HKL2MAP 0.3a-beta User Guide
Contents
1. SHELXE phasing and density modification Current status of data preparation substructure solution and phasing Filename for data collected at high energy remote Figure 3 HKL2MAP Main window after aM AD experiment has been selected and names of files containing diffraction data have been entered File Display Appearance Anomalous CC vs Resolution HREM PEAK HREM INFL PEAK INFL Figure 4 The Graphics Window for the results of SHELXC after choosing the plot of Anoma lous CC vs Resolution 16 4 3 Running SHELXD The name of the file containing substructure structures factors is automatically forwarded to the SHELXD part of the interface Also the interface tries to suggest a high resolution cut off for the data used for finding the substructure The user has to supply the expected number of sites and the type of heavy atom in the crystal In this case 8 Se sites are requested and SHELXD is started by pressing the Run SHELXD Button File Tools Project name jat 77 SHELXC prepare aF or FA data from experiment 00 00 13 SHELXD find heavy atoms CCmax 56 58 Try 16 100 00 08 30 Jiat_fank jiat_fains Browse Find 8 heavy atoms of type Se Use data from 333 to 30 resolution Allow sites on special positions yes no Limit number of tries to 100 5 PDB out Ma Browse more options view graphics Run SHELXD SHELXE phasing and density modifi2. GetExecutablePath for shelxc gt Path is Users tschneider A shelxi NewShelx 110901 intelmac shelxc gt Version is 2011 1 GetExecutablePath for shelxd gt Path is Users tschneider A shelxi NewShelx 110901 intelmac shelxd gt Version is SHELXD 2011 5 gt This is an MP enabled version of SHELXD running on 2 threads GetExecutablePath for shelxe gt Path is Users tschneider A shelxi NewShelx 110901 intelmac shelxe gt Version is 2011 1 GetExecutablePath for coot gt Path is usr local xtal coot64 bin coot gt Version is 0 6 1 GetExecutablePath for xterm gt Path is usr X11 bin xterm gt Version is Please have a look and check whether everything makes sense After closing the Welcome Window the Main Window of HKL2MAP becomes visible Fig ure 1 At the top of the Main Window a Menu Bar and an input line for the project name are located For the different programs called by HKL2MAP the interface provides Panels that can be openend and closed by clicking the respective boxes The bottom part of the Main Window consists of a Status Window containing the output generated by the called programs and a line displaying brief context sensitive help messages 3 2 Definition of Project In the context of HKL2MAP we consider an attempt to phase a structure with a certain set of parameters as a Proj
3. Yes of course 3 5 2 Estimation of the solvent content The solvent content of the crystal under consideration is calculated by assuming a volume of 140 A per amino acid residue SC residues 140 volume a u whereby volume a u is the volume of the asymmetric unit 3 5 3 Auto tracing If the CC after the first auto tracing round is gt 25 the structure is usually solved 3 5 4 Graphical Display with Coot XXX To be written Ctrl Left Mouse to translate the electron density 3 5 5 SHELXE OUTPUT CC Eobs Ecalc The CC between Eobs and Ecalc printed for the heavy atoms substructure is calculated on all data as apposed to the CC in SHELXD If this value is gt 15 the situation is good Note that this number depends a lot on resolution cut offs 3 5 6 CC against native If the CC after the first auto tracing round is gt 25 the structure is usually solved 3 6 How to continue To move the structure factors and phases into the CCP4 world here is template script bin csh f phs2mtz 12 C shell script to convert phs file produced by SHELXE into mtz format For running this script you need to have the CCP4 programs f2mtz and cad in your path Before running the script you have to adjust the unit cell parameters and the space group to the appropriate values C Thomas R Schneider 22 Oct 2004 EEE 2 EEE HHH echo echo Converting 1 echo
4. Reset View E Display Manager DOME A FERIA MX Successfully read coordinates file Users tschneider A shelxi src jial_i pdb Molecule number 1 created E 5 Figure 9 Electron density map for TE II with auto traced model superimposed as displayed by Coot 20 5 Sulphur SAD phasing of Zn free insulin 5 1 Diffraction Data The data used here originate from a test experiment on a crystal of Zn free Insulin using the Cu K radiation emitted from the rotating anode in the Department of Experimental Oncology at the European Institute of Oncology in Milan Italy The crystal was grown following the instructions distributed by the Sheldrick laboratory for the BIOXHIT test crystal project The crystal belonged to space group 1213 with unit cell parameters of a b c 78 3 A a 6 y 90 0 135 deg of data were collected in increments of 1 deg The data were processed with DENZO and SCALEPACK The scaled but unmerged data keyword no merge original index in SCALEPACK were stored in SCALEPACK sca format The crystal used contained one molecule of insulin 51 residues per asymmetric unit One molecule of insulin contains 3 disulphide bridges 5 2 Starting HKL2MAP and Entering the Project Name HKL2MAP can be started from a UNIX command line hkl2map amp After closing the Welcome Window the Main Window of HKL2MAP becomes visible Fig ure 10 At the top of the Main Window a Menu Bar and an input line for the project na
