Section 3 2 10 to display the results 14
61. range you selected by dragging over the graph is filled into the integration range text boxes e You may manually alter the integration limits within the text boxes When you are done editing the value the program will calculate the nearest actual data point value and display that Then use the Redo button 119 DCDT User Manual DCDT User Manual aa to force a new calculation with the modified range Calculated properties The following properties are calculated and displayed along with their estimated standard deviation e The total concentration area under the curve in instrument units o this is not calculated when the Normalize option Section 3 2 5 4 is on the case shown above since it is the same as the fraction of the total e The fraction of the total sedimenting material fraction of the total area under the curve for the full range o this will always be 100 if the Full range option is checked e The number weight z and z 1 average sedimentation coefficients S Sp S and Si Each property is calculated for both the g s and g s distributions is weighted by the sedimentation coefficient raised to the appropriate power see equations below For the so called true averages the 9 s distribution is weighted by powers of the molecular mass Computing the true averages would require an exact relationship or model to assign a molecular mass to each sedimentation coefficient aa The averages computed and di
62. results Report fit results Report fit results Report fit results Report fit results Report fit results Report fit results Report fit results Analysis page 11 201 Compute confidence intervals button 150 153 Create reports group box 153 154 Fit statistics 154 Fitted parameters 148 149 Fixed or constrained parameters 149 Graph bootstrap or Monte Carlo results 155 158 High cross correlation warning 154 155 View parameter cross correlations button 150 page 147 148 DCDT User Manual DCDT User Manual aa Graph fit results page 158 164 Analysis page 2 Set meniscus amp data region Set meniscus amp data region Set meniscus amp data region Set meniscus amp data region Set meniscus amp data region Set meniscus amp data region Set meniscus amp data region Set meniscus amp data region Data displayed options 100 101 Fringe jump removal options 99 100 Good data begin X cm from meniscus 96 97 Good data end at radius 97 98 ID points button 101 102 Jitter removal options 98 99 Meniscus position 96 Zoom meniscus button 101 Set meniscus amp data region page 94 96 Analysis page 3 Convert to s 20w 105 108 112 115 121 124 Select scans to analyze of sections up down spinner 109 110 Select scans to analyze Auto adjust button 104 Select scans to analyze Peak broadening limit 104 105 Select scans to analyze Use true mean
63. scans to analyze Calculate average dc dt curve Calculate g s distribution Fit distribution as N species Select fitting model 3 C Users John Philo Desktop Philo D Derive g s distribution Load raw scans Set meniscus amp data region Select scans to analyze Calculate average dc dt curve Calculate g s distribution ms 4 uy gt Any open analysis window Section 3 1 3 can be selected as the active analysis and any available step within that analysis can be selected by clicking on the corresponding node within the tree Steps which are not yet available are shown as gray inactive The topmost node in the tree provides access to the g s overlay graph Section 3 1 5 whenever more than one non minimized analysis window has calculated a 9 s distribution kOJ The Navigation Tree can optionally be closed to provide more room for analysis windows Its visibility can be controlled using an entry in the View menu Section 3 1 1 3 When the width of the Navigation Tree panel is altered by clicking the mouse at the edge and dragging that width will be remembered the next time the program is started Navigation tree context menu em 84 DCDT User Manual SR DCDT User Manual Right clicking on any branch of the tree with the mouse will bring up the context menu shown above This allows you to 1 save the analysis to disk close this analysis window close all analysis windows except this one collaps
64. subscripts refer to the temperature and solvent conditions b buffer w water The density and viscosity of water at 20 C are 0 998234 g ml and 1 002 centipoise respectively 20 w raw 123 DCDT User Manual DCDT User Manual aa If all those data are not available or cannot be estimated e g by SEDNTERP then the conversion cannot be made The program will optionally automatically compute a temperature correction to the partial specific volume using the same formula used in SEDNTERP reference 4 Vz V4 4 25 x10 T 20 This option is controlled by the check box on the lower frame and when it is checked the input box for the vbar at 20 C is disabled but the computed value is displayed The viscosity values are to be entered in centipoise Since SEDNTERP displays these values in poise to facilitate copying and pasting numbers from SEDNTERP whenever a number is entered that is less than 0 1 the program will assume it is a value in poise and multiply it by 100 The program automatically fills in the experiment temperature based on the temperature recorded in the header of the last scan used in calculating the dc dt data but since the temperature may drift during the run you may wish to adjust that value manually Please note that changing the temperature on this form overrides the temperature recorded with the scans and also alters the value used elsewhere in the program e g for conversions to buoyant or true ma
65. such a test the peak for a real species would be broadened but it should not disappear so the qualitative result peak or no peak is definitely enhanced by using more scans even though the shape of the peak may be significantly distorted How to adjust the slider controls This tutorial section will use some real absorbance data for a monoclonal antibody sample run at 45 000 rpm as an example First we ll do it the hard way totally manually 22 DCDT User Manual SR DCDT User Manual Adjust scans used in computing de dt Seansused 60 of 60 lt oe broadening i ers First scan 00001 RA1 Lastscan 00060 RA1 1 16 Scans 00001 RA1 20 COO6O RA1 from CXLAWINXUDAT Aisnsibodyi vv 8 Q Q 5 irs 7 T ay me Xs Ny nae t Vey igo cg i E VEA gis a Pehla ede Wat eal Gitte Male agr a tb va Ben TA re as 1 If you have loaded scans covering the entire range of boundary movement then you will almost surely want to move the top slider to the left to reduce the total range of boundary movement and improve the resolution sharpen the peaks The image above shows the initial state when all 60 scans covering the entire run were loaded Note that the individual curves from each scan pair are quite divergent and the average dc dt curve heavy black line is flat on top and very broad These are signs that the time span number of scans used is much too broad Note too that the peak broadening limit
66. the case shown species 2 is constrained to have twice the molecular mass of species 1 species 2 is a dimer See Constrain M or s ratios among species Section 3 2 6 6 4 This is an example of a parameter the zero offset that is being held fixed at zero in this case That is the program default for this parameter See Should I fit the offset Section 5 2 1 9 Convergence control Section 3 2 8 4 These two values are used in determining when a fit has converged The default values work well in most situations Lower values will produce more iterations causing the fitter to come ever closer to the best possible fit but those extra iterations will probably not produce changes that are significant compared to the statistical precision of the parameter values Higher settings on these values may help the fitter reach convergence on particularly long and difficult fits 6 Adjust parameter upper lower limits Section 3 2 8 5 This brings up a dialog box for altering the default upper and lower bounds on each parameter Generally changes in these values are not needed 137 DCDT User Manual DCDT User Manual a Cancel changes Set alter fitting parameters Cancel changes button in the on line documentation This is an Undo button that restores all values and settings as they were when this analysis page was first entered 3 2 8 1 Parameter value text boxes Parameter Value Co 1 0 837 s 1 S 3 600 Use thes
67. the initial state when all 110 scans covering the entire run were loaded The graph shows the individual dc dt curves calculated from each of the 55 scan pairs as dashed lines and the average of all 55 of those curves as a heavy black line Basically at this stage the results are quite poor because we are doing the calculations using scans from the entire run including those very early in the run when the boundary is still near the meniscus and the separation is quite poor Remember for 9 s analysis we are trying to capture a snapshot of the distribution in the cell at one particular time during the run If we do the dc d t calculations using all the scans that is just like trying to take a picture of a runner with the camera shutter locked open all the details get blurred out Thus in this case the 55 dc dt curves are quite divergent because we are including way too many scans Note that because the first scan pair black dashed line includes data at the earliest times it provides information about species out to nearly 500 S but none of the other scan pairs give data beyond 60 S Note too that the peak broadening limit Section 3 2 3 2 says that peaks for all species larger than only 0 22 kDa are being significantly broadened Typically we will want to analyze scans covering 15 20 of the total boundary movement The selection of the optimal subset of scans to analyze is a complex topic that is covered in depth in Tutorial How to s
68. the zero level for the 9 s distribution This may be Buia necessary to compensate for uncorrected jitter in interference data and or other effects that are causing shifts of the zero level If uncorrected such shifts may cause the 9 s distribution to go below zero or remain above zero in regions away from the peaks 124 DCDT User Manual SR DCDT User Manual A Since this manual zero adjustment could bias the results its use on a routine basis is not recommended When the button is pressed a red horizontal bar that will define the new zero level is displayed on the graph of g s The bar can be dragged up and down using the mouse At the end of the drag operation the new zero position is used to recalculate and re display the g s distribution The Undo button will restore the zero level to the original experimental value When one or more peaks are fitted to g s data which have been manually re zeroed the amount of the zero shift will be printed in the report of the fitting results Gal Bringing the 9 s curve to zero at any X axis position can only be done approximately due to the fact that the dc dt value at any value of s contains contributions from the plateau regions of all species with lower values of s Thus two or more iterations of pushing the Reset zero button may be necessary to bring that point completely to zero particularly if the chosen s value is to the right of a significant peak Because the tr
69. vertical line displayed on the 2D graph the dc dt data and 9 s distribution Due to the mathematics of the data transformations the data points near zero sedimentation coefficient in the g s distribution are often quite noisy anyway so excluding those points may be desirable simply for that reason Gal Failure to exclude this region will cause significant errors in the low sedimentation coefficient region of Obviously if you find that you often need to adjust this value then you may need to revise the default value via the Preferences dialog Section 3 1 1 4 2 3 2 2 3 Set meniscus amp data region Good data end at radius good data end at radius 7 10 cm This setting determines the upper radius limit on raw scan data to be used in calculating dc dt curves and 9 s distributions This setting is used to exclude the region near the cell base where solutes are accumulating That region must be excluded because the theory treats the cell as semi infinite On the other hand choosing too low a radius will cause loss of potentially valuable data The upper radial limit can be altered either by directly entering a number in the text box or by dragging the right hand green vertical marker displayed on the 2D graph to a new position For low molecular weight solutes back diffusion from the cell base may extend out to 7 cm or even further In such cases you must cut out the data influenced by the cell base because the calculations
70. want to use the Load only every Nth scan Section 3 2 1 8 option to reduce the total scans loaded at least initially Later after you have found an appropriate subset for analysis you can go back and load every scan from the beginning to end of that subset to get the maximum signal noise e At a minimum you will want to select at least 4 scans 3 Click Ok in the File Open dialog to load these scans Loading may require 10 seconds or more depending on the number of scans and possibly network speed if you are accessing through a network The progress bar will advance as the files load 6 DCDT User Manual SR DCDT User Manual Scans to be used in g s calculation 00016 IP1 00017 1P1 00018 1P1 00019 1P1 00020 1P1 00021 IP1 00022 IP1 00023 1P1 00024 1P1 T Load only every Nth scan N 2 S Sample description Run Clavene simulation to 5 e e Q sock Res ES At this point all the scan names will show in the Scans currently loaded in memory Section 3 2 1 6 and Scans to be used in g s calculation Section 3 2 1 7 list boxes and your description of that sample will appear near the bottom of the page in the sample description area Section 3 2 1 9 4 Push the Next button Section 3 1 3 1 to move to the Section 3 2 2 Set meniscus amp data region page Section 3 2 2 and see a graph overlaying all of the scans you just loaded Set the meniscus position and define the region containing the
71. 00085 IP3 00086 IP3 00087 IP3 00088 IP3 00089 IP3 00090 IP3 00091 IP3 00092 IP3 00093 1IP3 00094 IP3 00095 IP3 00096 IP3 00097 IP3 00098 IP3 00099 1IP3 00100 IP3 00101 IP3 00102 1P3 00103 1P3 x Interference Fluoresence Load new scan files Replace scans for this sample Add more scans for this sample Delete selected scans Data loading options T Load only every Nth scan N 2 IV Despike spikethreshold 0 200 op 00093 IP3 00094 IP3 00095 IP3 00096 IP3 00097 IP3 00098 IP3 00099 IP3 00100 IP3 00101 IP3 00102 1P3 00103 IP3 v Sample description Run Claverie simulation paana a to generate data PKR K296R 0 1 mg ml E lt lt Less Back Next Then push the Delete selected scans Section 3 2 1 5 button the rogue scan will be deleted from memory and you can continue with your analysis 183 DCDT User Manual DCDT User Manual a 5 Frequently asked questions 5 1 About g s distributions 5 1 1 What is the difference between s and s The usage of the symbol s is intended to indicate that this is an apparent sedimentation coefficient That is it is one derived from the transformation from radial coordinates to sedimentation coefficient coordinates through the relation In T pe A o t where Tn is the radius of the meniscus The designation as apparent will be clearer if we use the following g s distribution as an example
72. 1 averages value 4 287 0 004S z 1 Note stated uncertainties are 1 sigma and sedimentation coefficients are uncorrected raw values Copy all values to Clipboard f G The results are displayed in a dialog box example shown above For more details about the options within this dialog box and how the calculations are done see Averages and peak areas dialog Section 3 2 5 3 If you want these calculations done only over a portion of the distribution for example one peak use the Set integration region button Section 3 2 5 2 instead 117 DCDT User Manual DCDT User Manual aa 3 2 5 2 Calculate g s distribution Set integration region This command calculates and displays the concentration and the number weight z and z 1 average sedimentation coefficients s S S and s for a selected portion of both the 9 s and g s distributions The integration region is selected by dragging with the mouse over a region on the graph only the sedimentation coefficient range is significant during the drag operation the Y values are irrelevant a a a a E k d Although in the end the Y range is irrelevant you must move the mouse sufficiently in the Y direction to change the cursor from the no symbol to a magnifying glass symbol before raising the mouse button to complete the drag operation Integration range Fu
73. 2 command rather than Save analysis as Section 3 1 1 1 3 The Clone button will copy the current analysis into a new window so it can be modified without affecting the current window The Calculator button is equivalent to the Mass diffusion calculator Section 3 1 1 4 1 entry in the Tools menu Section 3 1 1 4 and pops up the Calculate Mass or D dialog box The Options button is equivalent to the Options dialog Section 3 1 1 4 2 entry in the Tools menu Section 3 1 1 4 and pops up the Program Options dialog box for setting user preferences The Help button is equivalent to the Index entry in the Help menu Section 3 1 1 6 and will bring up the 74 DCDT User Manual SR DCDT User Manual index for the program Help file 3 The display of the Toolbar is optional It can be turned off to provide more screen area for Section 3 1 3 Analysis windows using an entry under the Section 3 1 1 3 View menu Section 3 1 1 3 3 1 3 Analysis window This program allows multiple analyses to be open at one time For example those might be 1 different time spans of data from a single cell and run 2 multiple cells from one velocity run 3 data from multiple velocity runs Each of those analyses has an independent analysis window within the main program screen area Each analysis window shows up to 11 different pages of controls and graphs depending on the steps within the analysis that have been completed and or which results you wish
74. 2 3 1 and a program wizard will make these adjustments for you This wizard will first try to find the time in the run when the major species are about in the middle of the cell and then adjust the number of scans used upward until the peaks start to broaden significantly e This automatic choice often produces acceptable results but cannot be guaranteed to make the best choices Further you may be particularly interested in species within a certain range of sedimentation coefficients but the wizard doesn t know that and might not choose the portion of the run that is optimal for those species So ultimately your intelligence is likely to be needed to truly obtain the optimum settings been selected each time the group of scans to be analyzed is altered using the sliders or the Auto adjust button Section 3 2 3 1 the program wizards re evaluate the best position for jitter removal and recompute the fringe shift corrections prior to re computing the dc dt curves Thus you may see a change in the jitter removal region if you return to the graph on the Set meniscus amp data region page Section 3 2 2 Gal Note that for interference data when the Auto jitter removal Section 3 2 2 4 option has The peak broadening limit Section 3 2 3 2 is an important guide to whether the number of scans being used slider 1 is too high Species with molecular masses higher than this limit will have their peaks significantly broadened Generally you will
75. 3 DCDT User Manual DCDT User Manual aa 3 2 9 3 Perform fit Simulate button The Simulate button forces the program to calculate the theoretical fit and the residuals using the current parameters and model as though a fit has been completed Those calculations will enable the display of the graphs on the Graph fit results page Section 3 2 11 By manually altering the parameters via the Set alter fitting parameters page Section 3 2 8 and then using the Simulate button you can generate theoretical curves for specific values graph those results and if desired even export the theoretical data 3 2 9 4 Perform fit Cancel button Cancel This will abort the current fit or single iteration stopping any calculations that are in progress The Back button Section 3 1 3 1 can then be used as needed to go to the Set alter fitting parameters page Section 3 2 8 to alter parameter guesses or change which parameters are being fitted or to go to the Select fitting model page Section 3 2 6 to change the fitting model before trying to fit again 3 2 9 5 Perform fit Status This status box shows status messages as the fit progresses e Waiting for input indicates the program is halted waiting for the Do Fit Section 3 2 9 1 1 Iteration Section 3 2 9 2 or Simulate Section 3 2 9 3 button e Calculating sums indicates the program is looping through the data points collecting the sum of squared residuals and partial derivatives that a
76. 4 scans By default despiking is always on The purpose of despiking is to remove the single point spikes that sometimes occur in absorbance scans often due to lamp miss firing These spikes are visible as single point spikes within the dc dt curve from one pair of scans and a corresponding smaller spike in the average dc dt curve as displayed on the Select scans to analyze page The despiking routine defines spikes as data points which differ from the preceding and following points by more than a threshold value and removes those data points from the data being processed not in the original scan file This spike threshold defaults to 0 2 OD but can be altered using the corresponding text box The default value works well in most situations but may fail to detect some small spikes The danger of setting the threshold too low is that false spikes may be detected for example normal random noise in the data or the top of the meniscus region All of the original raw data is always retained in memory and will be stored by the Save Analysis As Section 3 1 1 1 3 command so you can alter this setting as desired and reprocess the data until you are satisfied with the results displayed on the Select scans to analyze page Section 3 2 3 Note that the despiking routine ignores a small region around the meniscus so the meniscus spike is not affected A If you want to see all the bad data in the graph on the Set menisus amp data regio
77. 4 3 2 3 2 Select scans to analyze Peak broadening limit 104 105 DCDT User Manual SR DCDT User Manual 3 2 3 3 Convert to s 20 w 3 2 3 4 Select scans to analyze Use true mean time button 3 2 3 5 of sections 3 2 4 Calculate average dc dt curve page 3 2 4 1 Calculate average dc dt X axis point spacing 3 2 4 2 Calculate average dc dt Set range for g s 3 2 4 3 Convert to s 20 w 3 2 4 4 Calculate average dc dt Reset zero amp Undo buttons 3 2 5 Calculate g s distribution page 3 2 5 1 Calculate g s distribution Show integrals over distribution 3 2 5 2 Calculate g s distribution Set integration region 3 2 5 3 Averages and peak areas dialog 3 2 5 4 Calculate g s distribution Normalize option 3 2 5 5 Convert to s 20 w 3 2 5 6 Calculate g s distribution Display options 3 2 5 7 Calculate g s distribution Reset zero amp Undo buttons 3 2 5 8 Calculate g s distribution Broad algorithm option 3 2 6 Select fitting model page 3 2 6 1 Select fitting model Number of species 3 2 6 2 Select fitting model Improved or classic methods 3 2 6 3 Select fitting model Data choice 3 2 6 4 Select fitting model Fit diffusion or mass 3 2 6 5 Select fitting model Weighting of data points 3 2 6 6 Select fitting model Constrain M or s ratios among species 3 2 6 7 Select fitting model Molecular mass units 3 2 6 8 Select fitting model Density and partial specific volume
78. 6 The vbar for all species was changed to 0 7326mL g 4 26 2012 20 49 40 Solvent density changed to 1 003000 g mL 4 26 2012 20 49 56 Lower limit of fitting range was set to 4 95 S 4 26 2012 20 50 02 Upper limit of fitting range was set to 7 15 S 4 26 2012 20 50 13 Initial guesses for the loading concentration and molar mass were automatically set to 6 000 S and 0 7653 OD for species 1 4 26 2012 20 50 16 Full fit started 4 26 2012 20 50 16 Fit Completed 4 iterations Results of fit Parameter Best Value Standard Error Starting Value Co 1 OD 0 8045 0 0011 0 7653 s 1 S 6 064 0 001 6 000 M 1 kDa 159 41 0 71 150 03 Parameters held constant Parameter Fixed Value offset OD S 0 00000 Fit statistics and convergence settings DCDT User Manual DCDT User Manual aa Final sum of squared residuals 0 0002945381 starting value 0 04889639 rms 0 001776434 for 89 data points and 86 degrees of freedom runs test Z 2 52 Convergence limit 0 1 parameter precision limit 0 05 The r m s residual corresponds approximately to 4 1E 3 OD Sedimentation coefficients are uncorrected raw values The diffusion coefficient corresponding to the best fit molecular mass is 3 50 F 1 species fit to g s data g s OD Svedberg Y exp Y fit 48 50 52 54 56 58 60 62 64 66 68 70 72 74 s Svedbergs residual 1 data fit 4 26 2012 20 50 47
79. 96 Broad algorithm 125 Broad g s algorithm 185 187 Buoyant mass 132 133 Calculate average dc dt Reset zero amp Undo buttons 115 Calculate average dc dt Set range for g s 112 Calculate average dc dt X axis point spacing 111 112 Calculate average dc dt curve page 110 111 Calculate g s distribution Broad algorithm option 125 Calculate g s distribution Display options 124 Calculate g s distribution Normalize option 121 Calculate g s distribution Reset zero amp Undo buttons 124 125 Calculate g s distribution Set integration region 118 119 Calculate g s distribution Show integrals over distribution 117 Calculate g s distribution page 115 117 Calculator 66 67 Cancel button 144 Cell base 97 98 Cell number 89 Citation 190 204 DCDT User Manual aa DCDT User Manual SR DCDT User Manual Classic method 127 Claverie simulation 93 94 Clone 63 Clone analysis to new window 63 Confidence limits 150 153 Constrained parameters 138 139 Constraints 129 132 138 139 Conventional g s algorithm 185 187 Convergence 139 Convergence control 139 Convert to s 20 w 105 108 112 115 121 124 Copy data 63 65 Copy report 153 154 Correlation matrix 150 Create reports 153 154 Cross correlation 189 190 Data choice 128 Data displayed options 100 101 Data loading options 92 93 Data range 134 136 Data simulation 57 60 93 94 Data type 89 Defaults 67 72 Delete selected scans
80. By default the scans are split into two groups first half and second half and pairs are formed from the corresponding members of each group Thus if there are 16 scans scan 9 is subtracted from scan 1 10 from 2 11 from 3 and 16 from 8 If the number of sections was instead set to 2 then the pairing is 1 5 2 6 3 7 4 8 9 13 10 14 11 15 12 16 When the number of scan pairs cannot be evenly divided by the number of sections then the last section will have more scan pairs than the other section s 3 2 3 1 Select scans to analyze Auto adjust button Auto adjust This button invokes a program wizard to automatically select the group of scans to be analyzed The wizard first calculated a time in the run when the main boundary is approximately in the center of the cell and selects 4 scans the minimum number centered around that time The wizard then starts increasing the number of scans monitoring the degree to which the dc dt curves from different scan pairs deviate from one another During this adjustment you will see repeated re calculations of the dc dt curves and re displays of the graph The wizard keeps increasing the number of scans until it is able to determine that the different scan pairs are deviating significantly an indication that the larger time span from the first to last scan is causing peak broadening and then selects a number of scans that should be a reasonable compromise between the conflicting
81. DCDT User Manual a DCDT User Manual DCDT User Manual Version 2 4 1 September 4 2013 DCDT User Manual SR DCDT User Manual Table of Contents 1 Getting Started 1 1 Program overview and organization 1 2 How to use this Help file 1 3 Standard and advanced modes 2 Tutorials 2 1 Tutorial Calculating the g s distribution 2 2 Tutorial How to select the scans to analyze 2 3 Tutorial Using the g s overlay graph 2 4 Tutorial Single species fit 2 5 Tutorial Multi species fits 2 6 Tutorial Simulating experiments with the Claverie simulator 3 Program elements controls and features 3 1 User interface 3 1 1 Main menus 3 1 1 1 File menu 3 1 1 1 1 Open saved analysis 3 1 1 1 2 Save analysis 3 1 1 1 3 Save analysis as 3 1 1 1 4 Clone analysis to new window 3 1 1 2 Edit menu 3 1 1 3 View menu 3 1 1 4 Tools menu 3 1 1 4 1 Mass diffusion calculator 3 1 1 4 2 Options dialog 3 1 1 5 Windows menu 3 1 1 6 Help menu 3 1 2 Toolbar 3 1 3 Analysis window 3 1 3 1 Next and Back buttons 1 2 23 3 4 5 20 20 27 27 31 31 44 44 57 57 60 61 61 61 61 62 62 62 63 63 63 65 65 65 66 67 67 72 72 72 74 74 75 75 77 77 78 DCDT User Manual DCDT User Manual a 3 1 3 2 More gt gt and lt lt Less buttons 78 3 1 3 3 Log page 78 83 3 1 3 4 User prompt area 83 3 1 4 Navigation tree 83 85 3 1 4 1 Derive g s distribution branch 85 86 3 1 4 2 Fit distribution as N species
82. DT User Manual SR DCDT User Manual Jitter removal Auto Manual The purpose of jitter removal is to correct for small fringe shifts vertical displacements of small fractions of a fringe that often occur from one interference scan to another The correction is done by selecting a region a range of radii where the scans are expected to align vertically and then forcing them to do so This type of matching is also called normalization in the Beckman Coulter software and fine adjusting in some versions of Walter Stafford s DCDT program A similar phenomenon also seems to arise when absorbance data are collected in pseudo absorbance mode two samples per cell In that case the vertical shifts typically occur in a few discrete jumps per run The jitter matching region is generally placed either in the air air region or a region just to the right of the meniscus Using a region to the right of the meniscus generally works better However this will only work correctly if the meniscus is completely cleared and there is no sedimentation of low molecular weight components that will produce signals in this region By default when new interference data are loaded the jitter wizard Section 6 7 tries to automatically determine the best matching region marks that region on the graph using two vertical gray bars to mark the region limits and then does the correction using that region The Auto button will show as depressed on
83. How should I cite this program 1 The primary citation for this program is o Philo J S 2006 Improved methods for fitting sedimentation coefficient distributions derived by time derivative techniques Anal Biochem 354 238 246 2 Because the original development of the dc dt method was by Walter Stafford please always also cite Walter Stafford s original paper o Stafford W F Ill 1992 Boundary analysis in sedimentation transport experiments A procedure for obtaining sedimentation coefficient distributions using the time derivative of the concentration profile Analytical Biochemistry 203 295 301 3 The program itself should be mentioned in your Methods section as DCDT by John Philo with the version number Specialized method citations The reference for the special multi segment dc dt calculations Section 3 2 3 5 is e Philo J S 2011 Limiting the sedimentation coefficient range for sedimentation velocity data analysis Partial boundary modeling and g s approaches revisited Anal Biochem 412 189 202 The reference for fitting to dc dt data rather than g s Section 3 2 6 3 and for the Use true mean time button Section 3 2 3 4 the broad algorithm is e Philo J S 2000 A method for directly fitting the time derivative of sedimentation velocity data and an alternative algorithm for calculating sedimentation coefficient distribution functions Analytical Biochemistry 279 151 163 190 DCDT User Man
84. Lastscan 00163IP1 149 ofsections 1 4 ood 3 Scans 001201P1 to 001631P1 trom CAXLAWINIXLIDATAWen0n2005 1 PKR_K296R 19 4 10946 0 0006 0 0005 de dt fringes sec o o 3 S N Ww 0 0001 0 0000 0 0001 00 06 10 15 20 25 30 35 40 45 50 55 G60 G5 70 75 80 85 s 20 w Svedbergs Two slider controls at the top of the page allow you to vary which scans are being used among all of those currently loaded in memory to optimize your analysis As you make adjustments the dc dt curves are immediately re calculated and displayed Q The top slider varies the total number of scans being used always an even number e Generally you will only want to use a small portion of the total run perhaps 10 20 e As you make adjustments the text boxes to the left of this slider indicate the number being used and 102 DCDT User Manual SR DCDT User Manual the total available G The lower slider will move that group of scans earlier or later in the run e Generally you will want to analyze a group of scans where the boundary is about in the middle of the cell e As you make adjustments the text boxes below this slider show the file names of the first and last scans Optimally selecting which scans to analyze takes some judgement and experience see Tutorial How to select the scans to analyze Section 2 2 for all the details and trade offs However now there is any easy way Simply use the Auto adjust button Section 3
85. Options Scansused 10 of 60 E b Peak ite broadening Posin widina 4 aT kAutoadjusta First scan 00025RAT Lastscan 00034RAT 197 Scans COO2SRA1 10 00O34 RA1 ftom CXLAWINXUDAT Aisnibodyi de dt OD sec s Svedbergs 00025 RA1 00030 RA1 00026 RA1 00031 RA1 00027 RAT 00032 RAI 00028 RA1 00033 RA1 QO0029 RA1 00034 RA1 average a Em Qa As you see this puts the main peak roughly in the center of the graph and sets the number of scans included to prevent that peak from being overly broadened In this case the resulting peak broadening limit is a bit above the actual molecular mass of that main peak Thus these are certainly good first pass settings but on the other hand by focusing on the main peak the wizard algorithm is picking scans that are too late in the run to cover the aggregates sedimenting faster than 11 S that are present in this sample So don t rely totally on the Auto Adjust wizard you need to put your own brain to work too to get the best results 2 3 Tutorial Using the g s overlay graph What data is needed to use the overlay graph The first requirement for using the g s overlay graph is that you need two or more open analysis windows Section 3 1 3 that contain computed g s distributions How do I generate the overlay graph To display the 9 s overlay graph you can either click on the top node of the Navigation tree Section 3 1 4 use th
86. Reference 3 198 Reference 4 198 Reference 5 198 Reference 6 198 Reference 7 198 199 Reference 8 199 Reference 9 199 Reference information About the bootstrap method 195 196 About the fitting algorithm 194 About the fitting functions 193 194 About the jitter wizard 195 About the meniscus wizard 194 195 About the Monte Carlo method 196 197 About this g s implementation 192 193 About using radial intensity scans 191 192 Symbols used 191 Replace scans for this sample button 90 Report fit results Compute confidence intervals button 150 153 Report fit results Create reports group box 153 154 Report fit results Fit statistics 154 Report fit results Fitted parameters 148 149 Report fit results Fixed or constrained parameters 149 Report fit results Graph bootstrap or Monte Carlo results 155 158 Report fit results High cross correlation warning 154 155 Report fit results View parameter cross correlations button 150 Report fit results page 147 148 Reports 153 154 Reset zero 115 124 125 Residual plot 158 164 RIN 98 99 210 DCDT User Manual SR DCDT User Manual Rogue scans 177 183 Rule of thumb 104 105 s 20 w 105 108 112 115 121 124 s 184 185 Sample description 93 Save 62 63 Save analysis 62 Save analysis as 63 Save analysis as 63 Scans currently loaded list box 91 Scans to be used in g s calculation list box 91 92 sections 109 110 Sedimentation coefficien
87. T User Manual aa You may see such a scan when you view the scans on the Set meniscus amp data region page Section 3 2 2 The image below shows an example where there is a fringe jump within the sample region Meniscus setting and data adjustments Meniscus at 5 904 cm good data begin 0 01 cm from meniscus good data end at radius 7 10 cm f Fringes 1 57 58 59 60 61 62 63 64 65 66 67 68 69 70 7 1 72 73 7A Radius cm Pz Less Back Next Because there may be dozens or even hundreds of scans in this display it can be difficult and frustrating to try to identify which is the bad one When there aren t too many scans you may be able to identify the rogue scan from the line color and pattern shown in the legend However by default this graph does not display a legend when there are more than 40 scans shown as is true above because the legend occupies too much space To force display of the legend right click on the graph and select a legend position from within the Legend Style sub menu as shown below 178 DCDT User Manual SR DCDT User Manual Meniscus at 5 904 cm good data begin 0 01 cm from meniscus good data end at radius 7 10 cm lear ase ose x llaa Legend Style Numeric Precision Plotting Method Data Shadows Grid Options Mark Data Points v Show Annotations 57 58 59 60 61 62 63 64 65 Unda Zoom Radius eN Maximize Help That results in the following graph fro
88. This is done by automatically switching the Fit Monitor to the Fitting parameters monitor tab Section 3 2 9 6 and displaying the value of that parameter with a bright yellow background 3 2 9 Perform least squares fit page This analysis page controls and monitors the non linear least squares fitting process e In most cases you will simply push the Do fit button Section 3 2 9 1 and watch the theoretical curve move toward the experimental data as the fit converges e However you are also provided options of proceeding one iteration at a time or aborting the process and of monitoring the actual parameter values and fit statistics rather than the graph This page is divided into two tabs 1 the Graphical fit monitor tab which will be shown and described below and 2 the Fitting parameters monitor tab Section 3 2 9 6 which is described in detail in its own Help topic The Do fit button Section 3 2 9 1 1 iteration button Section 3 2 9 2 Simulate button Section 3 2 9 3 and Cancel button Section 3 2 9 4 are common to both tabs as are displays of the fit Status Section 3 2 9 5 and the current iteration number Both tabs are available in both Standard and Advanced modes Section 1 3 although the Fitting parameters monitor tab Section 3 2 9 6 will be of more interest to advanced users 141 DCDT User Manual DCDT User Manual a o d wm B v 2 d D G a oS D gt Y exp Y fit 2 3 5 6 s Svedber
89. a the background color of the corresponding text box is set to light yellow e f you have previously set a range for fitting g s data and then change to fitting the dc dt data or vice versa the program will assume you wish to use the same range and sets this range for you Alternatively you can directly type the desired sedimentation coefficient range into the text boxes at the upper left How to set the initial guesses for peak positions 135 DCDT User Manual DCDT User Manual aa A magenta vertical bar will be displayed for each species 2 in the image above which you may drag with the mouse to mark an initial guess for the sedimentation coefficient of that species usually the center of the peak e f this is new data a program wizard will automatically find the highest peak and mark this as the position of the first species e You are allowed to drag the markers over top of one another the positions will be sorted in order of increasing sedimentation coefficient and assigned to species numbers in that order When are these new guesses applied The two radio buttons within the Species properties updating group box govern whether new guesses for concentration and D or M will be made based on the currently marked peak positions when the Next button is used or alternatively whether the current values from a previous fit should be retained e By default whenever the magenta peak position markers are dragged t
90. able movement to the Graph fit results page Section 3 2 11 Thus by manually modifying parameters on the Set alter fitting parameters page Section 3 2 8 and then using the Simulate button Section 3 2 9 3 you can generate theoretical plots species plots etc for any desired set of parameters not necessarily ones that give good fits 6 Do fit button The Do fit button Section 3 2 9 1 tells the fitter to proceed without halting after each iteration This is the normal mode and thus this button is given the focus by default when you enter this page so you can initiate the fit by pressing the Enter key Q 1 iteration button The 1 iteration button Section 3 2 9 2 is used to manually step through the fitting procedure one iteration at a time This can be a useful learning exercise but generally this would only be used when you are having trouble obtaining a fit and want to carefully monitor what is happening step by step Cancel button The Cancel button Section 3 2 9 4 will abort the current fit stopping any calculations that are in progress The Back button Section 3 1 3 1 can then be used as needed to alter parameter guesses or the fitting model before trying to fit again Fitting parameters monitor tab Section 3 2 9 6 Select this tab to monitor the actual values of the parameters and the decrease of the sum of squared residuals as the fit progresses 3 2 9 1 Perform fit Do fit button This button will allow the fit to
