All-atom Contact Analysis Tutorial, Introduction

9 pages

English

All-atom Contact Analysis Tutorial, Introduction

Shaej - Bubba

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9 pages

English

Le téléchargement nécessite un accès à la bibliothèque YouScribe
Tout savoir sur nos offres

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Extrait

Description

Structure Validation WorkshopUsing All-Atom Contact Analysis for ModelDiagnosis & RepairThis three-part tutorial is intended as a self-paced (although we use the practicals forworkshops), self-guided introduction to using all-atom contacts in macromolecule building andrefinement. While you will be introduced to a powerful toolset, there are other tools available, andwe invite you to explore them on our website (http://kinemage.biochem.duke.edu/).Parts of the practical -- The tutorial lessons:1. Add H, calculate contacts, and assess geometry by running the MolProbity web service.2. Repair of problem sidechains using the interactive KiNG/Probe system3. Local backbone adjustments using KiNGThe HTML pages, associated image files and datafiles (coordinate files, kinemage files, andmaps) comprising this tutorial can be downloaded as a zip archive.Reprint PDFs & Other"The penultimate rotamer library." Proteins, 2000 New rotamer library (the one of stereochemduke.odb)"Structure validation by C-alpha geometry: phi, psi and C-beta deviation." Proteins, 2003 updates Ramachandran plot contours and introduces the C-beta-deviation measure"New tools and data for improving structures, using all-atom contacts." All-atom contact methodology applied to crystallography; from Methods In Enzymology,Vol. 374"A test of enhancing model accuracy in high-throughput crystallography." JSFG, 2005 Statistics showing large improvements using these tools.All-atom contact ...

Informations

Publié par	Shaej
Nombre de lectures	35
Langue	English

Extrait

Structure Validation Workshop

Using All-Atom Contact Analysis for Model Diagnosis & Repair

This three-part tutorial is intended as a self-paced (although we use the practicals for workshops), self-guided introduction to using all-atom contacts in macromolecule building and refinement. While you will be introduced to a powerful toolset, there are other tools available, and we invite you to explore them on ourwebsite (http://kinemage.biochem.duke.edu/).

Parts of the practical --The tutorial lessons:

1.Add H, calculate contacts, and assess geometryby running the MolProbity web service. 2.Repair of problem sidechainsusing the interactive KiNG/Probe system 3.Local backbone adjustmentsusing KiNG

The HTML pages, associated image files and datafiles (coordinate files, kinemage files, and maps) comprising this tutorial can be downloaded as azip archive.

Reprint PDFs & Other

"The penultimate rotamer library." Proteins, 2000New rotamer library (the one ofstereochemduke.odb) "Structure validation by C-alpha geometry: phi, psi and C-beta deviation." Proteins, 2003updates Ramachandran plot contours and introduces the C-beta-deviation measure "New tools and data for improving structures, using all-atom contacts."All-atom contact methodology applied to crystallography; from Methods In Enzymology, Vol. 374 "A test of enhancing model accuracy in high-throughput crystallography." JSFG, 2005Statistics showing large improvements using these tools. All-atom contact analysis tutorialThis intro page and the three practicals above (but not screen shots nor data files).

Typographical conventions used in these worksheets:

filenamesare in bold, command-line inputsare in fixed-width, execution of the command is implied command-line prompts are indicated as shell> menu commands > menu-pulldownsare in italics, note the '>' separator to indicate sub-menues. dialog-box inputs & selections are by context.

Suggestions for your own use later: Try MolProbity on your own structures

Upload your PDB-format file into MolProbity. Add hydrogens, check geometry, and look at the contacts, as done in Part 1 above. If the structure is not too big, try making a Multri-crit kinemage. (Check out your competitor's structures, too!) As a minimum, any structure should have its Asn/Gln/His flips corrected before deposition. If the MolProbity statistics look good already, or if these correction procedures are used to make them so, those statistics can be reported asREMARK 42 in the PDB header, which will soon be available as an output from MolProbity.

