BIOEQS-Graphic User Interface USER MANUAL - Abcis
Contents
1. button2. have three options to move the species box around 1 Double click the box to drag it around any where you want Be careful not to drag it out of the form space It can happen Once the species box is no longer visible you will have to push the Clear All and start anew 2 Doing a single click will select the species box Note that at the time there is no apparent change to the look of the species box you selected You can move a species box up or down using the PAGE UP and PAGE DOWN buttons on your keyboard after selecting the species You can also press ALT any arrow key LEFT UP DOWN and RIGHT to move the species around Note that if you have any elements inside the box they will move with the box only if you use the single click option 3 The last option is to single click the species box and then press ENTER this will bring up a text box where you can type the exact energy you want After typing the exact DeltaG you want press ENTER The box will move to the exact energy you want Notice that the horizontal position is preserved To delete a species simply select the species and hit delete key To create an element Click on the Element box to create an element you ll notice that there are three different elements each with a different color and shape You should always start using the orange circle since it corresponds to element on the tabbed forms In the same manner the
3. some Do not hesitate to e mail catherine royer cbs cnrs fr We will try to fix them Model Making Tool HOW TO Version 1 00 Date 5 21 07 When you first load the model making tool window you should have a clean slate On the left hand side you can choose the range of DeltaG you want to use You need to simply type in the minimum and the maximum value on the appropriate boxes because the area 1s being scaled to these values Below the DeltaG max box is the of experiments you plan to analyze or simulate Type in the number of experiments you plan to analyze or simulate You will be able to change this number on the main form if you need to This option will fill out the main form of experiments also Basic interface principles As you get used to the interface you will be able to move around and do things differently I recommend creating the species that you need first then filling them with the appropriate elements corresponding to your experiment The basic interface uses single click mostly One exception is a double click done on the species box to drag the species box around Note that the value of the energy displayed on the species box as it is dragged corresponds to that of the top part of the box Fig 1 The startup form for the model making tool Model Making Tool To create a Species Click on the button Create a Species A box will appear right below this button You
4. 00 Below are examples of the two data files to be analyzed The first column corresponds to the different concentrations of the element being titrated into the solution in this case element 1 protein The second column corresponds to the value observed experimentally at that concentration and the third column is the estimated uncertainty on that value Note that the formatting of the numbers of the two data files is not exactly the same The only things tat are important are 1 that the first column be expressed in the units specified on the first page micromolar in our case such that the titration was done between 0 1 pM and 1 micromolar 2 All of the numbers must be real i e have a decimal point cgfbp2 dat 0 00010 750 2 0 0 00030 765 2 0 0 00050 765 2 0 0 00075 77 0 2 0 0 001 71 5 20 0 0015 71 5 20 0 002 780 2 0 0 003 790 2 0 0 004 795 2 0 0 006 805 2 0 0 007 82 0 2 0 0 0085 83 5 2 0 0 012 845 2 0 0 018 86 0 2 0 0 027 88 0 2 0 0 036 885 2 0 0 054 90 0 2 0 0 072 93 5 2 0 0 1 94 0 2 0 0 14 945 2 0 0 18 93 5 2 0 0 3 95 0 2 0 0 5 95 5 2 0 0 8 97 5 2 0 1 00 96 5 2 0 cgnfs2 dat 0 100000E 03 75 7046 2 000000 0 300000E 03 75 9593 2 000000 0 500000E 03 76 2580 2 000000 0 750000E 03 76 6791 2 000000 0 100000E 02 77 1407 2 000000 0 150000E 02 78 1472 2 000000 0 200000E 02 79 2214 2 000000 0 300000E 02 81 4406 2 000000 0 420000E 02 84 0421 2 000000 0 600000E 02 87 5135 2 000000 0 700000E 02 89 1448 2 000