5. dist tcl Users trs bin hkl2map tcl To make HKL2MAP run on your particular operating system you may need to remove the first two lines of the script usr bin env wish The following for non MAC usr bin wish such that usr bin wish becomes the first line of the script If HKL2MAP still does not work ask your IT guys whether wish is available on your system and if so where it lives To add the chosen location to your PATH add the following line to your cshrc file setenv PATH Users trs bin PATH On the MAC you need to make sure that the runtime libraries for the SHEL X programs can be found For this add the following line to your cshrc file here the libraries reside in Users trs bin setenv DYLD_ LIBRARY PATH Users trs bin 2 5 Windows I do not have a windows mashine in operation myself I am grateful for any hints on this 3 General Notes 3 1 Starting HKL2MAP Once HKL2MAP is available in the path of executables it can be started from the command line hkl2map amp The first thing that HKL2MAP will do is to check what type of executables are available and print the respective information to the terminal window trs laptop2 A shelxi exercise tschneider hkl2map KEKE KKK KKK KKK KKK KKK KK KKK KKK KKK KKK KKK AKAAR x HKL2MAP Version 0 23 0 KKKKKKK KKK KKK KKK KKK RARA KKK KKK KKK KKK KKK KKK KKKKKKKKKK OS Darwin 10 8 0 1386 i386 Tcl Tk Version 8 5 Windowing system aqua
6. if e 1 then echo The file 1 is available else echo echo exit endif Check for programs foreach v f2mtz cad echo n Checking for v if which v grep ci not found then echo not found echo Exiting echo exit else echo found OK endif end Run f2mtz echo echo Running f2mtz f2mtz hklin 1 hklout l r mtz gt t_ 1 r_f2mtz log lt lt END cell 96 0 120 0 166 1 90 0 90 0 90 0 symmetry C2221 labout H K L FP FOM PHIB SIGFP CTYPOUTHHHFWPO END grep Number of Reflections t_ 1 r_f2mtz log grep Space Group t_ 1 r_f2mtz log The file 1 is not available did you use the extension echo n Resolution Range grep A t_ 1 r_f2mtz log Run cad echo mm echo Running cad cad hklinl 1 r mtz hklout l r cad gt t_ 1 r_cad log lt lt eof cad LABIN FILE 1 1 FP E2 FOM E3 PHIB E4 SIGFP ABOUT El FP E2 FOM E3 PHIB E4 SIGFP eof cad Report echo echo gt Logs are in t_ 1 r_x log echo gt Data are in l r cad echo mm 13 3 7 Background literature XXX To be written 14 4 MAD phasing of TE II 4 1 Diffraction Data The data used for this tutorial originate from a four wavelengths MAD dataset on a Se Met deriva tive used to solve the structure of Escherichia coli thioesterase II 1 TE ID In addition to the four wavelengths MAD data a native data set to 1 9 A resolution wa
7. HKL2MAP 0 3a beta User Guide Thomas R Schneider European Molecular Biology Laboratory Hamburg Outstation webapps embl hamburg de hkl2map Contact thomas schneider at embl hamburg de September 21 2011 Contents 1 Intro 14 HKE2SMADS 22 Sra ae Be ES ar EGE ae et A 4 1 2 Organization of this guide 2 ee eee 4 2 Installation 5 2 1 Current Versions gas sec auf yeah alain Sana Aliens dd T 5 2 2 HKL2MAPtarball 2 2 2 ee eaa eae a a e E 5 2 3 Other programs required 2 22 2 Con 5 2 4 Installation on Linux and MAC o EEE 5 2 5 Windows cc A re a s BO 6 3 General Notes 7 3 1 Starting HKL2MAP Coon nn 7 3 2 Definition of Project 54 p 2 28 da RdA 7 3 3 SHEEXE 3 aes ay acd a un Pte en ee oe ee eS 9 3 3 1 What type of experiment should I choose 9 3 3 2 Loading data e a a ee SS 9 3 3 3 Graphics lt I sig I gt vs resolution a a 9 3 3 4 Graphics Completeness vs resolution 2 22 22 2222 9 3 3 5 Graphics lt d sig d gt vs resolution ns a enter 9 3 3 6 Graphics CC AF AF vs resolution lt lt 10 3 4 SHELEXD gt ps 4 a a lt a E ds Relate A oi 10 3 4 1 The Number of sites to request o e 10 3 42 The resolution limit for SHELXD 11 3 43 Special position flag for SHELXD 11 3 4 4 Number oftrials rad e A 11 3 45 SHELXD s figures of merit o nen 11 3 4 6 Min
8. NFL gt LREM for a typical Se Met experiment 3 3 6 Graphics CC AF AF vs resolution To a first approximation the anomalous differences measured at different wavelengths should be related by the same constant factor for all reflections coming from the same crystal Therefore for ideal data the correlation coefficient between anomalous differences measured at a wavelength i and at another wavelength j should be 1 0 or 100 In the real world this is of course usually not the case Nevertheless the value of the correlation coefficient for data grouped in resolution bins is a very useful indicator of the accuracy of the measurements Generally the correlation coefficient goes down for increasing resolution A correlation coefficient of less than 30 is a good criterion for truncating the data at the respective resolution Beware that the correlation coefficient can be artifically high if both data sets are not statistically independent or suffer from the same systematic errors For SAD data the correlation coefficient can not be calculated between anomalous differences collected at different wavelengths Instead it is calculated by comparing different measurements of the same anomalous differences note that this can only be done with unmerged data 3 4 SHELXD SHELXD is a program for the ab initio solution of crystal structures It can be used for solving entire structures from the diffraction intentisities alone or for solvi