Alternative procedures for running locally

To run the functionalities of Part 1 locally on your own system (Linux, Unix, Mac OSX, etc.) rather than on the web, download KiNG or Mage; and Prekin, Probe, Reduce, Flipkin, and Clashlist from our web site athttp://kinemage.biochem.duke.edu/and put them in your path. (Note: KiNG is the only program that requires JAVA. Mage is an older C program that will do everything here that KiNG does except the backbone corrections. So in the commands given here, either Mage or KiNG will do.)

Run them with the following commands: To add and optimize H atoms,

reduce -build 1xyz.pdb >1xyzH.pdb shell>

To run an automated check for amide and His flips, and produce animated kinemages: flipkin 1xyzH.pdb >1xyzNQflip.kin shell> flipkin -h 1xyzH.pdb >1xyzHisflip.kin shell>

Look at it in Mage: mage 1xyzNQflip.kin shell> Make a kinemage graphics file of the structure by: prekin -lots 1xyzH.pdb >1xyzH.kin shell>

Add all-atom contacts with: probe 1xyzH.pdb >>1xyzH.kin shell>

Then look at it in Mage.

For a clustered list of clashes, and an overall clashscore, run clashlist 1xyzH.pdb >1xyzHclcl.txt

All-atom contacts and rotamers in XtalView and O

Our "penultimate" rotamer library is available as a drop-in files from our web site for XtalView and O (also available from the O web site). XtalView and Mi-fit can invoke our all-atom clashes. Our Website has a macro for invoking all-atom contact dot display in O, and instructions for its use.

HTML README files for related software

Reduce flipkins O - probe

Probe kincontours O - rotamers

For help, questions, or bug reports later, contact: Dave or Jane Richardson e-mail: dcrjsr AT kinemage.biochem.duke.edu

http://kinemage.biochem.duke.edu/teaching/aca2005/

clashlist

Jane & Dave Richardson

next: Practical 1

Structure Validation Workshop

Using All-Atom Contact Analysis for Model Diagnosis & Repair

Scope and Objective

In this part one of the tuturial, you will use the MolProbity web service to add hydrogens to an example PDB structure, 2SIM. You will assess the model's quality via flip-state of HIS, GLN, and ASN sidechains, with Ramachandran plots, comparison against a rotamer database, a graph of C-beta deviations and also make and download from the webservice a kinemage file for the all-atom contact analysis ofPart 2.

Part 1: Running the MolProbity web service to add H, calculate contacts, and assess geometry (done on file2sim- a neuraminidase at 1.6Å resolution).

Prepare 1. Make a working directory for this practical, if you don't already have one. 2. Open a browser configured with Java 1.3 or later (e.g. a recent Mozilla or Netscape). Go to the web site at http://kinemage.biochem.duke.edu/, and choose MolProbity on the navigation bar. In the intro page, choose to use the KiNG viewer then proceed (with the standard MolProbity version for now - you can try the new alpha-test site later if you want). 3. On the main page, enter "2sim" in the field for choosing a PDB file and upload. Check what is reported about the file, and continue. 4. Back on the main page you should see a Java thumbnail kinemage of the C-alphas colored N to C; drag with the mouse to rotate it and admire the beta-propeller structure with a bound dansyl ligand.

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Add hydrogens & evaluate Asn/Gln/His flips

