Refinement/Structure Validation
Structural Molecular Biology Laboratory, ChemM230D

Ramachandran plot from a high quality model.
Suggested Reading Materials

1) Electron density map intepretation by T. A. Jones and M. Kjeldgaard, Methods in Enzymology, Vol 277 (1997) pages 173-207.  This link is a 17 page PDF file

2) Refmac5 manual by Garib Murshudov, University of York and CLRC, Daresbury Laboratory and Alexei Vagin, Martyn Winn, Roberto Steiner, Eleanor Dodson, Maria Turkenburg, Kim Henrick, Liz Potterton.

3)Verification of Protein Structures: Patterns of Nonbonded Atomic Interactions by C. Colovos & T. O. Yeates.  Protein Science (1993), 2, 1511-1519.  Describes ERRAT program.

4) Assessment of protein models with three-dimensional profiles by Roland. Luthy, James U. Bowie, & David Eisenberg. Nature (1992), 356, 83-85.

5) powerpoint presentation

6) Quality Control Server

7) UCLA Saves Server


Assignment & Procedures
5th Assignment: 
Structure Refinement Table
due at the end of lab period.
Objective: To produce a crystallographic structure refinement  table typically found in structural biology journals. 

Method: Copy the format of the table on the left.  Substitute the data from your pdb file for the data in the table.

A typical Table 3, adapted from Blaszczyk et al.,
  Crystallographic and Modeling Studies of RNase III Suggest a Mechanism for
     Double-Stranded RNA Cleavage, Structure, Vol. 9, 1225-1236, December 2001.

Part One: Refinement, difference Fourier map calculation, and model building.
Objective: To refine the PMSF complex structure and model the PMSF inhibitor in the Fo-Fc electron density map.

Strategy: Use the coordinates of the native proteinase K structure output by ARP/wARP as a starting point for the refinement of the PMSF complex.  Refine using maximum likelihood restrained refinement for 5 cycles. Calculate difference Fourier maps (fo-fc and 2fo-fc) and look for large peaks in the fo-fc density.  One of these peaks should correspond to PMSF, the others may be due to water molecules, side chain movements, or bits of the model that were not built by ARP/wARP previously.

Procedures: Login to Bernal, Laue, or Bragg. Change directories to your working directory. Type the word "ccp4i" to start the CCP4 programs GUI. Define yourself as the user in the "Directories & Projects Dir" button.  Select the options as shown in the example on the right.   Check the R-work and R-free.  Begin an O session by typing "ono" and in the graphic window type "@difmacro".  This command will read in the refined proteinase K model and a few libraries that will aid in model building.  Tear off the following sub-menus form the "menus" menu: Objects, User, Fake Dials.  You should be able to see three objects: the proteinase K model (prok1); the 2fo-fc difference Fourier map (2fofc); and the fo-fc difference Fourier map contoured in positive(pos/blue) and negative (neg/red) contour levels.  You will see a sidechain movement near Trp A9; fix it with RSR-rotamer. You will see a missing dipeptide between A171-A174 (why would this be missing from the model?); build it in with "build_resi prok1 a172 f" followed by "build_resi prok1 a173 f". Adjust the conformation by "grab residue". Lastly, you will see some density for the PMSF.  Read in pms.pdb and position it near the active site serine.  Write out coordinates for your modified protein (prok_pmsf1.pdb) and coordinates for you pmsf molecule (pms.pdb).  Catenate the pmsf coordinates to the bottom of prok_pmsf1.pdb and use this file for the next round of refinement.

pmsf bondsPMSF

PMSF binds to active site serine residue

refmac5 rigid body refinement

Refmac5 GUI used for initial refinement of the proteinase K structure against the PMSF complex data and produce difference Fourier maps.

Part Two: Refine your PMSF complex
Objective: To refine the PMSF that you have modeled.



Procedures: Refine using the PMS dictionary file and the model containing pmsf coordinates.

Mike's spiel.


Refmac5 refinement window.
Part Three: Structure vaildation.
Objective: Check the quality of your model with Ramachandran,Errat, and Verify3D plots.

 Run procheck, errat, verify3D or just do it all with the SAVS server.









Fill in the blanks with Proteinase K data listed in your PDB file.

Instructor's preparations

Back to CHEM M230D course syllabus 

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