9. SEED HKLF END a ttt ttt ttt ttttttttttttttttttttt SHELXC for insulin finished at 19 09 13 on 20 Sep 2011 Ma a ttt ttt ttt 1 5 0 1 100 1 3 Figure 11 HKL2MAP Main window after aSAD experiment has been selected the names of files containing been launched OO diffraction data and the space group have been entered and the SHELXC job has hkl2map Version 0 2j 0 SHELXC statistics X hkl2map Version 0 2d SHELXC statistics File Display Appearance E AA A P A Self Anomalous CC vs Resolution lt d sig gt vs Resolution Self Anomalous CC lt d sig gt Di NAT 90 NAT SAD 80 sn 70 2 7 60 50 40 137 30 20 10 0 Resolution 0 Resolution A inf 200 100 67 50 40 33 29 25 22 20 inf 20 0 100 67 50 40 33 29 25 22 2 0 a b Resolution A 2 10 Chi 2 2 39 Figure 12 The vs Resolution Graphics Window for the results of SHELXC after choosing a the plot of d and b the plot of Anomalous CC vs Resolution SHELXD panel which becomes visible when the More Option Button is pressed 23 hkl2map Version 0 2j 0 Fie v coot Project name insulin 00 00 02 N H i i erpe N Fa in insulin_fa hkl Browse A Ins in insulin_fa ins Browse Find 3 heavy atoms of type S Use datafrom 999 to 2 8 A resolution Allow sites on special positions yes no Limit number of tiesto 100 PDB out ins
10. cation 3 Current status of data preparation substructure solution and phasing SHELXC SHELXD fSHELXE original SHELKE inverted Figure 5 The Main Window after entering the numbers necessary for starting a SHELXD job The output from SHELXD is shown to the Status Window at the Bottom of the Main Window Again a graphical display of the results can be activated by pressing the View Graphics Button As opposed to the SHELXC related plots the plots describing SHELXD are automatically up dated to reflect the current status of the SHEL XD run and can be inspected at any time during the SHELXD run One useful plot is the plot of peak height vs peak number for the current best solution Fig ure 6 In favourable cases this plot shows a clear drop indicating the last significant site of the substructure File Display Appearance Site Occupancy vs Peak Number Figure 6 The Graphics Window showing a plot of peak heigt vs peak number A clear drop in peak height is seen between peak 8 and 9 A SHELXD run can be interrupted at any time using the Finish SHELXD Button 17 4 4 Running SHELXE The names of the files containing the native data the substructure structure factors and the best sites found by SHELXD are automatically filled into the respective fields of the SHELXE panel of the interface Before calculating phases with SHELXE the fractional solvent content for convenience a widget for auto
11. correlation coefficient between 3 3 and 4 0 A caused by diffraction measurements being affected by the presence of ice rings The textual output of SHELXC is echoed in the bottom part of the Main Window The plots shown in the Graphics Window can be written in postscript format using the Make Postscript Option in the File Menu of the Graphics Window 5 4 Running SHELXD Truncating the data to be used for substructure solution at 2 8 as suggested above will not allow to find the individual sulphur atoms in disulfide bridges ca 2 0 distance Therefore SHELXD will first search for 3 super atoms which then will be split into two independent sulphur atoms The splitting is triggered by asking SHELXD to resolve 3 disulphide bridges in the part of the 22 hkl2map Version 0 2j 0 File iv coot Project name insulin Y 00 00 02 Prepare Fa data from SAD y experiment Y Native in ins1 sca Browse it Y HA in ins1 sca Browse Cell a 78 3 b 78 3 c 78 3 alpha 90 beta 90 gamma 90 Space group name or number 1213 confirmed V z a Caz Native out insulin hkl Browse j A Fa out insulin_fa hkl Browse more options view graphics RunSHELXC SHELX SYMM SYMM SYMM SYMM SFAC UNIT SHEL PATS C SHELXD SHELXE original SHELXE inverted LER Y 1 2 2 1 2 X 1 2 1 2 2 X 1 2 Y Z 1 2 X SE 768 999 2 5 FIND 8 MIND NTRY
12. e stopping 7 Acknowledgements Thanks to Zbyszek Dauter for providing the TE II data The making of this tutorial was in part supported by the EU funded projects AUTOSTRUCT QLRI CT 2000 00398 and BIOXHIT References 1 J Li U Derewenda Z Dauter S Smith and Z S Derewenda Crystal structure of the es cherichia coli thioesterase ii a homolog of the human nef binding enzyme Nat Stru Biol 7 555 559 2000 26