1. Scroll down to see the available tools (some are grayed-out until H atoms are added). Choose the Add H option, which will now run the Reduce program on your file; don't change any of the advanced options. This process takes 10-20 seconds on 2sim, since the analysis optimizes entire local H-bond networks. 2. When done, a report is shown on those Asn, Gln, and His sidechains which Reduce decided should be flipped by 180 degrees, including scores and their differences. Continue, and Flipkin will now run Reduce with the opposite N/Q/H orientations and make kinemages to show you the alternatives. When done, you are offered those kinemages for either viewing or download. 3. Choose to view the Asn/Gln flips. When it has loaded in the Java graphics window, pull down the list of views and go in turn to each one marked with an asterisk (those that were flipped). You may want to enlarge the window. Animate between the two orientations by pressing "a" on the keyboard (or the Animate arrows in bottom of the right panel) evaluate the pale green H-bonds vs the red clashes, and decide whether you agree with Reduce's choice. All N,Q,H have flip green/red (good/bad) pairs: in the Views marked with a star the green ones have been flipped by Reduce. Asn 206 is a no-brainer, while Gln 194 is a bit subtler. Close the browser window containing the graphics display to view the flip score report. The check boxes give you the option of undoing a flip, in the rare but definitely possible case where you feel the algorithm made a misjudgment. These are all sound choices in 2sim, however, so just continue (choose "Regenerate H..." but nothing will be done) and return to the main page.

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Assess Geometry - Phi,Psi

1. Our updated geometrical-validation tools (in the green box) can be run either before or after adding hydrogens. 2. Click on 'Ramachandran plots'. The Java kinemage comes up showing all residues. Animate to the general-residue case, with a green contour around the favored area that includes 98% of the high-resolution, low-B data in an updated reference set and a blue contour around the allowed region that includes 99.95% of the high-quality data. 2sim has two residues outside the allowed contour, which constitute outliers that should be examined with real care

(Ser 230 is especially bad, but can be fixed). 3. Animate thru the other 3 cases (Gly, Pro, and pre-Pro), which show no outliers for 2sim. Close the window with the text Ramachandran report.

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Assess Geometry - Rotamers

1. Sidechain conformations cluster quite closely around local minima in the multidimensional chi-angle space. We use a smoothed plot of values found for high-resolution, low-B residues to define a contour that encloses 99% of that good data. Residues outside of that contour may be genuine (e.g. if they have enough compensating H-bonds to keep them there), but they should be examined individually. 2. On the main page, choose the 'Rotamer analysis' option in the green box, for a list of all residues in the structure with dubious sidechain rotamers. For 2sim over 8% of the sidechains are worse than the 1% level, which is much more than it should be at this resolution. 3. Look at the listed chi values; can you see for many of them why they are unfavorable? We will work on Thr 159 in Part 2. Return to the MolProbity main page.

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Assess Geometry - C-beta

1. We have found that the deviation of the model Cbeta atom from ideal position is a single quantity that encapsulates much of the critical information from bond-angle deviations. 2. On the main page, choose the 'C-beta scattergram plot' option in the green box, for a plot of all the Cbeta deviations in the structure shown on top of an ideal-geometry residue. 3. 2sim has about 8 or 10 residues with anomalously large Cbeta deviations. Click on a few of those points to identify them - many are branched-Cbeta residues; we will work on Thr 159 in Part 2. 4. Close the browser window containing the scatterplot.

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Calculate All-Atom Contacts

1. Now choose the 'Find all-atom contacts' option in the yellow box, which will run the Probe program on the entire structure, in either a graphics or a list mode. 2. First run the 'clashlist' version. 2sim has a relatively poor clash score, which is why it makes a good example (at that excellent resolution, people sometimes believe that the data will take care of everything). The clustered list can be used to find places that need attention. 3. Contact dot kinemages are generated by selecting the radio-button for Probe on this page. In this case, you should also check the top three boxes in the right-hand column of advanced properties: H-bonds, VDW contacts, and C-beta deviations will be added to the kinemage graphic files. An option to be used later, if you want, is to generate a "Multi-Crit" kinemage. This option is available in the blue-boxed "Other Tools (under development)" section on the main page. 4. Choose to view the sc-sc and sc-mc contacts. (If your browser was not able to run King, you could download this big file and view it in Mage or KiNG locally on your own computer). 5. Turn off wide contacts, close contacts, small overlaps, water, and H-bonds (but not H's) to get an overview of the problem areas. In front near the middle is a cluster of 3 bad clashes (red) near a very big Cbeta deviation (gold ball).