91. ad new scan files Section 3 2 1 2 button to bring up a multiple File Open dialog box 5 DCDT User Manual DCDT User Manual aa Date modified mi 00049JP1 000651P1 i Recently Changed 1000501IP1 000661P1 E Desktop 00067 1P1 ap Recent Places WH Computer JP 000541P1 _ 000701P1 E Documents _1000391P1 000551P1 00071 IP1 _ 000871P1 _ 000721P1 d AOO Pictures BD Music 000421P1 000581P1 000741P1 E Searches 00075 1P1 a 0X _1000761P1 amp 00045 JP1 d 00077 1P1 00093IP1 00109 JP1 00046 IP1 00078 1P1 O00 IP1 001101P1 1000471P1 _00079 1P1 000951P1 00048 1P1 J 000961P1 Folders A 4 Wt a Public Filename 00110JP 1 00001 1P1 000021P1 00003 intcell 1 ip3 opon fr _Cancoi Unlike other DCDT implementations and version 1 xxx of this program you will usually want to load scans covering the entire range of boundary movement and then later you will choose a subset of those scans that you are going to actually analyze perhaps 20 of the total So generally you will want to highlight the file names for all the scans click on the first scan hold the Shift key down and click on the last scan e f you know the data acquisition went long after all the sample was pelleted you probably want to omit those scans where the cell is empty e f you have hundreds of interference scans and a relatively slow computer you may
92. al information is retained so it can later be restored if needed e Note however that not all AUC analysis software follows this convention and in particular Peter Schuck s SEDFIT and SEDPHAT do not so if you want to use DCDT to edit scans for those programs you must permanently delete the bad data points By this UConn convention any data point for which the value of the third column in the data is negative will be ignored when the file is read For absorbance data this third column normally contains either zero when no replicates are used or the standard deviation of the absorbance value when multiple flashes are being averaged For interference data there normally is no third column entry However this software checks each line of data for a third column and will ignore any data point where a third column exists and is negative Thus to mark a data point as bad the entry in the third column is set to a negative value by adding a third column for that line with 1 as the value in the case of interference data For absorbance data simply changing the sign of the standard deviation allows you to later restore the original raw data if needed without loss of information by changing the sign back to positive again 4 5 How to identify and remove rogue scans The interference optical systems may sometimes produce rogue scans Needless to say you don t want to use these aberrant scans in your analysis 177 DCDT User Manual DCD
93. ansformation from dc dt to 9 s involves dividing each point in dc dt by its corresponding s value the effects of re zeroing 9 s at a certain s value will be to shift the 9 s data more strongly at lower s values and less strongly at higher s values As a consequence picking a re zeroing point far to the right in the data may have undesirable effects at small s values 3 2 5 8 Calculate g s distribution Broad algorithm option Broad algorithm This button will toggle on or off the use of the alternate broad algorithm for calculating the g s distribution and the average dc dt curve This choice primarily affects only the estimates of the error bar for each data point See What is the difference between the broad and conventional g s algorithms Section 5 1 3 for a detailed explanation 3 2 6 Select fitting model page This page is used for selecting the theoretical model which will be used to fit the data number of species choice of fitting to g s or dc dt curves as well as certain options which affect the way the data and fitting parameters are treated Each control or group of controls on the image below is a clickable hotspot that will lead to further information or just use the numbered list below the image 125 DCDT User Manual DCDT User Manual a Fitting options Datachoice __ Fit diffusion or mass Numberofspecies 2 fita s data C fitD foreach species ee C
94. arameter confidence limits 68 3 This sets the default confidence level for parameter confidence limits Usually this is the only setting on this tab the user will want or need to alter Fit converged when parameters change less than 0 05 The fit iterations will halt when the last iteration changes all parameters less than this amount See convergence control Section 3 2 8 4 Fit converged when variance change lt of statistical limit 10 This provides a second criterion for fit convergence halting the iterations when the improvement of the fit is no longer statistically significant See convergence control Section 3 2 8 4 Parameter cross correlation defined as high 97 cross correlations between this level and the very high setting will produce a mild warning on the Report fit results page Section 3 2 10 and in printed reports See What does high parameter cross correlation mean Section 5 2 4 Parameter cross correlation defined as very high 99 cross correlations above this setting will produce a stronger warning on the Report fit results page Section 3 2 10 and in printed reports See What does high parameter cross correlation mean Section 5 2 4 Maximum number of fit iterations before quitting 60 This defines the maximum number of iterations before the fit halts with a warning message Generally if a fit has not converged within 60 iterations it probably never will converge and this limit is to p
95. artial specific volume must be known Navigate to the Select fitting model page Section 3 2 6 shown above Set the number of species Section 3 2 6 1 using the up down spinner or by entering the number directly in the text box beside it o Generally you would increase the number of species by one for each round of fitting unless you have independent information on how many species are present If you have already fitted these data and set the other aspects of the fitting model appropriately you may skip steps 4 6 In the Function computation group box select a function computation method classic or improved Section 3 2 6 2 and from the Data choice Section 3 2 6 3 group select whether you are going to fit the 9 s or the dc dt data In the Fit diffusion or mass Section 3 2 6 4 group box choose whether you prefer to use diffusion coefficient as a fitting parameter or M the ratio of s D instead If you are using M then choose the molecular mass units Section 3 2 6 7 and enter the solvent density and partial specific volumes Section 3 2 6 8 if needed In the Weighting of data points Section 3 2 6 5 group box choose the type of Section 3 2 6 5 weighting for the experimental points if any For the improved method Section 3 2 6 2 theoretical weights the default are always preferred DCDT User Manual SR DCDT User Manual C If you believe these species are small oligomers of a common subunit you ma
96. as ASCII text to File gt lead to a submenu as shown below that determines which data to export 63 DCDT User Manual DCDT User Manual a dc dt Data all scan pairs Average dc dt Curve g s Distribution g s Distribution Data Being Fitted Calculated Fitting Function Residuals Species Table When writing to disk a standard File Save dialog allows you to specify a file name and the folder for the file By default the file extension will be dat but you can also select txt or any other extension When the data is exported the columns are separated by tab characters and rows are ended by a carriage return and line feed Scan data from graph This menu selection will export the scan data signal versus radius from the graph shown on the Set meniscus amp data region page Section 3 2 2 For interference scans this will give the data after removal of jitter and fringe jumps if that is what is currently shown on the graph This menu item is only enabled when this graph is currently being displayed If the graph shows N scans then the data table will have 2N columns with the first two columns holding the x and y values for the first scan the 3rd and 4th columns holding the x and y values for the second scan etc Note that copying the data into a large text string can take a minute or more when the number of scans is large An hourglass is displayed while the text string is being created Because absorba
97. ata that is fitted but to extend the theoretical plots over the entire data range 3 Species plot for fitted range area This plot is like 1 above but is limited to the sedimentation coefficient range that was actually fitted 159 DCDT User Manual DCDT User Manual a Select graph type from list Species plot for fitted range area w print Copy AddtoLog Scans 00027 RA 10 CCOSE RA1 tom C sediidstsiantibody 0 4 on a s OD Svedberg 52 54 s6 s s Svedbergs As noted under 1 above in this example the fitting range was deliberately limited to the range from 3 to 6 88 S to avoid fitting data that is strongly influenced by the presence of other species This allows obtaining accurate properties for the major species antibody monomer without having to accurately model the other species 4 Species plot for fitted range line This plot is like 2 above but is limited to the sedimentation coefficient range that was actually fitted 160 DCDT User Manual SR DCDT User Manual Select graph type fromlist Species plot for fitted range ine m Scans 00027 RA1 10 COOSS RA1 ftom Cisedirassianmbody g s OD Svedberg 36 38 40 42 44 46 42 so s 54 s6 s 60 62 64 66 6s 70 s Svedbergs data species 1 5 Overlay of data and fitted curve This plot overlays the data points that were fitted shown as circles with the best fit curve as a solid line This is
98. ation will make the boundaries and the peaks in dc dt and g s broader which makes the apparent D larger and therefore the apparent M smaller Thus if the apparent M is more than 5 below the correct value it is likely that your sample is heterogeneous or self associates If this is due to self association you should see shifts in s values by changing sample concentration A second important criterion is to ask whether the results make hydrodynamic sense Is a species sedimenting faster than is theoretically possible for its molecular mass f f lt 1 Is a species sedimenting much more slowly than is reasonable for its molecular mass f f very high With regard to whether the rms residual is consistent with the instrumental noise this may be difficult to judge because of the raw data have been substantially transformed in deriving the dc dt and 9 s distributions As an aid in making this judgement when a fit report is printed a calculation is made to back transform the observed rms residual and calculate an approximate equivalent noise level in the original scans Causes of bad fits A number of causes of bad fits are discussed under How to avoid common mistakes Section 4 1 Another factor that can cause boundary broadening and thus overestimates of D is scratches or roughness on the inside of the centerpiece 5 2 4 What does high parameter cross correlation mean Parameter cross correlation values near 1 or 1 indicate that
99. averagesvalue 3 972 0003S z 1 averagesvalue 4231 0 003S Properties extrapolated to t 0 from g s Loadingconcentaton Fraction oftotal 100 00 0 00 Number averages value 3612 0 009S Weightaveragesvalue 3 780 0003S zaverages valve 4 022 0 003S 2 1 averages value 4 287 0 006S Note stated uncertainties are 1 sigma and sedimentation coefficients are uncorrected raw values Copy all values to Clipboard This dialog box is used to display the results from either the Show integrals over distribution Section 3 2 5 1 or Set integration region Section 3 2 5 2 commands within the Calculate g s distribution page Section 3 2 5 Note that the values calculated for the g s distribution are model independent and are valid for Y interacting as well as non interacting systems The values from the g s distribution involve extrapolation back to the initial loading concentration and this extrapolation assumes each species is independent a mixture Integration range The Integration range controls at the top govern the range of sedimentation coefficients used in the calculations e If the Full range option is checked then the entire range for the distribution is used This is selected automatically when you use the Show integrals over distribution Section 3 2 5 1 command to bring up this dialog box e When you use the Set integration region Section 3 2 5 2 command the
100. aw scans Data loading options Data loading options l Load only every Nth scan N 2 iW Despike spike threshold 0 200 OD This group of controls specifies optional parameters that alter how scan files are loaded If the Load every Nth scan check box is activated checked then only every Nth scan among the scans selected within the File Open dialog will be loaded when new scans are read This applies to the Load New Scan Files button Section 3 2 1 2 Replace Scans for this Sample button Section 3 2 1 3 and the Section 3 2 1 4 Add More Scans for this Sample button Section 3 2 1 4 The value of N default 2 can be altered using either the up down spinner or by directly typing a number within its text box Normally hundreds of scans can be loaded and processed if desired limited only by available memory sy Thus the use of this option is almost never really required and for absorbance scans it is never 92 DCDT User Manual SR DCDT User Manual recommended This option is primarily intended to speed up data processing for situations where 1 a very large number of interference scans have been acquired and there is very little boundary movement between successive scans and 2 the signal noise of those scans is sufficiently high that averaging the dc dt data from a large number of scan pairs is not required T The Despike options are only visible in Advanced mode Section 1 3 and only active for absorbance
101. ay be fairly far off when peaks are poorly resolved o The fit should converge as long as the initial guesses for s and M are not too far off and provided there really is sufficient information in the raw data to uniquely define that species However good initial guesses can make the difference between converging or not converging and will always make it converge faster 2 You might want to fix certain parameters rather than floating them allowing the fitter to vary them This can be very helpful at the early stages of a complex multi species fit but should not be needed here with only 2 species plus a constraint 3 When you are done push the Next button to go to the Perform least squares fit page Section 3 2 9 In this case we will simply accept the guesses made by the program wizard F Fit the data When you enter this page the Graphical fit monitor tab Section 3 2 9 is initially shown displaying an overlay graph of the data and the theoretical curve resulting from the initial guesses with a residual plot below 51 DCDT User Manual DCDT User Manual a Status waiting for command cA i T D wb keraton number 0 g s fringes Swedberg Graphical fit monitor gt 2 amp gt 1 0 1 5 2 0 2 5 30 35 40 4 5 5 0 55 6 0 65 7 0 7 5 8 0 s Svedbergs data species 1 species 2 sum residuals Fitting parameters monitor A Less Back
102. be aggregates at or beyond 12 S present and those will not be analyzed with these settings The image above shows the results after moving the lower slider such that the group of 10 scans being analyzed starts 9 scans earlier At this earlier time we can assess species out to nearly 17 S A close inspection by zooming in on the graph which you can t do here would show that the average dc dt curve is still dropping out to the limit near 17 S which means this sample does contain traces of aggregates out to at least 17 S If we want to include those we would need to start the analysis at least this early in the run On the other hand if our primary interest was to measure the properties of the major peak we should probably go back to the earlier setting and perhaps even somewhat later in the run 5 Once you have decided on the right position within the run lower slider position you may need to re adjust the upper slider Overall with that adjustment you are trying to find a compromise between loss of signal noise as you use fewer scans and loss of resolution due to peak broadening as you use more scans Now let s do it the easier way using the Auto Adjust button If we start from the same initial state shown in the first image all 60 scans analyzed and simply push the Auto 26 DCDT User Manual SR DCDT User Manual Adjust button Section 3 2 3 1 the wizard will produce the result shown below Adjust scans used in computing de dt
103. between these two states depending on the current mode Within this Help file information that is specific for the Advanced mode will be indicated as shown below or simply by the Einstein symbol This is text concerning a feature that is only available in Advanced mode 3 DCDT User Manual DCDT User Manual aa By default when the program is installed each new window is opened in standard mode That behavior can be turned off using an entry on the General Options tab of the Options dialog Section 3 1 1 4 2 4 DCDT User Manual SR DCDT User Manual 2 Tutorials 2 1 Tutorial Calculating the g s distribution This tutorial will walk you through computing the 9 s distribution using a set of interference data as the example This tutorial will be done in standard mode Section 1 3 Load from disk the scans to be analyzed 1 Begin on the Load raw scans page Section 3 2 1 of a new or existing analysis window Section 3 1 3 You may wish to Section 3 2 1 1 preselect the data type and cell number Section 3 2 1 1 for the data you wish to load In this example interference data from cell 2 are pre selected Scans currently Scans to be used in vJ Preselect data type and cell loaded in memory g s calculation Data type Cell number Absorbance Interference 2 Fluoresence Date loading options Load only everyNthscan N 2 Sample description Run Claverie simulation to generate data 2 Push the Lo
104. branch 86 3 1 5 g s overlay graph 86 87 3 2 Analysis pages 88 3 2 1 Load raw scans page 88 89 3 2 1 1 Load raw scans Preselect data type and cell check box 89 3 2 1 2 Load raw scans Load new scan files button 89 90 3 2 1 3 Load raw scans Replace scans for this sample button 90 3 2 1 4 Load raw scans Add more scans for this sample button 90 3 2 1 5 Load raw scans Delete selected scans button 90 91 3 2 1 6 Load raw scans Scans currently loaded in memory list box 91 3 2 1 7 Load raw scans Scans to be used in g s calculation list box 91 92 3 2 1 8 Load raw scans Data loading options 92 93 3 2 1 9 Load raw scans Sample description 93 3 2 1 10 Load raw scans Run Claverie simulation to generate data button 93 94 3 2 2 Set meniscus amp data region page 94 96 3 2 2 1 Set meniscus amp data region Meniscus position 96 3 2 2 2 Set meniscus amp data region Good data begin X cm from meniscus 96 97 3 2 2 3 Set meniscus amp data region Good data end at radius 97 98 3 2 2 4 Set meniscus amp data region Jitter removal options 98 99 3 2 2 5 Set meniscus amp data region Fringe jump removal options 99 100 3 2 2 6 Set meniscus amp data region Data displayed options 100 101 3 2 2 7 Set meniscus amp data region Zoom meniscus button 101 3 2 2 8 Set meniscus amp data region ID points button 101 102 3 2 3 Select scans to analyze page 102 104 3 2 3 1 Select scans to analyze Auto adjust button 10
105. button 90 91 Density 133 134 Derive g s distribution branch 85 86 Despiking 92 93 Diffusion 66 67 128 Display options 124 Do fit button 143 Edit data 91 176 177 Edit menu 63 65 Error ellipse 150 153 Export 175 176 F statistics 150 153 FAQs 185 187 Broad g s algorithm 185 187 Citing this program 190 Fitting g s versus dc dt data 188 189 205 DCDT User Manual DCDT User Manual aa g s versus g s 185 Good fit 189 High parameter cross correlation 189 190 s versus s 184 185 Should fit the offset 187 File menu 61 62 File open 62 File save 62 Fit control check boxes 138 Fit distribution as N species branch 86 Fit statistics 154 Fit status 144 Fitted parameters 148 149 Fitting parameters monitor 141 143 144 146 Fitting parameters monitor tab 144 146 Fixed or constrained parameters 149 Fluorescence scans 57 60 89 Frequently asked questions How should cite this program 190 Should fit the offset 187 What constitutes a good fit 189 What does high parameter cross correlation mean 189 190 What is the difference between g s and g s 185 What is the difference between s and s 184 185 What is the difference between the broad and conventional g s algorithms 185 187 Why do get different answers when fitting g s versus dc dt 188 189 Fringe jump removal options 99 100 Fringe jumps 99 100 g s overlay graph 27 31 86 87 g s 185 Geometric mean time 108 109
106. button the axes will be rescaled to show you this region If the region you have defined is very small the zoom is not enabled as indicated by the cursor shape You can undo this zoom and return to the previous scales for the axes by entering a Z or z from the keyboard or right clicking on the graph and using the Undo Zoom entry on the pop up menu 47 Note that by default the axis scales will be rounded to even values after you zoom Sometimes this P behavior is undesirable and it can also prevent you from zooming to very high magnification This scale rounding behavior can be turned off by right clicking on the graph and selecting the Round Axis Scales After Zoom entry to toggle it off 4 4 How to remove bad data points from scan files 176 DCDT User Manual SR DCDT User Manual Generally it is not necessary to manually remove individual bad data points from a scan file because those can be temporarily removed by the despiking routine see Load raw scans Data loading options Section 3 2 1 8 If you do want to edit a scan file to remove bad data perhaps for use by other software methods for doing so are discussed below If you do need to manually remove bad data point s this is very easy to do 1 If needed switch to Advanced mode Section 1 3 2 Go to the Load raw scans page Section 3 2 1 and be sure the Despiking option Section 3 2 1 8 is turned off so that all data points are visible Advance to the
107. by the button remaining in the down state then pressing the button will turn off the conversion and revert to raw s values 112 DCDT User Manual SR DCDT User Manual G Conversion to s 20 w Values for experimental conditions partial specific volume ml g 0 7198 solvent density g ml 1 003070 solvent viscosity cp 1 0185 Temperature C 20 0 Partial specific volume at 20 C FI calculate temperature correction partial specific volume ml g 0 7198 ratio s 20 w s raw 1 02920 ratio D 20 w D raw 1 01647 E It is generally desirable to present and report s values rather than raw uncorrected sedimentation coefficients Conversion from raw s to S compensates for the factors such as buffer viscosity which are relevant only to a particular experiment giving the sedimentation coefficient that molecule would have if measured in water at 20 C Because of the solvent independent nature of s it describes quantitatively the fundamental hydrodynamic properties of the molecule and it is this value which is most useful in comparing the sedimentation behavior of different molecules A The partial specific volume that is entered on this form and used in converting to s scale is applied uniformly to the entire g s distribution Therefore if you believe your sample contains species with different vbar values for example different polypeptides or both PEGylated and non PEGylated forms of th
108. c Documents Visual Studio Projects DCDT Help Source HelpStudio3 8EABF9F3 CF47 45CE 80D1 1C75FF48FC72 form to calculate the corresponding diffusion coefficient Enter the run time elapsed time for the first data set the time interval between data sets the total DCDT User Manual SR DCDT User Manual 11 number of data sets 1000 maximum and the rotor speed See Tip below For dc dt analysis you will want the interval between sets to be fairly small perhaps 60 s to simulate interference data or 240 s to simulate absorbance data in a 4 hole rotor Enter the calculation interval in seconds This is the time increment between successive calculations of the concentration distribution o Smaller intervals will give a more accurate but slower simulation Faster moving boundaries will require smaller intervals for low speeds or small sedimentation coefficients larger intervals are fine A 1 sec interval should give good accuracy in most cases but for sharp boundaries to get accurate boundary widths diffusion coefficients you may need to go to 0 1 s The minimum interval is 0 01 sec maximum 50 sec o For highly accurate simulations of conditions giving sharp boundaries you will need a small interval and the simulation will likely require up to a minute to complete Absorbance simulations require smaller intervals because the radial spacing is larger but they calculate faster at a given interval for the same reason Enter th
109. c dt ftting this calculation is done by multiplying the observed r m s residual by the mean time interval between scan pairs and the square root of half the number of pairs For 9 s fitting it is done by computing the root mean square value of the product of the residual for each point and the corresponding sedimentation coefficient and then multiplying this by the mean time interval between scan pairs times the square root of half the number of pairs and then dividing this result by the mean effective sedimentation time for the scans If the Print report button is selected then a standard Windows printer dialog box will open allowing selection of the printer to be used for the report this will by default be the printer set as the default report printer via the Options dialog Section 3 1 1 4 2 The graph will be in color if that printer is a color printer The size of the text font used for reports whether printed or sent to the Clipboard may be altered using the Options dialog Section 3 1 1 4 2 Caution Although the Send report to Clipboard button does not actually generated printed output when a graph is to be included the program must have access to a valid printer in order to size the graph correctly relative to the text If the default report printer is not valid either because it is not currently connected or no printer with that name exists you will get a message asking for a valid printer which could be a PDF printer or other virtua
110. can get the label to display by increasing the axis maximum or decreasing the minimum a tiny amount perhaps 1 part in 100 000 which makes that tick mark clearly within the bounds of the graph Doing this has no visible effect on the graph scaling but solves the tick label display issue Clicking on an axis title or the graph title or subtitle both located above the graph will bring up small text entry dialog box that allows editing of the corresponding axis label similar to that shown below Customize axis title e Edit the X axis label Enter a space character for a blank label Radius cm Note that a completely empty entry is interpreted as the Cancel command so if you want a blank label simply enter a space character These graph title and sub title can also be changed through the Customization Dialog discussed below Right click context menu customizations Right clicking with the mouse on any graph will bring up a context menu that provides access to numerous graph options and customization features 173 DCDT User Manual DCDT User Manual a Viewing Style Border Style gt Font Size Legend Style UY Numeric Precision Plotting Method Data Shadows Grid Options Mark Data Points Display Mouse Coordinates V Round Axis Scales After Zoom Maximize Customization Dialog Export Dialog Help Print Graph to Default Printer The Display Mouse Coordinates item toggles on and o
111. cies with sedimentation coefficient s may be defined by s s A ih 20 gls odir e where s is the center of the peak A is the total area under the curve the loading concentration and o is the width of the Gaussian 193 DCDT User Manual DCDT User Manual aa Note that many definitions of the Gaussian function employ a definition of the width of a Gaussian which is twice that used here this definition is used for consistency with the one used by Walter Stafford The width of the Gaussian o is directly related to D reference 3 Section 7 3 through the relation ey ae Ine 2 O mO Ot 2 D where r is the meniscus position is the rotor angular velocity and 2 t is the integrated product of and time as recorded in the header of each scan When fitting to the dc dt data the fitting function is the analytical time derivative of the modified Fujita MacCosham function reference 1 Section 7 1 The modified Fujita MacCosham function is an approximate solution of the Lamm equation and is used for direct boundary fitting in SVEDBERG This function gives good results for proteins of 5 kDa and above 6 5 About the fitting algorithm This program uses a modified Gauss Newton iterative least squares fitting routine essentially corresponding to the preferred method as described by Johnson amp Faunt 1992 reference 2 Section 7 2 The user may select whether all data points are equally weighted or to a
112. concentration in instrument units In this case image above we now can see that there are some bad data points near zero S Clearly g s should not go negative a negative concentration This problem is probably caused by including data too close to the meniscus 14 DCDT User Manual SR DCDT User Manual GiSeumngeforaista 042 to sse S Scans 00035 IP1 to 0005011 trom C Usersivonn PhilloiDesitop Pnilio DataPro defdt fringes sec el More To clean this up we can just go Back Section 3 1 3 1 one step push the Set range for g s button Section 3 2 4 2 and drag the left magenta bar to set the minimum range to 0 42 S as shown above Alternatively we could navigate back to the Set meniscus amp data region page Section 3 2 2 and move the left hand green marker further away from the meniscus e Note that when the range has been reduced from the maximum possible that value is displayed in the text back with a white background rather than the yellow background used to indicate computed values DCDT User Manual DCDT User Manual aa weight average s 2 570 0 006 total c 1 437 0 002 Scans 00035 P1 to 000501 trom C Usersivonn PrilioiDesktop Pnilio Data Proty o Ny o in o io pe w a T w gt 7p B 2 0 4 a wo a Ss iu 9 _ wee For Q Beck es EA After that range adjustment the Next button brings us this result shown ab
113. cular mass of species 3 is not constrained but it could still be linked to that of species 1 perhaps as a trimer or tetramer and thus the group of radio buttons for linking the mass of species 3 to another species is still active An alternative type of mass constraint is also available For constant f f ratio mass scaling the mass of the constrained species is calculated assuming this species has the same hydrodynamic shape same frictional coefficient ratio relative to an anhydrous sphere f f as the species to which it is linked This constraint is therefore equivalent to the assumption used when calculating the c s distribution in Peter Schuck s program SEDFIT When the f f ratio is constant the mass scales with the sedimentation coefficient to the 3 2 power 131 DCDT User Manual DCDT User Manual aa Thus if species 2 is linked to species 1 with this type of constraint we have 3 S M M x a 1 To use this constant f f ratio mass scaling simply enter 1 as the mass ratio in the text box Sedimentation coefficient constraints o On the right side the sedimentation coefficient of species 2 is also constrained to be a multiple of that for species 1 The ratio of sedimentation coefficients for the constrained species is entered into the text box The table at lower right shows theoretical ratios for oligomers of hard spheres The table has room only for oligomers up to tetramer but ratios for pentamers hexamers
114. culated you may wish to save the summed data to disk If you are going to add noise you will probably not want to add it to each species but rather add it only to the last species If you added the same amount of noise to each species the total r m s noise will grow as the square root of the number of species 60 DCDT User Manual SR DCDT User Manual 3 Program elements controls and features 3 1 User interface 3 1 1 Main menus 3 1 1 1 File menu 2 New Analysis Window Ctrl N B3 Clone Analysis to New Window Close Current Analysis Close All Windows E7 Open Saved Analysis Ctri 0 a save LUTTE ANAIYSIS A C Users John Philo Desktop Philo Data PKR_K296R cell3 dcdt C Users John Philo Desktop Philo Data PKR_K296R cell2 dcdt C Users John Philo Desktop Philo Data PKR_K296R celll dcdt C Users Vohn Philo Desktop Philo Data sim_antibody_4 species 4species_fixed_ff0 dcdt C Users VJohn Philo Desktop Philo Data sim_antibody_4 species 4species dcdt C Users John Philo Desktop Philo Data Y1232 TRAP dcat New Analysis Window start a fresh analysis in a new Analysis window Section 3 1 3 This menu command is duplicated by a button on the Toolbar Section 3 1 2 Shortcut CTRL N Clone Analysis to New Window copy the current analysis into a new analysis window so it can be modified without affecting the current window This menu command is duplicated by a button on the Toolbar Section 3 1 2 Close Current Analysis W
115. d mode Section 1 3 otherwise they will start in Advanced mode Section 1 3 e Always start in Scan Preselect mode default checked if checked when loading scans for analysis the File Open dialog box will show only scans from the cell and data type that are pre selected e Font size for reports 12 the font size in points to be used in printed reports of fitting results Report text is always printed in the Arial font e Printer for reports Windows default printer The text box shows the name of the current printer to be 71 used for reports of fitting results To alter this default use the Browse button to open a Print dialog box and select from the drop down list of available local or network printers DCDT User Manual DCDT User Manual aa 3 1 1 5 Windows menu The Windows menu provides control over the display of the analysis windows Section 3 1 3 and the g s overlay graph Section 3 1 5 1 New Analysis Window aa Wind Close All Cascade Arrange Icons 1 C Philo Data PKR_K296R cell1 00120 00163 IP1 2 C Philo Data PKR_K296R cell2 00120 00163 1P2 3 Analysis1 New Analysis Window This will create a new empty analysis window Section 3 1 3 and is equivalent to the File New in the File menu Section 3 1 1 1 or the New button on the Toolbar Section 3 1 2 Show g s Overlay Window This will open the 9 s overlay graph if it is not yet open or bring i
116. d are now much less important because computers have gotten so much faster since the XL I was first released in 1995 How to speed up data acquisition for absorbance data The scan rate for absorbance data is more complex than for interference data It depends on a number of instrument settings including the scan mode step or continuous radial step size and number of replicates Below is a detailed discussion of the issues involved but the bottom line is that the default settings of continuous mode 003 step size and 1 replicate work well in most situations so generally it is probably best to just stick with the default One common misconception is that the scan rate is inversely proportional to the radial step size It is also 168 DCDT User Manual SA DCDT User Manual important to realize that the radial step size settings are only approximate The actual steps vary from position to position and scan to scan and even the overall average step size does not necessarily match this setting Another confusing issue is whether or not to ask for replicates From strictly a signal noise perspective it can be advantageous to use replicates as discussed in some detail below However the use of replicates with continuous mode is not recommended In continuous scan mode the slit drive motor moves continuously If replicates are used in this mode a single average radius position is assigned to each averaged data point and that radius assi
117. d arise from a truly polydisperse sample containing many components covering a broad range of sedimentation coefficients between 0 and 5 S in which case there really could be a significant amount of a species with a true sedimentation coefficient of 1 2 S On the other hand this distribution might also be broad primarily because of diffusion In that case the non zero value at 1 2 S might not be an indication that there is any real species with s 1 2 and instead the value of 9 s at 1 2 S may arise totally from the influence of diffusion on species having other sedimentation coefficients 184 DCDT User Manual SR DCDT User Manual That is diffusion may make it only appear that there is material sedimenting at 1 2 S because it makes the boundaries broad and therefore spreads the contribution of each species over a range of sedimentation coefficients Indeed this is true in this case since this is actually the result from a Claverie simulation for a 18 kDa protein at s 1 88 plus 5 dimer at s 2 82 Hence the terminology apparent sedimentation coefficient or s arises from the fact that the influence of diffusion has not been removed from these distributions and the resultant uncertainty in interpretation However when the 9 s or dc dt distributions are fitted as a mixture of species the values of sedimentation coefficient returned by the fit represent the values for true individual species and thus should no longer be desig
118. d the first sample in that run an easy approach to generating the appropriate comparisons is to first note which scans were used in for that first analysis this is displayed in the title bar at the top of that analysis window Then simply load only that same range of scans for each of the other cells The quantitative test for reversible association is comparing the weight average sedimentation 4 coefficients at different concentrations see Show integrals over distribution Section 3 2 5 1 If the weight average value increases with concentration reversible association is present How can I use the overlay graph in a publication or presentation To use this result in a presentation one other thing you would probably want to do is modify the figure legend to indicate the actual protein concentrations The default legend uses the captions of each analysis window 29 DCDT User Manual DCDT User Manual aa You can easily modify the legend text for each distribution by using the up down spinner control to select the data set and typing a new description into the adjacent text box as was done to generate the graph below A Overlay distributions normalized g s per Svedberg 00 05 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 s Svedbergs o 0 11 mg ml O 0 32 mg ml lt 1 1 mg ml Graph style Graph all Print Export E seing C Lines Edit legend text 1 1 mamil JE Points and lines You also can easily modif
119. ded but if necessary you can reduce the range of data that will used in calculating the g s distribution trimming off one or both ends by using the Set range for g s button Section 3 2 4 2 or manually altering the limits in the text boxes e Itis not uncommon to see some bad data points near zero S which come from the data points near the meniscus This is especially true for interference data if the reference buffer is not a good match to the sample e If you see the curve go below zero at the right end that probably means your data are still being influenced by the accumulation of solutes at the base of the cell Use the Back button Section 3 1 3 1 twice and move the cut off at the cell base green bar further to the left Now push the Next button Section 3 1 3 1 to move to the Calculate g s distribution page Section 3 2 5 Calculate and display 9 s optionally adjust axis scaling 13 DCDT User Manual DCDT User Manual aa weight average s 2 555 0 006 total c 1 447 0 002 Scams 00035 1 to 000501 trom C 1Usersivionn Philio Desktop Pnilio DataPro o in 9 d S i pe w a a w gt wn D A E ao Fi wo ma 0 1 we Fer Q Bock es EA This page calculates the g s distribution and displays it along with error bars The title line of the graph shows the weight average sedimentation coefficient integrated over the distribution and the total loading
120. del page Section 3 2 6 shown below 31 DCDT User Manual DCDT User Manual aa Density and partial specific volumes solvent density g ml 1 030000 Use the same partial specific volume for all species partia specific volume o partial spe partial specifi partial specifi 2 If needed set the number of species to 1 it will probably already be at 1 Be sure to enter the correct solvent density and partial specific volume if you want accurate molecular masses 3 Push the Next button to advance to the Set initial guesses on graph page Section 3 2 7 shown below 32 DCDT User Manual SR DCDT User Manual Fitting range es fame Make new concentration and peak width guesses based on new peak positions 7 Keep previous peak position concentration and peak width values 0 35 0 30 0 25 a s OD Svedberg 4 The wizard should automatically find the top of the main peak in the data and enter that as the initial guess for the sedimentation coefficient The Next button should already be highlighted so just hit Enter to advance to the Perform least squares fit page Section 3 2 9 This shows you a graph below with an overlay of the data being fitted circles and the theoretical function corresponding to the initial guesses for sedimentation coefficient mass and concentration made by the wizard In this case those guesses are fairly good ones The bott