13. ect The project name specified in HKL2MAP will be used as a prefix for all files generated by HKL2MAP or the SHELX programs called by it File Tools metae 22222 SHELXC prepare aF or FA data from experiment SHELXD find heavy atoms 7 SHELXE phasing and density modification J Current status of data preparation substructure solution and phasing SHELXE original SHELXE inverted Click here to expand or shrink the module Figure 1 HKL2MAP Main window after starting the program hkl2nap Version 0 1 ya Help Project name jjia1 SHELXC prepare F or FA data from experiment Prepare Fa data from SAD experiment Native in Browse HA in Browse cll af bf cf amaf beta gamma Space group name or number confirmed _1 File Tools Native out jjiat hkl Browse Fa out Jjia1 _fa hkl Browse more options view graphics run SHELXC SHELXD find heavy atoms SHELXE phasing and density modification Current status of data preparation substructure solution and phasing SHELXD SHELXI SHELXE inverted A de Figure 2 HKL2MAP Main window after entering the project name Once the project name has been entered all input fields of the interface concerning input and output filenames are loaded with default values derived from the project name and the SHELXC Panel is opened Figure 2 3 3 SHELXC SHELXC is a program t
14. ed 186 reflections with d gt 2 102 and 0 in range 2 102 gt d gt 2 102 added ii Density sharpening factor set to 0 90 Fourier grid 256 x 256 x 66 0 000 lt z lt 0 250 92 point spherical net set up with radius 2 42A 32 extra Fourier layers will be generated Overall CC between Eobs from delF and Ecalc from heavy atoms 43 64 lt wt gt 0 217 for initial phases lt wt gt 0 319 after including heavy atoms lt wt gt 0 218 Contrast 0 408 Connect 0 629 for dens mod cycle 1 lt wt gt 0 224 Contrast 0 492 Connect 0 608 for dens mod cycle 2 lt wt gt 0 233 Contrast 0 654 Connect 0 669 for dens mod cycle 3 lt wt gt 0 242 Contrast 0 735 Connect 0 702 for dens mod cycle 4 Pseudo free CC 41 49 lt wt gt 0 250 Contrast 0 788 Connect 0 722 for dens mod cycle 5 Figure 7 The Main Window after starting SHELXE The progress of the phasing calculations can be followed in the Status Window or more conve niently by activating the graphical view of the SHEL XE output by pressing the View Graphics 18 Button An important measure for phase quality is the Contrast between the variance in the electron density in the protein region and in the solvent region for a given phase set Using the Graphics Window activated via the View Graphics Button in the SHELXE Panel a continously updated plot of the value of the contrast vs cycle number can be displayed and the dec
15. ed 0 7 0 6 Pseudo free CC 0 5 72 75 04 38 73 residues 0 3 50 0 2 27 0 1 0 0 Cycle 0 10 20 30 40 50 60 70 80 Figure 14 Plot of contrast as a function of cycle number with starting phases calculated based on the original red and the inverted blue substructure for the partical structure against the native data of 53 13 23 6 Various 6 1 Tips and Tricks 6 1 1 How can I import data from mtz format Use Time Gr nes program mtz2sca This program can be obtained from http shelx uni ac gwdg de tg mtz2x mtz2sca download php Usingmtz2sca you can eas ily convert mt z files into sca files that can be read by HKL2MAP 6 2 Known issues 1 110827 Coot 0 6 1 The hat file written by SHELXE 110819 contains two END state ments This is valid SHELXE syntax The sites between the END statements are sites considered useless by SHELXE Such a hat file crashes coot Therefore the current version of HKL2MAP does not automatically load the hat file into COOT when COOT is started 2 110929 Tcl Tk on MACs On MACs the initial directory for file browsing may be a strange place This is not a bug but a features of the MAC Apparently there is some switch in the system preferences which controls this I was not able to find that switch 3 Finishing SHELXE upon request when auto tracing is activated can take quite a while because SHELXE will finish the current cycle of auto tracing and the subsequent density modification befor
16. imum distance between SHELXD sites 11 3 3 SOHBEXE A A en end Sg e Sale 12 3 5 1 Invert substructure 2 Coon 12 3 5 2 Estimation of the solvent cOnteMt o o 12 30 37 Autostr cihs ur wre Pea ak ed ee eR 12 3 5 4 Graphical Display with Coot 2 222 2 CE nn 12 3 5 5 SHELXE OUTPUT CC Eobs Ecalc o 12 3 5 6 CC against native a 3 6 How to contin er 2 34 var rar ae a ne a e e 3 7 Background literature Como oo 4 MAD phasing of TE II 4 1 Diffraction Data 2 2 ee 4 2 Running SHEEXC ata A Sa i 4 3 Running SHELXD ceste daah won hk aa ee a o ee a 44 Running SHELXE nn 4 5 Displaying Electron Density o o e 000000 5 Sulphur SAD phasing of Zn free insulin 3 1 Diffraction Datars 05 2 2 2 0 a he aed bee da Be Se A as 5 2 Starting HKL2MAP and Entering the Project Name 5 3 Running SHELXC oia a as bark nn pr a el 94 Running SHELXD sa 1 aout rt yee en Er 5 5 Running SHELXE ver a ne ae ee ed ee e 6 Various 6 1 Tips and Tricks 80 o a en a ee ee ee 6 1 1 How can I import data from mtz format 6 2 Known issues Li SM SA ni ea ck die Se See ee 7 Acknowledgements 15 15 15 17 18 19 21 21 21 21 22 24 26 26 26 26 26 1 Intro 1 1 HKL2MAP HKL2MAP is a graphical user interface for macromolecular phasing The current version con nects seve