(If you have lost track of "front", go to menu item Views/1 Unnamed view). Use a right-mouse-click or turn on PickCenter to center on that gold ball (it should be Thr 159, with a Cbeta deviation of .496Å), and zoom way in. The clashes surround the Thr methyl group, which is much too close to a backbone NH and 2 waters. Rotate to look down the Ca-Cb bond, to see that the chi1 angle is eclipsed. This residue looks very bad by many independent criteria (it was also on the bad rotamer list), but we will easily repair it in Part 2.

Download

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Close the browser window containing the KiNG graphics, return to the main page, and scroll down on the main MolProbity page to where the files are listed. Download2SIMH.pdband2SIMH-contact.kininto your working directory, logout from MolProbity, and exit the browser.

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http://kinemage.biochem.duke.edu/teaching/aca2005/

previous: Introduction

Jane & Dave Richardson

next: Part 2

Structure Validation Workshop

Using All-Atom Contact Analysis for Model Diagnosis & Repair

Part 2: Repair of problem sidechains using the interactive KiNG/Probe tools

This part 2 of the All-atoms Contacts tutorial presents two practicals in rebuilding two problematic side chains. The first is Thr 159 found in 2SIM, for which you prepared the contact-dot kinemage in Part 1 of the tutorial. This repair is done without reference to an electron density map (structure factors were not deposited) and so, changes to the model are limited to placing atoms in the same general area as the beginning model. The second practical is of a model in refinement/rebuilding with electron density and so, changes to the model can be greater with another round of refinement to check the proposed rebuilds.

A: 2SIM Thr 159 - View in KiNG

1. Get the programs: If you have not done so already, download and install the program KiNG (Probe will be included in the KiNG package) for your system, from the site atkinemage.biochem.duke.edu/software/. Instructions for installation are available from a link on the KiNG download page. 2. To work with the file,2SIMH-contact.kin, you just downloaded from MolProbity at the end of part 1: 1. If in MS Windows: double-click to launch KiNG (which should be in your working folder with the PDB and *.kin files), and open the contact kin file from the File Menu. 2. If in Linux: At the shell command-line type: king2SIMH-contact.kin shell> which assumes that KiNG is in your PATH. One of the following commands will add the location of KiNG to your PATH declaration in your shell setup file if you are using either the csh, tcsh or bash shells. echo 'setenv PATH /actual/full/path/in/which/I/put/it:$PATH' >> ~/.tcshrc (or shell> .cshrc) echo 'export PATH=/actual/full/path/in/which/I/put/it:$PATH' >> ~/.bash_profile shell> 3. If on MacOSX: drag & drop the contact kin file onto the KiNG icon.

3. Enlarge the graphics window, if desired, but leave room for later small working windows. 4. Choose menu itemEdit>Find pointand enter '159 cb' into the box , with pickcenter on. Zoom in until only half a dozen residues show, and narrow the clipping slab to see clearly. (zoom with right-click + vertical mouse drag; slab with right-click + horizontal drag; translate in Z with middle click + horizontal drag in the upper region of the display) Turn off all the Cbdev balls (the c-beta button), now that you know this one is big, and make sure H's are turned on. The 3 bad clashes surround the Thr methyl, bumping a backbone NH and 2 waters. Rotate in 3D to look down the Ca-Cb bond, to see that chi1 is eclipsed. Now you will repair this sidechain by finding a good rotamer in ideal geometry that has good contacts while occupying the same region of space as the original model.

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Set Up Rotamers and Torsions

1. Turn off the 'dots' button to remove the original contact dots. 2. The rotamer library is invoked in KiNG via the menu item:Tools >Structural biology>Sidechain rotator. When invoking this tool, another tool, the Model manager, will prompt you to open the PDB file for your model. Answer that query (2SIMH.pdb) Move on by ID'ing the residue of interest (middle click on any side-chain atom of Thr159). A pop-up window will appear allowing sidechain dihedral adjustments and rotamer selection.