121. derivative technique you are trying to take a snapshot of what is in the cell at a particular time during the run Thus typically only a fairly small portion of the scans from the run 15 20 are analyzed to produce a particular 9 s distribution Using too many scans is like leaving the shutter on a camera open too long the result is a blurred smeared out distribution This tutorial concerns how to optimally select 20 DCDT User Manual SR DCDT User Manual which group of scans should be used Below three drop down text blocks discuss differences from earlier implementations and present the principles behind deciding at what point in the run the analysis should be done and how many scans should be included in that analysis Then the tutorial describes how to actually interact with the two slider controls on the Select scans to analyze page Section 3 2 3 in order to achieve those goals This link jumps immediately to that How to tutorial section Section 2 2 What s new Previous users of DCDT and users of the Beckman Coulter software or other DCDT implementations will find that data selection procedures are quite different in this version In other implementations the data selection takes place primarily when the scans are selected for loading from disk That can make make data selection awkward and often requires several re starts In this version you generally will want to initially load scans encompassing the ent
122. dialog Section 3 1 1 4 2 for setting user preferences defaults for various program options Each of these menu items is mirrored by an icon on the Toolbar Section 3 1 2 65 DCDT User Manual DCDT User Manual aa 3 1 1 4 1 Mass diffusion calculator This View Menu command or its equivalent button on the Toolbar Section 3 1 2 will bring up a small calculator like form shown below which 1 calculates solute molecular masses given values for sedimentation and diffusion coefficients solute partial specific volume solvent density and temperature 2 calculates diffusion coefficients if the mass and sedimentation coefficient are known EET r Use s and M values from fit Species1 Species2 s Svedbergs 3 599 calculated D Ficks 5 710 ybar mlg 0 730 solvent density g ml 1 00535 temperature C 202 mass kDa 57 77 Ok k d This form is a handy way to determine the molecular mass corresponding to the s and D parameters from a fit where D is used as the fitting parameter or to find the D value corresponding to a molecular mass when M has been used as a fitting parameter without changing the fitting model Section 3 2 6 and re fitting How it works This dialog box implements the Svedberg equation sRT D i vp where R is the gas constant Vv Kelvin is the partial specific volume p is the solvent density and T is temperature in The user
123. do not account for the build up of solutes there Failure to remove this region will cause the dc dt and g s curves to go negative near the upper limit of sedimentation coefficients The default radial limit is controlled by a setting within the dc dt Options tab of the Options dialog Section 3 1 1 4 2 Example showing problems to avoid Failure to exclude this region will cause significant errors in the high sedimentation coefficient region of the dc dt data and 9 s As an example the graph below shows the dc dt curves for individual scan pairs from some data for a sample that unexpectedly contained some low mass fragments from proteolysis 97 DCDT User Manual DCDT User Manual aa 6 7 8 s Svedbergs Since the low mass material has a much higher diffusion coefficient it accumulates over a region at the cell base which extends to radii significantly below the default cutoff of 7 15 cm The result is a negative spike or tail at the high sedimentation coefficient end of the dc dt curve from each scan pair An important but often missed point is that although these regions of bad dc dt data represent only a small fraction of each individual curve the different sedimentation times for each scan pair mean that these bad data occur at different sedimentation coefficients Thus if these 4 data sets were averaged together the entire region above 12 3 S would be contaminated with bad data about 1 6 of the total curve
124. download htm 7 2 reference 2 Johnson amp Faunt 1992 Johnson M L And L M Faunt 1992 Parameter estimation by least squares methods Methods Enzymol 210 1 37 7 3 reference 3 Stafford 1997 Stafford W F III 1997 Sedimentation velocity spins a new weave for an old fabric Curr Opin Biotechnol 8 14 24 7 4 reference 4 Durschlag 1986 H Durchschlag 1986 In Thermodynamic Data for Biochemistry and Biotechnology H J Hinz ed Springer Verlag New York Chapter 3 p 45 7 5 reference 5 Stafford rule of thumb This rule of thumb is described in a document by Walter Stafford posted on the RASMB web site at http www bbri org dcdt Rule pdf 7 6 reference 6 Stafford 1999 W F Stafford Ill 1999 Analysis of reversibly interacting macromolecular systems by time derivative sedimentation velocity Methods in Enzymology 323 302 325 7 7 reference 7 Stafford 1994 Stafford W F III 1994 Boundary analysis in sedimentation velocity experiments Methods Enzymol 240 198 DCDT User Manual SR DCDT User Manual 478 501 7 8 reference 8 Philo 2000 Philo J S 2000 A method for directly fitting the time derivative of sedimentation velocity data and an alternative algorithm for calculating sedimentation coefficient distribution functions Analytical Biochemistry 279 151 163 A pre print of this article is available for downloading from the program download site as the fil
125. e http jphilo mailway com dcdtplus http jphilo mailway com dcdtplus pdf pdf 7 9 reference 9 Behlke amp Ristau 2002 Behlke J and Ristau O 2002 A new approximate whole boundary solution of the Lamm differential equation for the analysis of sedimentation velocity experiments Biophys Chem 95 59 68 7 10 reference 10 Garcia de la Torre J and V A Bloomfield 1981 Hydrodynamic properties of complex rigid biological macromolecules Theory and applications Q Rev Biophys 14 81 139 7 11 reference 11 Efron amp Tibshirani 1986 Efron B and Tibshirani R 1986 Bootstrap methods for standard errors confidence intervals and other measures of statistical accuracy Statistical Science 1 54 77 7 12 reference 12 Johnson amp Faunt 1994 Johnson M L and Straume M 1994 Comments on the analysis of sedimentation equilibrium experiments In Modern analytical ultracentrifugation acquisition and interpretation of data for biological and synthetic polymer systems T M Schuster and T M Laue eds Birkh user Boston pp 37 65 7 13 reference 13 Philo 2011 Philo J S 2011 Limiting the sedimentation coefficient range for sedimentation velocity data analysis Partial boundary modeling and g s approaches revisited Anal Biochem 412 189 202 7 14 reference 14 Stafford 1992 Stafford W F Ill 1992 Boundary analysis in sedimentation transport experiments A procedure for obtaining sed
126. e all branches of the tree except the one that was clicked expand any collapsed branches in all analyses ON E a alter the font size used to display the tree to either make the tree easier to read or to make it more compact 3 1 4 1 Derive g s distribution branch This branch of the Navigation Tree Section 3 1 4 contains five sub branches corresponding to the 5 steps in calculating a g s sedimentation coefficient distribution Each of those steps corresponds to one page of the Section 3 1 3 analysis window 1 85 Load raw scans page Section 3 2 1 o load scan files to analyze or numerically simulate a velocity run using the Claverie simulator Set menisus amp data region page Section 3 2 2 o set the meniscus position and the radial region for data to be used in calculating the g s distribution O for interference scans when needed make adjustments to the removal of jitter and fringe jumps Select scans to analyze page Section 3 2 3 o use scroll bars to select the appropriate time window in the run to use for analysis or just push the Auto Adjust button Section 3 2 3 1 o optionally change sedimentation coefficient scale to s 20 w units Calculate average dc dt curve page Section 3 2 4 o display final average dc dt curve o optionally adjust sedimentation coefficient range used for calculating g s distribution Calculate g s distribution page Section 3 2 5 o display graph of final 9 s distributio
127. e analysis By default a small region to the right of the meniscus is excluded because the data there are usually distorted by optical artifacts It is important to exclude the region near the base of the cell high radii where solutes are accumulating and therefore it may be necessary to drag the right hand green marker further to the left as is true for these data for a protein of fairly low molecular mass The image above shows this analysis page after that adjustment e The positions of the green markers can also be altered by typing values in the text boxes near the top center and top right of the page See Good data begin X cm from meniscus Section 3 2 2 2 and Good data end at radius Section 3 2 2 3 for more information 5 Push the Next button Section 3 1 3 1 to move to the Select scans to analyze page Section 3 2 3 Calculate the dc dt curves for each scan pair and select the subset of scans to be analyzed 10 DCDT User Manual SR DCDT User Manual a Analysis1 00001 00110 1P1 o l Paba Adjust scans used in computing de dt Options Scans used 110 of 110 lt gt Peak Posion wihinnn 4 C rs Firstscan O00001 IP1 Lastscan 00110 1P1 0 222 Scans 00001 1F1 2 00110P1 fom CiUsersonn PhiioiOesicopiPhiio DOstaProtyY arsaa S Ma nran ag N a emir pn GI tae a Wy A devdt fringes sec s Svedbergs ees F More Q Back Next A The image above shows
128. e button is pressed a red horizontal bar used to designate the new zero level is displayed on the graph of the average dc dt curve The bar can be dragged up and down using the mouse At the end of the drag operation the new zero position is used to recalculate and re display the average dc dt curve The Undo button will restore the zero level to the original experimental value When one or more peaks are fitted to dc dt or 9 s data which have been manually re zeroed the amount of the zero shift will be printed in the report of the fitting results 3 2 5 Calculate g s distribution page This page shows the 9 s distribution and provides options to instead display the g s distribution or the integral distribution G s It also provides commands to integrate specific regions or the entire distribution and display the resulting average sedimentation coefficients Each control on the image below is a clickable hotspot that will lead to further information or just use the numbered list below the image 115 DCDT User Manual DCDT User Manual aa Show integrals over distribution z P M x Display options anual adjustment convert 520 0 With error bars 5 G s oli Broad algorithm 3 812 0 003 ITAN EnOp2005 1PKR_KI R Set integration region weight averag 1 to 001631P1 from COXLAW 0 55 0 50 0 45 g gt D gt 9 wo w o mn gt ad on normalized g s per Sv
129. e drawback of this approach is to accurately find the desired confidence limit a large number of rounds N is required If for example N is only 100 then it is not possible to distinguish a parameter frequency of 2 5 the desired value for the lower wing for 95 confidence from a frequency of 2 or 3 That is if you wanted the confidence probability to be accurate to 0 1 it is necessary to have N gt 2000 What happens after have selected these options The Ok button will proceed to actually computing the confidence and Cancel will abort that process During the subsequent computations a small dialog box is displayed If you have selected the Search error ellipse option you will see a dialog box like that below showing which confidence limit is currently being evaluated an estimate of remaining time and a progress bar 152 DCDT User Manual SR DCDT User Manual This may take a while Varying parameters independently working on parameter Co 3 lower limit total time remaining 4 sec For either the Bootstrap or Monte Carlo options the dialog box shown below which indicates the current simulation round an estimate of remaining time and a progress bar This may take a while Working on simulation round 165 total time remaining 10 sec The Cancel button on these dialog boxes will abort the calculations leaving the previous confidence limits intact if they exist What if want to re comp
130. e entry under the View menu Section 3 1 1 3 or use the entry under the Windows menu Section 27 DCDT User Manual DCDT User Manual aa 3 1 1 5 When the overlay window is first opened it automatically graphs all the available data from non minimized analysis windows Section 3 1 3 minimized windows are ignored so you can exclude certain distributions without having to close that analysis window After the overlay window is opened you can force updates of the graph using the Graph all button The example below shows a comparison graph for the same protein stock at 0 11 0 32 and 1 1 mg ml The intent is to get a quick qualitative picture of any changes arising from reversible self association Ay Overlay distributions mn o g s fringes Svedberg o o 02 ao oo os 10 15 20 25 30 35 40 45 50 55 6 amp 0 65 70 75 80 8 amp 5 5 Svecbergs o CA 10429031 10946 083 00120 _00164P3 o C4 10429031 109460elt2 00120_00163 P2 C41042903 1109460ell1 00120_001631P1 5 Graph style Graph all Print Export Points C Lines Edit legend text C 1042903 1 10946 cell3 00120 00164 IP3 fi Points and lines The problem with the graph above is that the large difference in sample concentration makes the scales of the three curves very different and therefore it is hard to directly compare them This is a situation where normalization of the distributions is very helpful After going to the Calculate g s
131. e focus will be moved to the meniscus entry text box Thus to confirm the value set by the wizard all you need to do is push the Enter key on the keyboard e If these are interference scans using the Enter key will then also trigger the jitter wizard Section 6 7 an automatic fringe jump correction Section 3 2 2 5 and an update of the graph after these systematic errors are removed If the wizard has not done a good job and rather than accepting its choice you immediately use the mouse to drag the meniscus marker to a new position those automatic systematic noise corrections will be postponed until the end of that meniscus drag operation e To obtain accurate and reproducible sedimentation coefficients you must set the meniscus position carefully and reproducibly To do this you may need to zoom in on the region around the meniscus see How to zoom with the mouse Section 4 3 4 In Advanced mode the Zoom meniscus button can do this for you 3 2 2 2 Set meniscus amp data region Good data begin X cm from meniscus 96 DCDT User Manual SR DCDT User Manual good data begin 0 01 cm from meniscus This setting is used to exclude the region near the meniscus where the data are distorted by optical artifacts As you adjust the meniscus position the left hand green marker moves along with it to maintain a constant separation This value can be altered either by directly entering a value in the text box or by dragging the left hand green
132. e make the number of data points in the dc dt and 9 s curves approximately match the number of data points in the scans which corresponds to the default for the ds setting in this program then it really doesn t matter if a scan takes twice as long provided it produces twice as many data points with an equivalent noise level Overall then what really counts as a figure of merit for signal noise is the product of the data rate points recorded in the scan file per sec times the value factor for those points inverse square of their noise level Again assuming the replicates actual reduce the noise level by the square root of the number of replicates then our figure of merit becomes data rate replicates and the relative signal noise for different conditions should scale as the square root of this figure of merit The table below shows some results obtained by John Philo All scans covered the region from 5 95 to 6 15 cm A series of 8 scans were done to get the average time per scan The scan files were examined to determine the typical actual number of data points recorded this varies from scan to scan and from this the average step size was computed The figure of merit data points replicates per sec was then calculated and the r m s noise level over the region from 6 0 to 7 0 cm was determined averaged over 4 of the 8 scans Figure of Merit Measured Radial Step Seconds per Actual of Average Step Mode Replicates da
133. e number of data points to cover the radial range between the inner radius and cell bottom Again more points gives more accuracy but a longer calculation o Sharper boundaries will require more radial points for accuracy The data files that are generated will have data at these radial positions If desired specify a nonzero value for the concentration dependence coefficient of the sedimentation coefficient o This algorithm assumes a linear dependence of s with concentration given by the formula S So 1 ke Where k is the coefficient entered on the form For globular proteins an empirical value for k is often taken to be 0 009 ml mg corresponding to c in mg ml In this program the units for k are per AU or per fringe The D value has no concentration dependence If desired specify a nonzero value for the random Gaussian noise to be added to the simulated curves Press the OK button to start the simulation The progress bar will show approximately how far the calculations have progressed and they can be aborted using the Cancel button When the simulation is done the data are automatically loaded into the internal data arrays just as though they had been read in as scan files from a disk You will be asked whether you wish to also save these results to disk as scan files so they could be reloaded at a later date o If you save the scans you may specify which folder to put them into using the File Save dialog box that app
134. e re done and any previously calculated 9 s curves or fitting results will be cleared The consequence of too many points is that there will be far more data points along the X axis than were present in the original scans which requires the use of interpolation to create new data points in al Setting ds too large will result in poor resolution setting it too small will yield too many data points 111 DCDT User Manual DCDT User Manual aa between the experimental ones Thus the data points in the average dc dt and 9 s curves will not be truly statistically independent successive data points will be highly correlated If these curves were subsequently used in fitting then the statistical estimates of the uncertainties in the parameters would be too small because those statistical calculations assume all data points are independent 3 2 4 2 Calculate average dc dt Set range for g s Set range for g s 0 25 tof11 215 S Fairly often you may want to exclude some data at low or high sedimentation coefficients from the 9 s calculation e The data at the smallest s values may be noisy or unreliable because of the presence of low molecular weight contaminants or sedimentation of salts or other buffer components e The data at the highest s values may need to be excluded if that region is being influenced by the buildup of solutes at the base of the cell However it is preferable to eliminate that problem by alteri
135. e same protein you should not apply this conversion you must work in raw sedimentation coefficient units e For similar reasons the partial specific volume entered on this conversion form is applied to all species that are fitted The actual scaling factors that will be applied to correct the raw sedimentation and diffusion coefficients to standard conditions are displayed in the panel nearest the bottom of the dialog box What about extrapolation to zero concentration This program does not attempt to correct for concentration dependence of sedimentation coefficients extrapolation to infinite dilution to give S55 w Often dc dt analysis is applied to samples at concentrations sufficiently low that concentration dependence can be neglected and if so the corrected results can be considered to be Ss w Values but the program will not explicitly designate them as such How this conversion affects the g s distribution and average dc dt curve 113 DCDT User Manual DCDT User Manual aa Please be aware that conversion to s also alters the magnitudes of the 9 s and g s distributions This arises because these distributions are defined such that the area under the curve gives the concentration Thus if the X axis is expanded or contracted as a consequence of conversion to the s20 w scale the Y axis must be contracted or expanded to keep the area unchanged Internally in the program the dc dt 9 s and g s data are
136. e text boxes to input starting guesses for the parameters or fixed values if that parameter is not being fitted e The units for concentrations are absorbance units or fringes sedimentation coefficients are in Svedbergs diffusion coefficients are in Ficks and masses are either in kDa or Svedberg Fick depending on your choice of molecular mass units Section 3 2 6 7 e You may enter values in scientific notation if you choose using formats such as 1 23E1 or 1 23e1 1 23D1 or 1 23d1 The ESCape key will return you to the previous value Warnings will be issued if you enter values outside of the current upper and lower bounds on that parameter 3 2 8 2 Fit control check boxes Fit this parameter v Yes v Yes These check boxes control whether a parameter will be varied iteratively during the fit floated or held constant at the value specified in the text box to the left fixed The text next to the check box will correspondingly read Yes or No k d TIP When a fit is having difficulty converging it is often helpful to temporarily fix one or more parameters so the fitter can get the other parameters into a reasonable range giving a rough fit of the experimental data Then return to the Set alter fitting parameters page Section 3 2 8 and turn on fitting of one more parameter and re fit continuing this process until all parameters are actively fitted The boxes are inactive grayed and read constrained for cons
137. e used whereas with the conventional algorithm the error bars actually increase in going from 12 to 16 scans panel A 0 10 0 08 amp g s AU Svedberg oS x 0 02 16 scans 16 scans j j 9 00 O 4 B 12 16 20 24 2B 32 36 400 4 8 12 16 20 24 2B 32 36 40 s Svedbergs s Svedbergs The broad algorithm may be switched on a case by case basis even after 9 s has already been calculated using a button on the Section 3 2 5 calculate g s distribution page 186 DCDT User Manual SR DCDT User Manual So should I use the broad algorithm Generally no Because the use of experimental weights Section 3 2 6 5 during fitting is no longer recommended and because the improved method Section 3 2 6 2 is now the default the issue of improved estimates for the uncertainties of the data points in the g s or dc dt data is no longer very important Probably the one circumstance where the broad algorithm will continue to be useful is when you want to calculate and report the weight z or z 1 average sedimentation coefficient Section 3 2 5 1 for the whole sample or a portion of the distribution The use of the broad algorithm will improve the accuracy of the estimates of the uncertainties for those quantities How the broad algorithm affects the average dc dt data The broad algorithm also calculates a revised average dc dt curve again providing an improved estimate of the error bars on dc dt when the
138. each of the scans currently in use and sets the meniscus at the arithmetic mean of those positions rounded to the nearest 0 0001 cm after averaging o This usually works well since conventionally for absorbance scans the meniscus position is considered to at the top of the positive spike When the sample OD is quite high the plateau is sometimes above the meniscus spike and this algorithm may fail to find the correct meniscus position in such cases o If you manually drag the meniscus marker you will then be asked whether you want the wizard to re 194 DCDT User Manual SR DCDT User Manual calculate the mean position of the spike This is helpful when the scans contain two positive spikes and the wizard has identified the wrong one as the true meniscus 2 For interference scans there is usually a slight negative dip at the sample meniscus Thus the logic positions the meniscus at the minimum fringe reading within the first 1 4 of the scan 3 For fluorescence scans there is not yet sufficient data to discern a pattern to allow automatic location of the meniscus Therefore at this time the program simply puts the meniscus at 6 0 cm or whatever is the first data point if the scan starts to the right of 6 0 cm 6 7 About the jitter wizard To make the jitter correction automatically the program tests two general regions using spans of 20 data points 1 a region in the air air space o if the scans start to the right of 5 83 cm t
139. ears This dialog box also allows you to create a new folder in which to store the simulation The scans are normally written in sequence starting with 00001 You will also be asked to provide a comment line for the scans Also please note that in order to provide an obvious meniscus position for simulations of conventional cells when the data are written to disk two extra data points to the left of the meniscus are added and the data point at the meniscus position is set to a value equal to the loading concentration This produces a spike at the meniscus similar to that seen in real absorbance data Finally you will automatically be taken to the Set meniscus amp data region page C Users Public Documents Visual Studio Projects DCDT Help Source HelpStudio3 14FCF551 188C 4C48 9E48 0B1B6957B1E6 to view a graph of the simulation you just created o Note that in simulated data the concentration at the cell base often builds up to very high values Thus the graph will be automatically zoomed to give a range from 0 to the loading concentration which means the region where solutes accumulate will go off scale sE Finding the right scan interval and number of scans to get the proper range of boundary movement can 59 take some trial and error Do some quick low accuracy simulations with a long calculation interval to DCDT User Manual DCDT User Manual aa get the time range correct and then do a final accurate simulation with a
140. ecies are assumed not to inter convert over the time course of the velocity run Thus this program is generally not appropriate for analysis of interacting systems unless the rates of the association dissociation process are very slow and the sample is effectively a mixture Further the fact that the program implements five species is not meant to imply that you will be successful at fitting that many to your data Resolving five species would only be possible for very high molecular mass species and even then it would likely be necessary to impose constraints Section 3 2 6 6 on their masses or sedimentation coefficients 3 2 6 2 Select fitting model Improved or classic methods Function computation Improved method Classicmethod hese radio buttons choose between alternative methods for computing the fitting functions They are shown only in Advanced mode The Classic method is provided primarily to provide backward compatibility with DCDT versions 1 xx The Improved method is recommended for all new analyses The differences between these approaches are discussed fully in About the fitting functions Section 6 4 e In the Classic method the fitted values for D or M are quite sensitive to the peak broadening that occurs as the scans used for analysis cover a larger and larger time span see reference 8 Section 7 8 The sedimentation coefficients are also subject to systematic errors but that is a smaller effect The impr
141. ed This causes the Ac At values that are calculated to be a poor approximation of the true derivative dc dt This topic is discussed further under peak broadening limit Section 3 2 3 2 which uses a rule of thumb calculation to 21 DCDT User Manual DCDT User Manual aa determine when too many scans are being included In the past when fitting peaks using done using the Section 3 2 6 2 classic method the peak broadening directly eroded the accuracy of the D or M values so selection of the number of scans was a critical parameter However when using the default new improved method the peak broadening effects do not compromise the accuracy of the fitted hydrodynamic parameters Nonetheless even with the improved method the peak broadening is still present and it can still compromise the resolution between species and thus the ability to obtain accurate properties for each species when multiple species are present Thus it is still generally best to avoid using so many scans that the peak broadening limit Section 3 2 3 2 is far below the molecular mass of the species in your sample In some circumstances it may be quite appropriate to accept significant peak broadening in return for higher signal noise For example if you are unsure whether a small peak in the distribution truly represents an additional species you may wish to add many more scans in the analysis to improve the signal noise and see whether this peak is still present In
142. ed in Walter Stafford s versions of DCDT and the WinDCDT version from the Univ of Connecticut by Jeff Lary and Walt Stafford The differences relate only to the treatment of the data near the meniscus and near the base of the cell 1 This implementation excludes data points at radii just to the right of the meniscus because the data near the meniscus are always distorted by optical aberrations and because the data in this region are also usually noisy o The width of this excluded region near the meniscus is determined by a meniscus clearance parameter with a default value of 015 cm This default value can be altered using the Options dialog Section 3 1 1 4 2 For interference data when the region used for jitter removal Section 3 2 1 4 is to the right of this excluded region near the meniscus then the data within the jitter alignment region will also be excluded from the dc dt calculations since these data have already been forced to be zero on average 2 This implementation also excludes data near and beyond the base of the cell based on a maximum radius 192 DCDT User Manual SR DCDT User Manual for inclusion initial default value 7 15 cm This default value can be altered using the Options dialog Section 3 1 1 4 2 the value can be manually altered for each analysis from the set meniscus amp data region page Section 3 2 2 A similar exclusion is used Walt Stafford and Jeff Lary s versions of DCDT but the value cann
143. edberg i ia on 00 05 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 20 w Svedbergs Below are brief descriptions of the controls on this page more details can be found by following the links Show integrals over distribution button Section 3 2 5 1 Push this button to calculate and display the sedimentation coefficient averages S S and s 1 and total concentration for the entire range of the g s distribution 2 Set integration region button Section 3 2 5 2 This button calculate and displays the sedimentation coefficient averages S S and s and concentration for a region of the g s distribution for example one peak That region is set by dragging with the mouse Normalize button Section 3 2 5 4 This button toggles on or off normalization scaling of the distribution so the entire area under the curve is 1 100 When toggled on the area under a peak represents the fraction of that species Convert to s 20 w button Section 3 2 3 3 This button toggles on or off the conversion of the X axis to Soo w units When toggled on it brings up a dialog box for entry of the property values needed to make this conversion 116 DCDT User Manual SR DCDT User Manual The controls below are shown and available only in Advanced mode Section 1 3 9 Display options Section 3 2 5 6 These options control which type of distribution is displayed g s g s or G s and w
144. elect the scans to analyze Section 2 2 But here we will simply let the program s Auto adjust wizard do the work for us by pushing the Auto adjust button Section 3 2 3 1 this is pre selected when you enter this page so actually you can just hit the Enter key DCDT User Manual DCDT User Manual aa Adjust scans used in computing de dt Scansused 16 of 110 lt pm t Peak ve ER nies broadening ay E oe Auto adjust First scan 00035IP1 Last scan 00050 1P1 119 Scans 000351 20 CCOSO IF1 fom C Users Jonn Prilio DesizopiPnilio OstaiFrotY de dt fringes sec 0 0 0 5 1 0 1 5 2 0 2 5 3 0 3 5 4 0 4 5 5 0 5 5 6 0 6 5 s Svedbergs 00035 IP1 00043 IP1 00036 IP1 00044 IP1 00037 IP1 00045 IP1 00038 IP1 00046 IP1 00039 IP1 00047 IP1 00040 IP1 00048 IP1 00041 1P1 00049 IP1 00042 IP1 00050 IP1 average es F Mor Q Back es EY As shown above after the Auto adjust Section 3 2 3 1 procedure average dc dt curve shows a nice peak that is roughly centered along the X axis and the individual dc dt curves from each scan pair now only 8 pairs all lie fairly close to that average The positions of the two slider controls now reflect the fraction of the total scans that is being analyzed upper slider and the position of that group of scans within the total scans that were loaded lower slider Note that the peak broadening limit Section 3 2 3 2 is now
145. ences between the registered and trial versions This version is converted to the registered version with the serial number that is supplied when you register WHAT DOES IT COST The software license costs 600 with a discount to 400 for academic and non profit institutions Upgrading from version 1 xx is 250 or 150 with the academic discount This is a site license and users may run the software on multiple computers ata single site A site means a single laboratory or a single Facility Analytical centrifuges housed in more than one department or building of a company or university or under the control of independent principal investigators by definition represent different sites and therefore each ofthose sites must purchase its own license Check for updates This opens the Download page in your web browser Program web site This opens the main DCDT web page in your web browser E mail author This opens an e mail for your questions comments or suggestions 3 1 2 Toolbar PRR Ar DN BEAL The Toolbar provides buttons as convenient alternatives for some of the menu commands The usual New Open and Save buttons are equivalents of the New Analysis Window Open saved analysis Section 3 1 1 1 1 and Save analysis as Section 3 1 1 1 3 items in the File menu Section 3 1 1 1 Once an analysis has been saved and assigned a file name the Save button becomes equivalent to the Save analysis Section 3 1 1 1
146. eport and save the fit to disk Computing confidence intervals and printing reports is done from the Report fit results page Section 3 2 10 It is highly recommended that you use the Save analysis as Section 3 1 1 1 3 command under the File Menu Section 3 1 1 1 or the equivalent Save button on the Toolbar Section 3 1 2 so you can quickly reload everything about this fit in the future and continue fitting with altered data or models if you choose to do so k d TIP 1 If you are uncertain whether a particular peak in your data represents a real species as opposed to noise or an artifact you may wish to increase the number of scans used in calculating dc dt by a factor of two or more to improve the signal noise ratio even if doing so results in significant broadening of the peaks The point of adding the additional scans is to see whether this additional peak persists through a larger portion of the experiment or instead just fades away as additional data are added Once you are sure of the qualitative conclusion that the species is real before actually fitting you will probably want to reduce the number of scans again to avoid excessive broadening D TIP 2 Sometimes samples contain a broad distribution of either very large or very small species aggregates or small contaminants Ignoring these species may distort the results for the species of interest yet trying to fit many many species is also unreasonable Under such circum
147. erent and neither is necessarily right or best under all circumstances The differences between fitting to 9 s versus dc dt data are far less when using the new improved method Section 3 2 6 2 than they were using the classic method Section 3 2 6 2 We will first discuss issues that are common to both the improved method and the classic method and then give a detailed and lengthy discussion that applies strictly to the classic method You can and should confirm the phenomenon discussed below for yourself by fitting to Claverie simulations Section 3 2 1 10 where the correct answer is known Differences between fitting g s versus dc dt with either improved or classic methods In multi species fits there may be some differences in sensitivity to minor components and resolving power Minor species that sediment faster than the main component will produce a more prominent peak in the dc dt data than in g s and conversely minor species that sediment slowly will be more prominent in g s than in dc dt In principle this effect is compensated by the fact that the signal noise for the minor component is equivalent in both distributions so fitting either should be equivalent However when the separation is poor the higher amplitude for the minor component may help the fitting so one option may be significantly better than the other Another fundamental difference between the fitting methods arises because the dc dt curves f
148. ever those differences will be negligible compared to the noise in the data How this conversion affects results from fitting When this conversion is used the sedimentation coefficients obtained by fitting will also be s values and the diffusion coefficients from fitting will be D values They will be marked as such on all program outputs and the values used to make the conversion will be printed along with the printed reports of fit results Switching between raw and s units will usually force a re calculation of the average dc dt curve and 9 s distribution because the X axis point spacing will change if that is being determined by the default Automatic option see Calculate average dc dt X axis point spacing Section 3 2 4 1 Thus making this switch will invalidate any previous fitting results How this conversion affects reported diffusion coefficients Activating this option will also cause raw diffusion coefficients to be converted to D values via the formula 293 15 Dy D a l Ny w T where T is the sample temperature Kelvin Data required for conversion Conversion to Ss requires knowledge of the solvent density at the experiment temperature the solvent viscosity h at the experiment temperature the solute partial specific volume vbar at the experiment temperature and the solute partial specific volume at 20 C through the relation 1 Vr Prb how where the
149. f species 1 a Fitted parameters Section 3 2 10 1 This table lists the best fit parameters the estimated uncertainty for that parameter the standard error or true confidence limits after those have been computed and the initial value for that parameter at the start of the fit procedure Fixed or constrained parameters Section 3 2 10 2 This table lists the values for parameters that were held constant during the fit and also any parameter constraints that were imposed View parameter cross correlations Section 3 2 10 3 This button will bring up a dialog box displaying the parameter cross correlation matrix See What does high parameter cross correlation mean Section 5 2 4 Compute confidence intervals Section 3 2 10 4 This button initiates a procedure to calculate more rigorous estimates of the uncertainties in the parameter values Generally you will only do this when you are satisfied this is your final result The button is disabled in the example above because this computation has already been completed Create report group box Section 3 2 10 5 Either of the two buttons will generate a report which documents every aspect of your analysis This report is then either printed or copied to the Clipboard in rich text format for pasting into a word processor or electronic laboratory notebook The Include Graph checkbox controls whether a summary graph showing an overlay of the data and the fit as well as a residual
150. f you right click on any scan name a pop up context menu will appear with options to 1 display the scan header information comment wavelength run time total number of data points etc 2 edit the scan file using NotePad If you choose to edit the scan file in NotePad the program will minimize itself and launch NotePad with that scan loaded The program then waits for NotePad to close When NotePad closes the program detects whether the file was altered and if so it automatically re loads the altered data For other data editing options see How to remove bad data points from scan files Section 4 4 3 2 1 7 Load raw scans Scans to be used in g s calculation list box This list shows the scan file names for the subset of scans currently designated to be used in calculating the g s distribution These are the ones actually used if the distribution has already been calculated 91 DCDT User Manual DCDT User Manual a Scans which are loaded into memory but which are being excluded are shown as a in the same position in the list as that scan appears to the left in the Scans currently loaded in memory list box Section 3 2 1 6 In the example shown scans 100 119 have been loaded but are being excluded If you right click on any included scan name a pop up context menu will appear with options to 1 display the scan header information comment wavelength run time etc 2 edit the scan file using NotePad 3 2 1 8 Load r