17. ing on the Browser Button As many accidents happen due to misunderstanding between programs about space groups HKL2MAP requires an explicit confirmation of unit cell constants and space group Confirmed Button before SHELXC can actually be started from the interface Once SHELXC is started by clicking on the Run SHELXC Button the interface is blocked for further input until the program has finished The output of the SHELXC run is directed to the Status Panel at the bottom of the Main Window After SHELXC has finished a graphical version of the SHELXC output can be activated by pressing the View Graphics Button Clicking this button will open a graphics window allowing to display various plots The type of plot to be displayed can be selected using the Display Menu in the Graphics Window The plots shown in the Graphics Window can be written in postscript format using the Make Postscript Option in the File Menu of the Graphics Window 15 File Tools Prepare Fa data from MAD experiment Native in home trs shelxijia hkl Browse Peakin homestrs shelxifjia_peak sca Browse Inn in fnomentrsfshelifia_imM sea Browse HRemin home rs shelxijia_hremsca Browse IRemin s Browse Cell a 86 000 b 120 000 c 166 130 alpha 30 000 beta 90 000 gamma 30 000 Space group name or number C2221 confirmed Native out Browse Browse more options view graphics run SHELXC
18. ion fail completely The low resolution cut off should be chosen in a way that overloaded data and reflections that were actually caught by the beamstop are excluded The high resolution cut off should be chosen such that not too much noise is included For multi wavelength experiments a good high resolution cutoff is where the correlationc coefficient be tween signed anomalous differences falls below 30For single wavelength experiments the data can be truncated where the lt d sig d gt falls below 1 4 1 5 The number of reflections after application of E value and or resolution cut offs should be at least 20 30 times larger than the number of expected sites 3 4 3 Special position flag for SHELXD For soaks sites can end up on special positions so the avoidance of special positions feature should be switched off 3 4 4 Number of trials Normally 100 trials is agood number For SAD data as they are sometimes quite weak and noisy it may be worth trying 1000 trials For very difficult cases 50000 tries can be used Large numbers of tries can be used especiallly when the MP enabled version of SHELXD is used 3 4 5 SHELXD s figures of merit CC corresponds to an weighted correlation coefficient between Fy and Ecate The fact that it is WEIGHTED by 1 sigma E creates the danger that its value can be strongly influenced by incor rect sigmas i e a reflection with an extremely small sigma may dominate that whole evalua
19. ision which of the substructure enantiomorphs results in the correct phases can be made while the phasing calculations are running hkl2map Version 0 2j 0 SHELXE statistics File J Display Contrast vs Cycle Contrast 15 original inverted 12 Pseudo free CC a3 51 45 77 19 0 6 residues 188 0 3 543 0 0 Cycle 0 10 20 30 40 50 60 70 80 Figure 8 The plot of contrast vs cycle number The contrast values for the original and the inverted substructure are shown in red and blue respectively Here the inverted substructure is the correct one The phasing job for the incorrect substructure red is stopped halfway When a clear difference between contrasts for the two enantiomorphs is seen the phasing job using the wrong enantiomorph can be interrupted by pressing the respective Finish Original Inverted Button to free the computing resources 4 5 Displaying Electron Density The electron density maps corresponding to the phase sets produced by SHELXE can be inspected by pressing the run coot Button corresponding to the required substructure enantiomorph in the SHELXE panel of HKL2MAP To display the result of the auto tracing load the pdb file lt proj gt pdb for the original sub structure or lt proj gt _i pdb for the inverted substructure into COOT For the case discussed here the filenames would be jial pdb or jial_i pdb XI Coot File Edit Calculate Draw Measures Validate HID About Extensions