5. 000 0 850000E 02 91 1752 2 000000 0 121000E 01 94 3463 2 000000 0 180000E 01 96 6444 2 000000 0 270000E 01 97 8643 2 000000 0 360000E 01 98 3334 2 000000 0 540000E 01 98 7077 2 000000 0 720000E 01 98 8609 2 000000 0 100000 98 9730 2 000000 0 140000 99 0462 2 000000 0 180000 99 0837 2 000000 0 300000 99 1323 2 000000 0 500000 99 1594 2 000000 0 800000 99 1740 2 000000 1 00000 99 1788 2 000000 One should save the answerfiles on the previous pages into any folder on the computer BIOEQS_GUI makes another temporary copy that it uses to run the program 4 Results By clicking the View and copy Results button in the Experimental Parameters page the program will copy the results of the bioeqsg dll to the same location as the answerfiles and will take you to the Results page All of the results parameters file as well as a file with data fit and residuals are saved into ascii output files that are stored in the same location as you put your answerfiles These files have the same name as the answerfile but instead of an extension called ans they have extensions of res and tmp respectively The returned values for all experiments are shown on this page including the global chi square chi squares for all the experiments and pertinent information The experimental fits are graphed by pressing the Graph Experimental Fits button Covariance Click on the covariance button in the Results tab to access a page w
6. 15 8 2000000E 01 model ans Xval 16 16 9 7000000E 01 model ans Xval fix 17 17 9 3000000E 01 model ans Xval 18 18 9 0000000E 01 model ans Xval fix 19 19 0 0000000E 00 model ans Xval fix 20 20 0 0000000E 00 model ans Xval 21 21 7 5000000E 01 Data type TIMEGS Filename C data cgnfs2 dat parameter type M L D logic initial value model ans deltaG 22 22 model ans deltaG 23 23 model ans deltaG 24 24 model ans deltaG 25 25 model ans deltaG 26 26 model ans deltaG 27 27 model ans deltaG 28 28 model ans deltaG 29 29 model ans deltaG 30 30 model ans Xval 31 31 model ans Xval 32 32 model ans Xval 33 33 model ans Xval 34 34 model ans Xval 35 35 model ans Xval 36 36 model ans Xval 37 37 model ans Xval 38 38 model ans Xval 39 39 model ans Xval 40 40 model ans Xval 41 41 model ans Xval 42 42 grstate parameters follow obs12 00 00 prot 0000 000000 2000 000000 0000 001000 0000 000000 0000 000000 291 00 obs11 00 00 prot 0000 000000 0050 000000 0000 001000 0000 000000 0000 000000 291 00 Above is a copy of the ascii answerfile generated by the graphic user interface of BIOEQS for the model specified above with two titrations at two different concentrations of the second element Below is a copy of the answerfile corresponding to the stoichiometric model that is embedded in the global answerfile and called model ans model ans file example Number of Free Energy Equations 9 Number of Possible Species 12 N
7. BIOEQS Graphical User Interface USER MANUAL ver 1 2 Catherine Royer Joseph Beechem and Tilman Rosales A see sasnasnnsacasoscscaseeancaginese eooc or iS Erose SSos Esse NT 2 Graphical User Interface Description oooooooosss 4 EM aida 4 ZIDOS CVC date ena aaa 4 JIExperniment Ypes tecla o Experimental ParameterS A A A A A A AE 5 Answertile example cerren as 6 AS e E E E A EE EE OE Eis 7 corpo dais iia 8 A voueedaie vageanaeenyheniynd sued vide R a wa aied aaa ashes Oo A iai 9 E E A A ae ee le A io 10 CONAM A E AEE 10 Se SMULAN ONS A O O E 10 General WATTHINOS caiakssinsccugnatorconvedavessucacvoaseacouesteedatesenusdousuacsuenadiorndenpedassesedsecsensescaueseuncsvonns 11 PEODLENES Aia gtr AR ees wig tase el A AAA AE A AAA E a caen Il Model Making Tool HOW TO did 11 Basic INSET ACE Principles ri AO Dm aceras 11 To create a SPECIES a 12 To reae a A a n chin E a R 13 Introduction Bioeqs is a data fitting software package designed for analysis of data for biomolecular interactions and conformational changes The fitting algorithm is a Marquardt Levenberg least squares algorithm typical of many available data analysis routines The uniqueness of BIOEQS lies in the solver used to calculate the observable values from the input parameters While most available programs use closed form analytical expressions for the binding isotherms BIOEQS uses a numerical algorithm based on constrained optimization with Lagrange multipliers t
8. blue square corresponds to element 2 and the triangle to element 3 Creating the first element also creates an image on the form to remind you that you have an element of that type somewhere on the form Again be careful not to accidentally drag an icon outside the boundaries A check to keep them inbounds is on the works To pick up an element do a single click on the element icon This attaches the icon to the mouse movement Do a single click anywhere on the form to drop the element icon For the program to count the element it has to be inside a species box The program simply checks what s inside the boundaries of the species box top bottom and sides This is very important the element icons need to be inside the species box To delete an element simply select the species and press delete key To create an isomer At this time the number of isomers is done by selecting the species box with one single click and pressing the number key on the keyboard 1 through 4 Only up to four isomers allowed per element This is a GUI limitation The number of isomers for that species is displayed on the box itself Fig 2 NOTE A species box changes color when double clicked but not when single clicked The box becomes white after hitting ENTER so that you can type in the exact energy you want Remember to press ENTER when done Model Making Tool If you get into trouble you can always delete all by pressing the Clear All