151. ff a display of the graph coordinates X Y values that correspond to the current mouse position when the mouse is positioned over the graph The Round Axis Scales After Zoom item toggles on and off whether after zooming in on a region of the graph see How to zoom with the mouse Section 4 3 4 the graph scales are first rounded rounding on or instead the graph displays the exact region that was zoomed rounding off Rounding avoids very odd values for the scale minimum and maximum but after zooming into a very small region it sometimes leads to a range that is much larger than you intended to display The Customization Dialog item brings up a tabbed dialog shown below that allows you to alter features such as line point styles and colors the plotting method for individual data sets points lines etc legend display legend position graph borders and graph background colors This customization dialog also provides a second means of customizing axis scaling but not tick marks as can be done by clicking directly on the axis and graph titles but not axis titles 174 DCDT User Manual SR DCDT User Manual Customization General Plot Subsets Axis Font Color Style i Graph Attributes Desk Foreground ERR REED oer mi To ele Shadow Col ee E Quick Styles Graph F d i aie ia Bitmap Gradient Styles _ Graph Background Light Medium Dark Table Foreground Inset Inset Inset C Shadow _ Shadow
152. ff0 rate mass scaling Predicted sedimentation coefficient ratios dimer 1 45 Cancel changes trimer linear 1 7 trimer triangular 1 86 on 200 tetramer square planar 2 20 Ter a tetramer tetrahedral 2 26 It would also be possible to constrain the sedimentation coefficient of our putative dimer to be an appropriate multiple of the monomer sedimentation coefficient but because sedimentation coefficients depend on conformation it is hard to predict precisely what that ratio should be However if a species is present only in a very low amount constraining its sedimentation coefficient may be necessary to get the fit to converge less than 1 For example suppose you had a protein where the native structure is a tightly associated dimer but your sample contains a small amount of a partially denatured incompetent non associating monomer In that situation you would want to constrain the monomer to be half the molecular mass of the dimer That is the reference species should be one that comprises a substantial fraction of the total and whose properties will be well determined by the fit rather than a minor component Gel Note that in some situations it is better to constrain a species to be a fragment of another mass ratio The Ok button will then close the dialog and set the constraint returning you to the Select fitting model page Section 3 2 6 When you return the Constrain M or s ratios among species chec
153. fficient of the whole g s distribution then it is important that the derived distribution function comes fully to zero at both the low and high sedimentation coefficient ends of the distribution This means the scans should be early enough that no species has reached the cell base yet late enough that all species have cleared the meniscus e If you are studying a system in which you are trying to resolve the properties of multiple individual components a mixture or a strongly interacting system that can be treated as a mixture then usually the best choice is a time period just before the most rapid component reaches the cell base However when there is a large spread in sedimentation coefficients it may also be worthwhile to run an additional analysis later in the run after the faster components are pelleted in order to obtain more accurate values for the sedimentation and diffusion coefficients of the slower sedimenting component s How many scans should be included in the analysis Adding more scans to the analysis involves a trade off between improving the signal noise ratio and the loss of resolution as a consequence of peak broadening A loss of resolution peak broadening occurs as more scans are added because the amount of boundary movement between the first and last scan used in the analysis becomes excessive Again using our snapshot analogy there is too much movement while the shutter is open so the picture is blurr
154. ficients would still be correct Thus it is not a good idea to start the rotor spinning and then wait for the temperature to equilibrate See How and why to equilibrate the temperature Section 4 2 2 o However in most cases it is quite acceptable to pause at low speed 3K to take a quick scan to check that the samples are loaded properly The exception to this would be for samples with very high s values that will sediment significantly even at low speed 2 Influence of cell base One common problem in dc dt analysis is failure to properly exclude the region at the cell base where solutes are accumulating e Optimally this region should be excluded at the time the data acquisition is set up and therefore it never appears in the data Proper adjustment of the Good data end at radius Section 3 2 2 3 setting should take care of this problem However for low mass solutes or different cell types this default may not exclude all the bad data resulting in negative dc dt and 9 s values at the high sedimentation coefficient region See Good data end at radius Section 3 2 2 3 for further discussion and an example graph 3 Insufficient sample volume A common cause of poor resolution of multiple species in an analysis is loading too little sample into the cell too little column height A longer sedimentation distance improves the physical separation between species having different sedimentation coefficients and also reduces the
155. fit deldt data fit M SID ratio instead of D Improved method Weighting of data points Classicmethod Theoretical Experimental None 3 mE Constrain M or s ratios among Set constraint values H species _Set constraintvalues Molecular mass units Density and partial specific volumes solvent density g ml 1 001360 C s D Svedberg per Fick JV Usethe same partial specific buoyant molecular mass kDa volume for all species temperature must be known R partial specific volume for EZE Temperature 7C 20 2 ail species ml g partial specific volume of molecular mass kDa species 2 ml g temperature solvent density partial specific volume of and solute partial specific volume species 3 ml g must be known partial specificvolume of species 4 ml g partial specific volume of species 5 ml g Below are brief descriptions of the controls on this page more details can be found by following the links Q Number of species Section 3 2 6 1 Set using the up down spinner or type a number into the text box allowable range 1 5 Molecular mass units Section 3 2 6 7 Choose among true mass buoyant mass or s D ratio Only relevant when the Fit M s D ratio instead of D Section 3 2 6 4 option is on always true in Standard mode Section 1 3 3 Density and partial specific volumes Section 3 2 6 8 Set the solvent density and sample partial specific volume s Only relevant w
156. g fitted x y pairs Calculated fit This menu selection will copy the theoretical values from the fitted functions x y pairs Residuals This menu selection will copy the residuals from the fit x y pairs Species table This menu selection will copy the total of all species followed by the contribution from each individual species x total species1 species2 This menu item is only enabled if 2 or more species were fitted 3 1 1 3 View menu a g s Overlay Window Toolbar x Navigation Tree The View menu provides one of several means to display the g s overlay graph Section 3 1 5 This menu item will only be active when two or more analysis windows that contain 9 s distributions are open enough data to compare and overlay This menu also allows you to toggle on or off the display of the Toolbar Section 3 1 2 and the Navigation Tree Section 3 1 4 Turning off the Toolbar Section 3 1 2 and or the Navigation Tree Section 3 1 4 will provide additional screen space for display of multiple analysis windows 3 1 1 4 Tools menu B Mass Diffusion Calculator l P Options The Tools menu contains submenus to bring up either of two tools 1 the Mass diffusion calculator Section 3 1 1 4 1 for calculating masses when the sedimentation and diffusion coefficients are known or diffusion coefficients when the mass and sedimentation coefficient are known 2 the Options
157. gion is barely cleared G Tip 2 If you have hundreds of interference or fluorescence scans at least initially to save time you may want to load only every second or third scan by using the Load every Nth scan Section 3 2 1 8 option If the Preselect data type and cell Section 3 2 1 1 option is on checked then the File Open dialog will initially show only those scan files that match that data type absorbance interference or fluorescence and cell number You can always change the data type and cell number that are being shown or display all scan files by picking appropriate choices from the Files of type drop down list near the bottom of the File Open dialog box The initial scan folder that will be displayed depends on settings within the File Locations tab of the Options dialog Section 3 1 1 4 2 You may choose either to always start in a particular folder for example the folder where your instrument deposits new data or to have the program remember the location of the last data that were loaded and to always start there 3 2 1 3 Load raw scans Replace scans for this sample button Replace scans for this sample This button is used to load a different set of scans for a particular sample retaining sample dependent settings such as the meniscus location It is the retention of those settings that distinguishes this operation from using the Load New Scan Files button Section 3 2 1 2 to replace the scans This but
158. gnment assumes the motion is uniform which is probably not true Thus the use of replicates with continuous scans can compromise the precision of the radius values which in turn compromises the precision of the sedimentation coefficients which is one of the key strengths of sedimentation velocity Signal noise considerations for different absorbance scan settings Is it better to have more data points with a higher noise level or fewer with a lower noise level In theory the value of each data point is inversely proportional to the square of its uncertainty For the moment let us assume that the number of data points per scan is independent of whether we use replicates If we ask for four replicates and actually obtain the two fold lower noise level for those points expected by theory then each of those points is worth four times as much as a data point with 1 replicate Thus if our scan with four replicates takes more than four times as long to produce we are worse off using replicates if our scan with four replicates takes less than four times as long we are better off using replicates even though we will have fewer scans to use in the analysis In practice the signal noise issue with replicates is even more complex because asking for replicates also affects the number of data points produced per scan In theory our ability to define the properties of individual species by fitting is proportional to the total number of data points If w
159. goals of good signal noise and low peak broadening Remember this automatic adjustment will not necessarily produce the true optimum settings but they should be at least a good starting point for your further adjustments Remember too that the wizard is not as smart as you are and can sometimes be fooled by noisy data 3 2 3 2 Select scans to analyze Peak broadening limit Peak broadening limit kDa 516 This displayed molecular mass value provides a means to test whether the time span between the first and last scans currently included in the analysis is too large to give an accurate approximation of dc dt for the species you are studying If too many scans are being used the peaks in dc dt and g s will be broadened resulting in poor resolution This will also result in incorrect estimates for D and M if such peaks are fitted using the Section 3 2 6 2 classic approach of fitting to Gaussian functions but does not cause such errors when using the new Section 3 2 6 2 improved method The rule of thumb Whether or not the time span is too large depends on the molecular mass s D ratio of the species that are sedimenting with higher mass species requiring shorter time spans and therefore fewer scans This limit is calculated using a rule of thumb test to determine the approximate highest molecular mass that can be present without experiencing significant broadening M max The rule of thumb that is used was devel
160. graph area e Note that for graphs containing large numbers of scans especially interference scans there can be a significant delay 10 sec or more between clicking the button and the display of the graph and also between clicking a data point and the display of its identity This is simply because there are tens to hundreds of thousands of clickable regions to be created and then identified The main purpose of this display mode is to help identify rogue scans see How to identify and remove rogue scans Section 4 5 or individual bad data points When the same data point is clicked two times in succession separate single clicks not a double click the program assumes this may be a bad data point that you want to remove You will be asked a series DCDT User Manual DCDT User Manual aa of questions about whether you want to remove that point only temporarily from memory or to permanently alter the scan file either reversibly or irreversibly See How to remove bad data points from scan files Section 4 4 for more details 3 2 3 Select scans to analyze page Overview This page displays a graph of the dc dt curves calculated from each scan as dashed lines as well as the average of all those individual dc dt curves as a thicker black line Adjust scans used in computing de dt Scansused 44 of 100 lt mim t Peak D AE T broadening Position within run limit kDa Use true mean time Firstscan 001201P1
161. gs 2 sum residuals The image above shows this analysis page immediately after entry from the Set initial guesses on graph page Section 3 2 7 for a two species fit 9 Fit overlay and species plot The upper graph panel displays an overlay of the data to be fitted displayed as circular points and the initial theoretical fit cyan line based on the initial peak positions that were set on the Set initial guesses on graph page Section 3 2 7 The graph also shows the individual theoretical contribution from each species blue and green lines which when summed give the cyan line Residual plot The lower graph panel displays a residual plot red line The vertical scale of this plot will change as the fit proceeds and the residuals grow smaller 3 Status box The Status box Section 3 2 9 5 will display status messages as the fit proceeds In this case the program is waiting for the Do fit button Section 3 2 9 1 1 iteration button Section 3 2 9 2 Simulate button Section 3 2 9 3 or Cancel button Section 3 2 9 4 to be pressed 4 Iteration counter The iteration counter simply displays the total number of iterations in this fitting session 0 in this case since fitting has not started 142 DCDT User Manual SR DCDT User Manual Simulate button The Simulate button Section 3 2 9 3 simply computes the theoretical curves based on the current parameter settings The primary purpose is that this will en
162. he Claverie simulator Section 2 6 3 2 2 Set meniscus amp data region page This page allows you to view a graph of the scan data set the meniscus position and set the radial limits defining the data to be used in generating g s distributions The radial positions or the meniscus and for the data range can be specified either directly in the text boxes at the top or by clicking and dragging the vertical red or green markers on the graph itself When this page is first displayed with new data the meniscus wizard Section 6 6 automatically attempts to set the meniscus position but manual adjustment is always needed for fluorescence scans and may also be needed for interference or absorbance scans Each control on the image below as well as the vertical position markers on the graph are clickable hotspots that will lead to further information or just use the numbered list below the image 94 DCDT User Manual SE DCDT User Manual e The bottom row of controls is visible only in Advanced mode Section 1 3 These controls allow you to zoom in on the meniscus region to make a more accurate placement although you can always zoom with the mouse to display the analyzed data region as a 3 D waterfall graph or to display whether the rotor temperature changed over the course of the run For interference scans two groups of controls are active to allow fine tuning the removal of jitter and fringe jumps Lastly the ID points button di
163. he first 20 data points in each scan are used o otherwise a region started at 5 83 cm is tested if the scan to scan variance is high at 5 83 cm probably because the reference meniscus is to the left of 5 83 cm the wizard compares regions starting at 5 82 5 81 and 5 80 cm and picks the best one lowest variance 2 the region to the right of the meniscus starting at the left green good data marker a region that will be constant if the meniscus is fully cleared at the time of analysis o the initial trial region begins 0 007 cm to the right of the green data marker o four additional regions each 20 points further to the right are also examined to see which of these five possible regions is best if the boundary is well clear of the meniscus generally a position further to the right closer to the boundary works best The wizard examines the deviations within each scan over these regions and picks the one where the variance is lower after removing the average Y value for that scan over that region 6 8 About the bootstrap method In the bootstrap with replacement reference 11 Section 7 11 method usually shortened to just bootstrap method for evaluating confidence limits data points are selected randomly from the original fitted data set to make a new data set with the same number of points The random selection means that some of the original points will be selected more than once and obviously others not at all That
164. he program assumes this means you will want new estimates for c and D or M so it automatically selects the Make new concentration and peak width guesses based on new marked positions option e If you instead want to force the program to retain the current values then manually select the Proceed using previous concentration and peak width values option How are the initial guesses for c and D or M computed An initial guess for the concentration is made based on the magnitude of the data at the marked peak position the peak height and a guess for D or M is made based on the peak width at half height Since the peak widths are used in calculating the D or M value these guesses will probably not be very accurate for peaks that are poorly resolved The peak width is also used in determining the guess for the concentrations so those values may also be significantly in error for poorly resolved peaks Nonetheless these initial guesses are generally more than adequate starting points and will usually allow the fit to converge 3 2 8 Set alter fitting parameters page This page is used to manually enter or alter the initial guesses for the fitting parameters for specifying whether those parameters will be fitted or held constant and for specifying the criterion for convergence of the fit In Standard mode Section 1 3 this page is skipped If you move to this page from the Set initial guesses on graph page Section 3 2 7 you will see the ini
165. he use of experimental weights is not recommended this option is offered primarily for backward compatibility with DCDT versions 1 xx How are the theoretical weights calculated Because each data point in the average dc dt curve is divided by its corresponding s value during the transformation from dc dt to 9 s the theoretical uncertainty for the g s values is much higher when the value of s is near zero the left end of the distribution than it is when s is large the right end of the distribution Thus the theoretical weight of each point is simply given by A x s 2 where the constant A is chosen so the sum of all weights equals N the total number of data points as is true when all points are weighted equally These theoretical weights generally give a much more realistic estimate of the relative precision of data from different regions of the 9 s distribution than the experimental ones and therefore this is now the default for fitting 9 s When fitting to the dc dt data the theoretical weight for all points is the same the same as for an unweighted fit so the theoretical weight option is disabled An unweighted fit is now the default when fitting to the dc dt data Consequences for reported fit statistics If you elect an experimentally weighted fit a chi squared value will be calculated and displayed with the fit results for both unweighted and theoretically weighted fits the sum of squared residuals will be calcu
166. hen it is checked the input box for the vbar at 20 C is disabled but the computed value is displayed The viscosity values are to be entered in centipoise Since SEDNTERP displays these values in poise to facilitate copying and pasting numbers from SEDNTERP whenever a number is entered that is less than 0 1 the program will assume it is a value in poise and multiply it by 100 The program automatically fills in the experiment temperature based on the temperature recorded in the header of the last scan used in calculating the dc dt data but since the temperature may drift during the run you may wish to adjust that value manually Please note that changing the temperature on this form overrides the temperature recorded with the scans and also alters the value used elsewhere in the program e g for conversions to buoyant or true mass units 3 2 4 4 Calculate average dc dt Reset zero amp Undo buttons M Manual adjustment Reset zero Undo o The Reset zero button is used to manually redefine the zero level for the average dc dt curve This may B be necessary to compensate for uncorrected jitter in interference data and or other effects that are causing shifts of the zero level If uncorrected such shifts may cause the g s curve to go below zero or remain above zero in regions away from the peaks Since this manual zero adjustment could bias the results its use on a routine basis is not recommended When th
167. hen the Fit M s D ratio instead of D Section 3 2 6 4 option is on always true in Standard mode Section 1 3 a The controls below are shown and available only in Advanced mode Section 1 3 Data choice Section 3 2 6 3 You may fit either to the 9 s distribution or the average dc dt data Fit diffusion or mass Section 3 2 6 4 Choose either D or molecular mass s D ratio as the second hydrodynamic parameter used to characterize each species 126 DCDT User Manual SR DCDT User Manual Improved or classic methods Section 3 2 6 2 Choose between alternative methods for computing the fitting functions The improved method is recommended for all new analyses the classic method is provided for backward compatibility with versions 1 xx B Weighting of data points Section 3 2 6 5 Use theoretical weighting recommended experimental weighting or no weighting Constrain M or s ratios among species Section 3 2 6 6 Use constraints to force species to behave as expected for a series of oligomers 3 2 6 1 Select fitting model Number of species 5 Number of species 2 i You may select 1 to 5 species by either typing the number into the text box or using the up and down arrows on the spinner control to increment the number up or down Gal It is important to keep in mind that for multi species fits 1 the different species are assumed to sediment independently behave as a simple mixture 2 the different sp
168. hether error bars are included on the graph Reset zero and Undo buttons Section 3 2 5 7 The Reset zero button allows manual adjustments of the zero level Use with caution Undo will remove the manual adjustment Broad algorithm option Section 3 2 5 8 This option toggles on or off the use of an alternative algorithm to calculate g s That alternative algorithm gives better estimates of the error bars for the points in the distribution when the peaks are significantly broadened due to excessive boundary movement during the selected scans 3 2 5 1 Calculate g s distribution Show integrals over distribution Show integrals over distribution This command calculates and displays the total concentration and the number weight z and z 1 average sedimentation coefficients s S S and s for the entire range of both the 9 s and g s distributions sed Coefficient Averages amp Peak ARSENE Integration range M FuilRange 0245 to 7980 s _ Redo Properties at time of analysis from 9 s Concentration a Fraction oftotal 100 00 0 00 Number averagesvalue 3 585 0 011S Weight average s value 3736 00035 Zaverage s value 3972 0 003S z 1 averages value 4231 0 0035 Properties extrapolated to t 0 from g s Loading concentason S1 Fraction oftotal 100 00 0 00 Number average s value 3612 0 0095 Weight average s value 3 780 0 0035 zaveragesvalue 4 022 0 003 S 2
169. ht click with the mouse on any graph to bring up a context menu that provides access to various graph options and customization features including the Export dialog 175 DCDT User Manual DCDT User Manual aa Viewing Style Border Style gt Font Size Legend Style Numeric Precision gt Plotting Method gt Data Shadows Grid Options gt Mark Data Points Display Mouse Coordinates V Round Axis Scales After Zoom Maximize Customization Dialog Export Dialog Help Print Graph to Default Printer The Export dialog menu item brings up a dialog box that allows 1 exporting the image to a file or the Clipboard 2 exporting the data that creates the graph 3 printing the graph to any available printer With regard to exporting data values note that the data contained in many of the graphs can be more easily copied to the Clipboard using the Edit menu Section 3 1 1 2 Exported image types supported include Windows metafile Windows bitmap BMP JPEG and portable network graphics PNG 4 3 4 How to zoom with the mouse To zoom in enlarge a section of the graph you can click the left mouse button at the corner of a region within the axes of an existing graph and then drag move while holding the left mouse button until you reach the opposite corner of the region you wish to be expanded As you drag an inverted area will indicate the region being marked When you stop dragging by releasing the mouse
170. icant changes in parameter values Specifying a larger value for them can help avoid wasted iterations especially in situations where the parameters are highly correlated Gal In any iterative fitting scheme the iterations can proceed indefinitely However if the fit is successful at some point the sum of squared residuals will approach a minimum value and the changes in parameters between successive iteration will be very small so further iterations are pointless Therefore it is necessary to have a rule to define when the fit has converged The first criterion for convergence is based on a percentage precision on the parameters Fitting continues until the changes in all parameters with each iteration are below this precision By default this program will define the fit to have converged if the change in all parameters is less than 0 05 but this can be altered from the Set alter fitting parameters page Section 3 2 8 The problem with using parameter precision as the only convergence criterion is that usually some of the parameters can be determined with much higher precision than others and many many iterations can be spent trying to get high precision on a parameter whose standard error will be very large In this program it is almost always possible to determine the sedimentation coefficients with much higher precision than the diffusion coefficient or molecular mass This program avoids this problem by also basing convergence on a seco
171. ile menu 61 62 Fit distribution as N species branch 86 g s overlay graph 86 87 Help menu 72 74 Log page 78 83 Mass diffusion calculator 66 67 More gt gt and lt lt Less buttons 78 Navigation tree 83 85 Next and Back buttons 77 78 Open saved analysis 62 Options dialog 67 72 Save analysis 62 Save analysis as 63 Toolbar 74 75 Tools menu 65 User prompt area 83 View menu 65 Windows menu 72 User prompt area 83 Vbar 133 134 View menu 65 View parameter cross correlations 150 Weight average sedimentation coefficient 117 118 119 119 121 Weighted fit 128 129 What constitutes a good fit 189 What does high parameter cross correlation mean 189 190 What is the difference between g s and g s 185 What is the difference between s and s 184 185 What is the difference between the broad and conventional g s algorithms 185 187 Why do get different answers when fitting g s versus dc dt 188 189 Windows menu 72 X axis point spacing 111 112 Z average sedimentation coefficient 117 118 119 119 121 Zoom 176 Zoom meniscus button 101 213 DCDT User Manual
172. imentation coefficient distributions using the time derivative of the concentration profile Analytical Biochemistry 203 295 301 199 DCDT User Manual 200 DCDT User Manual aa DCDT User Manual BA DCDT User Index of sections up dow Manual n spinner 109 110 1 iteration button 143 About the bootstrap method 195 196 About the fitting algorithm 194 About the fitting functions 193 194 About the jitter wizard 195 About the meniscus wizard 194 195 About the Monte Car lo method 196 197 About this g s implementation 192 193 About using radial intensity scans 191 192 Absorbance scans 57 60 89 Add more scans for t Adjust parameter up his sample button 90 per lower limits button 139 141 Advanced mode 3 4 Analysis page 1 Load raw scans Load raw scans Load raw scans Load raw scans Load raw scans Load raw scans Load raw scans Load raw scans Load raw scans Load raw scans Add more scans for this sample button 90 Data loading options 92 93 Delete selected scans button 90 91 Load new scan files button 89 90 Preselect data type and cell check box 89 Replace scans for this sample button 90 Run Claverie simulation to generate data button 93 94 Sample description 93 Scans currently loaded in memory list box 91 Scans to be used in g s calculation list box 91 92 Load raw scans page 88 89 Analysis page 10 Report fit results Report fit
173. indicate this sample consists of about 90 of a 58 kDa monomer at 3 56 S plus about 10 dimer at 5 45 S The first thing we want to know if whether this monomer dimer mixture model actually gives a good fit of the data One way to examine the quality of fit more closely is to push to the Next button to generate various graphs of the fitting results 53 DCDT User Manual DCDT User Manual a ic AL Analysis1 00120 00164 1P3 babea See rh pm tom Scans 0012003 to 0016403 trom C iWsersivonn PhilloiDesktop Philio DataPKR_K296R 0 14 a s fringes Svedberg 0 02 0 00 0 02 1 0 15 2 0 25 3 0 35 40 45 5 0 5 5 6 0 6 5 7 0 75 8 0 s Svedbergs data MBM species1 MBM species 2 sum ad Alles Cbs A The image above shows a species graph summarizing this fit There are no obvious regions where these two species do not explain the experimental data fairly well except possibly in the region from 1 4 to 2 2 S 54 DCDT User Manual SR DCDT User Manual r m s deviation 2 35E 3 Y exp fit 35 40 45 50 55 s Svedbergs Similarly the residual plot above suggests this is statistically a fairly satisfactory fit of these data except for the region below 2 2 S The deviations below 2 2 S might indicate there is a trace of a third slowly sedimenting component but note that these are interference data and thus the low sedimentation coefficient region is subject to systematic errors if
174. indow close the active analysis window Close All Windows close all open analysis windows Open Saved Analysis Section 3 1 1 1 1 61 DCDT User Manual DCDT User Manual aa restores results and or fits previously saved to disk This menu command is duplicated by a button on the Toolbar Section 3 1 2 Shortcut CTRL O Save Analysis Section 3 1 1 1 2 update analysis previously saved to disk This menu command is duplicated by a button on the Toolbar Section 3 1 2 Shortcut CTRL S Save Analysis As Section 3 1 1 1 3 assign file name and save dc dt and g s curves and fitting results if any in a permanent record on disk Exit exits program after reminder to save unsaved or altered analyses to disk if there are any Recent Files list of 6 most recently saved or restored analysis files click on these entries to restore that analysis in a manner equivalent to the Open Saved Analysis Section 3 1 1 1 1 command 3 1 1 1 1 Open saved analysis This command will restore the data results and states of various program variables from dc dt or g s calculations and or fitting results that were previously saved using the Save Analysis As Section 3 1 1 1 3 command This menu command is duplicated by a button on the Toolbar Section 3 1 2 The command brings up a standard Windows file dialog that allows the user to specify the file name and folder of a previously saved dcdt file saved by version 2
175. ints This will work well if all the scans are taken after the meniscus region has cleared 3 Middle half compares the scans in the region from 1 4 to 3 4 of the sample column This is the default method when the program is initially installed 4 Outer eighth makes the comparison only for the high radius portion This will work well if all the scans have a well defined plateau region provided the scans do not include too much data from the region where sample is accumulating at the bottom of the cell The outer end of the range used for this comparison is limited by the good data end at radius setting A default automatic method will automatically be used whenever this analysis window is first entered after new scans have been loaded Whenever an automatic method has been used the Auto button within this group will be depressed If a different method is selected via the drop down list box while the Auto button is depressed the new method will be calculated and applied to the data displayed on the graph Manual fringe jump removal The Manual button initiates a procedure where the position used for fringe jump compensation is specified by double clicking on the graph at the desired radial position The actual region used in the calculation is then a group of 11 points centered around that position This manual setting will change the Auto button to the off raised position Once the Manual button is pushed this operation must be c
176. ious saved using the Save analysis as Section 3 1 1 1 3 command This command is enabled when the previously saved version is altered 62 DCDT User Manual SR DCDT User Manual 3 1 1 1 3 Save analysis as This command will write out a file that contains all the data raw scans dc dt and g s curves and any fit results that have been obtained up to and including the last calculation or fit that was performed This provides a permanent record of your work that can be quickly reloaded if further processing is needed or to print graphs and reports and to serve as an archival record of your work The command brings up a standard Windows file dialog that allows the user to specify the file name and folder The user can specify a filename of any length the program will automatically add the extension of dcdt to that name This command does not become active until after at least the individual dc dt curves have been calculated Select scans to analyze page Section 3 2 3 The data file is written in a proprietary binary format All scans which have been loaded into memory are saved independent of whether that scan is presently included in the analysis The scan data require only 8 bytes of storage per data point much more compact than the original ASCII scan files The path to this saved results file will be added to the top of the recent files list at the bottom of the File menu Section 3 1 1 1 3 1 1 1 4 Clone analysis t
177. ire range of boundary movement and then use the two slider controls on the Select scans to analyze page Section 3 2 3 to select a portion of those scans for analysis This makes it very easy to tune into the region you want without needing to start over and load different sets of scans When during the run should the analysis be done The general rule is that optimal resolution separation of species occurs just as the fastest sedimenting species reaches the base of the cell Actually it is not truly the base of the cell that counts but rather the region near the base where solutes accumulate Thus if your sample contains only one species the optimal situation is when the last scan used in the analysis was taken just before the plateau region disappears If a given sample contains species whose sedimentation coefficients differ by more than a factor of 2 this means that there is no time during the run at which all species are still present in the cell and at which the properties of the slowly sedimenting component s can be determined optimally Under these circumstances there may be no single best set of scans for the analysis and determining which scans should be analyzed depends on the nature of the sample and what you are trying to learn e If you are studying an interacting system self association mixed association or ligand binding which you are going to characterize by calculating the weight average sedimentation coe
178. is each bootstrap data set is really a subset of the original data but some points are repeated This new data set is then fitted and the new fitted parameters are stored in an array This whole process is repeated N times and then for each fitted parameter the array of N values is used to evaluate the confidence limits Note that in the implementation used here some random noise is added to the best fit parameters before each round so that each bootstrap round begins from slightly different starting values to avoid biasing the results What are the advantages and drawbacks of this approach The principal advantage of the bootstrap method is that it makes no assumptions about the magnitude or randomness of the noise for the data being fitted Thus for example if the residuals for the original best fit show a systematic pattern that pattern is retained by the data subsets created during the bootstrap rounds A further advantage over the Monte Carlo method 66F1FDB7 2169 4D76 9FCO 5F 18A94F 5F05 is that in real experiments the noise level of the raw data is often not constant across a scan or for scans at different 195 DCDT User Manual DCDT User Manual aa times in the run That is particularly true for fluorescence scans where the noise magnitude is usually substantially higher in the plateau regions than in the low concentration regions to the left of the boundary That same phenomenon also exists for absorbance scans but the cha
179. izard 194 195 6 7 About the jitter wizard 195 6 8 About the bootstrap method 195 196 6 9 About the Monte Carlo method 196 197 7 Bibliography 198 7 1 reference 1 Philo 1997 198 7 2 reference 2 Johnson amp Faunt 1992 198 7 3 reference 3 Stafford 1997 198 7 4 reference 4 Durschlag 1986 198 7 5 reference 5 Stafford rule of thumb 198 7 6 reference 6 Stafford 1999 198 7 7 reference 7 Stafford 1994 198 199 DCDT User Manual 7 8 reference 8 Philo 2000 7 9 reference 9 Behlke amp Ristau 2002 7 10 7 11 7 12 7 13 7 14 reference 10 reference 11 reference 12 reference 13 reference 14 Efron amp Tibshirani 1986 Johnson amp Faunt 1994 Philo 2011 Stafford 1992 DCDT User Manual aa 199 199 199 199 199 199 199 200 DCDT User Manual SR DCDT User Manual 1 Getting Started 1 1 Program overview and organization Below is a picture to illustrate various parts of the program user interface followed text explaining more about the program design and operation See How to use this Help file Section 1 2 for information about expandable sections and other features of this Help file The See Also section at the bottom or use the button at upper left will lead you to tutorials and other information to help get you started DCDT baa file Ect View Jools Windows Help Nevigebon Tree mw gls Ovarlay Graph 4 CA
180. ize above 003 did not decrease the time per scan So is there really one optimum absorbance scan setup for 9 s analysis Not necessarily Certainly the conventional wisdom that continuous mode is faster than step mode generally holds true If we strictly pay attention to the theoretical figure of merit then the conclusion would be that continuous mode 003 cm step size and 4 replicates is best at least among the conditions tried here However this ignores the fact that using replicates in continuous mode may cause errors in radial position and compromise the accuracy of the sedimentation coefficients Further the figure of merit criterion also assumes the overall noise in the 9 s distribution is dominated by random statistical noise in the absorbance measurement In practice the variations in dc dt from different scan pairs seem to be larger than expected due to random deviations implying the existence of some source of systematic noise A direct comparison was made of using 4 replicates versus 1 with continuous mode and a 003 step size on samples with good signal noise OD of 0 8 The number of scans used in each analysis was chosen to cover a nearly equivalent time span so the degree of band broadening was kept equivalent Although in theory the experiment using 4 replicates should have given a lower noise level for the 9 s distribution as well as more data points in that distribution the actual result was that the
181. k box will be checked to remind you that one or more constraints is active 48 DCDT User Manual SR DCDT User Manual Data choice Fit diffusion or mass hate of pee frals data fit D for each species a a ea fitdeldt data ft M 5D ratio instead of D Improved method nary eget Classic method Theoretical Experimental None J Constrain M or s ratios among species Molecular mass units Density and partial specific volumes solvent density 1 001360 D Svedberg per Fick z 5o J Use the same partial specific buoyant molecular mass kDa volume for all species temperature must be known f R of Temperature C 20 3 species 1 mlg 9 7231 partial specifi f tam BEd 0 7231 temperature solvent density and cific vo o solute partial specific volume must s miig 0 7231 be known 0 7231 0 7231 m A Less CQ Back Nex A D Set the initial guesses for sedimentation coefficients and the range of s values to be included in the fit The Next button will bring you to the Set initial guesses on graph page Section 3 2 7 49 DCDT User Manual DCDT User Manual a a s fringes Svedberg 0 02 0 00 0 02 1 0 1 5 2 0 2 5 3 0 3 5 4 0 4 5 5 0 5 5 6 0 6 5 7 0 7 5 8 0 s Svedbergs E el i Less C Back New AJ When you first enter this page with new data a program wizard will find the top of the main peak and use that position as the initial
