BCH 6744C: Macromolecular Structure Determination by X-ray Crystallography. Practical 5 Refinement and Structure Function Analysis

Transcription

1 BCH 6744C: Macromolecular Structure Determination by X-ray Crystallography Practical 5 Refinement and Structure Function Analysis

2 Introduction The ultimate goal of most structural analyses is to relate the information obtained to the function of the macromolecule under investigation. You have crystallized your protein, collected diffraction data for it, processed that data, phased the reflections, using the method of molecular replacement, and done preliminary refinement of your model. In this final practical, you will further refine your model using CNS 1 and then explore the structure with O 2 to see how it relates to the functional properties of lysozyme. You will also check the quality of you model, using the program Procheck 3 and you will make figures of your model and active/functional sites using the program Bobscript Brunger, A.T., P. D. Adams, G. M. Core, W. L. Delano, P. Gross, R. W. Grosse-Kunstleve, J.-S. Jiang, J. Kuszewski, N. Nilges, N. S. Pannu, R. J. Read, L. M. Rice, T. Simonson, and G.L. Warren. Crystallography and NMR system (CNS): A new software system for macromolecular structure determination Acta Crystallogr.D54: Jones, T.A., Zou, J.-Y., Cowan,S.W. and Kjeldgaard,M. (1991). Improved methods for building protein models in electron density maps and the location of erros in these models. Acta Cryst. A47, Laskowsky, R.A., MacArthur,M.W., Moss,D.S. and Thornton, J.M. (1993). PROCHECK: a program to check the stereochemical quality of protein structures. J.Appl. Cryst. 26, Esnouf, R.M Bobscript: an extensively modified version of MOLSCRIPT that includes greatly enhanced coloring capabilities. J. Mol. Graph. Model. 15,

3 Refinement of Model (now based on your observed Data) At this point in the practical you should have a first model of your lysosyme that represents your observed diffraction pattern better than the model you used for molecule replacement. WHY? This should be saved as start1.pdb. Step 1. Conversion of your model pdb file to CNS format and generate a structure file. >cns_edit generate.inp -TO EDIT THE INPUT FILE Input - pdb file name is start1.pdb Output - generate1.mtf generate1.pdb Save editted file as generate1.inp >cns < generate1.inp > generate1.log & - TO RUN THE JOB Step 2. Crystallographic simulated annealing refinement (anneal.inp) For most refinement procedures, you now need to run an annealing refinement, which as discussed in Lecture 15, is a procedure whereby the heating of the molecule is simulated, to displace side-chains etc from local minima, then the molecule is allowed to cool, returning atoms to positions that should be the global energy minima. But because you ran anneal in Practical 4 and you used a very good initial model, your lysozyme side-chains most likely fit well into density already. If that is the case, skip this step, if not then run anneal as you did last week. Use the generate1 files generated in Step 1. Step 3. Crystallographic conjugate gradient minimization refinement - All atoms, positional. >cns_edit minimize.inp - TO EDIT THE INPUT FILE Input - generate1.mtf generate1.pdb fobs.cv (you already have this file from last week) Space group is P (tetragonal) Cell Parameters a=79.097, c= CHANGE ACCORDING TO YOUR CELL PARAMETERS Resolution Use default settings. Output - minimize1.pdb Save editted file as minimize1.inp >cns < minimize1.inp > minimize1.log & - TO RUN THE JOB Step 4. Restrained, individual B-factor refinement >cns_edit bindividual.inp - TO EDIT THE INPUT FILE Input - generate1.mtf minimize.pdb fobs.cv Space group is P (tetragonal) Cell Parameters a=79.097, c= Use default settings. Output - bindividual.pdb Save editted file as bindividual1.inp >cns < bindividal1.inp > bindividal1.log & - TO RUN THE JOB 3