20. matic calculation of the solvent content can be activated by pressing the Estimate Solvent Content Button has to be entered Also the user has to supply the information whether or not the native crystals contained the substructure atoms or not The interface allows to specify which enantiomorph of the substruture should be used for phas ing As an alternative to testing the enaniomorphs one after the other the respective jobs can be run simultaneouly by choosing try both enantiomorphs Dual CPU computers are particularly convenient to run the jobs for the two enantiomorphs in parallel hkl2map Version 0 2j 0 Project name jia1 00 00 02 CCmax 61 9 Try 84 100 00 00 43 y Contrast 0 735 Cycle 5 5 20 00 00 31 Native in jia1 hkl Browse Fain jiat_fa hki Browse SHELXD out jia1_fa res Browse Phase structure and refine density for 20 cycles 4 Use fractional solvent contentof 0 661 Estimate the solvent content Native data do include heavy atoms Extend diffraction data to a A native data extend to 1 90 A Y Run 3 cycles of autotracing Y interrupt calculations for incorrect enantiomorph after 4 cycles Invert heavy atom substructure for phasing try both enantiomorphs v Phases ori jia1_m5_s0 661 phs runcoot Browse Phasesinv jia1_m4_s0 661_i phs runcoot Browse more options viewgraphics finishing finish inverted SHELXE original SHELXE invert
21. me are located For the different programs called by HKL2MAP the interface provides Panels that can be openend and closed by clicking the respective boxes The bottom part of the Main Window consists of a Status Window containing the output generated by the called programs and a line displaying brief context sensitive help messages 5 3 Running SHELXC To run SHELXC first the type of experiment has to be chosen After the choice has been made the SHELXC panel changes and presents input fields appropriate for the respective type of exper iment For the present example SAD has been selected and in response the interface generates input fields for filenames for files containing the diffraction data for a native and a SAD data set The filenames can be typed in or files can be selected using a browser that is actived by clicking on the Browser Button Please note that for this case the anomalous data and the native data were collected on the same crystal and are in fact in the same data set As many accidents happen due to misunderstanding between programs about space groups HKL2MAP requires an explicit confirmation of unit cell constants and space group Confirmed Button before SHELXC can actually be started from the interface Once SHELXC is started by clicking on the Run SHELXC Button the interface is blocked for further input until the program has finished The output of the SHELXC run is directed to the Status Panel at the bottom
22. ng substructures of heavy atoms and or anomalous scatterers against the appropriate substructure structure factors In the context of HKL2MAP SHELXD is used to solve substructures 3 4 1 The Number of sites to request This number should be as close to the reality as possible For Se Met derivatised crystals it is usually possible to have a rather accurate idea on how many sites one should expect If Se incorporation has been checked for the protein preparation the uncertainties in the number of sites originate from not knowing the number of molecules in the assymetric unit and not knowing whether or not all Methionine sidechains are ordered Most people consider Seleniums in N terminal Methionines as disordered For halide soaks it is usually advantageous to start with a relatively small number as spurious sites will be included in the calculation of figures of merit and render these meaningless in extreme cases For phasing sites with and occupancy of less than 20 30 are probably meaningless For disulphide bridges if resolution is higher than 2 1 A one can look for single atoms With data worse than 2 4 A it is better to look for super atoms and then split these Between 2 1 Aand 2 4 A try both options 10 3 4 2 The resolution limit for SHELXD The resolution limits are very important parameters for SHELXD Direct methods are very sensi tive to inaccurate data a few badly measured reflections can make a substructure determinat
23. o analyse diffraction data originating from SAD MAD SIRAS MIRAS experiments It prints some useful statistics to access the quality of the data and extracts substruc ture structure factors DeltaF s or FA s that represent the substructure of the heavy atoms or the anomalous scatterers only These substructure structure factors can then be used by SHELXD to find the substructure atoms SHELXC also estimates an angle alpha the angle between the complex structure factors of the protein atoms FP and the heavy atoms FH or the anomalous scatterers FA This angle will later be used by SHELXE to start the calculation of phases for the protein Generally it is recommended to read scaled but unmerged data as the statistics generated from these data will be more reliable 3 3 1 What type of experiment should I choose The first time around you will probably choose the type of experiment that you intended to do in the first place However if it turns out that the data are not of the expected quality one can divert to other strategies A typical example would be a MAD experiment where the crystal suffered from radiation damage and only the data set collected first can be used in a SAD approach 3 3 2 Loading data On input SHELXC can read diffraction data in SHELX hkl or in SCALEPACK sca formats Depending on the type of experiment chosen the appropriate fields will be displayed for defining filenames 3 3 3 Graphics lt I sig I g