182. keyboard movement keys while the SHIFT key is pressed and then press either the Print or Copy buttons Copying of the selected portion can also be done using the standard Windows CTRL C shortcut key combination The Magnification up down spinner allows you to increase or decrease the size of the text and images being displayed within the rich text box that displays the Log This is sometimes useful because 1 The rich text box sometimes fails to detect that the images are larger than its width and therefore it should display a scroll bar along the bottom 2 The size of images imbedded in rich text is absolute which can cause problems when the analysis is saved on one computer and then opened on a computer with a different display resolution The Return button simply hides the Log page and brings the current analysis page back to the foreground ding graphs to the Log Typically the various graphs generated during intermediate steps of generating the g s distribution as well as the graphs that can be generated and displayed on the Graph fit results page Section 3 2 11 or by the Graph bootstrap or Monte Carlo results Section 3 2 10 8 dialog are considered temporary results and are not placed into the Log However if for example one of those graphs illustrates why further refinement of a fit is needed it may be appropriate to document that finding in the Log before proceeding with such refinements Therefore buttons are provided on both
183. l printer if you aren t currently connected to a real printer Also note that the graph produced by this rich text copy operation will be in color whether or not this printer is actually capable of color 3 2 10 6 Report fit results Fit statistics m Fit statistics Sum of squared residuals 1 295815E 3 Degrees of freedom 543 RMS residual 3 840659E 3 This area of the Report fit results page Section 3 2 10 will report a statistical summary of the fit For experimentally weighted fits the values for chi squared the degrees of freedom the rms residual and the reduced chi squared will be displayed For unweighted or theoretically weighted fits as shown here the sum of squared residuals the degrees of freedom and the rms residual will be displayed 3 2 10 7 Report fit results High cross correlation warning 154 DCDT User Manual SR DCDT User Manual Q WARNING there is 1 parameter cross correlation gt 0 97 This area of the Report fit results page Section 3 2 10 is used to display a warning whenever some of the parameters are highly cross correlated See What does high parameter cross correlation mean Section 5 2 4 There are two levels of warning one for high correlation and one for very high correlation The numbers of correlations exceeding each level is listed unless there are none at that level e The default levels that trigger those warnings are 0 97 and 0 99 respectively but these
184. lable This page displays the best fit parameter values the values for parameters that were held constant or constrained the fit statistics and buttons for printing a report Each area or control on the image below is a clickable hotspot that will lead to further information or just use the numbered list below the image View parameter Species Parameter Best Value 95 0 Confidence Region Starting Value cross correlations 1 Co OD 0 9634 0 9599 0 9688 0 9634 1 s 5 6 197 6 190 6 203 6 300 Compute confidence 1 M kDa 149 60 148 03 150 94 170 00 inieonie 2 CofOD 0 0196 0 0123 0 0253 0 0196 Create Reports 2 s S 8 953 8 724 9 218 8 953 Include Graph 2 M kDa 298 36 76 38 2658 72 299 19 3 CofOD 0 0093 0 0064 0 0123 0 0093 3 s S 11 281 10 690 11 849 11 280 Send report to 4 CofOD 0 0061 0 0049 0 0079 0 0061 cipboard 4 s S 13 722 13 380 14 242 13 721 Graph Monte Carlo results Weighted SSQ 1 299198E 3 Degrees of freedom 543 Fit statistics Parameter or Constraint Value offset OD S 0 00000 i M constraint 1 M 3 3 x M 1 hi residual 3 805992E 3 M constraint 2 M 4 4 x M 1 Un weighted SSQ 8 010524E 3 A WARNING there is 1 parameter cross correlation gt 0 97 147 DCDT User Manual DCDT User Manual aa The image above shows the results from a 4 species fit where the masses of species 3 and 4 have been constrained as trimer and tetramer o
185. lated and displayed 3 2 6 6 Select fitting model Constrain M or s ratios among species C Constrain M or s ratios among Ses species _Set constraintyalues 129 DCDT User Manual DCDT User Manual aa Parameter constraints are used with multi species fits when there is reason to believe two or more species represent oligomers of a common monomer The Set constraint values button brings up a dialog box that allows the sedimentation coefficients and or masses of the different species to be linked together and constrained to the ratios expected for oligomers By constraining these ratios the number of fitted hydrodynamic parameters is greatly reduced often resulting in more accurate values for the other parameters that are fitted Whenever constraints are currently active the Constrain M or s ratios among species check box will be checked Clearing this check box will inactivate all constraints Setting this check box when no constraints are active will bring up the dialog to set constraints The actual dialog box and procedure for establishing the constraints is described in detail below but first it may be useful to explain the intended use for these constraints Why are constraints useful As an example a common degradation pathway for proteins upon storage is the formation of disulfide linked dimers or higher oligomers and thus many protein samples could contain a small amount of dimer If the fraction of dimer is quite small
186. lays 1 the returned best fit values for the fitted parameters o Note that the units applicable to the fitting parameters are shown these will depend on other program choices and options such as whether the g s distribution has been Section 3 2 5 4 normalized 2 an estimate of the error uncertainty for each parameter o When the fit is first completed that error estimate will be a simple estimate of the standard error based on the covariance matrix o After the Section 3 2 10 4 Compute confidence intervals button Section 3 2 10 4 has been used this area will instead show the true confidence intervals with the confidence level shown in the column heading 3 The starting value for each parameter the initial guess at the start of that fitting session o This is provided primarily to document the initial conditions of the fit o Note that fits saved from version 1 xx of this program did not store that information so this column will not be displayed 3 2 10 2 Report fit results Fixed or constrained parameters Fixed or Constrained Parameters Parameter or Constraint Value offset AU S 0 00000 M constraint 1 M 3 3 x M 1 M constraint 2 M 4 4 x M 1 This table lists 1 The values for parameters that were held constant during the fit parameters whose values were not optimized during the fit In this example the baseline offset has been held fixed at zero 2 Equations summarizing any parameter constrain
187. librating temperature will also cause problems Many people have been taught to bring the rotor to 3000 rpm and wait for temperature equilibrium before going to the final rotor speed This approach will cause problems for nearly all analysis methods that attempt to derive diffusion coefficients or masses from velocity data including this one e To handle diffusion these methods must assume instantaneous acceleration to the final rotor speed If any significant amount of sedimentation occurs at low speed the derived diffusion coefficients and masses will be compromised but a quick scan at 3000 rpm to check for proper cell loading should not be a problem for species lt 10 MDa The trick to proper temperature equilibration start a run at zero rotor speed This will turn on the diffusion pump without spinning the rotor e Remember the instrument only turns on the infrared thermometer to read the actual rotor temperature when the vacuum drops below 100 microns so it is essential to get the diffusion pump working to get proper temperature equilibration e Setting the speed to zero can only be done from the instrument control panel not from the computer through the control software 1 first push the Speed button then push 0 and Enter 2 then hit Enter then Start A Caution This trick creates a small problem If you need to release the vacuum when running at a speed of zero the instrument fails to force you
188. ll Range 1 728 b 3552 S Properties at time of analysis from g s Concentration 0 9370 0 0018 fringes Fraction of total 76 09 0 09 Number average s value 2 516 0 010S Weight average s value 2 601 0 008 S z average s value 2 685 0 011 S z 1 average s value 2 764 0 021S Properties extrapolated to t 0 from a s Loading concentration 1 0925 0 0021 fringes Fraction of total 76 01 0 08 Number average s value 2 529 0 009 S Weight average s value 2 614 0 008 S z average s value 2 697 0 013 S z 1 average s value 2 776 i 0 025 S Note stated uncertainties are 1 sigma and sedimentation coefficients are s 20 w values Copy all values to Clipboard The results are displayed in a dialog box example shown above For more details about the options within this dialog box and how the calculations are done see Averages and peak areas dialog Section 3 2 5 3 118 DCDT User Manual SR DCDT User Manual If you want these calculations done over the entire range of the distribution use the Show integrals over distribution button Section 3 2 5 1 instead 3 2 5 3 Averages and peak areas dialog ised Coeffient Averages amp Deak Arse Integration range M FullRange 0245 to 7980 s5 Redo Properties at time of analysis from 9 s Concentration a Fraction oftotal 100 00 0 00 Number average s value 35854 00118 Weight average s value 37384 00035 z
189. ll change if that is being determined by the default Automatic option see Calculate average dc dt X axis point spacing Section 3 2 4 1 Thus making this switch will invalidate any previous fitting results How this conversion affects reported diffusion coefficients Activating this option will also cause raw diffusion coefficients to be converted to D values via the formula 293 15 Dy Ding ae P o l Ny w T where T is the sample temperature Kelvin 20 w Data required for conversion Conversion to Sg requires knowledge of the solvent density at the experiment temperature the solvent viscosity h at the experiment temperature the solute partial specific volume vbar at the experiment temperature and the solute partial specific volume at 20 C through the relation 1 Vr Prb how where the subscripts refer to the temperature and solvent conditions b buffer w water The density and viscosity of water at 20 C are 0 998234 g ml and 1 002 centipoise respectively Soo w raw If all those data are not available or cannot be estimated e g by SEDNTERP then the conversion cannot be made 114 DCDT User Manual SR DCDT User Manual The program will optionally automatically compute a temperature correction to the partial specific volume using the same formula used in SEDNTERP reference 4 V V 4 25 x10 7 20 This option is controlled by the check box on the lower frame and w
190. lot outside the fitted region we can now visualize the fact that this single species does not provide a good fit outside that fitted region and also get a good estimate of the aggregate content If instead the aggregate region had been included in the fit the fitter would broaden the single species peak lower estimated mass in order to reduce the residuals in the aggregate region and we would actually get a much poorer estimate of the aggregate content as well as poor values for the properties of the main peak This graph displays a small title line that lists the first and last scan files included in the analysis and the folder from which they were loaded 2 Species plot for entire data range line This plot is also a species plot for the entire data range like 1 but the individual species are shown as simple line plots rather than filled areas While the area plots are pretty when you are trying to look closely at whether the fit really follows the data well the area plots can sometimes obscure the experimental data so this type of plot is often better for that purpose Select graph type from lst Species plot for entire data range line w cerint copy Add to Log Scans 00027 _RA1 10 COOSS RA1 tom Cosedtnidavsianibody g s OD Svedberg 0 1 2 3 4 5 6 7 8 9 10 11 12 s Svedbergs data species 1 See the discussion under plot 1 above about why it is often useful to limit the range of d
191. lume using the same formula used in SEDNTERP reference 4 Vz V4 4 25 x10 T 20 This option is controlled by the check box on the lower frame and when it is checked the input box for the vbar at 20 C is disabled but the computed value is displayed The viscosity values are to be entered in centipoise Since SEDNTERP displays these values in poise to facilitate copying and pasting numbers from SEDNTERP whenever a number is entered that is less than 0 1 the program will assume it is a value in poise and multiply it by 100 The program automatically fills in the experiment temperature based on the temperature recorded in the header of the last scan used in calculating the dc dt data but since the temperature may drift during the run you may wish to adjust that value manually Please note that changing the temperature on this form overrides the temperature recorded with the scans and also alters the value used elsewhere in the program e g for conversions to buoyant or true mass units 3 2 3 4 Select scans to analyze Use true mean time button Use true mean time This button is only visible in Advanced mode Section 1 3 and this option is not recommended for general use Briefly this option is sometimes helpful when you will be doing multi species fits to 9 s distributions and for data where the peaks have been severely broadened and distorted because the range of boundary movement is very large for example a la
192. m which you may be able to see that scan number 92 file 00092 ip3 is the bad one 179 DCDT User Manual DCDT User Manual a FA Analysis1 lol x Meniscus setting and data adjustments Meniscus at 5 904 cm good data begin 0 01 cm from meniscus good data end at radius 7 10 cm a ETE el PT e Fringes 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 7 4 Radius cm cccsours cocs1P3 ccosaiP3 coossuP3 cocesiP3 cccss r3 coeseur3 000871P3 Osas cocssue3 ccosolP3 coostie3s coosziF3 ccosauF3 ccos4iP3 000351P3 00uP3 000971P3 ccoseP3 coossuP3 O0100IP3 001011P3 00102IP3 00103IP3 CO104F3 CO10SP3 CO10EIP3 001073P3 CO10EIP3 CO10SUP3 001103P3 001111P3 00112IP3 001134P3 00114IP3 CO11SIP3 001161P3 CO1ITIP3 001181IP3 001191P3 001204P3 001211P3 00122IP3 001231P3 001241P3 00175IP3 00176IP3 001271P3 00123P3 001291IP3 Pes Less Back Next However an alternate procedure that works even when there are hundreds of scans is to push the ID points button Section 3 2 2 8 This turns every point in every scan into a clickable object and places a window above the graph that will display the scan file name and X Y data values for any point that is clicked as sho
193. meter 1 M 1 NA J Parameter 2 Co 1 v Frequency Aouanbal4 aayeynung Corresponding graphs for the other fitting parameters can be obtained using the drop down list labeled Parameter 1 Note that this example shows the results from a rather large number of bootstrap rounds 2000 and consequently the width of the bins in the histogram can be small and the overall shape of the distribution is fairly well defined With lower numbers of bootstrap or Monte Carlo rounds the width of the bins must be larger in order to have enough events in each bin to give a statistically meaningful result The program automatically adjusts the number of bins displayed to try to account for this statistical issue TIP The number of bins used for generating the histogram can be altered by clicking on any of the blue bars with the mouse Using fewer bars will tend to average out the statistical fluctuations but at a cost of fewer positions to define the true shape of the distribution Parameter cross correlation graphs Clicking the Two parameter cross correlation plot radio button will generate a scatter plot like that shown below where each point represents a pair of parameter values for each round of bootstrap or Monte Carlo simulation The vertical and horizontal red lines mark the best fit value for that parameter 156 DCDT User Manual SR DCDT User Manual i Graph Bootstrap Results reson Graph type Single para
194. meter histogram Parameter selection Parameter 1 M 1 NA Parameter 2 Co 1 a E Readoutdata values X 0 27 0 28 0 29 Co 1 Copy Print Addtolog ok For this example the data points are clearly clustered in an elliptically shaped region tilted roughly 30 degrees to the left of vertical This pattern shows that the mass values are moderately highly correlated with the concentration values a negative correlation in this case As the mass value is increased or reduced from the best fit value it is possible to partially compensate for that change and minimize the increase in the variance of the fit by simultaneously decreasing the concentration parameter That is the two parameters cannot be determined completely independently If the points are very tightly clustered along a line corresponding to a correlation coefficient near 1 or 1 this is an indication that too many parameters are being fitted the information content of the data is not sufficient to determine all the parameters independently In some cases the non linearity of the fitting may produce clustering along a curve rather than a line In any situation where the distribution is very tightly clustered along a diagonal line or a curve the fitting results are probably not trustworthy and it may be appropriate to reduce the number of parameters by introducing parameter constraints Section 3 2 6 6 or by reducing the number of species
195. n DCDT User Manual DCDT User Manual aa o optionally integrate distribution to calculate peak areas and sedimentation coefficient averages o optionally normalize Section 3 2 5 4 the distribution to facilitate comparisons of samples at different concentrations 3 1 4 2 Fit distribution as N species branch This major branch of the Navigation Tree Section 3 1 4 for each analysis window holds the sequence of steps needed to set up and complete the fitting of either the 9 s or dc dt data 1 Select fitting model page Section 3 2 6 o Select the fitting function number of species fitting of D or molecular mass and weighted or unweighted fits Also select the molecular mass units and enter the partial specific volume and solvent density to give the correct molecular mass values For multi species fits you can optionally constrain M or s ratios among species 2 Set initial guesses on graph page Section 3 2 7 o enter initial guesses for the sedimentation coefficients of each species from a graph of the data being fitted and establish the range of sedimentation coefficients to be included in the fit F 3 Set Alter fitting parameters page Section 3 2 8 o review and optionally alter which parameters are being fitted and the initial guesses for the fitting parameters set criteria for fit convergence o this page is shown only in Advanced mode Section 1 3 4 Perform least squares fit page Section 3 2 9 o perform the fi
196. n coefficient that molecule would have if measured in water at 20 C Because of the solvent independent nature of s it describes quantitatively the fundamental hydrodynamic properties of the molecule and it is this value which is most useful in comparing the sedimentation behavior of different molecules A The partial specific volume that is entered on this form and used in converting to s scale is applied uniformly to the entire g s distribution Therefore if you believe your sample contains species with different vbar values for example different polypeptides or both PEGylated and non PEGylated forms of the same protein you should not apply this conversion you must work in raw sedimentation coefficient units e For similar reasons the partial specific volume entered on this conversion form is applied to all species that are fitted The actual scaling factors that will be applied to correct the raw sedimentation and diffusion coefficients to standard conditions are displayed in the panel nearest the bottom of the dialog box What about extrapolation to zero concentration This program does not attempt to correct for concentration dependence of sedimentation coefficients extrapolation to infinite dilution to give Dw Often dc dt analysis is applied to samples at concentrations sufficiently low that concentration dependence can be neglected and if so the corrected results can be considered to be sage values but the prog
197. n marker are not displayed on this 3D graph e The 2D button displays the normal 2 dimensional graph e The T button displays a graph of the recorded rotor temperature versus elapsed run time useful to check whether the temperature was truly stable during this run The range of temperature data displayed corresponds to all the scans in memory not just those currently being used in the analysis See also How and why to equilibrate the rotor temperature Section 4 2 2 3 2 2 7 Set meniscus amp data region Zoom meniscus button This button will zoom the 2D graph to show only a radial region 2 3 mm wide centered around the current setting of the meniscus position This is intended to help position the meniscus more accurately This command does not alter the Y axis scales and thus it may still be necessary to manually zoom with the mouse to increase the vertical magnification This is a toggle button and will remain down to alert you that the graph is currently in zoomed meniscus mode You can unzoom either by toggling the button off or by hitting the Z key while the graph has the focus 3 2 2 8 Set meniscus amp data region ID points button 101 This button will cause the 2D graph of the raw data to be displayed as individual data points rather than continuous lines and each data point becomes a clickable object When a data point is clicked the corresponding scan file name and x y data values are displayed within a box above the
198. n of the printer orientation and use of color The Export button will bring up a dialog that allows export of the graph image in several formats as a file or to the Clipboard This export function can also export all of the data values rs The radio buttons within the Graph Style box allow easy choice of different plotting styles points lines or both points and lines The Edit Legend text box and up down spinner allow quick alteration of the description of each distribution to help create graphs for presentations or publication 87 DCDT User Manual DCDT User Manual a 3 2 Analysis pages 3 2 1 Load raw scans page This page allows you to load data from scan files and is the starting point for any new analysis Each control on the image below is a clickable hotspot that will lead to a separate page with further information or just use the numbered list below the image Note that the Next button will not be active unless at least 4 scans have been loaded FA C 042903 110946 cell3 00120 00164 1P3 Scans currently Scans to be used in V Preselect data type and cell loaded in memory g s calculation Data type Cell number 00100 IP3 PPT 00101 1P3 00102 IP3 Interference 3 fe 00103 1P3 00104 IP3 00105 1P3 00106 1P3 00107 IP3 C Fluoresence Load 2 files Replace this sample Add more scans for this sample Delete 9 scans Data loading options F Load only every scan
199. n page Section 3 2 2 for example if you want to edit the raw scans to eliminate bad points then you will want to turn despiking off 3 2 1 9 Load raw scans Sample description This text display area shows the sample description from the comment line of the last scan that was loaded the information that was entered for that cell during data acquisition or the description of the last Claverie simulation if simulated data are currently loaded 3 2 1 10 Load raw scans Run Claverie simulation to generate data button A Claverie simulation uses finite element methods to solve the Lamm equation This approach can be give highly accurate simulations of velocity experiments but is computationally intensive The purpose of providing this simulator is to provide a tool for what if explorations and tests of the accuracy of the fits Some examples of questions that can be approached this way are 1 Is the difference between the mass value returned by fitting g s and an expected result really significant or just due to inaccuracy of the method 2 To what extent will adding more scans to your analysis cause broadening of the peaks and therefore inaccurate D and M values 3 What would the data and residuals look like if my sample contained a small amount of a second species but was analyzed as a single species This Claverie simulator code kindly provided by David Cox and Walter Stafford allows you to generate accurate data for these
200. nated as s or apparent values You should be aware that some scientists in this field also use the s symbol and apparent sedimentation coefficient terminology to indicate raw sedimentation coefficients i e values which are not s 20 w values that have been corrected to standard conditions and or which have not been extrapolated to zero concentration 5 1 2 What is the difference between g s and g s The g s g hat distribution reflects the composition of the sample at the time the scans used in the analysis were taken Thus the total area under the curve represents the plateau concentration at that time in the run and the concentration of any one species in a mixture area under any particular peak is its concentration in the plateau region at that time The 9 s distribution is determined by extrapolating the g s distribution back to time zero accounting for the radial dilution effects Thus the total area under the 9 s curve represents the loading concentration and the concentration of any one species in a mixture area under any particular peak is its loading concentration The g s distribution is therefore the more fundamental distribution and is more closely tied to the raw experimental data but it is the 9 s distribution that is more commonly presented Gal For interacting systems reversible self association or mixed association the composition will in general change during the velocity run re
201. nce scans have different numbers of data points some of the columns in the data table will have fewer entries than others Table positions with non existent data are copied as empty strings dc dt data all scan pairs This menu selection will copy the dc dt data from each scan pair If there are N scan pairs then the data table will have 2N columns with the first two columns holding the x and y values for the first scan pair the 3rd and 4th columns holding the x and y values for the second scan pair etc Note that copying the data may take 10 seconds or more when the number of scans is large Because individual dc dt curves have different numbers of data points some of the columns in the data table will have fewer entries than others Table positions with non existent data are copied as empty strings Average dc dt curve This menu selection will copy the average dc dt curve for the scans currently in use 3 columns with x y and standard deviation values g s distribution This menu selection will copy the 9 s distribution table 3 columns with x y and standard deviation values g s distribution This menu selection will copy the g s distribution table 3 columns with x y and standard deviation values The menu items below are only enabled after a fit or fit simulation has been completed 64 DCDT User Manual SR DCDT User Manual aly a Data being fitted This menu selection will copy the actual data values bein
202. nd test the size of the changes in the sum of squared residuals and in general it is this criterion which halts the iterations first Statistics tells us that a change in the value of a parameter will not be statistically significant unless that change increases the sum of squared residuals by a factor of approximately 1 N where N is the number of data points being fitted Thus once successive iterations of the fit produce a fractional drop in the sum of squared residuals that is smaller than 1 N further iterations will produce refinements in the parameters that are not statistically significant The actual implementation is that the fit will continue until the fractional drop in the sum of squared residuals is less than 1 N times the convergence factor Thus if the convergence factor is 0 01 the default value the fit continues until the changes are 100 times smaller than the smallest change that is likely to be statistically significant For multi species fits or in situations where the parameters are very highly correlated it may be worthwhile to increase the values of the convergence factor or parameter precision factor to reduce computation time 139 DCDT User Manual DCDT User Manual aa 3 2 8 5 Adjust parameter upper lower limits button Adjust parameter upper lower limits This button will bring up the dialog box below for altering the default upper and lower limits bounds on parameter values DCDT Fitting Parameter Bo
203. ndering an extrapolation to time zero invalid Therefore only the g s distribution should be presented or used in calculating averages over the distribution for such samples For non interacting systems mixtures the g s distribution is preferred since one usually wants to know the composition that was initially loaded into the cell 5 1 3 What is the difference between the broad and conventional g s algorithms The program offers two alternative ways of calculating 9 s 1 the conventional algorithm developed by Walter Stafford reference 7 Section 7 8 2 the broad algorithm developed by John Philo reference 8 Section 7 8 This alternative broad algorithm is primarily provided e for backward compatibility with DCDT versions 1 xx and e to give better estimates of the precision of the weight and z average sedimentation coefficients Section 3 2 5 1 from the 9 s distribution in some circumstances Selection of the broad algorithm is only available in Advanced mode Section 1 3 bel 185 DCDT User Manual DCDT User Manual aa Fitting to 9 s or dc dt calculated using the broad algorithm is implemented only for the classic method Section 3 2 6 2 not for the new improved method Section 3 2 6 2 How do the algorithms differ The conventional algorithm first averages together the dc dt data from different scan pairs then calculates g s from that average and then calculates 9 s from g s The
204. ng the Good data end at radius Section 3 2 2 3 value from the Set meniscus amp data region page Section 3 2 2 By default the initial range for calculating 9 s is the full range of the average dc dt curve If you want to reduce that range you can either 1 manually enter new values into the text boxes or 2 push the Set range for g s button to display two magenta vertical lines on the graph marking the range Drag those lines with the mouse to alter the limits Whenever the current upper or lower limit is at the full limit of the average dc dt data the background color of the corresponding text box becomes a light yellow as a visual indication of that state 3 2 4 3 Convert to s 20 w This option will convert sedimentation coefficients for dc dt and g s curves as well as fitted values to the scale S20 w El Note that s will also sometimes be written in this document as s 20 w to avoid problems in generating subscripts Convert to s 20 w The Convert to S20 w buttons are present on the Set meniscus amp data region page Section 3 2 2 the Calculate average dc dt curve page Section 3 2 4 and the Calculate g s distribution page Section 3 2 5 When the data are in raw sedimentation coefficient unit pressing this button will bring up a dialog box shown below for entry of the data needed to perform the conversion discussed in more detail below If this conversion has already been done as indicated
205. nge in the noise magnitude is smaller than for fluorescence scans One drawback of this method is that it operates on the 9 s or dc dt data not the original raw scans Thus it cannot directly evaluate whether the data transformations themselves introduce any statistical bias Reference 13 Section 7 13 shows that statistical bias resulting from the data transformation is in practice not a significant issue 6 9 About the Monte Carlo method In the Monte Carlo method for evaluating confidence limits first a set of theoretical raw scans is generated based on the number of species and the parameters from the best fit Then random noise at a specified rms level is added to those theoretical raw scans and new average dc dt data and a new 9 s distribution are calculated and then a new data set is created covering the same range of sedimentation coefficients as the original data This new data set is then fitted and the new fitted parameters are stored in an array This whole process is repeated N times and then for each fitted parameter the array of N values is used to evaluate the confidence limits Note that in the implementation used here some random noise is added to the best fit parameters before each round so that each Monte Carlo round begins from slightly different starting values to avoid biasing the results HHow is the magnitude of the random noise to be added determined One challenge for applying the Monte Carlo method to
206. ngs up the dialog box shown below a dealt iia aia i i a Set Molecular Mass or Sedimentation Coefficient Constraints Molecular s D ratio constraints Sedimentation coefficient constraints Radio buttons set the species to which Ratio of Radio buttons set the species to Ratio of that molecular mass s D is linked Molecular which that sedimentation coefficient Sedimentation Masses is linked Coefficients Species 1 2 2 000 none 2 1 45 Species 2 1 2 000 none C 1 1 45 Enter 1 for constant f0 rate mass scaling Predicted sedimentation coefficient ratios dimer 1 45 Cancel changes trimer linear Ls trimer triangular 1 86 Clear mass constraints Clear s constraints tetramer linear 2 00 tetramer square planar 2 20 eo ae tetramer tetrahedral 2 26 To force species 2 to be a dimer simply click on the radio button labeled 1 within the Species 2 group box to indicate that species 2 is constrained as a multiple of species 1 as shown below You can then enter the appropriate ratio in the text box to the right the default in this case is already 2 000 47 DCDT User Manual DCDT User Manual aa Radio buttons set the species to which Radio buttons set the species to that molecular mass s D is linked which that sedimentation coefficient is linked Species 1 G none 2 Species 2 Species 2 _ none 1 2 none J1 Enter 1 for constant
207. not unusual that only a small number of scans can be used before significant peak broadening occurs At 45K rpm the boundary for an antibody 150 kDa is moving quite rapidly compared to the time between scans so to keep our snapshot from being blurry we can only keep our shutter open a short time 25 DCDT User Manual DCDT User Manual a as Analysis2 00017 00026 RA1 h Adjust scans used in computing de dt Options Seansused 10 of 60 lt ama r Peak sie broadening Posi wihinnn lt ae n 00017 RA1 Lastscan 00026 RA1 105 Scans 00017 RA1 10 C0025 RA1 from CXLAWINIXUDAT Aisniibody deidt OD sec 0 2 4 6 8 10 12 14 16 18 20 s Svedbergs O0017 RA1 00022 RA1T 00018 RA1 00023 RA1 00019 RA1 00024 RAI 00020 RA1 00025 RA1 00021 RA1 00026 RA1 average essed F More Q Back nex A 4 After getting the top slider in roughly the right range you also may also want to move the lower slider left or right to analyze data earlier or later in the run to cover higher or lower sedimentation coefficients A general rule of thumb is that the main peak should be roughly in the middle of the graph However that rule may not produce a sedimentation coefficient range that includes all of the species that are present in your sample In the image above the average dc dt curve is not flat over the region from 9 to 12 S suggesting there may
208. ntly sedimenting components Such fitting may involve up to 6 steps which are grouped to form the fit distribution as N species branch Section 3 1 4 2 within the navigation tree Section 3 1 4 Wizards and Standard Mode make it easy but more options are there when needed Although many options and opportunities to fine tune the analysis are available whenever possible the program uses intelligent wizards to choose initial settings and parameters for you The program even pre selects the buttons you will probably need to hit next In Standard mode Section 1 3 shown here and the initial default many options are hidden to keep things simpler but you can easily switch to Advanced mode Section 1 3 using the More gt gt button Section 3 1 3 2 when you need more power Analysis the easy way 1 Load the raw scans 2 Push the Enter key to confirm the choices the program has made Pushing Enter 10 times will take you all the way from raw data to a g s distribution and on through fitting your sample as a single species to give you its sedimentation coefficient and estimated molecular mass Yes it is that easy True for a final result you will probably want to go back and tweak things a bit like exactly which scans are analyzed For an accurate molecular mass of course you also have to stop and input the exact solvent density and partial specific volume However the automatic choices should be more than good enough to tell you
209. nu or using the familiar icons on the Toolbar Analysis window An analysis document within DCDT is contained within an Analysis window Section 3 1 3 In the example above three analysis windows are open which happen to be 3 different cells from the same run in this case Each analysis window concerns the analysis of one sample In each analysis window Section 3 1 3 you can work through the analysis in a step by step manner and different controls and or graphs are shown as different pages within the window at different steps Navigating within and between analysis pages 1 DCDT User Manual DCDT User Manual a Movement among the steps pages within one analysis window can be accomplished using either the Next button and Back button at the lower right or by right clicking on the analysis window with the mouse and using the Go to step popup menu ar DCDT also provides an Explorer style Navigation tree Section 3 1 4 along the left side This navigation tree provides an easy mechanism to move both between different analysis windows and also to specific pages steps within each analysis window Five steps are needed to complete derivation of the g s distribution and those are grouped to form the derive g s distribution branch Section 3 1 4 1 within the navigation tree Section 3 1 4 Once a g s distribution has been computed you may optionally fit that distribution as a mixture of one to five independe
210. o that you don t want to include the region near the base of the cell where solutes are accumulating Corollary to Rule 2 adjust the regions being scanned so you don t waste time taking data for regions of the cell that you can t include in the analysis How to speed up data acquisition for interference data While interference scanning is inherently much faster than absorbance there are still things that can be done to maximize the scan rate and these may be particularly important if your instrument control computer has a slow processor and you are scanning many cells or trying to measure samples at very low concentrations The display of the scans within the data acquisition program the GUI takes a significant amount of time particularly if you set N gt 1 on the Overlay last N scans option under the Options button The absolute maximum scan rate is obtained by setting N 0 on the Overlay last N scans option which results in no display of the new data The drawback is of course that you cannot as easily monitor the course of the run You can however monitor the experiment by displaying scans using some other program such as the plotting commands in the Beckman software or John Philo s public domain program XLGraph The extent to which scan rate is affected by the graphing of the incoming data depends on the computational and graphics speed of your computer These settings are more important for slower computers an
211. o new window This command will open a new analysis window Section 3 1 3 containing an exact copy of the current analysis window That copy includes all the raw data in memory not the original scan files on disk calculated dc dt and g s data settings etc This menu command is duplicated by a button on the Toolbar Section This allows you to alter the analysis while preserving an open copy of your original analysis For example you might want to analyze scans early or later in the run and then overlay that result with the earlier one in the g s overlay graph Section 3 1 5 The name of the cloned window is derived by adding an underscore character and a sequence number to the name of the current analysis Thus the first cloned version of a window called cell2 would be cell2_1 An alternative way of altering an analysis while preserving a saved original version is to make the alterations and then save the new version under a new name using the Save analysis as Section 3 1 1 1 3 command However with that approach you might accidentally overwrite the old version if you forget to alter the name 3 1 1 2 Edit menu These commands export data from the current analysis as text tables The data can either be copied to the Clipboard so these values can be pasted into some other program or written to disk as text ASCII files These Copy or Write menu items under the Edit menu a Copy Data to Clipboard z il Write Data