9. d 1 and element 3 In these cases the first column in the data file must correspond to the different concentration points of the titration The other three types of experiments are thermodynamic in nature They are Folding in which the first column of the data file corresponds to the different denaturant concentration points in Molar units Pressure in which the firs column of the data file corresponds to the pressure points in bar and Temperature in which the firs column of the data file corresponds to the temperature points in Kelvin After that one must specify the constant conditions of the experiment i e the concentraiton of the elements that do not change during the experiment and the thermodynamic conditions that do not change denaturant concentration O by default pressure atmospheric by default and temperature in Kelvin Finally the data filename for each experiment must be entered Experimental Parameters The third page Experimental Parameters is where the fitting parameter initial guesses are entered Depending upon the type of experiment there are different parameters First there are the thermodynamic parameters For titrations there will be one AG of formation per complex For denaturant folding experiments there will be a AG and an m value of formation per complex for pressure experiments there will be a AG and a AV of formation per complex and for temperature experiments there will be a AG and a AH of formation per co
10. d obs12 correspond to observable values that are the result of the weighted average of the values for all of the species containing element 1 usually taken to be protein for obspr element2 usually taken to be the first ligand for obs11 and element 3 usually taken to be the second ligand for obsl2 The observable value at each data point in the experiment is the weighted average for the fractional population of each of the species at that data point given the free energy parameters and concentrations and for the relative quantum yield of each species Q 1 for all species by default The second type of observable xprot xfrl and xfrl2 correspond to the fractional population of a particular species with respect to total element l protein xprot total element 2 ligand 1 xfrl1 or total element 3 ligand 2 One must of course have a physical observable that reports specifically on this species If one chooses xfrll for example then after that one must specify which of the ligand 1 element2 containing species is being designated xfrll 03 for example would correspond to twice liganded dimer M2L2 in our example Then one must also specify which site isomer is being reported on by the observable In most cases there will only be one so one puts in xfrl1 03 01 3 Experiment type BIOEQS supports 6 experiment types the first three 1 element 2 element and 3 element correspond to titrations by element 1 protein element 2 ligan
11. f points desired For titrations the step size is 0 1 log units The step size for the other experiment types is a linear increment given by End Value Start Value No of points Pressing create files will create the experiment file at the selected location with a dat extension The program requires the same input as the regular analysis When all the variables are filled in i e Master Table press Calculate to create the simulations files Pressing the View and copy results button takes you to the Results page where you can also graph the simulated experiments by pressing the Graph experimental fits button General warnings The regional settings on the computer need to have the decimal place indicator set as a period and not a comma THIS IS VERY IMPORTANT Every time the program runs it will attempt to change the regional settings When the program is closed by pressing the EXIT button it will return the settings to their original values If the program crashes or is terminated these settings will not be restored You can always change them back by going to the Control Panel gt Regional and Language Settings and selecting Customize Here the decimal descriptor and the digit grouping descriptor can be changed It is recommended that any previous versions of this program be removed from the user s computer before installing a newer version of the program Problems Surely there will be