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Evaluate Contacts, and Choose

1. To update contact dots automatically as you adjust the sidechain, check the 'Probe dots' box in the Model manager window. Turn off the old 'dots' button, if you have not already done so in KiNG's side panel. 2. The dialog box proposes a command line for Probe: remove the words "not water" so you can evaluate the H-bonds to the waters, press either the "Enter" or "Return" keys to accept, and Probe will return the contact analysis for the present conformation, updated as you change it. 3. Try all 3 rotamers (p, t, or m for plus, trans, or minus chi1), looking for one with good contacts that occupies about the same space as the original model. The p rotamer fits quite well, but its Og1 does not H-bond with the backbone NH of Ile161. Try changing chi1 to form that H-bond, while adjusting/rotating the OH (2nd torsion) to maintain its water H-bond. Forming the sc-mc H-bond causes a clash of one of the methyl hydrogens with the carbonyl oxygen of Val157. KiNG does not do methyl rotations. So, the trick here is to balance forming the Thr159 Og1 to Ile161 H--N H-bond against the nascent clash -- while imagining a methyl rotation of up to 10 degrees from stagger to relieve the clash. 4. The choice between the central-value p rotamer and this modified conformation should be determined by their fit to the

electron density, but unfortunately the structure factors were not deposited. However, both the library rotamer and its modified conformation are in the same local minimum and both are enormously better than the original. 5. There is no need to save the changes for this lesson. However, When you have resolved the clashes to your satisfaction, you can accept your new Thr159 model: 'Release' the checked out residue from both tools and accept. If desired, save the modified pdb file in the Model Manager. 6. Quit from KiNG.

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B: Cth-833 Arg B 54 - View in KiNG

1. Since this is a new SECSG structure not yet available from the PDB, make sure you have the files cth833_0410011335_refmac1_qcH.pdbandcth833_refmac1_qcH-contact_map.kinin your working directory, and open the kinemage file in KiNG. 2. Choose the view for 'b 54 Arg-Asp', which showed up in MolProbity's clashlist. Turn on the 2Fo-Fc map (here preloaded for just the sections you'll need, but normally accessed thru the structural biology function in KiNG). Move around, to see that both sidechains fit their density pretty well but make 2 serious clashes. Turn on H-bonds and small overlaps, to see that the Arg H-bonding is OK to the water but has poor geometry to the Asp. Now let's see if you can do better.

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Choose & Optimize an Arg B 54 Rotamer

1. As you did in A above, turn off the 'dots' button, choose menu item:Tools >Structural biology>Sidechain rotator. When invoking this tool, another tool, the Model manager, will prompt you to open the PDB file for your model. Answer that query (cth833_0410011335_refmac1_qcH.pdb); because the PDB file contains alternate conformations, KiNG will also ask which set of conformations to use (in this case answer 'B'). Move on by ID'ing the residue of interest (middle click on any side-chain atom of Arg 54). Check the 'Probe dots' box in the Model manager window, and again delete 'not water' from the probe command line, accepting that command change by pressing the "enter" or "return" keys. 2. You want to see if there is another rotamer capable of fitting the density but with better interactions. To narrow down the 27 choices for Arg, look down the Calpha-Cbeta vector to see if the chi1 choice is clear; in this case the density shows that Cg must lie opposite the backbone NH, which means a trans chi1. Try each one of the listed rotamers that starts with t, making a note of all that position the guanidinium near the right place and in nearly the right plane (there should be 2, one of which is similar to the original conformation and the other has the guanidinium 'flipped' over. Choose that flipped rotamer, and use the chi rotations to optimize its fit in the density and its H-bonds while minimizing any clashes. Use Views 5 & 6 to check fit from all directions. Are you convinced that this conformation is definitely better than the original? 3. For one more cross-check, go to the 'a 54 Arg-Asp' view and turn on the original dots, to see how this residue was fit in the other subunit. 4. Quit from KiNG.

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http://kinemage.biochem.duke.edu/teaching/aca2005/

previous: Part 1

Jane & Dave Richardson

next: Part 3

Structure Validation Workshop

Using All-Atom Contact Analysis for Model Diagnosis & Repair

Part 3: Local backbone adjustments using KiNG

(done on file1mo0).