212. of parameter space where convergence will be better and therefore a can be increased back toward 1 Further since it is much faster to calculate 146 DCDT User Manual SR DCDT User Manual variances in order to test values for a than it is to carry out a full iteration it is usually worthwhile to try keeping a large in order to move more rapidly to convergence Therefore the rule applied is that when a from the preceding round is lt 0 005 it is set to 0 02 for 0 005 lt a lt 0 25 it is multiplied by 4 and for 0 25 lt a lt 1 it is set to 1 Exception 1 occurs when the sequential reductions of a during the attempt to find a lower variance reduce a below 0 001 This means the fitter is having major trouble That may be because too many species are being included in the fitting model or because one or more of those species are poorly resolved or in very low abundance In that circumstance the program tries making a negative taking the parameters in the opposite direction since even small positive values of a just make things worse If a negative a lowers the variance that value is accepted otherwise the sign of a is inverted again and that positive value accepted If a negative value of a is used 5 iterations in a row the program assumes convergence has been reached 3 2 10 Report fit results page The Report fit results page is displayed automatically upon completion of a fit or you can navigate to it whenever fit results are avai
213. of rounds is 200 or less Because in reality for non linear fits the parameter distribution may be asymmetric around the mean two standard deviations are calculated one for those parameters that exceed the mean value and another for those that are less than the mean value Those two standard deviations are then used to calculate an upper and lower confidence limit respectively and those values are not in general symmetric about the mean e It is also sometimes true that the mean parameter value from the bootstrap or Monte Carlo rounds is significantly shifted away from the corresponding best fit parameter When this is true specifically when the shift is greater than 20 of the standard deviation the confidence limits are also shifted to make the mean match the best fit value Observed parameter frequencies With this choice no statistical assumptions are made about the shape of the parameter distribution Instead the N parameter values are first sorted and then to find the confidence limit the list is searched inward from the beginning or end the lowest or highest observed values until the appropriate confidence frequency has been exceeded For example for 95 confidence probability and N 1000 the confidence limits correspond to frequencies of 2 5 giving 5 total for the upper and lower wings of the distribution which is 25 data points out of 1000 Therefore the confidence limits are the 25th and 975th values in the sorted list Th
214. om panel of the graph shows the residual plot corresponding to those initial guesses 33 DCDT User Manual DCDT User Manual a Status waiting for command Iteration number KT foom Cancel 0 35 0 30 0 25 0 20 g s 0D Svedberg Y exp Y fit s Svedbergs data Species 1 residuals Graphical fit monitor Fitting parameters monitor wel Fe More E Back Nex A 5 The Do fit button Section 3 2 9 1 is automatically selected so all you have to do hit Enter again to complete the fit As the fitter iterates to improve the fit the graphs will update but this will probably happen too quickly for you to see 6 When the fit converges it will automatically advance to the Report fit results page Section 3 2 10 shown below This page shows the final best fit parameters Section 3 2 10 1 the values for any parameters that were held fixed Section 3 2 10 2 and summarizes the fit statistics Section 3 2 10 6 34 DCDT User Manual SR DCDT User Manual CofOD 0 8558 s S 145 65 m STi O a Nee A 7 You may wish to print a report and or push Next again to display various graphs The default species plot area graph is shown below 35 DCDT User Manual DCDT User Manual a a As Analysis1 00034 00047 RA1 Fala See gaoh ye om Spaces pinions daarne rsi e CE evo bon Scans 00034 RA1 to 00047 RA1 from C iUsersionn PrilioiDeskopiPhlio Da
215. ompleted by double clicking on the graph until it is complete other controls will be inactive All these methods are implemented in the same manner as the corresponding methods in Walter Stafford s original DCDT program The default method can be altered via the dc dt Options tab within the Section 3 1 1 4 2 Options dialog Section 3 1 1 4 2 3 2 2 6 Set meniscus amp data region Data displayed options Data displayed 20 Comestea 30 Lr This group of three buttons controls options for the type of graph and what data is displayed e The Corrected button is only active for interference data and only after the meniscus has been set This options displays the scan data after the jitter and fringe jumps have been removed This 100 DCDT User Manual SR DCDT User Manual button acts as a toggle switch with the uncorrected raw scans shown when it is off up e The 3D button will bring up a 3 dimensional waterfall graph of the scans In this graph only the portions of the scans within the analysis region between the green vertical bars on the 2D graph is shown The viewing angle for the 3D graph can be rotated using the scroll bars on the right and bottom of the graph the axes are suppressed during the rotation operation o This 3D display really has no function for data analysis it is simply an alternate method of displaying the data and the motion of the boundaries The positions of the meniscus and other regio
216. on coefficients used in computing the 9 s distribution that is do not use the Set range for g s Section 3 2 4 2 procedure on the Calculate average dc dt curve page Section 3 2 4 for this purpose Change the fitting range not the 9 s distribution itself C Push the Next button to go to the Set alter fitting parameters page Section 3 2 8 3 Optionally alter the initial guesses or which parameters are being fitted The page for altering the initial guesses for the parameter values or changing which parameters will be fitted 38 DCDT User Manual SR DCDT User Manual is shown below as Analysis1 00034 00047 RA1 r Fit this Parameter Value parameter Co 1 OD 0 8107 lV Yes s 1 S 6 250 lV Yes M 1 kDa 177 82 lV Nes offset OD S 0 00000 T No Convergence control Convergence factor 10 Adjust ter Parameter precision 0 05 i alia Cancel changes p Aj Less Q Back Ne A A You may wish to alter the initial guesses for the sedimentation coefficient and D or M if you think the estimates made from the graph are poor The estimates for D or M may be fairly far off when peaks are poorly resolved e The fit should converge as long as the initial guess for s is not too far off more than a factor of 2 but good guesses will always make it converge faster B You might want to fix the values for certain parameters rather than floating them allowing the fitter to vary
217. onable direction for the increment vector but not necessarily the right magnitude Thus the idea is to test how the variance changes along this direction and pick a value of a that will at least reduce the variance When each fit begins a is initially set to 1 At each iteration the variance is first calculated for a new set of parameters using an initial value of a based on that from the preceding iteration see rule 1 below If these new parameters give a lower variance than that for the previous parameter set these parameters are accepted and the next iteration is begun If instead the new parameters give a higher variance then the value of a is reduced by factors of two until finally a lower variance is obtained unless the value of a falls below 0 001 which triggers exception 1 below Once this lower variance is obtained this variance together with those for the preceding values of a are used to fit a parabolic function of variance versus a and thereby to predict what value for a will give the minimum variance This predicted value for a is then used to calculate the parameter set for the next iteration of fitting Although these procedures are intended to guarantee that the variance will drop at each and every iteration occasionally that will not be true Rule 1 adjusts the starting value for a when a from the preceding iteration is small In this circumstance it is hoped that this previous iteration succeeded in moving into a region
218. oped by Walter Stafford reference 5 It states that the time difference Dt between the first and the last scan used should not exceed the following value 104 DCDT User Manual SR DCDT User Manual p 160 mM RPM 1000 where t is the average of the times for first and last scan M is the molecular mass in kilodaltons and RPM is the rotor speed This formula assumes a solvent density of 1 and a partial specific volume of 0 725 ml g At The elapsed times used in this calculation are actually derived from the z values stored with each scan and the rotor speed rather than the elapsed time since the wt values reflect the true effective sedimentation time This rule can also be re arranged to calculate for any group of scans the highest mass that will not be significantly broadened M maw given by 160 t 0375 3 a At RPM 1000 1 p Please note that just because a certain species has a mass lt M max this does not mean that its peak will have no broadening This rule of thumb was originally developed by Walter Stafford for use in qualitative analysis to assess when the peaks would be visually broadened ie Just because a certain species exceeds M max does not mean that the dc dt or g s data are invalid Indeed in some circumstances it may be appropriate to strongly violate this rule of thumb For example if you are unsure whether a small peak in the data truly represents an additional species you may wish to
219. or each species do not return to zero at high sedimentation coefficients whereas the individual peaks in 9 s are non zero only over a finite range When fitting to dc dt a poor fit of a slowly sedimenting component can lead to a error in the high s tail from that component that will affect the results for the faster sedimenting components Differences between fitting g s versus dc dt using the classic method For a full and detailed discussion of the accuracy and advantages disadvantages of both approaches when using the the classic method Section 3 2 6 2 see reference 8 Section 7 8 That discussion is summarized below In general when the time span of the scans is short and peak broadening is minimal fitting to dc dt will give more accurate values for s and D or M This is true primarily because certain approximations are involved in transforming from dc dt to 9 s and in part because the function used for fitting the dc dt data is a better approximation to the true shape of the curves than is the Gaussian function used in fitting the g s data Fitting to 9 s tends to underestimate D and overestimate M by a few percent and in some circumstances these errors can be as large as 10 In theory the accuracy for s D and M when fitting to dc dt is 1 but in real experiments the boundaries are always a bit broader than expected for reasons not fully understood so even under optimal circumstances D usually comes back a few
220. ore Load Raw Scans Set Meniscus amp Data Region Select Scans to Analyze Calculate Average dc dt Curve Calculate g s Distribution Select Fitting Model Set Initial Guesses on Graph Set Alter Fitting Parameters Perform Least Squares Fit Report Fit Results Graph Fit Results or buttons to toggle between Standard and Advanced modes Section 1 3 4 a progress bar that indicates the fractional completion of data loading or lengthy calculations and a User Prompt area Section 3 1 3 4 that displays instructions or other information This panel is part of every analysis page s The panel containing the Next Back and More Less buttons progress bar and user prompt can optionally be moved to the top of the analysis window Right clicking the mouse within this panel will bring up a context menu that will toggle this panel between top and bottom locations Switching between analysis windows When multiple analysis windows are open you can switch to a different analysis window by either clicking on that window with the mouse using the Navigation tree Section 3 1 4 or using the Windows menu Section 3 1 1 5 About window names New analysis windows are initially named Analysis1 Analysis2 etc The current name for each window is shown at its top in its Title Bar area and also in the corresponding root node within the Navigation tree Once an analysis has been saved to disk see Save Anal
221. ore in this folder option specifies that the File Save dialog always begins in a particular specified folder such as the user s My Documents folder When this option is selected the Browse button is active and may be used to select the default folder o The Store in same folder as scan files option has the File Save dialog start in the folder from which the last scans were loaded That approach has the advantage of keeping the saved analysis together with the raw data Other options tab i Program Options Fitting M Model Defaults F Fitting Options de dt Options File Locations Other Options Default Sample amp Solvent Properties Window size Initial size of analysis windows of default 100 Complexity versus ease of use v Always start in Standard mode Scan loading v Always start in Scan Preselect mode Printed reports Font size for reports points 12 Printer for reports Canon MF4200 Series UFRII Browse Ok Cancel e Initial size of analysis windows default 100 this sets the size of newly created analysis windows and other program dialog boxes as a percentage of the program default size Smaller sizes will allow more analysis windows to be open without crowding larger sizes will enhance readability font size increases with window size Allowed range 50 150 of normal e Always start in standard mode default checked if checked new analysis windows will be in Standar
222. ot be altered by the user 3 Another way in which this implementation is different concerns how the dc dt curves from each scan pair are combined into an average dc dt curve Scan pairs from later in the run will yield dc dt curves that begin and end at lower sedimentation coefficients than those from earlier in the run Thus at the extremes of the sedimentation coefficient range there are data points from only one of the scan pairs which makes defining the average value and standard error for those points problematic o In this implementation the program computes the average dc dt curve only for the range of sedimentation coefficients that is covered by at least two of the scan pairs Because there are fewer values to average near the extremes of the sedimentation coefficient range you will find that the error bars for those points are generally larger 6 4 About the fitting functions The fitting function that is used depends on the choice between the improved or classic methods Section 3 2 6 2 and the choice of fitting to either the 9 s distribution or the average dc dt curve i The classic method is provided primarily to provide backward compatibility the improved method is recommended for all new analyses Function calculation for the improved method The improved method is based around calculating the actual raw sedimentation boundaries that would occur for the current sum of individual species at the exact time poin
223. oundaries then you may wish to test the hypothesis that the poor result is due to the presence of a small amount of oligomer This can be done by first constraining both the M and s value of the second species and fitting If this gives a good fit and reasonable values for the monomer parameters then you could conclude that the hypothesis is satisfied At that point you would probably also want to try releasing the constraint on the oligomer s value re fitting and noting whether the fitted dimer s value is still consistent with the assumed oligomer size If this is also true you would have still more confidence that your hypothesis is true See Tutorial Multi species fits Section 2 5 for an example Why are constraints sometimes needed The need for constraints arises when 1 The separation between two species is too poor to uniquely define all the properties of both of them 2 Only a small amount of a species is present and either the signal noise or the separation is too poor to permit uniquely determining all three of its properties c s and D 3 You are more concerned about obtaining accurate values for certain properties but others are less important The use of constraints if they are appropriate ones can often greatly increase the precision of the remaining fitted parameters The constraint dialog box 130 DCDT User Manual SR DCDT User Manual The dialog box used for establishing or removing the constraints is sh
224. ove This is basically a final result but to create a final distribution for presentation or publication you would probably want to convert these raw sedimentation coefficients to the s scale 16 DCDT User Manual SR DCDT User Manual Full Range 1 728 to 3 552 S Properties at time of analysis from g s Concentration 0 9370 0 0018 fringes Fraction oftotal 76 09 0 09 Number average s value 2 516 0 010 S Weight average s value 2 601 0 008 S z average s value 2 685 0 011 S z 1 average s value 2 764 I 0 021 S Properties extrapolated to t 0 from g s Loading concentration 1 0925 0 0021 fringes Fraction of total 76 01 0 08 Number average s value 2 529 0 009 S Weight average s value 2 614 0 008 S z average s value 2 697 0 013 S z 1 average s value 2 776 I 0 025 S Note stated uncertainties are 1 sigma and sedimentation coefficients are s 20 w values Pushing the Convert to s 20 w Section 3 2 3 3 button brings up the dialog box shown above where you enter the correct partial specific volume solvent density and solvent viscosity 17 DCDT User Manual DCDT User Manual aa __Showintegrisoversistrbution _Setintagratongegion Normaize Cameram weight average s 2 646 0 006 total c 1 437 0 002 Scans 00035 P1 to 000501 trom C Usersivonn PhilioiDesktop Pnilio Data Proty a t v F TF w gt i wn w i
225. oved methods eliminate those systematic errors The Improved method is the default for all new analyses 127 DCDT User Manual DCDT User Manual aa 3 2 6 3 Select fitting model Data choice Data choice fit g s data C fit de dt data These radio buttons determine whether the data points to be fitted are from the 9 s distribution or from the average dc dt curve In Standard mode Section 1 3 the 9 s data are automatically chosen For a discussion of the advantages and drawbacks of fitting to dc dt versus g s see Why do get different answers when fitting g s versus dc dt Section 5 2 2 3 2 6 4 Select fitting model Fit diffusion or mass Fit diffusion or mass _ fitD foreach species fit M s D ratio instead of D pa Internally DCDT always uses sedimentation coefficient s and the diffusion coefficient D as the fitting parameters which characterize the hydrodynamic properties of each species However instead of using D as a fitting parameter it is often desirable to instead use the ratio s D a quantity which is proportional to molecular mass M In fact fitting to mass rather than D is the initial program default when installed but that can be altered via the Options dialog Section 3 1 1 4 2 One significant advantage of fitting M rather than D is that this allows using constraints on the mass values for different species for example fitting to a mixture of monomer and dimer with the
226. ow integrals over distribution Section 3 2 5 1 button the same calculations are done averaging over the entire range of the distribution 19 DCDT User Manual DCDT User Manual aa weight average s 2 646 0 006 08 Scams 00035 IF1 to 000501 trom C WUsers onn PhilloiDesktop Pnilio DataPro 0 7 _ 0 6 w o 05 gt w 0 4 p amp 0 3 vu o N 0 2 o Cc 0 1 0 0 0 1 00 05 10 15 20 25 30 35 40 45 50 55 60 65 s 20 w Svedbergs V More Back Next The one remaining thing you might wish to do is to normalize Section 3 2 5 4 the distribution divide by the total concentration so the area under the curve is exactly 1 That is quite useful if you want to compare distributions for samples at different concentrations on the g s overlay graph Section 3 1 5 see Tutorial Using the g s overlay graph Section 2 3 To normalize simply push the normalize button Section 3 2 5 4 and you will get the result shown above e The normalized curve of course looks the same only the Y axis scaling is different For normalized data the total concentration is not displayed since it is always 1 000 At this point you are done with this analysis unless you wish to fit the peak s as individual independent species See Tutorial Single species fit Section 2 4 and Tutorial Multi species fits Section 2 5 2 2 Tutorial How to select the scans to analyze Normally when you the time
227. own below In this example the fitting model includes three species so the controls related to species 4 and 5 are inactive grayed fi Molar mass s D ratio constraints 2 Set Molar Mass or Sedimentation Coefficient Constraints babae Sedimentation coefficient constraints Radio buttons set the species to Ratio of Radio buttons set the species to Ratio of which that molar mass s D is linked Molar which that sedimentation coefficient Sedimentation Masses is linked Coefficients Species 1 2 000 none 3 2 1 45 Species 2 Species 2 none 1 2 000 none 1 1 45 4 000 j 2 26 Predicted sedimentation coefficient ratio dimer 1 45 Cancel changes e trimer linear 1 75 hd trimer triangular 1 86 Son Clear mass constraints Clear s constraints tetramer linear 2 00 ine mer square planar 2 20 Clear all constraints tetramer tetrahedral 2 26 Mass constraints The image shows an example of a 3 species fit where species 2 is believed to be a dimer of species 1 Hence on the left side the molecular mass of species 2 is set up to be constrained to be exactly twice that of species 1 whatever that may be The desired ratio of masses for the constrained species is entered into the text box Once the constraint link is established to species 1 species 1 cannot itself be linked to other species if there were any so its linking box becomes inactive As shown the mole
228. p a dialog box shown below for entry of the data needed to perform the conversion discussed in more 105 DCDT User Manual DCDT User Manual aa detail below If this conversion has already been done as indicated by the button remaining in the down state then pressing the button will turn off the conversion and revert to raw s values Conversion to s 20 w Values for experimental conditions partial specific volume ml g 0 7198 solvent density g ml 1 003070 solvent viscosity cp 1 0185 Temperature C 20 0 Partial specific volume at 20 C 7 calculate temperature correction partial specific volume ml g 0 7198 ratio s 20 w s raw 1 02920 ratio D 20 w D raw 1 01647 ae It is generally desirable to present and report s values rather than raw uncorrected sedimentation coefficients Conversion from raw s to S compensates for the factors such as buffer viscosity which are relevant only to a particular experiment giving the sedimentation coefficient that molecule would have if measured in water at 20 C Because of the solvent independent nature of s it describes quantitatively the fundamental hydrodynamic properties of the molecule and it is this value which is most useful in comparing the sedimentation behavior of different molecules A The partial specific volume that is entered on this form and used in converting to s scale is applied uniformly to the entire g s dis
229. portant for obtaining accurate sedimentation coefficients you may wish to zoom the graph around the meniscus position You can do that manually by dragging the mouse over the region on the graph left click and drag 8 DCDT User Manual SR DCDT User Manual Meniscus setting and data adjustments 5 9428 cm good data begin 0 015 cm from meniscus good data end at radius 7 150 cm Meniscus at Fringes Be eee fe More Q Beck J hes 7 3 For interference scans as soon as a meniscus position is set the default method for fringe jump removal will be applied the jitter wizard Section 6 7 will determine the best position for removing jitter and remove it and the graph will alter to display the corrected data as shown above At this point the scans should align vertically so the boundary movement can be seen but in some cases adjustments may be needed See jitter removal Section 3 2 2 4 and fringe jump removal options Section 3 2 2 5 for more information e Note that it is normal that the scans do not overlay in the air air space to the left of both meniscii because of the fringe jumps DCDT User Manual DCDT User Manual aa Meniscus setting and data adjustments Meniscus at 5 9428 cm good data begin 0 015 cm from meniscus good data end at radius 7 040 cm Fringes eed F More C Back New AJ 4 The region between the vertical green bars defines the good data that will be used in th
230. pply either theoretical or experimental weights That choice is made within the Select fitting model page Section 3 2 6 There are three modifications to the standard Gauss Newton method all of which occur at the end of each iteration 1 Each new parameter is tested to be sure it falls within defined upper and lower bounds If that parameter would exceed the bounded limit it is set at the limiting value When this occurs a yellow warning box appears next to that parameter on the Fitting parameters monitor tab Section 3 2 9 6 of the Perform least squares fit page Section 3 2 9 2 An increment reduction factor Section 3 2 9 6 1 is applied to insure that the new set of parameters actually gives a lower variance and to improve the rate of convergence The current value of the increment reduction factor is also displayed on the Fitting parameters monitor Section 3 2 9 6 3 If any constraints Section 3 2 6 6 have been set on the fitting parameters those constrained parameters are updated to reflect the new values of the parameters to which they are linked 6 6 About the meniscus wizard 1 For absorbance scans the program logic first locates a starting meniscus position by finding the highest OD value within the leftmost 20 of the last scan being used in the analysis trying to find the top of the positive spike at the meniscus It then examines a region within 0 02 cm of that initial position to find the top of the positive spike for
231. r programs 175 176 How to identify and remove rogue scans 177 183 How to optimize data acquisition for g s analysis 167 170 How to print graphs 172 How to remove bad data points from scan files 176 177 How to zoom with the mouse 176 207 DCDT User Manual ID points button 101 102 Improved method 127 Increment reduction factor 146 147 Initial guesses 134 136 Intensity scans 191 192 Interference scans 57 60 89 98 99 99 100 Jitter 98 99 Jitter removal options 98 99 Jitter wizard 195 Load every Nth scan 92 93 Load new scan files button 89 90 Load raw scans Add more scans for this sample button 90 Load raw scans Data loading options 92 93 Load raw scans Delete selected scans button 90 91 Load raw scans Load new scan files button 89 90 Load raw scans Preselect data type and cell check box 89 Load raw scans Replace scans for this sample button 90 Load raw scans Run Claverie simulation to generate data button 93 94 Load raw scans Sample description 93 Load raw scans Scans currently loaded in memory list box 91 Load raw scans Scans to be used in g s calculation list box 91 92 Load raw scans page 88 89 Log page 78 83 Mass diffusion calculator 66 67 Meniscus 96 96 97 Meniscus clearance 96 97 Meniscus position 96 Meniscus wizard 194 195 Menus Edit menu 63 65 File menu 61 62 Help menu 72 74 View menu 65 Window menu 72 Mistakes 166 167 Molecular mass 66 67 128 Molecular mas
232. rage dc dt curve and then calculating as indicated in the preceding paragraph What are the advantages and drawbacks of this approach The principal advantage of the Monte Carlo method is that it evaluates the confidence limits based on noise in the raw scans and noise that is truly random Its main drawbacks are 1 we don t usually know the true noise level of the raw scans 2 the noise level may not be constant across each scan or from scan to scan o in particular note that for fluorescence scans the noise magnitude usually is substantially lower in the region to the left of the boundary than in the plateau region for that reason the bootstrap method 196 DCDT User Manual SR DCDT User Manual 629DC392 391 2 4327 B60E 9D26F 1441468 may be more appropriate for fluorescence data o the noise in absorbance scans is also usually significantly lower in the low OD regions to the left of the boundary than in the plateau region 3 we generally don t know whether the experimental noise is truly random except when we are fitting simulations 197 DCDT User Manual DCDT User Manual aa 7 Bibliography 7 1 reference 1 Philo 1997 Philo J S 1997 An improved function for fitting sedimentation velocity data for low molecular weight solutes Biophysical Journal 72 435 444 This article can also be downloaded as an Adobe Acrobat document called sveddoc2 pdf from the program download page http www jphilo mailway com
233. ram will not explicitly designate them as such 122 DCDT User Manual SR DCDT User Manual How this conversion affects the g s distribution and average dc dt curve Please be aware that conversion to s also alters the magnitudes of the 9 s and g s distributions This arises because these distributions are defined such that the area under the curve gives the concentration Thus if the X axis is expanded or contracted as a consequence of conversion to the s20 w scale the Y axis must be contracted or expanded to keep the area unchanged Internally in the program the dc dt g s and g s data are always kept in raw units the conversion is only applied as graphs are generated and values are displayed and printed Generally this is transparent to the user but with regard to setting the ds intervals for the average dc dt and 9 s curves it is not possible to have conveniently rounded intervals between data points in both raw and s scales Consequently switching between raw and s _ units will usually force a re calculation of the average dc dt curve and 9 s distribution because the X axis point spacing will change if that is being determined by the default Automatic option see Calculate average dc dt X axis point spacing Section 3 2 4 1 Thus the average dc dt curve and 9 s distribution will be slightly different in the different units the difference is not simply a multiplicative factor for the X axis How
234. re needed to calculate improved parameters e Inverting matrix indicates the matrix inversion needed for calculating improved parameters is being done e Adjusting increment indicates that the new parameters are being tested to be sure the sum of squared residuals is improved During this time the increment reduction factor Section 3 2 9 6 1 may change e Converged last sum indicates that convergence has been obtained and a final set of sums needed for calculating parameter errors is being accumulated 3 2 9 6 Fitting parameters monitor tab This tab is used to monitor the actual values of the fitting parameters as they are updated during the fit 144 DCDT User Manual SR DCDT User Manual 6 Analysis2 00023 00032 RA3 ol em f Status waiting for command Iteration number 1 Simulate Do fit Cancel Parameter Value Value Co 1 0 4902 0 4958 s 1 3 729 3 575 M 1 33 05 36 93 Co 2 0 5385 0 5225 s 2 7 279 7 325 M 2 140 31 147 32 Sum Errors 2 4 438425E 3 3 250641E 2 Increment Reduction Factor 1 0000 H Graphical fit monitor Fitting parameters monitor The image above shows the analysis page after the first iteration of a fit which was initiated via the 1 iteration button Section 3 2 9 2 Fit control buttons and displays These controls are shared with the Graphical fit monitor tab Follow this link Section 3 2 9 for descriptions and links to further de
235. recision lowest o are weighted most heavily 2 The Theoretical weights option not available prior to version 2 0 assigns a weight to each point in the g s distribution on a theoretical basis assuming the uncertainties for all points in the original raw scan files are equivalent This is now the program default when fitting to 9 s distributions Why experimental weights are not recommended The drawback to experimental weights is that the o value for each data point is determined from the differences among the dc dt curves for individual scan pairs that went into computing the average dc dt curve The problem with that approach to estimating o is that the amplitude and width of each dc dt curve varies with time during the run and unless the scans cover a very narrow time span much of the variation among the dc dt curves for individual scan pairs is due to those systematic changes rather than noise in the raw data This is discussed in detail in reference 8 Section 7 8 Consequently the o values are often grossly over estimated particularly when the number of scans grows larger and particularly at the tops of the peaks Thus when experimental weights are used it is often the case that the most important data points near the top of a peak are assigned very low weights It is also true that as you add more scans into the analysis to improve the signal noise ironically the estimated o values often increase Because of these phenomena t
236. refer to the fitted data if you have reached this page after doing a simulation Section 3 2 9 3 rather than a fit then the plots show the results of the simulation 1 Species plot for entire data region area This type of plot shows each fitted species as an area graph and extends the plot over the entire sedimentation coefficient range not just the range that is fitted In this example below the area plot of the fit to the antibody monomer region highlights the fact that this one species does not fit the high sedimentation coefficient region due to the presence of aggregates The area to the right of the single species peak between the theoretical fit and the experimental data is directly a measure of the aggregate content z Species plot for entire data range area plot It is also apparent that there is material sedimenting in the region from 0 5 to 3 5 S that is not accounted for by this single species fit this sample contains both antibody fragments and detergent micelles that sediment in that region Because of the presence of those other species for this fit the fitting region was limited to 158 DCDT User Manual SR DCDT User Manual sedimentation coefficient between 3 S and 6 88 S the latter is about midway down the right side of the peak That was done deliberately in order to get a good fit to the monomer properties by excluding regions strongly influenced by these other species By extending the p
237. relative influence of diffusion on each species It is recommended that you load enough sample to give a meniscus position lt 6 0 cm about 430 microliters of sample in a 12 mm charcoal epon cell 4 Cell leakage Another common cause of bad fits and strange results is slow sample leakage which is easily detected by shifts of the meniscus position to higher radii over the course of the run Slow sample leakage usually results from 166 DCDT User Manual SR DCDT User Manual evaporation via a leak at the red cell plug gasket rather than a liquid leak through the window centerpiece interface gt 5 Wavelength shifts With absorbance data you really cannot take data at different wavelengths during the same run e Because the monochromator never returns to exactly the same wavelength if you use different wavelengths then the apparent concentration will vary from scan to scan because the extinction coefficient will be slightly different as the wavelength varies 1 2 nm from scan to scan This effect can most easily be seen as a jumping up and down of the plateau region from scan to scan If you need data at different wavelengths it is highly recommended that you do each wavelength as a separate run 6 Drifting sample temperature Another instrumental problem that can cause bad fits and poor or inconsistent results is changes in sample temperature during the run e t is important to thoroughly equilibrate the rotor tempera
238. revent infinite or near infinite loops Fitting model defaults tab 69 DCDT User Manual DCDT User Manual aa e Fit diffusion or mass fit M this default governs whether to fit the diffusion coefficient for each species or the species molecular mass the s D ratio See Select fitting model fit diffusion or mass Section 3 2 6 4 e Molecular weight units true molecular mass this default sets the units to be used for molecular mass See Select fitting model molecular mass units Section 3 2 6 7 File locations tab e Default location for scan files remember last location These options govern which folder is initially displayed in the File Open dialog when new scan files are loaded o The Always start in this folder option specifies that scan browsing always begins in a particular specified folder For example this might be a network drive or a folder where all experiments for an individual XL I user are stored When this option is selected the Browse button is active and may be used to select the default folder o The Remember last location and start there option has the program remember the last folder from which scans were loaded and start there 70 DCDT User Manual SR DCDT User Manual e Default location for saved analyses store with scans These options govern which folder is initially displayed in the File Save dialog during the Save Analysis As procedure o The Always st
239. rge range may be needed because the signal noise of the data is very low What does it do This button toggles between two alternative algorithms for calculating the dc dt curves resulting from each pair of scans that is included in the analysis When a pair of scans is subtracted the result is a table of Ac versus radius Those radius values can be converted to apparent sedimentation coefficients s using the relation r s In Im where Fn S the radius of the meniscus However since we are using a pair of scans which ot value for wt should be used In the original algorithm developed by Walt Stafford reference 7 Section 7 7 and in versions 1 x of this program the wt value used in the formula above is the harmonic mean of the wt values for the two scans 108 DCDT User Manual SR DCDT User Manual When the Use true mean time option is on this program instead uses the true mean the arithmetic mean of the f values for the two scans in the pair Why is the true mean option sometimes useful The use of the harmonic mean can significantly distort the shapes of the average dc dt and 9 s curves when the range of boundary movement is quite large when the peaks are severely broadened In particular the use of the harmonic mean results in 9 s distributions that do not fully return to zero on the right hand side of a peak falsely making it appear there is a small amount of faster sedimenting components present With
240. rrent graph onto the Clipboard in Windows enhanced metafile format The Print button will send this graph to the printer The Add to log button will add the current graph at the end of the Log page Section 3 1 3 3 The Readout data values checkbox will turn on a readout of the graph coordinates corresponding to the current mouse position This could for example be used to find the lower and upper 90 confidence limits for the actual distribution without assuming a Gaussian shape by locating the mass at the points where the cumulative frequency magenta line reaches 5 and 95 respectively giving the desired 10 probability that the true value lies outside the confidence interval 3 2 11 Graph fit results page This analysis page displays nine different types of graph of the fit results fitted data or theoretical fitted curves The type of graph is selected from a drop down list at the top of the page Also located at the top of the page are convenient buttons for printing the graph Print button copying the graph image to the Windows Clipboard as a high resolution metafile so it can be pasted into another document Copy button and adding the graph image at the end of the Log page Section 3 1 3 3 Add to Log button The drop down sections below show examples of each type of graph all from the same analysis of an antibody sample with brief comments about the intended use of that plot type Note that although these descriptions
241. s 3 2 7 Set initial guesses on graph page 3 2 8 Set alter fitting parameters page 3 2 8 1 Parameter value text boxes 3 2 8 2 Fit control check boxes 3 2 8 3 Constrained parameters 105 108 108 109 109 110 110 111 111 112 112 112 115 115 115 117 117 118 119 119 121 121 121 124 124 124 125 125 125 127 127 127 128 128 128 129 129 132 132 133 133 134 134 136 136 138 138 138 138 139 DCDT User Manual DCDT User Manual aa 3 2 8 4 Convergence control 139 3 2 8 5 Adjust parameter upper lower limits button 139 141 3 2 9 Perform least squares fit page 141 143 3 2 9 1 Perform fit Do fit button 143 3 2 9 2 Perform fit 1 iteration button 143 3 2 9 3 Perform fit Simulate button 144 3 2 9 4 Perform fit Cancel button 144 3 2 9 5 Perform fit Status 144 3 2 9 6 Fitting parameters monitor tab 144 146 3 2 9 6 1 Increment reduction factor 146 147 3 2 10 Report fit results page 147 148 3 2 10 1 Report fit results Fitted parameters 148 149 3 2 10 2 Report fit results Fixed or constrained parameters 149 3 2 10 3 Report fit results View parameter cross correlations button 150 3 2 10 4 Report fit results Compute confidence intervals button 150 153 3 2 10 5 Report fit results Create reports group box 153 154 3 2 10 6 Report fit results Fit statistics 154 3 2 10 7 Report fit results High cross correlation warning 154 155 3 2 10 8 Report fit results Graph bootstrap or Monte Carlo results