12. here you are able to perform rigorous confidence limit testing of the recovered parameters You can do up to 20 different parameters at a time The number of points per fitting parameter has been set to 100 The analysis requires as input the fitting parameter selected from a drop down list the starting and ending chi square values and the interval desired After pressing the Analyze button the returned values are displayed in the output table The amount required for the calculation will vary depending on the total number of variables A graph of the returned chi square vs fitting parameter is also displayed A blue and a green line may also be plotted showing the 0 67 and the 0 9772 confidence level for the chosen experiment If the 0 67 or the 0 9772 confidence levels are outside the graphing range an OUT OF BOUNDS will be displayed color coded to the appropriate confidence level 5 Simulations The simulation options can be accessed by checking the simulation box on the model page Simulations are performed in a similar manner as the normal analysis You can add noise to the simulations by giving a seed for the noise generator and the percent noise No noise is added if the seed is set to zero The files required for the simulation can be created in the experiment page or the users can simply use their own files The user can input the filename for the experiment the starting and ending value when needed and the number o
13. mplex We note that the temperature experiments have not been debugged for the user interface version of BIOEQS so use at your own risk Note that in the example below the model has been proposed such that protein binding to DNA is cooperative AG7 gt than 2 x AG5 but DNA binding disfavors ligation AG6 AGS lt AG1 This is carried through for all of the free energies of complex formation The second type of fitting parameter corresponds to those associated with the observables If the observable is of the type weighted average of one of the elements obspr obsl1 or obsl12 then each species obsl2 in our example must be given a value Xval that corresponds to the value that would be observed if 100 of the element is in that species For species that do not contain the element the value should be fixed at zero In our example the observable is a the fluorescence anisotropy of a dye covalently attached to the DNA element 3 ligand 2 so the observable is obsl2 All species that do not contain DNA M ML Mo M2L M2L2 should have their Xvals set to O and fixed In our example we set the free DNA to a number close to what is observed at the beginning of the titration 75 milli anisotropy units and that for the dimer bound DNA to a value close to that observed at the end of the titration Unless there are obvious bumps in the curve it is best to fix the intermediates somewhere in between In the example below it turned out tha
14. n the Figure below The free energies for the transitions between species for example that of binding of a second ligand to the once liganded dimer can be calculated by subtracting AG from AG3 The species are listed in matrix form Species Species Stoichiometry Stoichiometry Stoichiometry symbol Element 1 Element 2 Element 3 ML MeL M2L gt M2 MD MLD M2D SININ BR Q PN MLD 9 M2L2D 10 M 11 L Oo jOo l N N WN N R e O e jo uw O O jO N ReRi iO O er ei r iO O O O 12 D The matrix is always terminated by an identity matrix with number of rows and columns equal to the number of elements The maximum number of elements dimensioned for BIOEQS is three The maximum number of species is 100 There is a final column in the model file in the stoichiometric matrix that specifies the number of site isomers for each species In the present example one could imagine that the DNA half sites for each monomer may not be equivalent in sequence Therefore the MD species and the MLD species may populate both of the site isomers corresponding to the two half sites Their population will be weighted according to the free energies of formation of the two isomers Unless one has an observable that actually is related to the differential population of each of the half sites DNA footprinting results for example it is best to consider that
15. o solve the set of free energy equations for a given model in terms of the species concentration vector given the mass balance constraints for the elements M L D AG ML ra AGs AG 2 AGe AGo MD AGs AGo MeL MLD M2D AG3 Male MLD MLD Any system can be described in terms of the elements that make it up For example for a protein that binds a small ligand and also DNA the three fundamental elements are monomeric protein M ligand L and DNA D One can combine these three elements to form a variety of different complexes of varying stoichiometries For example one can imagine monomer bound DNA MD DNA bound by liganded monomer MLD dimer bound DNA M2D monomer bound by ligand ML unliganded dimer M2 and once M2L and twice bound by ligand M2L2 DNA bound by dimer with only one site liganded M2LD and the full complex of fully liganded dimmer bound to DNA M gt L2D This makes a total of nine complexes each having a corresponding free energy of formation from the three free elements The total number of species is 12 corresponding to the 9 complexes plus the 3 free elements Such a system is necessarily defined by only 9 free energy relations AG species elements These 9 free energies can be defined in many ways The BIOEQS convention is to define them always in terms of free energies of formation for example the free energy of formation of the twice liganded dimer from free ligand and free monomer is AG3 i