Not all disallowed contacts can be resolved by rotamer selection and sidechain dihedral adjustments; sometimes, local backbone adjustments are needed to sample a broader space. This lesson looks at a set of clashes about Ile120 of 1mo0, a C. elegans triose phosphate isomerase. We use KiNG - a Java-encoded kinemage viewer - to select an appropriate rotamer and adjust the mainchain and sidechain.

Survey the problem

1. The files1mo0H.pdb,1mo0H-contact.kinand1mo0.omap.tgzare available on the software CDs and are probably in your working directory already; if not, add them. The first two were produced in MolProbity, and the 1mo0 2Fo-Fc map was previously downloaded from the EDS site (http://fsrv1.bmc.uu.se/eds/). 2. In your working directory, start KiNG, and open the 1mo0H-contact.kin file from within KiNG. (KiNG keeps its own current directory so it can find all of its helper programs; in the setup here you will need to navigate up and back down again to your own working directory each time you open a file from KiNG. 3. Lower the display overhead on your computer by clicking off components of the kinemage (your choice, but try it without wide & close contacts, small overlap and others). 4. Center the display on Ile A120 by using theEdit>Find point...menu choice. Adjust zoom and slab to inspect clashes about Ile A120. (zoom with right-click + vertical mouse drag; slab with right-click + horizontal drag; translate in Z with middle click + horizontal drag in the upper region of the display) 5. Load the 2Fo-Fc electron density map via theTools>Structural biology >Electron density mapsmenu item.

Try rotamers

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1. The rotamer library is invoked in KiNG via the menu item:Tools >Structural biology>Sidechain rotator. When invoking this tool, another tool, the Model manager, will prompt you to open the PDB file for your model. Answer that query (1mo0H.pdb) and a further question about alternate conformations (answer 'A' this time!) and move on by ID'ing the residue of interest (middle click on any side-chain atom of Ile A120). A pop-up window will appear allowing sidechain dihedral adjustments and rotamer selection. 2. To update contact dots automatically as you adjust the sidechain, check the 'Probe dots' box in the Model manager window. Turn off the old 'dots' button in KiNG's side panel. 3. Try the available rotamers to see if any look promising to alleviate the clashes seen for the residue. Probe takes a second or two to update the contacts. 4. Pay particular attention to the "pt" rotamer, which shows a high degree of clashes, but note that the clashes are all on one face of the residue. This is a key observation leading to resolution of the clashes.

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Chiropraxis: the backrub

1. Since all clashes are to one face of the residue in the pt rotamer, if we could rotate the entire sidechain away from that face, then we might get to a position from which we can eliminate the clashes. Such a motion requires movement of the backbone to swing the Cbeta. KiNG's backbone tool allows for such "chiropraxis" of the backbone by rotation about the axis between Ca(i-1) and Ca(1+1), called a "backrub", plus rotation of the two individual peptides. 2. To rebuild this trouble spot in the 1mo0 model press the "Backrub" button in the rotation dialog box. Uncheck the box for idealizing the flanking sidechains (if they already fit their density well, it's better to keep them as is). Turn on the probe dots and/or ED map and adjust the pt rotamer to remove the clashes, keep fairly good geometry and satisfy the ED map; mainly use the C-alpha hinge tool, but also you can fine-tune the rotamer chi angles.

3. Measure phi, psi, tau, chi1 and chi2 of your new Ile A120 (use theTools>Measure angle & dihedraltool). Compare these measures to those of Ile B120 (get there withEdit>Find point..., or the Views menu).

Save your new model

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When you have resolved the clashes to your satisfaction, accept your new Ile A120 model: 'Release' the checked out residue from both tools and accept. If desired, save the modified pdb file in the Model Manager.

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http://kinemage.biochem.duke.edu/teaching/aca2005/

previous: Part 2

Jane & Dave Richardson