242. s axis titles and graph titles are hotspots When passing over these hot spots the cursor changes into a hand with pointing finger single clicking will bring up a small dialog box for editing and customizing those features 172 DCDT User Manual SR DCDT User Manual G Customize axis scale and tick marks Maximum value Z2 Minimum value 59 Major tick interval 0 2 Minor tick interval 0 1 Show minor ticks Apply Ok Cancel Clicking on an axis brings up the dialog shown above which allows alterations of the scale range minimum and maximum the intervals between major and minor tick marks and whether minor ticks are displayed note that the graphs generated by DCDT normally do not show minor tick marks If the graph is currently zoomed then this dialog will change the axis of the zoomed graph and won t alter what is displayed when the graph is un zoomed with the z key Gal The major tick marks the ones which can have their values labeled are always positioned starting from zero not necessarily from the start of the axis Thus in the example above even though the X axis of this raw data plot starts at 5 9 cm because that value is not divisible by 0 2 the labeled major tick marks will be at 6 0 6 2 6 4 rather than at 5 9 6 1 6 3 fy TIP Sometimes a tick label which should be present at the beginning or end of an axis does not display P due to round off error When this happens you
243. s particular example there is a high in absolute value negative correlation between the concentration and mass parameters for species 2 The negative correlation between the concentration and sedimentation coefficients of species 1 with the concentration of species 2 are also quite strong 0 9363 and 0 9362 although not quite strong enough to flag in this case For this sample species 2 a dimer of an antibody is present at a level of only 2 and there is simply insufficient information in the data to determine the concentration sedimentation coefficient and mass accurately for species 2 This is a case where it would be better to constrain the mass of species 2 which will greatly improve the precision of the estimates for the concentration and sedimentation coefficient of not only species 2 but also species 1 3 2 10 4 Report fit results Compute confidence intervals button Compute confidence intervals This button will initiate computation of confidence intervals for the parameters a much more statistically rigorous way to express the uncertainties in the best fit parameters but time consuming to calculate You are first presented with a dialog box for setting the desired confidence level and selecting a computation method as shown below The default level of confidence that will be shown in this dialog 68 3 95 etc can be altered via the Options dialog Section 3 1 1 4 2 The Ok button initiates the confidence level
244. s units 132 133 Monte Carlo 155 158 Monte Carlo method 150 153 196 197 208 DCDT User Manual aa DCDT User Manual SR DCDT User Manual More gt gt and lt lt Less buttons 78 More gt gt and Less lt lt buttons 78 Navigation tree 83 85 Next and Back buttons 77 78 Next button 77 78 Normalize button 121 Number of species 127 Number average sedimentation coefficient 117 Offset 187 Open 62 Open saved analysis 62 Options dialog 67 72 Out of bounds 139 141 Overlay plot 158 164 Overview 1 2 Parameter bounds 139 141 Parameter constraints 129 132 Parameter cross correlation 150 154 155 Parameter errors 150 153 Parameter limits 139 141 Parameter precision 139 150 153 Parameter value text boxes 138 Partial specific volume 133 134 Peak broadening limit 104 105 Perform fit 1 iteration button 143 Perform fit Cancel button 144 Perform fit Do fit button 143 Perform fit Simulate button 144 Perform fit Status 144 Perform least squares fit page 141 143 Pitfalls 166 167 Preferences 67 72 Preselect 89 Print graph 175 176 Print graphs 172 Print report 153 154 Program citation 190 Program organization 1 2 Program overview and organization 1 2 209 DCDT User Manual DCDT User Manual aa Pseudo absorbance 98 99 191 192 Reference 1 198 Reference 10 199 Reference 11 199 Reference 12 199 Reference 13 199 Reference 14 199 200 Reference 2 198
245. scenarios which you can then use for fitting comparing the fitting results to the known correct answers It also allows for concentration dependent sedimentation coefficients something not dealt 93 DCDT User Manual DCDT User Manual aa with by the fitting functions and thus permits you to see how concentration dependence will affect the results It does not however include concentration dependence of diffusion coefficients The dialog box used to control and run the simulations is shown below as it appears while a simulation run is in progress This run corresponds to the default parameters a quick moderate accuracy simulation of ovalbumin at 60K rpm Claverie Simulation Control s Svedbergs D Ficks Loading conc OD Meniscus radius cm Inner radius cm Cell bottom cm Run time for Ist scan s Time between scans s Total number of scans RPM Calculation interval s 3 540 7 89 1 0000 6 0000 6 0000 7 2000 180 180 50 60000 2 0 Y Axis 1 601 oo 0 0000 01 0 0050 Number of data points c dependence of s per OD Gaussian noise rms X Ans Absorbance E Add to previous Claverie data P Interference _ Fluoresence Note to simulate conventional cells set meniscus radius inner radius to simulate synthetic boundary cells set meniscus radius gt inner radius Can For instructions on how to use this simulator see Tutorial Simulating experiments with t
246. smaller time interval If you are going to be summing data for more than one species remember you will want to pick a run time that is appropriate for all the species Step by step multi species simulation Multi species simulations are done by summing single species simulations This feature is governed by the Add to previous Claverie data check box near the bottom of the form This check box becomes active when Claverie data are currently in memory It is only possible to sum simulations that generate compatible data sets same radii start time etc That is in summing simulations the only parameters changed between species are their s and D values and concentration The steps for a multi species simulation are 1 Perform a simulation for the first species o You may save the results to disk if you want to keep that data but it is not necessary to save to disk 2 Immediately after the first simulation invoke the Claverie simulation command again 3 Enter the desired concentration sedimentation coefficient and diffusion coefficient for the next species gt Be sure to check the Add to previous Claverie data check box it is automatically cleared when the form is loaded Run the simulation for the next species Note that the graph being displayed shows only the current simulation being generated not the sum If there are additional species return to step 2 por ot OA ON After the last species has been cal
247. specific volume and solvent density If those values are not well known it may be preferable to work in units of buoyant molecular mass H When you are done push the Next button to go to the Set initial guesses on graph page Section 3 2 7 2 Set initial guesses for peak positions and the range of s values to be included in the fit 37 DCDT User Manual DCDT User Manual aa This page below has the same appearance in both Advanced and Standard modes Fitting range Species properties updating Make new concentraton and peak width guesses based on new peak positions s Keep previous peak position concentration and peak width values 0 35 0 30 0 25 0 05 a s OD Svedberg in B gt o 0 00 s Svedbergs E Less Back A If needed drag the magenta markers indicating the initial guess for the sedimentation coefficient to the proper position The program wizard automatically sets this at the highest point in the curve B Since the Standard mode analysis of these data above indicated the presence of some aggregates this time we ll try narrowing the range to just the center of the main peak As shown above the the green markers that define the left and right ends of the data to be fitted have been dragged to positions marking roughly 50 of the maximum peak height If you want to reduce the influence of other species you should not do this by narrowing the range of sedimentati
248. splayed are the so called formal averages where the 9 s distribution The Broad algorithm option Section 3 2 5 8 may give more accurate estimates of the standard deviations of these computed quantities Other features A Copy all values to Clipboard button is provided to copy all the results to the Windows Clipboard so they can be pasted into another document Individual values may be copied by highlighting them with the mouse and using the CTRL C copy keyboard shortcut The note at the bottom of the form will indicate whether the sedimentation coefficient averages represent uncorrected raw values or Ss values How are these values calculated The average sedimentation coefficients and the concentrations are given by Eo o J g s 4s a g s s ds fg d 5 c g s ds where the integrations run over the entire range of sedimentation coefficients covered by the distribution Thus the results will only be valid thermodynamic quantities if the distributions are calculated at a time such that all species are included The error bars on these quantities are calculated from the estimated uncertainty for each data point in the sedimentation coefficient distribution functions Og which derives from the differences in dc dt data for different scan pairs through the following formulae derived from the standard propagation of error rules 120 DCDT User Manual SR DCDT User Manual paa o ds s 9 s ds
249. splays the scan data as individual clickable data points for read out and or removal of bad points Meniscus aS and data adjustments 7 100 cm Meniscus at 5 9195 cm good data begin 0 015 cm from meniscus good data end at radius Jitter removal Fringe jump removal Data displayed D oom auto huo i coneced meniscus 0 8 0 6 Fringes 0 4 0 2 0 0 6 8 7 0 7 2 5 8 TY 6 2 6 4 6 6 Radius cm Controls on this page Meniscus position red marker on graph Section 3 2 2 1 2 Good data begin X cm from meniscus left green marker on graph Section 3 2 2 2 Good data end at radius right green marker on graph Section 3 2 2 3 f ii The controls below are shown and available only in Advanced mode Section 1 3 95 DCDT User Manual DCDT User Manual aa Zoom meniscus button Section 3 2 2 7 o Jitter removal options Section 3 2 2 4 gray markers on graph interference data only O Fringe jump removal options Section 3 2 2 5 interference data only Data displayed options Section 3 2 2 6 ID points button Section 3 2 2 8 SNotes about some features of the graph display To keep the graph legend from occupying too large a fraction of the graph the legend is suppressed when more than 40 scans are displayed as shown above Also as shown above when more than 40 scans are displayed the sequence of colors and line styles that is normally used by the graphing routines is
250. ss units 3 2 5 6 Calculate g s distribution Display options m Display options _ with eror bars ae cn This group of controls governs which type of sedimentation coefficient distribution is displayed and whether errors bars 1 standard deviation are shown on the graph e When the G s button is depressed the integral distribution big G of s is displayed rather than the differential distribution little g of s This G s distribution measures the fraction of the sample that sediments with sedimentation coefficients less than or equal to each value of s o Note that this G s distribution is calculated directly from the 9 s distribution by integration and is not corrected for diffusion i e it is not equivalent to the G s distribution obtained by the van Holde Weischet method G s is always normalized the values are fractions and range from 0 to 1 e When the g s button is depressed the g s data is displayed rather than 9 s e When both the G s button and g s button are depressed the integral distribution G s is displayed e The With error bars button is a toggle switch that turns on display of error bars when it is depressed o Error bars are not displayed for the integral distributions 3 2 5 7 Calculate g s distribution Reset zero amp Undo buttons M Manual adjustment gt Reset zero Undo 5 The Reset zero button is used to manually redefine
251. stances try limiting the range of s values used in the fit to exclude contributions from such species If this does not work consider modeling the group of large or small species as a single species with some average s value and a very high diffusion coefficient The drawback to the latter approach is that the broad peak used to model the group of species is likely to overlap the peak s for the species of interest and therefore the accuracy of the returned properties for those major peaks may be poor 2 6 Tutorial Simulating experiments with the Claverie simulator This tutorial first covers how to simulate a sample containing a single species and then how to do multi species simulations Step by step single species simulation 57 DCDT User Manual DCDT User Manual a 58 Claverie Simulation Control s Svedbergs D Ficks Loading conc OD Meniscus radius cm Inner radius cm Cell bottom cm Run time for Ist scan s Time between scans s Total number of scans RPM Calculation interval s Number of data points c dependence of s per OD Gaussian noise rms 3 540 7 89 1 0000 6 0000 6 0000 7 2000 180 180 50 60000 2 0 601 0 0000 0 0050 Y Axis 1 X Ans Absorbance Interference _ Fluoresence Note to simulate conventional cells set meniscus radius inner radius to simulate synthetic boundary cells set meniscus radius gt inner radius F Add to pre
252. subtracted For a given level of instrument noise then the signal noise for any particular Ac At computation will grow approximately linearly with time What ultimately limits this increase in signal noise is the need to keep the boundary movement from becoming excessive causing broadening of the peaks in the distribution and loss of resolution between species The maximum time span that can be covered without broadening is related to the mass es of the solute s as discussed under peak broadening limit Section 3 2 3 2 This leads us to Rule 1 Rule 1 the first thing to do to optimize signal noise is to use the largest time span possible between the first and last scan in the analysis while staying within the restrictions necessary to avoid peak broadening Once this criterion is met the overall signal noise is improved by including and averaging more scans within this maximum time span and will improve roughly as the square root of the number of scans assuming the noise is random This leads to Rule 2 Rule 2 scan as fast as possible so more scans can be included in the analysis For both absorbance and interference data the time to take a scan is roughly proportional to the radial range being scanned A common mistake is to leave the radial range set at the default values of 5 8 to 7 3 cm This results in a significant fraction of the instrument time being spent taking data above the meniscus and beyond the base of the cell Remember to
253. suppressed since it becomes impossible to distinguish individual scans when there are so many and instead the colors are varied to produce a rainbow effect Note that when more than 40 scans are shown you can force display of a legend by right clicking on the graph and selecting a legend position from within the Legend Style submenu The legend text size is reduced when there are 20 or more scans shown Note also that if you wish to export the scan data being displayed on this graph for use in another program whenever this page is being displayed that can be done using the Scan data from graph items on the Edit menu Section 3 1 1 2 3 2 2 1 Set meniscus amp data region Meniscus position Meniscus at 5 904 cm This setting defines the radial position of the meniscus The value can be altered either by directly entering a value in the text box or by dragging the red vertical line displayed on the 2D graph e For absorbance scans if you manually drag the meniscus marker on the graph you will then be asked whether you want the wizard to re calculate the mean position of the positive spike the mean of the actual spike positions for every scan that has been loaded This is helpful when the scans contain two positive spikes and the wizard has identified the wrong one as the true meniscus Upon first entry to this analysis page after new scans have been loaded the meniscus wizard Section 6 6 will try to locate the meniscus for you and th
254. t 184 185 Select fitting model Constrain M or s ratios among species 129 132 Select fitting model Data choice 128 Select fitting model Density and partial specific volumes 133 134 Select fitting model Fit diffusion or mass 128 Select fitting model Improved or classic methods 127 Select fitting model Molecular mass units 132 133 Select fitting model Number of species 127 Select fitting model Weighting of data points 128 129 Select fitting model page 125 127 Select scans to analyze of sections up down spinner 109 110 Select scans to analyze Auto adjust button 104 Select scans to analyze Peak broadening limit 104 105 Select scans to analyze Use true mean time button 108 109 Select scans to analyze page 102 104 Set defaults 67 72 Set initial guesses on graph page 134 136 Set integration region 118 119 Set meniscus amp data region Data displayed options 100 101 Set meniscus amp data region Fringe jump removal options 99 100 Set meniscus amp data region Good data begin X cm from meniscus 96 97 Set meniscus amp data region Good data end at radius 97 98 Set meniscus amp data region ID points button 101 102 Set meniscus amp data region Jitter removal options 98 99 Set meniscus amp data region Meniscus position 96 Set meniscus amp data region Zoom meniscus button 101 Set meniscus amp data region page 94 96 211 DCDT User Manual Set range for g s
255. t optionally 1 iteration at a time and monitor the fit progress either through graphs or a display of the numerical values of the fitting parameters 5 Report fit results page Section 3 2 10 o view a display of the best fit parameters their uncertainties the fit statistics and optionally print a report 6 Graph fit results page Section 3 2 11 o view various graphs of the fit results and data and optionally print or export them 3 1 5 g s overlay graph The g s overlay graph is used to compare distributions for different samples or for the same sample analyzed at different times in the run See Tutorial Using the g s overlay graph Section 2 3 86 DCDT User Manual SR DCDT User Manual ty Overlay distributions l lol x io Qo normalized gis pe co Ss o o gt a 8 N gt o n Graph style Points Lines Edit legend text C 042903 110946 cell3 00120 00164 IP3 Points and lines Qo The Overlay graph is enabled whenever there are two or more analysis windows open where g s distributions have been computed F J Any minimized minimized analysis windows shrunk to an icon are ignored however which makes it possible to add or subtract curves from the graph without closing or opening analysis windows The Graph All button will update the graph with whatever distributions are currently available 9 The Printbutton will bring up a Print dialog box to allow selectio
256. t to the foreground if it is minimized or hidden by analysis windows This command is only available when two or more analysis windows contain 9 s data Close All Windows This provides a quick way to close all analysis windows prompting you to save any containing unsaved results and the 9 s overlay graph freeing up screen space for new analyses Cascade This is the usual Windows command to make all the open windows have a uniform size and arrange them is an overlapping cascade Arrange Icons This is the usual Windows command that arranges the icons corresponding to any minimized windows along the bottom of the document area Window List The bottom of this menu contains a list of all open windows with a check mark next to the active window You can activate a different window and bring it to the front by clicking its name or by pressing the corresponding number on the keyboard 3 1 1 6 Help menu 72 DCDT User Manual SR DCDT User Manual A Contents Ctri Fl Index Search About DCDT Registration 2 Check for Updates Program Website E mail author Lee Contents This brings up the Table of Contents tab for this Help file and opens the Program Overview and Organization Section 1 1 page Index This brings up the Index tab for locating specific topics in the Help file Search This brings up the Search tab for locating specific words or phrases that are not in the Index About DCDT
257. t values for the solvent density p and sample partial specific volume V Those values are only relevant and needed when you have chosen to use molecular mass the s D ratio as the second hydrodynamic parameter for the fit within the Fit diffusion or mass Section 3 2 6 4 group box and when you have chosen units of true molecular mass within the Molecular mass units Section 3 2 6 7 group box e When checked the Use the same partial specific volume for all species check box causes the program to assign the same to all species as would be true for example if they are all oligomers of a single type of monomer This is the default state of the program o If your sample contains more than one chemical species uncheck the box and then fill in the correct values for the other species o If you later turn on the Use the same partial specific volume for all species check box the value entered for species 1 will automatically assigned to all the other species e When the program starts p and V are set to default values These default values may be altered using the Options dialog Section 3 1 1 4 2 e When you alter p or V on this page those values will also be used in other calculations applied to this analysis window Section 3 1 3 such as converting raw sedimentation coefficients to s 20 w values 3 2 7 Set initial guesses on graph page This page allows you to 1 set the sedimentation coefficient range of the data to be fitted
258. ta being fitted This plot shows only the actual experimental data that were fitted seston eto 0 45 0 40 0 35 0 30 0 25 0 20 0 15 g s OD Svedberg 0 10 0 05 36 38 40 42 44 46 46 50 s2 54 s6 s 60 62 64 66 6s 70 s Svedbergs 164 DCDT User Manual SR DCDT User Manual 165 DCDT User Manual DCDT User Manual aa 4 How to 4 1 How to avoid common mistakes Problems with strange results or bad fits usually arise either from a violation of the assumptions that are inherent in this type of analysis or instrumental problems Below is a list of mistakes and pitfalls to avoid 1 Wrong assumptions It is essential to keep in mind that fitting the 9 s or dc dt data to a sum of species requires certain assumptions about the nature of the sample and the way in which the experiment is conducted 1 the sample has no significant concentration dependence of s or D values 2 if there are multiple species they do not interact or inter convert or if they do inter convert that process occurs slowly compared to the time of the velocity run 3 solution non ideality is minimal 4 the rotor is directly and rapidly accelerated to run speed o With regard to the last assumption regarding rotor acceleration it is not possible to obtain correct diffusion coefficients or molecular masses for experiments where the rotor speed is altered after significant sedimentation has occurred although the sedimentation coef
259. ta rate Noise OD Size cm Scan Data Points Size cm replicates r m s continuous 003 1 84 566 0021 6 7 00420 continuous 005 1 64 363 0033 5 7 00424 continuous 010 1 55 184 0065 2 1 00399 continuous 003 4 157 758 0016 19 4 00213 continuous 005 4 157 512 0023 13 1 00212 continuous 010 4 157 238 0050 6 1 00202 step 003 1 96 380 0032 4 0 00386 step 005 1 73 239 0050 3 3 00396 step 010 1 59 121 010 2 1 00383 step 003 4 183 337 0036 7 4 00219 169 DCDT User Manual DCDT User Manual aa step 005 4 109 227 0053 8 3 00207 step 010 4 77 120 010 6 2 00202 Some observations from the above results are e Increasing the radial step size above 003 does not reduce the time per scan proportionally to the number of data points produced and always results in a lower figure of merit e For the same nominal radial step size step mode always produces fewer data points than continuous mode i e continuous mode produces smaller step sizes than the value requested e With 1 replicate step mode always takes longer and also produces fewer data points than continuous mode e Using 4 replicates actually does lower the noise level by very close to the expected factor of 2 slightly less than a factor of 2 for the continuous mode e Using 4 replicates never increases the time per scan by as much as a factor of 4 and often by less than a factor of 2 e With 4 replicates in continuous mode increasing the step s
260. taiantiiogyi 0 35 0 30 0 25 o 8 a s OD Svedberg an 0 00 eg lt x 0 1 2 3 4 5 6 z 8 9 10 11 12 13 14 s Svedbergs data MMM species 1 ad Choe Cbea 8 If asingle species does not fit the entire range of data well for example notice the divergence of the data and the fit above 8 5 S on the graph above due to the presence of irreversible aggregates in this sample you may wish to go back to the Set initial guesses on graph page Section 3 2 7 and narrow the range of data to be fitted to just the central portion of that single peak thus excluding other minor components that might be present More details and Advanced mode Below is a more detailed and general procedure for a single species analysis in advanced mode Section 1 3 It looks more complicated than it is this takes only a minute or two 1 Select an appropriate fitting model A Navigate to the Select fitting model page Section 3 2 6 In advanced mode Section 1 3 it looks as shown below 36 DCDT User Manual SR DCDT User Manual Fitting optons E Data choice Fit diffusion or mass Number of species 1 fit a s data ft D for each species Function computation fit de dt data fit M s D ratio instead of D Improved method heal dary ok iia ie Classic method Theoretical C Experimental None Constrain M or s ratos among species Set constraint values Molecular mass units Density
261. tails B Current value column This column shows the parameter values for the current iteration of the fit Comparisons to the Previous value column to the right will show whether this parameter is changing rapidly or converging to a definite value e Before the first iteration is completed these values will correspond to those entered or computed as the initial guesses e f a parameter tries to go outside the current upper or lower limits the background color of its text box will flash momentarily bright yellow It will stay yellow once that parameter reaches the limit Note that when parameters go out of bounds the program will switch from the Graphical fit monitor tab to show this Fitting parameters monitor tab if needed 145 DCDT User Manual DCDT User Manual aa Previous value column This column shows the parameter values from the previous iteration of the fit e Before the first iteration is completed this column will be empty 4 Chi squared or sum of squared residuals This row displays the total chi squared or the sum of squared residuals calculated for the current and previous iteration depending on whether an experimentally weighted theoretically weighted or unweighted fit Section 3 2 6 5 was selected 5 Graphical fit monitor tab Section 3 2 9 Select this tab to monitor fit overlay and residual plots as the fit progresses QO Increment reduction factor The increment reduction factor Section 3 2 9 6 1
262. the improved fitting algorithm the fitted hydrodynamic parameters will be accurate no matter whether the harmonic mean or true mean is used but under conditions of severe peak broadening the peaks will usually be less broadened and distorted when the true mean is used That reduction of broadening can be helpful for multi species fits When should this option not be used This option is only helpful for multi species fitting using the improved fitting algorithm You should definitely not use it if your primary interest is in the g s distribution itself or calculating properties such as the weight average sedimentation coefficient from it It s use for those purposes would result in sedimentation coefficients that are less accurate 3 2 3 5 of sections a of sections 1 lt gt This control is only visible in Advanced mode Section 1 3 and this option is definitely not recommended for general use Briefly this option is sometimes helpful when you will be fits to g s distributions for samples containing more than one species to help avoid severe broadening of the peaks because the range of boundary movement is very large for example a large range might be needed because the signal noise of the data is very low The reasons why this option was developed are discussed and some examples of its use can be found in reference 13 Section 7 13 a PDF of this article can be obtained from John Philo What does this do By defa
263. the Graph fit results page Section 3 2 11 and the Graph bootstrap or Monte Carlo results Section 3 2 10 8 to add those graphs to the Log when it is desirable to permanently document them Loading saved fits lacking a Log When a fit saved using an earlier program version which lacked the Log page feature is loaded a new Log will be created describing the old fit listing the various parameters such as the fitting region the names of the scan files etc An example of such a case is shown below 82 DCDT User Manual SR DCDT User Manual 2 i C XLIDATA antibody cell1_1species 00027 00038 RA1 o amp Log is not present in this analysis stored by program version 2 3 4 A report of the prior analysis will now be added to the log Fitting g s data to a 1 species model Fitting from s 3 700 to 6 880 Svedbergs Comment The g s data were derived from 12 scans at 45000 rpm 20 C and a wavelength of 276 nm using a point spacing of 0 02 S The root directory for the scans was C sedfit data antibody The scans used were from the following files 00027 RA1 00028 RA1 00029 _RA1 00030 RA1 00031 RA1 00032 RA1 00033 RA1 00034 _RA1 00035 RA1 00036 RA1 00037 RA1 00038 RA1 The meniscus position is 5 957 cm Molecular masses are based on a sample temperature of 20 C a solvent density of 0 998234 g ml and solute vbar 0 73 ml g The data were fitted using the improved method Results of fit be
264. the best fit value for that parameter is extremely sensitive to the value for another parameter Thus these two parameters cannot really be independently determined and neither is truly well defined 189 DCDT User Manual DCDT User Manual aa Gel Technically the high correlation means that the increase in the sum of squared residuals that arises when a parameter is moved away from its optimum value can be almost completely compensated by simultaneously adjusting the value of the other parameter It is not at all uncommon for the concentration parameters in multi species fits to be highly negatively correlated and this is generally not a cause for concern High cross correlations for the other parameters is however usually a significant concern and probably means that the data contain insufficient information to determine all the parameters uniquely This condition might be relieved for example by reducing the number of species or by using the procedure The likelihood of high negative correlations between species concentrations arises because it is generally true that it will be possible to largely compensate for the effect of decreasing the concentration of one species by adding the same amount to another species i One important way to reduce parameter cross correlation in multi species fits is to reduce the number of fitting parameters by constraining the ratios of molecular masses or sedimentation coefficients between species 5 3
265. the critical chi squared level is based on equation 35 in Johnson amp Straume 1994 reference 12 Section 7 12 This approach is relatively fast to calculate It s major drawback is that it is based on certain statistical models and assumptions including an assumption that all the data points are statistically independent Further there is some disagreement among different sources about exactly how the critical chi squared level should be calculated Bootstrap with replacement In this method data points are selected randomly from the original fitted data set to make a new data set with the same number of points but due to the random selection some data points will be selected more than once This new data set is then fitted those parameters are stored and the process is repeated many times to build up a distribution of parameter values The confidence limits are then calculated from that parameter distribution based on options as described below The up down spinner next to this radio button sets the number of bootstrap rounds default 500 See About the bootstrap method Section 6 8 for more details 151 DCDT User Manual DCDT User Manual a Monte Carlo In the Monte Carlo method random noise is added to a set of theoretical scans that are calculated based on the current best fit parameters Those simulated scans with noise are then used to calculate a new average dc dt and 9 s curves and then a new data set for fitting This
266. the jitter is not perfectly removed See also What constitutes a good fit Section 5 2 3 Often though a more significant guide to whether this is a reasonable fit is based on whether the results make scientific sense For example is the returned monomer mass reasonable based on a known sequence mass or mass spectrometry data Is the sedimentation coefficient reasonable for that mass In this example we find that the ratio of the dimer to monomer sedimentation coefficients is 1 53 That is somewhat higher than the 1 45 ratio for a dimer of spheres but certainly seems to fall within a range that is hydrodynamically reasonable H If you were using constraints on M or s values you may wish to relax the constraints one by one and refit Relaxing the constraint is one way to test whether the hypothesis that is implicit in the constraint is really true If the species are well resolved and none of their concentrations are too small it should be possible for the fit to converge even with one or all the constraints removed particularly when you start with values giving a reasonably good fit If the constrained parameter s do not change much when the constraints are removed and especially if the confidence interval for the unconstrained parameter includes the value implied by the 55 DCDT User Manual DCDT User Manual aa constraint you can be reasonably confident that your hypothesis that these species are oligomers is true With regard to
267. the program for example for conversions from raw s to s 20 w About s 20 w units If conversion to s 20 w units is currently active the sedimentation and diffusion coefficients will be s 20 w and D 20 w values the vbar value will be value at 20 C the solvent density will automatically be set to that of water at 20 C and cannot be altered and the temperature will be set to 20 C and cannot be altered While conversion to s 20 w units is active changing the vbar on this form will not alter the vbar value used elsewhere in the program 3 1 1 4 2 Options dialog The Options item on the Tools menu Section 3 1 1 4 will bring up the Program Options dialog box which uses multiple tabs to list various user preferences for program defaults The options on each tab will be described below with the default setting at program installation noted in square brackets The Cancel button will close the dialog without altering any settings The Ok button will save any new settings which in general apply only to newly opened analysis windows Default sample amp solvent properties tab 67 DCDT User Manual DCDT User Manual aa e Sample partial specific volume 0 73 ml g e Solvent density 0 998234 g ml the default is the density of water at 20 C e Solvent viscosity 1 002 cp the default is the viscosity of water at 20 C Note that these values are in centipoise not poise as used in SEDNTERP and SEDFIT
268. the very wide confidence interval on that parameter In this sample at this concentration there is probably some reversible dimer present as well as an irreversible one and the dynamic interconversion of the reversible dimers might be affecting the calculated masses This too is a good lesson be cautious about interpreting the results unless you are certain your sample is truly behaving as a mixture 56 DCDT User Manual SR DCDT User Manual l If the fit is still unsatisfactory you may wish to try increasing the number of species and fitting again It is certainly not always easy to judge whether systematic residuals really represent another species or are just due to experimental error Based on simulations in principle under favorable circumstances one should be able to resolve three species without knowing anything in advance about the hydrodynamic properties of any of them if all are present at levels over 10 20 Even under favorable circumstances however determining four or five species is probably impossible unless you know and fix during the analysis the properties of one or more of them Also remember that failure to obtain a good fit or implied molecular masses that make no sense may indicate that the species are not truly independent and that they interact and or inter convert J When you are satisfied with the fit you will probably want to determine the true confidence intervals for the parameters print out a r
269. them This may be helpful at the early stages of a complex multi species fit but will probably not be needed for a single species fit To fix a parameter un check its box in the Fit this parameter column and the value will be held at whatever value is entered in the Value text box C A baseline offset parameter is available but should not be used routinely See Should I fit the offset Section 5 2 1 D A program wizard automatically chooses upper and lower limits for each fitting parameter to keep the values bounded within a range that makes physical sense Usually those limits do not need to be manually adjusted but if needed you can do so using the Adjust parameter upper lower limits button Section 3 2 8 5 E When you are done push the Next button to go to the Perform least squares fit page Section 3 2 9 39 DCDT User Manual DCDT User Manual a 4 Fit the data When you enter this page the Graphical fit monitor tab Section 3 2 9 is initially shown displaying an overlay graph of the data and the theoretical curve resulting from the initial guesses with a residual plot below B v 2 b ao a a a Y exp Y fit Gam lies Gaede A Rather than completing the fit automatically using the Do fit button Section 3 2 9 1 let s try stepping through one iteration at a time using the 1 iteration button Section 3 2 9 2 The image below shows the result of the first iteration
270. tial guesses that were computed based on your inputs there Each control or group of controls on the image below is a clickable hotspot that will lead to further information or just use the numbered list below the image This particular example is that for a fit involving 2 species and where the molecular mass of species 2 is being constrained to be a multiple of that of species 1 136 DCDT User Manual SR DCDT User Manual Fit this Parameter Value Parameter Cof1 fringes 0 242 IV Yes s 1 S 3 549 lV Yes M 1 kDa 59 15 V Yes Co 2 fringes 0 028 Y Yes 2 S 5 395 V Yes 6 1 M 2 kDa 113 30 constrained offset fringes S 0 0000 T No Convergence control Convergence factor 10 Parameter precision 0 05 Cancel changes Below are brief descriptions of the controls on this page more details can be found by following the links Parameter value text boxes Section 3 2 8 1 Input or alter the initial guess for parameters being fitted or the desired value for that parameter for parameters being held constant Fit control check boxes Section 3 2 8 2 When checked that parameter will be fitted floated when unchecked that parameter is held constant fixed Constrained parameters Section 3 2 8 3 The values for parameters that are constrained are neither fitted nor fixed Their values cannot be altered from this dialog they are computed based on the values of other parameters In
271. time button 108 109 Select scans to analyze page 102 104 Analysis page 4 Calculate average dc dt Reset zero amp Undo buttons 115 Calculate average dc dt Set range for g s 112 Calculate average dc dt X axis point spacing 111 112 Calculate average dc dt curve page 110 111 Analysis page 5 Averages and peak areas dialog 119 121 Calculate g s distribution Calculate g s distribution Calculate g s distribution Calculate g s distribution Calculate g s distribution Calculate g s distribution s Calculate g s Analysis page 6 202 Broad algorithm option 125 Display options 124 Normalize option 121 Reset zero amp Undo buttons 124 125 Set integration region 118 119 Show integrals over distribution 117 distribution page 115 117 Select fitting model Constrain M or s ratios among species 129 132 Select fitting model Data choice 128 Select fitting model Density and partial specific volumes 133 134 Select fitting model Fit diffusion or mass 128 Select fitting model Improved or classic methods 127 Select fitting model Molecular mass units 132 133 DCDT User Manual SR DCDT User Manual Select fitting model Number of species 127 Select fitting model Weighting of data points 128 129 Select fitting model page 125 127 Analysis page 7 Set initial guesses on graph page 134 136 Analysis page 8 Adjust parameter upper lower limi