16. t all of the anisotropy values of the DNA bound by liganded protein were a bit lower than in absence of ligand and so this is also apparent in the Xval values specified for these species If the observable corresponds to the fractional population of a particular species then only two parameters are given the value observed when the species fractional population is 0 and that observed when it is 100 These numbers are called Sval starting value and Eval ending value In all cases the observable can be any type of numerical value 1 e radioactive counts on a gel fluorescence anisotropy ellipticity intensity of any spectroscopic signal Only raw intensity values need not be corrected for quantum yield differences if they exist Answerfile example Total number of experiments 2 Data type TIMEGS Filename C data cgfbp2 dat parameter type M L D logic initial value model ans deltaG 01 01 5 0000000E 00 model ans deltaG 02 02 1 2000000E 01 model ans deltaG 03 03 1 7000000E 01 model ans deltaG 04 04 7 0000000E 00 model ans deltaG 05 05 9 0000000E 00 model ans deltaG 06 06 1 3000000E 01 model ans deltaG 07 07 2 0000000E 01 model ans deltaG 08 08 2 4000000E 01 model ans deltaG 09 09 2 7000000E 01 model ans Xval fix 10 10 0 0000000E 00 model ans Xval fix 11 11 0 0000000E 00 model ans Xval fix 12 12 0 0000000E 00 model ans Xval fix 13 13 0 0000000E 00 model ans Xval fix 14 14 8 5000000E 01 model ans Xval fix 15
17. there is only one site isomer One also specifies on this page the concentration scaler for the elements 1 2 and 3 Denaturant concentration is always in molar units Graphical User Interface Description 1 Model The Graphical User Interface for BIOEQS BIOEQS_GUD consists of 4 pages The first page is called Model and allows the user to input information about the model Number of elements Number of species Definition of element names M for monomer L for ligand D for DNA for example The stoichiometry of each species The number of site isomers for each species The relative quantum yield of each species the highest being 1 HALA NS If an answerfile already exists for the model to be used it can be loaded rather than recreated One must also specify the solver This should be EQS for all systems except for monomer folding experiments by denaturant temperature or pressure The second page called Experiment allows the user to specify how the experiment was done The user must specify 1 What is the observable for the experiment 2 What kind of experiment is it 3 The concentration of the elements not being titrated 4 The denaturant concentration if applicable otherwise taken to be zero 5 The pressure if applicable otherwise taken to be atmospheric 6 The temperature in Kelvin 7 The data file name 2 Observable There are 6 possible types of observables allowed in BIOEQS The first three obspr obsl1 an
18. umber of Elements 3 Concentration Scaler 3 mM 6 uM 9 nM 6 Solver EQS KOKK K K K K K K K K K K K K K K K K K K K K K K K K K K K K K K K K K K K K K K K K K K K K K K K K K K K K K K K K K K K K K K K K K K K K K K K K 01 01 00 01 00 00 00 01 00 02 02 00 01 00 00 00 01 00 03 02 00 02 00 00 00 01 00 04 02 00 00 00 00 00 01 00 This is the stoichiometric matrix with the 05 01 00 00 00 01 00 01 00 number of site isomers in the last column 06 01 00 01 00 01 00 01 00 07 02 00 00 00 01 00 01 00 08 02 00 01 00 01 00 01 00 09 02 00 02 00 01 00 01 00 10 01 00 00 00 00 00 01 00 Note here the identity matrix for the elements 11 00 00 01 00 00 00 01 00 in the first 3 colmuns and last 3 lines 12 00 00 00 00 01 00 01 00 FAS FAS FAS 2g E K E E K K K K K K K K 2g K K K K K K K K K K K K K K K K K K K K K K K K K K K K K K K K K K K K K K K K K K K K K K K K K K FK K K K 2 FK K K 01 0 00 0 00 0 00 02 0 00 0 00 0 00 03 0 00 0 00 0 00 04 0 00 0 00 0 00 05 0 00 0 00 0 00 06 0 00 0 00 0 00 07 0 00 0 00 0 00 08 0 00 0 00 0 00 09 0 00 0 00 0 00 10 0 00 0 00 0 00 11 0 00 0 00 0 00 12 0 00 0 00 0 00 This matrix is no longer used PEs FAS 2S 2g E K K K K K K K K K K K K K K K K K K K K K K K K K K OO FK K K K 2k 2 K K 01 1 00 This last vector is the quantum yield vector 02 1 00 03 1 00 04 1 00 05 1 00 06 1 00 07 1 00 08 1 00 09 1 00 10 1 00 11 1 00 12 1
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