24. of the Main Window After SHELXC has finished a graphical version of the SHELXC output can be activated by pressing the View Graphics Button Clicking this button will open a graphics window allowing to 21 FAAA a IALA X hkl2map Version 0 24 isin eieiei Project name insulin ES FA data from experiment Prepare Fa data from SAD experiment mate a BrOWS Ham AAA AAA Browse cell af bf ef apha taf gamma Space group name or number confirmed _1 Native out insulin hkl Browse Faout insulin_fa hkl Browse more options view graphics run SHELXC J SHELXD find heavy atoms 7 SHELXE phasing and density modification 7 j Current status of data preparation substructure solution and phasing sueno SHELXD SHELXE original original SHELXE inverted inverted 4 Figure 10 HKL2MAP Main window after starting the program display various plots The type of plot to be displayed can be selected using the Display Menu in the Graphics Window Figure 12 The signal to noise ratio for the anomalous signal as shown in the plot of d vs resolution Fig a goes below 1 0 at ca 2 2 A indicating that most of the data could actually be used for structure determination However the anomalous correlation coefficients show a sharp drop around 2 7 2 9 A indicating that the data should better be truncated at this resolution In addition the there is a sharp dip in the anomalous
25. ral programs from the SHELX suite to guide the user from analyzing scaled diffraction data SHELXC via substructure solution SHELXD and phasing SHELXE to displaying an electron density map COOT HKL2MAP can handle SAD SIR SIRAS or MAD data HKL2MAP can be obtained from thomas schneider at embl hamburg de The author does not take any responsibility for the correct function of HKL2MAP or any of the programs called by it Reports on faulty unexpected or destructive behaviour to the above email address will be appreciated Information about the SHELX suite of programs can be found at shelx uni ac gwdg de SHELX Information about COOT can be found at http 1mb bioch ox ac uk coot Please cite the following references when you report the use of HKL2MAP HKL2MAP Pape T Schneider TR 2004 HKL2MAP a graphical user interface for phasing with SHELX programs J Appl Cryst 37 843 844 SHELXC Sheldrick GM 2003 G ttingen University SHELXD Schneider TR Sheldrick GM 2002 Substructure solution with SHELXD Acta Cryst D58 1772 1779 SHELXE Sheldrick GM 2002 Macromolecular phasing with SHELXE Z Kristallogr 217 644 650 CooT Emsley P Lohkamp B Scott WG Cowtan K 2010 Features and development of Coot Acta Cryst D66 486 501 1 2 Organization of this guide Installation on linux MAC Windows and how to start the program are described in Section 2 General guidelines for setting the parameters accessible b
26. s collected on the S Met form of the protein The data were kindly provided by Zbyzsek Dauter The structure of TE II was determined in space group C 222 with unit cell constants of a 96 0 A b 120 0 A c 166 1 A a B y 90 0 The data were processed with DENZO and SCALEPACK The scaled and merged data were stored in SCALEPACK sca format code A dmin A filename hrm 0 9747 2 5 jia_lhrem sca pk 0 9787 2 5 jia_peak sca ip 0 9793 2 5 Jia infl sca Im 0 9801 2 5 jia_lrem sca nat n a 1 9 jia hkl Table 1 Overview of data sets used in this tutorial jia hkl is in SHELX hkl4 format i e contains h k 1 I o I in asci format The refined structure of TE II pdb code 1C8U contains 2 x 4 Se atoms in a total of 2 x 285 amino acids The solvent content is ca 66 More information about the structure and its deter mination can be found in Li et al 2000 Nat Stru Biol 7 555 4 2 Running SHELXC To run SHELXC first the type of experiment has to be chosen Figure 3 After the choice has been made the SHELXC panel changes and presents input fields appropriate for the respective type of experiment For the present example MAD has been selected and in response the inter face generates input fields for filenames for files containing the diffraction data for a native and four MAD data sets The filenames can be typed in or files can be selected using a browser that is actived by click
