BCH 6744C: Macromolecular Structure Determination by X-ray Crystallography. Practical 4 Phasing and Model Building

Transcription

1 BCH 6744C: Macromolecular Structure Determination by X-ray Crystallography Practical 4 Phasing and Model Building

2 Introduction The phase problem is one the major rate limiting steps in X-ray crystallography, the other being the ability to grow high resolution diffraction crystals from your purified sample. This problem arises because we are unable to measure the phase angle of the diffracting waves collected from the Bragg planes in a crystal when exposed to X-rays. In this practical you will use one of the methods discussed in Lecture 11, Molecular Replacement, to obtain initial phases for your lysozyme data, that you can use to calculate an electron density map for the atoms in the crystal that generated the diffraction data that you processed last week. The model that you will use is from the PDB data base, accession number 1HEW, filename 1HEW.pdb. You will use a package of computer programs, Crystallography Nuclear Magnetic Resonance Systems (CNS) 1, to accomplish your goal of phasing. This is a comprehensive package of programs that handle the different steps involved in structure determination and model refinement, using the methodologies of X-crystallography and NMR. Due to time restrictions you will only use some of the subroutines available for these types of manipulations, but you are advised to review the list of input files available via the web based GUI to get an idea of the programs capabilities by typing cns_web on the SGIs. The input files for the subroutines that you will use in this practical will also use a GUI for editing. Once you have obtained an electron density map, you will use the graphics program O 2 to view your map and interpret the electron density and converting from a CNS to an O format using the program Mapman 3. The 1HEW model that you will use for phasing has been modified with interactive substitutions and deletions. You will be expected to look through the density and adjust the structure based on your calculated density. Remember that the phase dominants the structure factors, thus your map is still very biased towards the 1HEW model that you used, but your map should be of high enough resolution and good quality to see where mutations have been made in the model. 1. 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, G.J. Kleywegt & T.A. Jones (1996). xdlmapman and xdldataman - programs for reformatting, analysis and manipulation of biomacromolecular electron-density maps and reflection data sets. Acta Cryst D52,

3 Phasing Lysozyme Diffraction data Step1. Conversion of model pdb file to a CNS format & Generate structure file for protein (dna/rna, water, ligands and/carbohydrates >cns_edit generate.inp Input - pdb file name is 1HEW.pdb Set the b factor flag to 15 and occupancy to 1. Output file names - generate0.mtf generate0.pdb Save edited file as generate0.inp >cns < generate0.inp > generate0.log & (to run job in the background) Step 2. Convert output hkl file from Scalepack to cns format file. >to_cns your-file.output fobs.hkl (line of command on terminal) Step 3. Setup test array for cross-validation (free R) using a random selection of data. >cns_edit make_cv.inp Input - fobs.hkl Space group is P (tetragonal) Cell Parameters a=79.097, c= (Hint - there is more to add!) 5% Output file name - fobs.cv Save edited file as make_cv0.inp >cns < make_cv0.inp > make_cv0.log & Step 4. Crystallographic rigid-body refinement - to phase data >cns_edit rigid.inp Input - generate0.mtf generate0pdb fobs.cv Space group is P Cell Parameters a=79.097, c= Use default settings for number of steps (20) and cycle (1). Output - file name - rigid0.pdb Save edited file as rigid0.inp >cns < rigid0.inp > rigid0.log & 3

4 Density Map Visualization and New Model Building Step 1. Make an electron density map using phase information from a model >cns_edit model_map.inp Input - generate0.mtf rigid0.pdb fobs.cv Calculate a 2Fo-Fc map Output file name- model0.map (route name is model0) >cns < model_map0.inp > model_map0.log & 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) model0.map (for map file name) cns (for type of map being read) Output - wr (for writing out map) m1(for the map being read) model0.omap (for output map file name) brix (for O map format) Quit - to exit program Step 3. Read model coordinates into the program O >o7 (use default settings to get to the graphics window) *Minimize your graphics window and return to unix window running O* >pdb_read rigid0.pdb hew (to read your coordinate file into O, with a molecule name hew) OR >pdb_read and follow prompts for file and molecule names. filename:rigid0.pdb molecule name:hew >mol hew (to activate the molecule that you have just read in and want to manipulate) >obj hew z ; end (to display the whole molecule on the graphics window) (If you want to display only a portion of the molecule between X1 and X2 you will type: >obj sect z x1 x2 end to display an object called sect with residues x1 to x2) >save (save new binary file as lyso.o) >s-l-s hew (to list the residues in you 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* 4

5 Step 4. Visualize model0.omap in O *Back to the Unix window running O* >read menu.o (to read in a user menu for manipulations in O) Activate menu under pull down menu icon on graphics window) >fm_file (follow prompts to enter calculated map file name and the map name in O) Calculated map file is model0.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* Activate maps under density pull down menu icon Step 5. Locate mutations in model, build and save new coordinate file *Switch on crystal eyes emitter and use crystal eyes to view molecule and density* Look at the polypeptide chain. Note how it fits into the density. 4 mutations have been made in the coordinate file F34G W62G W63G R128 has been deleted. The density for the correct type of residues should be evident. *In the graphics window* Use mutate under the Build pull down menu to mutate residues back to what they should be. Each time you do this, your molecule will be deleted. Redefine in the unix window: >obj hew z ; end (to re-display molecule again in the graphics window). *Back to the Unix window* Use mut_ins, move_zone and merge_atoms to create coordinates for the deleted residue: >mut_ins (on the Unix window and follow prompts) molecule name: hew after which residue:127 new residue name and type: 128 arg >move_zone (on the Unix window, then follow instructions on graphics window) Double click on a neighboring ARG, e,g, X, on the graphics window Use right-hand-side dials to move residue into density to R128, leave it there. * Back to Unix window* >merge_atoms (on the Unix window and follow prompts) molecule name and which residue to merge from:hew X molecule name and which residue to merge to:hew 128 5

6 Step 5. Contd.. *Back to the Graphics window* Click no to de-select move_zone manipulation and NOT save coordinates of moved arg residue X. You now have residue R128 and the coordinates for the neighboring ARG X is restored. You are done! NOTE If this does not work, make a mock.pdb file with just the coordinates for any R residue, read it into O with odb-read and move it into the density at residue 128. *Back to Unix window* pdb-write start1.pdb (to output your new pdb file) save (to update binary.o file) lyso.o stop (to exit from program and save all your work). ******NEVER use QUIT to exit if you want to save work.******* If you finish early - explore your unit cell. E.g. Use crystallographic symmetry to see where all your molecules in the units are. >pdb-readi start1.pdb hew2 >mol hew2 obj hew2 z ; end *On the Unix window >sym_setup (follow directions to set up symmetry for your molecule hew2). Molecule name: hew2 Cell parameters: enter your cell parameters Symmetry: P >sym_sphere (follow directions to display symmetry molecules) Molecule name:hew2 Name for symmetry molecules:sym Radius for symmetry:50 (as an example) >>>This will generate a number of lysozyme molecules related by crystallographic symmetry. *Back to graphics window* Look to see where your molecules are. Is it consistent with the space group? Can you see how symmetry related molecules interact? 6

7 Practical 4 - Assignment The structure of lysozyme, published in 1965, was the first enzyme structure ever determined. This offered the possibility of a molecular level of explanation for its mechanism. 1. What is the function of lysozyme in vivo. 2. Which polypeptide residues are essential for its catalytic activity? Keep a copy for next week. 3. Identify additional residues on the lysozyme molecule involved in another biological function, e.g. an antigenic site involved in antibody binding during neutralization, list them. Keep a copy for the next week. 7