272. time derivative analysis is that the rms residual of a fit to 9 s or dc dt data does not directly tell us the rms noise level of the original raw scans Therefore this implementation uses an iterative process First an estimated level of noise is used for N rounds of Monte Carlo simulation The mean rms residual from these Monte Carlo rounds is then compared to that from the original best fit If these two values differ by more than 2 the user is asked whether to re adjust the noise level for the Monte Carlo to try to better match the best fit If so then the noise is scaled up or down another N rounds of Monte Carlo are performed and the noise levels are again compared This iterative process then continues until noise levels agree within 2 or the user is satisfied that some poorer match is sufficient Usually a match within 2 is achieved with one or two iterations For the dc dt data it is fairly trivial to estimate the noise in the raw scans since it is reasonable to assume that the noise in the average dc dt curve is that of the raw data times the square root of 2 from the subtraction of a pair of scans divided by the average time difference between the scan pairs For the 9 s data the noise in the raw scans is estimated by first assuming that the noise in g s varies inversely with sedimentation coefficient the pattern expected if the noise in the raw scans is random using that assumption to calculate the apparent noise in the ave
273. time span is grows longer This revised average dc dt curve is back calculated from the average g s distribution as described in reference 8 Section 7 8 Because the broad algorithm gives smaller error bars on dc dt it also tends to improve the precision of parameters derived by fitting to dc dt using the classic method the parameters will have lower uncertainties This improvement in precision is greatest for sedimentation coefficients and loading concentrations there is little improvement in the precision of D or M The broad algorithm also somewhat reduces the systematic errors in D and M arising from peak broadening i e it improves their accuracy For a more complete discussion of this issue see reference 8 Section 7 8 One complication arising in back calculating dc dt from g s is that the range of sedimentation coefficients used in calculating g s and 9 s is often less than the full range of the dc dt data This complication is handled by using the conventional algorithm to calculate the average dc dt for the regions that were excluded from the 9 s calculations 5 2 About fitting 5 2 1 Should fit the offset The offset parameter allows for some error in defining the zero level of the g s or dc dt data Whether or not to allow a non zero offset is largely a matter of scientific judgement If you have no reason to believe there is any uncertainty in the zero level then you probably should keep the offset at zero
274. tion for each point of the average is calculated The range of sedimentation coefficients used for the average corresponds to the range of 110 DCDT User Manual SR DCDT User Manual values that are present in at least 2 of the individual dc dt curves Controls on this page 3 Set range for g s button Section 3 2 4 2 Push this button to manually set the sedimentation coefficient range for the g s calculation by dragging position markers on the graph 2 Range limit text boxes these text boxes display and allow you to manually alter the sedimentation coefficient range for the g s calculation Convert to s 20 w button Section 3 2 3 3 This button toggles on or off the conversion of the X axis to Soo y units When toggled on it brings up a dialog box for entry of the property values needed to make this conversion a The controls below are shown and available only in Advanced mode Section 1 3 Show error bars button This button toggles on or off the display of error bars for each data point on the graph The error bars are shown as vertical gray line segments extending from each point they are not visible in the graphic above because for these data they are smaller than the data points 9 X axis point spacing options Section 3 2 4 1 These control and display the sedimentation coefficient interval between adjacent data points Reset zero and Undo buttons Section 3 2 4 4 The Reset zero button allows manual
275. to see Qo Those different pages occupy the area that is outlined and washed out within the image below The washed out area shows the first page the Load raw scans page Section 3 2 1 a page used for loading the scans to be analyzed 75 DCDT User Manual DCDT User Manual aa ES Analysis1 00120 00163 IP2 i gt curen gt Preselect data type a el in memor Data type ber 00110 P2 In 2 3 p Load new scan file FA OE heplace sca rt sampile Sa ea antral Sart z sed 1 5 24 m 74 IDa 4 1DD Delete selected a 20 1F2 20 IF2 ata loading 29 P2 29 P2 Loa every N can N 2 e sips 3 1P2 90133 1P2 4 P2 Y 00134 IP2 hi Moving from step to step Movement among the steps pages within one analysis window can be accomplished using either Q A the Next button and Back button Section 3 1 3 1 B the Navigation tree Section 3 1 4 or at the lower right C by right clicking with the mouse on the analysis window in between the buttons and other controls and using the Go to page pop up menu shown below This context menu also has commands for saving this analysis to disk closing this analysis window or closing all analysis windows except this one 76 DCDT User Manual SR DCDT User Manual C GotoPage S Close This Analysis Close All But This Analysis Additional tools along window bottom Along the bottom of each window is a panel containing the M
276. ton is only active after scans have been loaded using the Load New Scan Files button Section 3 2 1 2 3 2 1 4 Load raw scans Add more scans for this sample button Add morescans for this sample This button allows you to load additional scans while retaining sample dependent settings such as the meniscus position The added scans can be from times either earlier or later in the run than those already loaded The scan files are sorted based on the run times at the end of this operation and any duplicate scans are removed This button is only active after scans have been loaded using the Load New Scan Files button Section 3 2 1 2 3 2 1 5 Load raw scans Delete selected scans button Delete selected scans This button will delete the scans whose names are currently highlighted in the Scans currently loaded in memory list box Section 3 2 1 6 If nothing is highlighted a beep is emitted but nothing else happens 90 DCDT User Manual SR DCDT User Manual This command is useful if you need to remove an entire scan where the data is bad for example an interference rogue scan that bears no resemblance to the others 3 2 1 6 Load raw scans Scans currently loaded in memory list box This list box shows the scan file names for all scans currently in memory for this analysis window including ones that might currently be excluded from the analysis Scans loaded in memory 00123 IP3 00124 IP3 00125 IP3 x I
277. trained parameters Section 3 2 8 3 3 2 8 3 Constrained parameters M 2 kDa 12 V constrained Users cannot input values for those parameters whose values are being computed via constraints see Constrain M or s ratios among species Section 3 2 6 6 The current values of those parameters are displayed with a light yellow background to indicate that these are computed quantities The corresponding Fit control check box Section 3 2 8 2 is also inactive since the parameter is automatically being fitted and reads 138 DCDT User Manual SR DCDT User Manual constrained as a reminder of that condition Note that the displayed value will not change if you manually alter the parameter to which this parameter is linked the constrained values are recomputed just prior to each fit iteration 3 2 8 4 Convergence control Convergence control Convergence factor 10 Parameter precision 0 05 The Convergence factor and Parameter precision factor are numerical factors used in determining when a fit has converged The criteria used to detect convergence are either 1 the fit iterations are producing insignificantly small changes in all the fitting parameters 2 the fit iterations are lowering the sum of squared residuals by an amount that is too small to be statistically significant Specifying a lower value for these factors will produce more iterations but those iterations will be less and less likely to produce signif
278. tribution Therefore if you believe your sample contains species with different vbar values for example different polypeptides or both PEGylated and non PEGylated forms of the same protein you should not apply this conversion you must work in raw sedimentation coefficient units e For similar reasons the partial specific volume entered on this conversion form is applied to all species that are fitted The actual scaling factors that will be applied to correct the raw sedimentation and diffusion coefficients to standard conditions are displayed in the panel nearest the bottom of the dialog box What about extrapolation to zero concentration This program does not attempt to correct for concentration dependence of sedimentation coefficients extrapolation to infinite dilution to give Dw Often dc dt analysis is applied to samples at concentrations sufficiently low that concentration dependence can be neglected and if so the corrected results can be considered to be sage values but the program will not explicitly designate them as such 106 DCDT User Manual SR DCDT User Manual How this conversion affects the g s distribution and average dc dt curve Please be aware that conversion to s also alters the magnitudes of the 9 s and g s distributions This arises because these distributions are defined such that the area under the curve gives the concentration Thus if the X axis is expanded or contracted as a consequence
279. ts button 139 141 Constrained parameters 138 139 Convergence control 139 Fit control check boxes 138 Parameter value text boxes 138 Set alter fitting parameters page 136 138 Analysis page 9 Fitting parameters monitor tab 144 146 Increment reduction factor 146 147 Perform fit 1 iteration button 143 Perform fit Cancel button 144 Perform fit Do fit button 143 Perform fit Simulate button 144 Perform fit Status 144 Perform least squares fit page 141 143 Analysis pages Calculate average dc dt curve 110 111 Calculate g s distribution 115 117 Graph fit results 158 164 Load raw scans page 88 89 Perform least squares fit 141 143 Report fit results 147 148 Select fitting model 125 127 Select scans to analyze 102 104 Set initial guesses on graph 134 136 Set meniscus and data region 94 96 Set alter fitting parameters 136 138 Analysis window 75 77 Auto adjust 20 27 Auto adjust button 104 Average dc dt curve 110 111 Averages and peak areas dialog 119 121 Back button 77 78 203 DCDT User Manual Bad data points 101 102 176 177 Bad scans 177 183 Baseline offset 187 Bibliography reference 1 198 reference 10 199 reference 11 199 reference 12 199 reference 13 199 reference 14 199 200 reference 2 198 reference 3 198 reference 4 198 reference 5 198 reference 6 198 reference 7 198 199 reference 8 199 reference 9 199 Bootstrap 155 158 Bootstrap method 150 153 195 1
280. ts corresponding to the experimental scans being analyzed Those theoretical raw scans are then processed to generate either average dc dt data or the g s distribution using the identical algorithms that were applied to the experimental data Because this approach exactly mimics the processing of the experimental data the peak broadening that inevitably occurs as the boundary movement between the first and last scans analyzed grows larger does not cause errors in the fitted properties for each species as it does for the classic method In essence this approach gives accuracy comparable to whole boundary fitting approaches while retaining the advantages of time derivative approaches The theoretical sedimentation boundaries are calculated using an approximate solution to the Lamm equation described by Joachim Behlke in reference 9 Section 7 9 that is also used in his whole boundary analysis program LAMM The actual function used is the first three terms in Eq 28 of that paper the terms which describe the moving boundary This function was shown to give errors for sedimentation coefficient of less than 0 2 and errors for D or Mor less than 0 5 for proteins of 2 kDa or larger and errors of 0 1 or below for all parameters for proteins larger than 10 kDa Function calculation for the classic method When fitting to the g s data the fitting function is a Gaussian A Gaussian function describing the 9 s curve for a single spe
281. ts that were imposed In this example the mass of species 3 is constrained to be 3 times the mass of species 1 species 3 is a trimer of species 1 and species 4 is constrained to be a tetramer of species 1 149 DCDT User Manual DCDT User Manual aa 3 2 10 3 Report fit results View parameter cross correlations button View parameter cross correlations This button will bring up a window showing the cross correlation matrix Parameters with high or very high correlations will be flagged with yellow or red backgrounds The graphic below shows this window Parameter Co 1 s 1 M t Co 2 2 M 2 Co 3 gt correlation with Co 1 EMN 0 9261 0 9207 0 9363 0 7029 0 9182 0 7359 correlation with s 1 0 9261 1 0000 0 8271 0 9362 0 7000 0 9221 0 7400 correlation with M 1 0 9207 0 8271 1 0000 0 8168 0 7351 0 7729 0 5771 correlation with Co 2 0 9363 0 9362 0 8168 1 0000 0 5127 0 9871 0 8690 correlation with 2 0 7029 0 7000 0 7351 0 5127 1 0000 0 4903 0 1893 correlation with M 2 0 9182 0 9221 0 7729 0 9871 0 4903 1 0000 0 8870 correlation with Co 3 0 7359 0 7400 0 5771 0 8690 0 1893 0 8870 1 0000 correlation with s 3 0 5889 0 5910 0 4416 0 7778 0 0532 0 7689 0 7586 correlation with Co 4 0 4176 0 4185 0 3057 0 5842 0 1188 0 5678 0 4973 correlation with 4 0 3216 0 3221 0 2321 0 4667 0 1324 0 4482 0 3345 In thi
282. ture Temperature is the key to obtaining reproducible and accurate sedimentation coefficients The raw sedimentation coefficient will typically increase by 2 4 per degree due primarily to the large temperature dependence of the viscosity of water Since sedimentation coefficients can be measured with a precision of 0 2 or better that means temperature control to 0 1 degree is necessary to avoid compromising the accuracy of the s 20 w values Di You can check the temperature stability for your experiment by displaying a graph of the recorded temperature versus elapsed time covering all the scans currently in memory by clicking the T button within the Data displayed options Section 3 2 2 6 box on the Set meniscus amp data region page Section 3 2 2 this requires being in Advanced mode Section 1 3 How can tell if the temperature was stable during my run A plot of the temperatures recorded during the run can be obtained from the Set meniscus amp data region page Section 3 2 2 the T button available in Advanced mode Section 1 3 What is required for good temperature control 1 Sufficient time to achieve temperature equilibration 2 Vacuum below 5 microns to avoid frictional heating of the rotor 7 Reduce the temperature equilibration time by putting the rotor and monochromator into the chamber P to equilibrate under vacuum while you are cleaning assembling and filling cells However the wrong method of equi
283. ture before starting the run and remember that you are not really reading the rotor temperature until the vacuum drops below 100 microns See How and why to equilibrate the temperature Section 4 2 2 e You can check the temperature stability for your run by clicking the T button within the Data displayed options Section 3 2 2 6 box on the Set meniscus amp data region page Section 3 2 2 to display a graph of the recorded temperature versus elapsed time this requires being in Advanced mode Section 1 3 7 Interference from buffer components Especially for interference scans you may see systematic deviations in the g s curves at low sedimentation coefficient values resulting from redistribution of salts or other low molecular weight components in the cell region near the meniscus e To minimize these problems the sample and reference column heights can be matched exactly if you use a synthetic boundary centerpiece or better still the Walter Stafford meniscus matching centerpiece design available through Spin Analytical http spinanalytical com and follow this procedure 1 Fill the cell as you would a conventional cell 2 Bring the rotor up to about 10K rpm to force sufficient solvent to cross through the capillary to equalize the volumes in both channels you can see this happen on the Raptor fringe display 3 Stop the run remove the rotor and turn it over several times to mix the sample channel This procedure gi
284. ual SR DCDT User Manual 6 Reference information 6 1 Symbols used OD diffusion coefficient D 20 w diffusion coefficient corrected to standard conditions 20 C water but not extrapolated to zero concentration molecular mass m foer oo Ce feas s 20 w sedimentation coefficient corrected to standard conditions 20 C water but not extrapolated ee zero concentration solvent solvent density partial specific volume angular velocity 27 x RPM 60 product of angular velocity squared times elapsed time in the run proportional to total sedimentation force 6 2 About using radial intensity scans In addition to normal absorbance scans this program can also directly load radial intensity scans including so called pseudo absorbance scans where two different samples were loaded in one cell one in the sample channel one in the reference channel When the program detects that you are trying to load intensity scans it brings up the following dialog box for you to select how you want the data to be handled 191 DCDT User Manual DCDT User Manual aa Convert scan data to True absorbance buffer in reference channel C Pseudo absorbance for sample channel Pseudo absorbance for reference channel i Write converted data as scan file in same folder Processing and handling of pseudo absorbance data When you select either of the pseudo absorbance options the program assumes a maxim
285. ult the scans are split into two groups first half and second half and pairs are formed from the corresponding members of each group Thus if there are 16 scans scan 9 is subtracted from scan 1 10 from 2 11 from 3 and 16 from 8 If the number of sections is instead set to 2 then the pairing is 1 5 2 6 3 7 4 8 9 13 10 14 11 15 12 16 This reduces the time span between the two scans in a pair by a factor of 2 and thus reduces the peak broadening by approximately a factor of 4 When the number of scan pairs cannot be evenly divided by the number of sections then the last section will have more scan pairs than the other section s This is done because peak broadening is less of an issue for the later scans Why is using multiple sections sometimes useful Peak broadening can significantly degrade the resolution between species as well as distorting the shapes of the average dc dt and 9 s curves This occurs when the range of boundary movement is quite large With the improved fitting algorithm the fitted hydrodynamic parameters will be accurate no despite the broadening but the broadening can still make it difficult to accurately measure the properties of poorly resolved peaks The peak broadening is also problematic when you are trying to focus your fit on a single peak in order to remove the influence of other minor components aggregates fragments impurities etc 109 DCDT User Manual DCDT User
286. ults Switching between raw and s units will usually force a re calculation of the average dc dt curve and 9 s distribution because the X axis point spacing will change if that is being determined by the default Automatic option see Calculate average dc dt X axis point spacing Section 3 2 4 1 Thus making this switch will invalidate any previous fitting results How this conversion affects reported diffusion coefficients Activating this option will also cause raw diffusion coefficients to be converted to D values via the formula 293 15 Dy D a l Ny w T where T is the sample temperature Kelvin Data required for conversion Conversion to Ss requires knowledge of the solvent density at the experiment temperature the solvent viscosity h at the experiment temperature the solute partial specific volume vbar at the experiment temperature and the solute partial specific volume at 20 C through the relation 1 Vr Prb how where the subscripts refer to the temperature and solvent conditions b buffer w water The density and viscosity of water at 20 C are 0 998234 g ml and 1 002 centipoise respectively 20 w raw 107 DCDT User Manual DCDT User Manual aa If all those data are not available or cannot be estimated e g by SEDNTERP then the conversion cannot be made The program will optionally automatically compute a temperature correction to the partial specific vo
287. um intensity value of 1000 and converts all the data points relative to that value using the formula OD log 1000 1 When the Write converted data as scan file in same folder check box is checked which is true by default as the intensity scan files are read the converted absorbance or pseudo absorbance values will be written out as a normal radial absorbance scan file into the same folder from which the intensity scans are being read For pseudo absorbance data the file name will be preceded by an S or R to indicate whether this data is from the sample or reference channel respectively That is pseudo absorbance scan file 00001 RI1 will generate new files S00001 RA1 or ROO001 RA1 If these converted files are later read by the program the only way it can distinguish that these were pseudo absorbance scans is the S or R as the first letter of the file name Therefore whenever scans with names starting with S or R are read a dialog box will ask you to confirm whether these are in fact pseudo absorbance data e The important functional difference between pseudo absorbance data and true absorbance data is that pseudo absorbance data exhibit systematic shifts somewhat analogous to jitter in interference scans Thus when the program recognizes pseudo absorbance data it turns on jitter correction Section 3 2 2 4 by default 6 3 About this g s implementation The calculation algorithms used here differ slightly from those us
288. umed to be 3 times the anhydrous sphere value corresponding to the lowest sedimentation coefficient the maximum M value is set as 10X that for an anhydrous sphere with the maximum sedimentation coefficient How to alter the limits The user is presented with the dialog box shown above containing a list of the current upper and lower bounds These can be manually altered as desired Note that the alterations will apply only to the current analysis window Section 3 1 3 they do not change how the program computes default values The OK button will accept any alterations that have been made and exit the form the Cancel button will leave the values unchanged What happens if a parameter goes outside the limits During each round of fitting the new parameters are tested against the limits If any parameters will go out of bounds the increments for all parameters are reduced proportionally using the increment reduction factor Section 3 2 9 6 1 until none will go out of bounds That procedure moves all parameters in the direction and in the relative proportions that should improve the quality of the fit However this bounding procedure may prevent the good parameters the ones not exceeding the bounds from ever reaching the values needed for an optimum fit i e often when any parameter is going out of bounds the fit will never converge Therefore it is often useful to warn the user that parameters are trying to go out of bounds
289. unds Parameter Lowerbound Upper bound con Mm S000 s 1 S 2 096 6600 M 1 kDa 453 75982 Coz 0 000 f 50 000 s 2 S 20 6600 M 2 kDa 4 53 759 82 Coa 0 0000 f 50 000 s 3 S 209 f 6600 M 3 kDa 4 53 75982 Co s 0 0 000 If 5o 000 s 4 S 2 096 6600 M 4 kDa 453 75982 Co s Qf 0 000 f 50 000 s 5 S 2096 f 6600 M 5 KDa 4 53 f 75982 offset S 2 0000 10 0000 x _Gancet_ What is the purpose of these limits The intent of bounding the parameters is to prevent the fitter from straying into areas where the parameters would make no physical sense Generally the fitter never needs to impose bounding and the user will not need to alter the default values for the bounds but in certain situations the program defaults may be inappropriate and could potentially prevent convergence How are the default limits calculated 1 Default limits for concentrations are calculated based on the data type absorbance interference or fluorescence 2 Default limits for sedimentation coefficients are set based on the range of values within the data 0 8 x the lowest value 1 5 x the highest value 3 Default diffusion coefficient or molecular weight limits are then calculated based on those sedimentation 140 DCDT User Manual SR DCDT User Manual coefficient limits and estimates of values for anhydrous spheres o For example the maximum diffusion coefficient is ass
290. ute the confidence limits with different choices You can always change your selection and re compute with a different confidence level or different computation method simply by pushing the Compute confidence button again 3 2 10 5 Report fit results Create reports group box Create Reports Fj Include Graph Send report to clipboard Either of the two buttons within this group box will generate a complete nicely formatted report defining what 153 DCDT User Manual DCDT User Manual a data were fitted how they were fitted the best fit parameters fit statistics etc That report is then either printed or copied to the Clipboard in rich text format so that it can be pasted into a word processor or electronic lab notebook This report is intended to document every aspect of your analysis All user choices and program defaults that affect the calculations of the data or the fitting procedure are listed in the report The Include Graph checkbox governs whether a summary graph showing an overlay of the data and the fit with a residual plot underneath as shown on the Graphical fit monitor tab of the Perform least squares fit page Section 3 2 9 will be included at the end of the report As an aid to judging the quality of the fit the program will translate the rms residual from the fit into an equivalent noise level for the original raw scans and include that value in the report e This calculation is only accurate to 20 For d
291. ved the fact that curve shapes are more accurate for dc dt fitting can lead to significantly enhanced ability to resolve the species and get accurate values for their properties It cannot be over emphasized that the best way to determine what these types of analyses can and cannot do and to assess their potential accuracy is to run Claverie simulations Section 3 2 1 10 of your experiments including noise and fit the simulated data 5 2 3 What constitutes a good fit Judging good and bad fits is somewhat of an art and ultimately relies on the accumulated experience of the experimenter However in general a good fit is one where e the residuals are small and randomly distributed they are not systematically above or below zero over substantial portions of the radial range e the rms residual is consistent with the intrinsic optical noise of the instrument e the returned values make scientific sense based on other information you have about this sample With regard to the last point one important criterion is whether the apparent molecular weight implied by the s D ratio is consistent with the sequence weight values from mass spectrometry or sedimentation equilibrium results For truly homogeneous non associating samples the apparent M from fitting using the improved method Section 3 2 6 2 should probably be within 2 5 of the correct value It is important to remember that the presence of extra species or self associ
292. ver because the temperature may drift during the run you may wish to adjust that value manually The program internally converts the temperatures from Celsius to Kelvin scales o Please note that changing the temperature on this page overrides the temperature recorded with the scans and also alters the value used elsewhere in the program e g for conversions from raw s to s 20 w 3 If you select true molecular mass go to the Density and partial specific volumes group Section 3 2 6 8 on the lower right side of the page and fill in the values for solvent density and the partial specific volume s To obtain accurate true molecular mass you must know the solvent density and solute partial specific volume accurately Note that changing the molecular mass units on this page will automatically alter the results displayed on the Report fit results page Section 3 2 10 if a fit has been completed 3 2 6 8 Select fitting model Density and partial specific volumes 133 DCDT User Manual DCDT User Manual aa Density and partial specific volumes solvent density g ml 1 001360 M Use the same partial specific volume for all species partial specific volume for all species ml g 0 7213 partial specific volume of species 2 ml g partial specific volume of species 3 ml g partial specific volume of species 4 ml g partial specific volume of species 5 ml g This group of controls is used to se
293. ves a slight dilution of the sample 4 Reload the rotor and re equilibrate the temperature When you come up to running speed the reference and sample meniscus will be exactly matched 4 2 How to optimize your experiments 4 2 1 How to optimize data acquisition for g s analysis There are a number of things that can be done to optimize the experimental setup to improve the results of time derivative analysis This section will first discuss what factors limit the signal noise and then describe specific things for optimizing either absorbance or interference data 167 DCDT User Manual DCDT User Manual aa What factors govern the signal noise of a dc dt analysis A common misconception is that using scans taken very closely together in time will limit the signal noise because the differences between successive scans are smaller In fact the faster scans are acquired the better the signal noise ratio It is important to remember that the algorithm does not subtract successive scans Rather the group of scans is broken into two halves and the corresponding members of each half are subtracted Thus if there are 10 scans scan 6 is subtracted from scan 1 scan 7 from scan 2 etc The resulting concentration differences Ac are divided by the time differences between the scan pairs At to give Ac At which will closely approximate the true derivative dc dt provided there is not excessive boundary movement between the two scans being
294. vious Claverie data Can Decide if you are simulating absorbance interference or fluorescence data and check the appropriate radio button at the lower right on the form o This choice affects the units of concentration the default number of radial points the allowed range for the loading concentration and added noise and the file names for the generated data sets For absorbance mode the default 601 data points corresponds to approximately the number of data points actually collected when using the default 0 003 cm point spacing for velocity scans For interference and fluorescence modes the default 1641 and 601 data points respectively correspond approximately to the radial spacing of of those optical systems Decide if you are simulating conventional cells or a synthetic boundary cell For a conventional cell the inner radius and the meniscus position are set to be equal for a synthetic boundary cell the meniscus is at a radius greater than that of the cell inner radius o Making the meniscus and inner radius equal for conventional cells essentially prevents diffusion of solutes to the region above the meniscus In a synthetic boundary cell solutes can diffuse to radii inside the initial boundary position Enter the desired concentration sedimentation coefficient and diffusion coefficient for the species being simulated o If you are trying to simulate a particular molecular mass use the Mass diffusion calculator C Users Publi
295. want to have this limit roughly equal to the highest mass present in your sample See Peak broadening limit Section 3 2 3 2 for more details 5 The Convert to s 20 w button Section 3 2 3 3 allows you to toggle the sedimentation coefficient scale between raw versus standardized s 20 w values J O The Use true mean time button Section 3 2 3 4 controls the assignment of the run time to each scan pair when calculating the dc dtcurves When this button is on down the algorithms use the true mean of the times for those two scans When this button is off up the default the algorithms use the harmonic mean as in the original Stafford implementation The true mean can give better results for multi species fits using the new improved method when the peaks are severely broadened but should not be used in other circumstances See Use true mean time button Section 3 2 3 4 for further details J O The of sections up down spinner Section 3 2 3 5 can be used to divide the scan pairing scheme into multiple sections for the purpose of reducing peak broadening at a penalty of lower signal noise ratio for the average dc dt curve and the g s distribution This option should only be used if the intent is to fit the data not if the g s distribution itself or averages over the distribution is the intended output of the analysis 103 DCDT User Manual DCDT User Manual aa How are scans paired when the dc dt curves are calculated
296. whether your sample is a monomer dimer trimer etc 1 2 How to use this Help file Within certain topics you will see expandable sections like the one below that when clicked expand to provide more details about that subject Getting context sensitive Help from within the program As is true for many Windows programs pushing the F1 key at any point in the program will bring up a Help screen relevant to that context page within the analysis window dialog box or control Remember too that virtually every button or other control will display brief tool tip help if you hover over that control with your mouse For pages with multiple expandable links you may also see a Show AIl Hide All link like the one at the right below that expands or contracts all such material within that page Special text sections 2 DCDT User Manual SR DCDT User Manual Short blocks of text that is specific for the Advanced mode Section 1 3 will be indicated by a colored block with the Einstein symbol as shown below This is text concerning a feature that is only available in Advanced mode Long blocks of text or whole topics specific to Advanced mode may be indicated simply by the Einstein icon Tips about easier or better ways to accomplish tasks will be indicated by a colored block with the idea symbol as shown below k d To best way to do X is to use the Y button and then enter Cautions about potential mistakes or pitfalls will
297. wn below Generating this display can take a fair amount of time so be patient 180 DCDT User Manual SR DCDT User Manual F Analysis1 iscus setting and data adjustments Meniscus at 5 904 cm good data begin 0 01 cm from meniscus good data end at radius 7 10 cm ee eee ee A Fringes 57 58 59 6 0 COCEOIFS COOSOIPS CO1COIFS m CO1101F3 7 0O1200F3 O 0ces1 Fs x 000S1 IFS F CO101F3 001111P3 001211P3 6 1 a 0122P3 62 63 64 65 66 67 68 69 70 71 4 0008S IFS O 000931P3 00103 IF3 Y CO11SIFS 001231IP3 Radius cm 7 O00s4uIP3 COOS4IFS 0O1041F3 CO114 IF3 0O124F3 COOSSIFS 4 000951P3 O 00105 1r3 X 001151P3 001251P3 m 000551P3 7 000S65uP3 gt 001061IP3 001161P3 001261FP3 000871P3 000971P3 001071P3 O 001171P3 001271P3 COOCEEIFS a 000961P3 T 001081P3 gt 001181P3 001281P3 72 7 3 F COossrs CCOSS IPS 001091P3 4 001191P3 O 001291P3 7 4 _ lt lt Less _ Back _Next Now if you click anywhere along the right portion of the rogue scan the easily identified region to the right of the fringe jump then as shown below the window above the graph identifies that the rogue scan is number 92 181 DCDT User Manual DCDT User Manual a ra Analysis1 Mm x iscus setting and data adjustments Meniscus at 5 904 cm good data begin 0 01 cm from meniscus good data end at radius 7 10 cm Saros
298. xist in the model will be automatically cleared You will not need to bring up this dialog box to clear them 3 2 6 7 Select fitting model Molecular mass units 132 DCDT User Manual SR DCDT User Manual m Molecular mass units s D Svedberg per Fick buoyant molecular mass kDa temperature must be known Temperature C 20 2 molecular mass kDa temperature solvent density and solute partial specific volume must be known This group of controls allows you to choose among three systems of units for molecular mass This choice is only relevant if you have chosen to use molecular mass the s D ratio as the second hydrodynamic parameter for the fit within the Fit diffusion or mass Section 3 2 6 4 group box Generally you will want to use units of true molecular mass and this is the initial program default However in some cases you may not know the values for both p and V and thus cannot calculate the true mass In other cases you may wish to work directly with the buoyant molecular mass or in units of the s D ratio Svedbergs per Fick To set the molecular mass units 1 Use the radio buttons to select among the three choices of units 2 To use units of buoyant molecular mass or true mass the sample temperature must be known o The program automatically fills in the sample temperature based on the temperature recorded in the header of the last scan used in calculating the dc dt data Howe
299. y the plotting style using the radio buttons within the Graph style group For other graph customization options such as adding a title see How to customize graphs Section 4 3 2 When you are satisfied with the graph there are buttons provided for printing it and for exporting the image or the actual data table to the Windows Clipboard or a file A heterogeneous mixed association example These overlay graphs can also be quite useful in studying heterogeneous associations The example below shows data for two different proteins when run by themselves and then data when the they are mixed to give the equivalent concentration of each protein Obviously the new peak near 7 S represents a protein protein complex and most of both proteins has been incorporated into complexes 30 DCDT User Manual SR DCDT User Manual Hist Overlay distributions ad a N a 0 08 g s AU Svedberg 8 o s Svedbergs mixture proteins 1 and 2 O protein1 protein 2 Print _ Export Graph style Le Points C Lines Edit legend text om proteins 1 and 2 DE Points and lines 2 4 Tutorial Single species fit This tutorial assumes you have already calculated the g s distribution see the procedure under Tutorial calculating the g s distribution Section 2 1 Here we will fit g s data for a monoclonal antibody First here is the easy way in Standard mode 1 Navigate to the Select fitting mo
300. y wish to constrain the ratios of the molecular masses and or sedimentation coefficients for the different species to values appropriate for oligomers The use of constraints and how to set them is discussed in detail under Constrain M or s ratios among species Section 3 2 6 6 Using these constraints reduces the number of fitting parameters and therefore makes the fit converge much more easily and rapidly and is particularly advantageous when the fraction of a particular species is low The use of constraints will also give lower uncertainties for the remaining fitting parameters However the danger of the constraints is that your assumptions may be wrong and if they are wrong the results will be nonsense even if it appears to be a good fit It is obvious that the 9 s distribution shown above shows a shoulder at roughly 5 0 5 5 S indicative of a second species but this species is in fairly low abundance and is poorly resolved from the main peak at 3 5 S The ratio of the sedimentation coefficient for this shoulder relative to the main peak is around the 1 4 1 5 range that is typical for dimers Therefore it makes sense to try constraining this second species to have the mass of a dimer twice the monomer mass whatever that might be In other words we will test the hypothesis that these data can be well fitted as a mixture of monomer and dimer To implement this constraint we push the Set constraint values Section 3 2 6 6 button which bri
301. ysis As Section 3 1 1 1 3 then that path and filename are displayed in the Title Bar As in the example shown above when the full path and file name are too long to display the path is abbreviated After scans have been loaded and selected for analysis the names of the first and last scan that are being used are also shown in that window s Title Bar IP3 scans 120 through 164 in the example pictured above 3 1 3 1 Next and Back buttons The I If the Next button is currently disabled this indicates the program is waiting for you to complete some operation on this page that is required before moving to the next step loading scans setting the meniscus position completing a fit etc The Back button will always be disabled on the first page Load raw scans page Section 3 2 1 77 DCDT User Manual DCDT User Manual a 3 1 3 2 More gt gt and lt lt Less buttons The and buttons toggle between Standard and Advanced modes Section 1 3 The button text changes to indicate whether More options will be shown when the button is clicked switch to Advanced mode or Less options will be shown switch to Standard mode 3 1 3 3 Log page As you perform an analysis DCDT creates a Log to record exactly what you have done and when it was done This Log can be displayed at any time using the button along the bottom of the Analysis window Section 3 1 3 x t Analysis1 00022 00033 RA1 GCIEJ 4 26 2012 20

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