Download Protein Structure Validation

Protein Structure Validation
Or
How to Know
When Your Model is Finished
Tristan Fiedler
ACA Summer School 2003
Protein Structure Validation
• Types of Model Building Errors
• Why Errors are Made
• How to Detect & Avoid Errors
• Available Tools & References
References
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Intl Tables for Cryst. Vol. F : Crystallography for Biological Macromolecules. Rossman & Arnold, eds. Ch. 21
Methods in Enzymology. Vol 277, Part B. Carter & Sweet, eds. 1977. Ch. 10
Crystallography Made Crystal Clear. Rhodes. 2000. Academic Press
Principles of Protein X-ray Crystallography. Drenth. 1994 Springer-Verlag
http://www.usm.maine.edu/~rhodes/CMCC
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http://www.sb.fsu.edu/~chapman/Classes/Crystallography/password/Structure_Assessment_files/v3_document.htm
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http://como.bio.columbia.edu/tong/Public/Replace/tf/contents/contents.html
http://www.biochem.ucl.ac.uk/~roman/procheck/procheck.html
http://xray.bmc.uu.se/gerard/embo2001/modval/03.html
http://www.bmsc.washington.edu/CrystaLinks/validation.html
http://kinemage.biochem.duke.edu/validation/valid.html
http://www.cmbi.kun.nl/whatif/
http://www.ccp4.ac.uk/html/sfcheck.html
http://xray.bmc.uu.se/usf/gmrp_man.html
http://xray.bmc.uu.se/~gerard/gmrp/gmrp.html
Times to Celebrate !
Protein - Crystal - Phases - Model - Paper
Backbone - Fit Sequence - Coarse Model - Optimize - Evaluate
Types of Errors
• Entirely Wrong Fold
• Local Fold Incorrect
– Individual Subunits
– Connectivity between 2o Structure
– ALWAYS consult Experimental Map
• Main Chain thru Side Chain (> 2.7 Å)
• Frameshift
– Loops
– 2n : May go undetected
• Side Chain Conformations
– Widespread initially
– Alter rotamer not torsion angles
– Usu. Corrected by Automated refinement
Why Are Modeling Errors Made?
• Phase Errors (MIR, MAD, MR) affect maps
– Better : Area Detectors, Cryo, Density Modif, Synchrotron Radiation
• Resolution
– w/o f errors, trace chain at > 4 Å
• Inexperience
• Pressure to Publish
• Structure Factors Not Deposited
– Difficult to Assess PDB Model Quality
• Good Enough R-factor …
Crystallographic R factor
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59% - randomly placed atoms
30 to 50% unrefined - essentially correct
>30% refined - incorrect structure
25 to 30% refined - minor errors
15 to 20% refined - essentially correct
0% - perfect / currently unattainable !
http://www.sb.fsu.edu/~chapman/Classes/Crystallography/password/Structure_Assessment_files/v3_document.htm
Improper R factor Manipulation
• Removal of Diffraction Data
– Resolution Limits
– I/s cutoff
• Reduce Weight of Restraints
• Increase # Parameters in Refinement
– Remove NCS averaging
– Inappropriate Temp. Factor Model
• “Overzealous Modelling” - Overfitting
– Alternate Conformations
– Excessive Solvent Modelling
• ~2 Å : 1 H2O per amino acid residue
• ~1 Å : 1.6 H2O per amino acid residue
• Isoelectronic Ions: Na+, NH4+
How to Detect & Avoid Errors
in Protein Modelling
Avoiding Errors in Modelling (1)
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Obtain highest quality data
Treat Model as Hypothesis
Verify Chemical & Biochemical Sense
Refer often to the Experimental Map
Use Info from Well-Refined Structures
– Engh & Huber Bond Lengths & Angles
– Implemented in “O” - OOPS
• Rotamer Libraries
• Main Chain Fragments
Avoiding Errors in Modelling (2)
• After each Refinement cycle, assess:
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RSR, omit maps, Ramachandran plot
Pep-flip (outlier > 2.5 Å)
Rotamer sc-fit (outlier > ~ 1.0 Å)
Hydrogen bonding (eg. H, N, Q, H2O)
Unusual B factors, bond lengths & angles
Deviations from planarity
Large Positional Shifts
Ramachandran Plot
http://xray.bmc.uu.se/gerard/embo2001/modval/03.html
phi (C[i-1]-N[i]-CA[i]-C[i]), psi (N[i]-CA[i]-C[i]-N[i+1])
Chi-1 Torsion Angle Distribution
http://xray.bmc.uu.se/gerard/embo2001/modval/03.html
Chi-1 Chi-2 Torsion Angle Distribution
Chi-2
Chi-1
N=47,000 residues
http://xray.bmc.uu.se/gerard/embo2001/modval/03.html
R factor Alternatives
• Free R (global)
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Brunger, 1992. Nature 355: 472
Much more reliable; Harder to manipulate
Use to monitor model improvement
Helps reduce over-fitting of data
Rfree - Rcryst < ~ 0.05 (good models)
Exclude ~ 5% of data (500 - 2000 reflections)
• Real Space R (local)
– Jones et al., 1991 Acta Cryst A 47: 110
– Compares observed & calculated density
– Subsets : residues, main chain, side chain
Tools for
Detecting & Correcting Errors
in Model Building
OOPS :
Efficient rebuilding of protein structures
• Real-space fit of model to density
– Maps : 2Fo-Fc, 3Fo-2Fc, SA-omit, NCS averaged
• Main Chain Geometry
– Pep-flip, phi/psi, peptide bond planarity
• Side Chain Geometry
– Rotamer fit, Ca chirality
• Temperature factors & occupancies
– User-determined cutoff values
• Mask Violations
– For Electron-Density Averaging
• Significant Shifts
– xyz, B, occ, phi/psi, chi1/chi2
PROCHECK
procheck filename [chain] resolution
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Covalent geometry
Planarity
Dihedral angles
Chirality
Non-bonded
interactions
• Main-chain hydrogen
bonds
• Disulphide bonds
• Stereochemical
parameters
• Parameter comparisons
• Residue-by-residue
analysis
http://www.biochem.ucl.ac.uk/~roman/procheck/procheck_run.html
Procheck
Sample
Output
Ramachandran
Plot
Quality
Peptide Bond
Planarity
Bad NonBonded
Interactions
Ca
Tetrahedral
Distortion
Main Chain
Hydrogen Bond
Energy
Overall G
Molprobity : Sample Output
http://kinemage.biochem.duke.edu/molprobity/help/tutorial.html
Be Careful
&
Good Luck !