27. t vs resolution This plot shows the signal to noise for the measured diffraction intensities different data sets in different colors versus resolution As diffracted intensities are less at higher resolution than at low resolution they become more difficult to measure so the plot signal to noise lt I sig I gt for the measurements should generally go down with increasing resolution 3 3 4 Graphics Completeness vs resolution For a good dataset the completeness should be above 95 for all resolution bins Sometimes the completeness is lower at low resolution this can be due to overloaded reflections being re jected or a too small rotation range For the substructure solution incomplete data are sometimes acceptable for phasing it is very important to have complete data 3 3 5 Graphics lt d sig d gt vs resolution This plot shows the signal to noise for the anomalous differences As the anomalous differences are measured as small differences between large numbers the differences in intensity between Friedel mates the signal to noise for the anomalous differences is usually not very high This quantity also goes down with increasing resolution Anomalous data contain useful information as long as the d calculated by SHELXC is higher than 0 8 If data have been collected at several wavelengths the lt d sig d gt should be higher for the wavelengths with larger anomalous signal e g PEAK gt HREM gt I
28. tion of the CC PATFOM is not on any absolute scale Also for low symmetry space groups it may be deceiv ingly high and for high symmetry space groups only few symmetry operations are checked in the Patterson minimum function so noise may dominate it may be deceivingly low many symmetry operations are checked in the Patterson minimum function one Zero is enought to result in a Zero PATFOM for a certain pair of atoms The occupancies obtained from SHELXD are renormalized such that the highest site has an occu pancy of 1 0 even if in reality the occupancy is only 0 5 This can lead to complications if even the highest occupancy site still has small occupancy e g in halide soaks After correcting for this overall scaling the occupancies are probably correct within plus minus 20 3 4 6 Minimum distance between SHELXD sites Typical minium distances for Se 3 5 A S S bonds 1 7 A Fe in Heme groups 6 A 11 3 5 SHELXE SHELXE is a program to rapidly derive a phase set for data set based on substructure structure factors and a substructure consisting of heavy atoms and or anomalous scatterers To find out what files SHELXE uses and what files it writes out have a look at the top part of the respective log file CPU times for tl2 correspond to trs laptop2 This is a 2 66 GHz Intel Core 2 Duo MAC BookPro 3 5 1 Invert substructure Will SHELXE deal properly with space groups that need changing when the structure is inverted
29. ulin_fa pdb Browse Enable Patterson search to find starting atoms yes no Minimum distance allowed between heavy atoms 1 0 Resolve 3 disulphide bridges Fix random number seed to allow reproduction of results yes no Exclude E valueslessthan 1 5 s Allocate memory for 4 x 1Mio reflections lessoptions viewgraphics runSHELXD SHELXC SHELXE original SHELXE inverted SYMM Y Z X SYMM Y 1 2 2 1 2 X SYMM 1 2 Y 1 2 2 X SYMM 1 2 Y Z 1 2 X SFAC SE UNIT 768 SHEL 999 2 5 PATS FIND 8 MIND 1 5 0 1 NTRY 100 SEED 1 HKLF 3 END tHtt tt SHELXC for insulin finished at 19 09 13 on 20 Sep 2011 a EFF FT H Ft Fttttt tttttt ttt ttt ttt ttt ttt tt ttt ttt ttt ttt Figure 13 Input parameters for SHELXD to find 6 sulphur atoms in three disulphide bridges using data to 2 8 A resolution The best solution has a CCau of 53 3 percent 5 5 Running SHELXE Starting SHELXE using the sites determined by SHELXD gives a CC Ene Ecalc of 25 22 indicating that things go well After a few cycles of density modification contrast and Pseudo free CC clearly indicate that the original substructure has the right hand for the phase calculation In fact after 3 rounds of auto tracing 50 out of 51 residues have been traced resulting in an CC 24 ABE hkl2map Version 0 2j 0 SHELXE statistics Fie Jos Contrast vs Cycle Contrast 09 original a invert
30. y via the HKL2MAP interface are given in section 3 along with references explaining the underlying methodology The remaining sections describe different phasing scenarios e MAD phasing e S SAD Elastase single sulphurs e S SAD Ins split super sulphurs I SAD Elastats soak Pt MIR Lysozyme 2 Installation 2 1 Current versions The applications in this guide have been run using Coot version 0 6 1 and SHELXC D E as avail able on the SHELX server on 19 08 2011 2 2 HKL2MAP tarball The installation will normally be done starting from a tar ball hk l2map 0 3a beta dist obtained either from the HK L2MAP website after registering or from Thomas via email 2 3 Other programs required To run HKL2MAP you need to have the following programs in your PATH e shelxc shelxd shelxe e wish This is the interface to the Tcl Tk toolkit the programming language in which HKL2MAPis actually written Most computers have wish in the PATH Especially if your are using the CCP4i interface you will have wish in your PATH automatically In case that wish is not available ask your friendly IT guys for help e coot To test whether for example the executable shelxc isin your PATH type which shelxc from the command line 2 4 Installation on Linux and MAC Unpack the tarball in a temporary location tar xvzf hkl2map 0 3a dist tgz Rename and copy the HKL2MAP executable to a convenient location here Users trs bin cp hk12map 0 3a
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