4 Step 5. Pick water molecules in electron density map >cns_edit water_pick.inp - TO EDIT THE INPUT FILE Input - generate1.mtf bindividal.pdb Space group is P (tetragonal) Cell Parameters a=79.097, c= fobs.cv Resolution limits Calculate an Fo-Fc map Residue name for picked waters should be HOH First residue should be reset to 200 Output - water_pick.mtf water_pick.pdb Save edited output file as water_pick1.inp >cns < water_pick1.inp > water_pick1.log & -TO RUN THE JOB Density Map Visualization and Model Building Analysis Step 1. Make an electron density map using phase information from a model >cns_edit model_map.inp - TO EDIT THE INPUT FILE Input - water_pick.mtf water_pick.pdb fobs.cv Calculate an 2Fo-Fc map Output - model1.map Save edited output file as model1_map.inp >cns < model_map.inp > model_map.log & -TO RUN THE JOB Step 2. Convert the map from CNS format to O format >mapman (line of command on terminal - then answer prompts) Input - re (for reading a map) m1 (for map) model1.map (for map file name) cns (for type of map being read) Output - wr (for writing out map) which map: m1(for the map being read) map file name: model1.omap (for output map file name) format : brix (for O map format) Quit - to exit program - YOU NOW HAVE A NEW MAP FOR O. Step 3. Read model coordinates into the program O >o7 - TO START THE PGM O Enter lyso.o when it asks for a O file name - RUNNING O AFTER THE FIRST TIME Then use CRs to get to the graphics window. 4

5 *Minimize your graphics window and return to unix window running O* >pdb_read water_pick.pdb lyso (to read your coordinate file into O, with a molecule name lyso) And use CR for the questions - i.e. use defaults. OR >pdb_read -And follow prompts for file and molecule names. Filename: water_pick.pdb molecule name:lyso And use CR for the questions -i.e use defaults >mol lyso (to activate the molecule that you have just read in and want to manipulate) >obj lyso z ; end (to display the whole molecule on the graphics window) If you want to display only a portion of the molecule between residues X1 and X2 you will type: >obj cat z x1 x2 end to display an object called cat with residues x1 to x2) (For example to display you catalytic residues in the molecule) >s-l-s lyso (to list the residues in your coordinate file) >cent_xyz xyz (highlight xyz coordinates from any residue in the list and click the middle mouse button to display it here). This will center the molecule at those coordinates. OR >cent_a x (x=residue number, to center on a residue) *Re-activate your graphics window to view the molecule* Step 4. Visualize model1.omap in O *Back to the Unix window running O* >fm_file (follow prompts to enter calculated map file name and the map name in O Calculated map file is model1.omap, map name in O can be anything - best to use default. >fm_setup (to set up 3 map files with different sigma levels, 1-3, in different colors). >fm_draw (to display maps on the graphics window). *Back to the graphics window* Structure Analysis Step 1. Compare your new coordinate file (water_pick.pdb) with the old (start1.pdb). Can you see any differences? HINT - Use C or ribbon or tapeworms representations to make things simple if you need to. Step 2. Read in the old map. Compare with the new map. Hint: Where can you look to see if there has 5 been any improvements? Ideally you should re-adjust your model again to fit the new density and go through the refinement cycle again - But we will not do that due to time restrictions.

6 Model Quality and Secondary structure assignment - PROCHECK This program will check the quality of the model that you have built with regards to main and side-chain torsion angles, bond angles, bond lengths etc and assign secondary structure. Step 1. Change directory to Procheck by typing cd../procheck (if you are in the Model sub directory) cp../model/water_pick.pdb. rlogin to the phage or parvo SGIs if you are on gemini or circo >procheck water_pick.pdb Line command to run program. This will generate numerous postscript files. You only need water_pick_01.ps and water_pick_06.ps. - The first is your Ramachandran plot and the 6th file will give you your secondary structure assignments. Step 2. Look at the first file >xpsview filename - To view the file on the screen. You will need the Ramachandran plot for your paper, plus the numbers about the % of the residues in the different regions. Step 3. Look at the 6th file For making secondary structure plots of your molecule, you will need to determine which residues are -strand, -helix and coil. Lucky for you DAVE has done this already and put it into your Bobscript area, input filename is lyso.in. Functional Analysis Step 1. Map the catalytic residues that you found out for home work onto the structure. >obj cat z x1 x2 end - If the residues are adjacent in the sequence OR >obj cat >z x1 x1 >z x2 x2 >end To display an object called cat with the residues highlighted on the graphics window. Any thing special about the location(s)/interaction(s). of the residues? Make notes for you paper. Step 2. Map an additional functional property on the molecule. Any thing special about the location(s)/interaction(s). of the residues? Make notes for your paper. Making Pretty Pictures Make figures of your model and active/functional sites using the program Bobscript 4. 6