MOLEC STRU DETER XRAY
MOLEC STRU DETER XRAY BCH 6744
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This 7 page Class Notes was uploaded by Kavon Huel on Friday September 18, 2015. The Class Notes belongs to BCH 6744 at University of Florida taught by Staff in Fall. Since its upload, it has received 22 views. For similar materials see /class/206967/bch-6744-university-of-florida in Biochemistry and Molecular Biology at University of Florida.
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Date Created: 09/18/15
BCH 6744C Macromolecular Structure Determination by Xray Crystallography Practical 4 Phasing and Model Building Introduction The phase problem is one the major rate limiting steps in Xray crystallography the other being the ability to grow high resolution diffraction crystals from your puri ed 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 Xrays 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 lHEW filename lHEWpdb You will use a package of computer programs Crystallography Nuclear Magnetic Resonance Systems CNS1 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 Xcrystallography 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 cnsiweb 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 02 to view your map and interpret the electron density and converting from a CNS to an O format using the program Mapman3 The lHEW 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 lHEW 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 AT P D Adams G M Core W L Delano P Gross R W GrosseKunstleve JS Jiang J Kuszewski N Nilges N S Pannu R J Read L M Rice T Simonson and GL Warren Crystallography and NMR system CNS A new software system for macromolecular structure determination 1998 Acta CrystallogrD54905921 2 Jones TA Zou JY CowanSW and KjeldgaardM 1991 Improved methods for building protein models in electron density maps and the location of erros in these models Acta Cryst A47 110119 3 GJ Kleywegt amp TA Jones 1996 XdlMAPMAN and XdlDATAMAN programs for reformatting analysis and manipulation of biomacromolecular electrondensity maps and re ection data sets Acta Cryst D52 826828 Phasing Lysozyme Diffraction data Step1 Conversion of model pdb le to a CNS format amp Generate structure le for protein dnarna water ligands and carbohydrates gtcns7edit generateinp 3 Input pdb le name is lHEWpdb Set the b factor ag to 15 and occupancy to 1 Output le names generate0mtf generate0pdb Save edited le as generate0inp gtcns lt generate0inp gt generate0log amp 3 to run job in the background Step 2 Convert output hkl le from Scalepack to cns format file gttoicns your leoutput fobshkl 3 line of command on terminal Step 3 Setup test array for crossvalidation free R using a random selection of data gtcns7edit makecvinp Input fobshkl Space group is P43212 tetragonal Cell Parameters a79097 c3809l Hint there is more to add 5 Output le name fobscv Save edited le as makecv0inp gtcns lt makecv0inp gt makecv0log 8 Step 4 Crystallographic rigidbody re nement to phase data gtcns7edit rigidinp 3 M generate0mtf generateOpdb fobscv Space group is P43212 Cell Parameters a79097 c3809l Use default settings for number of steps 20 and cycle 1 Outpu le name rigid0pdb Save edited le as rigid0inp gtcns lt rigid0inp gt rigid0log amp 3 Density Map Visualization and New Model Building Step 1 Make an electron density map using phase information from a model gtcns7edit modelmapinp a m generate0mtf rigid0pdb fobscv Calculate a 2FoFc map Output le name mode10map route name is modelO gtcns lt modelmap0inp gt modelmap0lo amp Step 2 Convert the map from CNS format to O format gtmapman 3 line of command on terminal then answer prompts m re for reading a map ml for map mode10map for map file name cns for type of map being read Output wr for writing out map mlfor the map being read mode10omap for output map le name briX for 0 map format Quit to eXit program Step 3 Read model coordinates into the program 0 gt07 3 use default settings to get to the graphics window Minimize your graphics window and return to unix window running 0 gtpdb7read rigid0pdb hew 3to read your coordinate le into 0 with a molecule name hew gtpdb7read and follow prompts for file and molecule names lenamerigid0pdb molecule namehew gtmol hew 3 to activate the molecule that you have just read in and want to manipulate gtobj hew z end a 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 gtobj sect 2 X1 X2 end 3 to display an object called sect with residues Xl to X2 gtsave 3 save new binary le as lysoo gtsls hew to list the residues in you coordinate le gtcentixyz 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 4 OR gtcent7a X 3 eresidue number to center on a residue Re activate your graphics window to View the molecule Step 4 Visualize mode10omap in 0 quotBack to the Unix window running 0quot gtread menuo to read in a user menu for manipulations in O Activate menu under pull down menu icon on graphics window gtfm7file 3 follow prompts to enter calculated map file name and the map name in 0 Calculated map file is mode10omap map name in O can be anything best to use default gtfm7setup 3 to set up 3 map les with different sigma levels 13 in different colors gtfm7draw 3 to display maps on the graphics window quotBack to the graphics windowquot Activate maps under density pull down menu icon Step 5 Locate mutations in model build and save new coordinate file quotSwitch on crystal eyes emitter and use crystal eyes to view molecule and densityquot Look at the polypeptide chain Note how it fits into the density 4 mutation have been made in the J39 file W63G R128 has been deleted The density for the correct type of residues should be evident quotIn the graphics windowquot 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 Rede ne in the unix window gtobj hew z end a to redisplay molecule again in the graphics window quotBack to the Unix windowquot Use mutins movezone and mergeatoms to create coordinates for the deleted residue gtmut7ins 3 on the Unix window and follow prompts molecule name hew after which residue 127 new residue name and type 128 arg gtmoveizone 3 on the Unix window then follow instructions on graphics window Double click on a neighboring ARG eg X on the graphics window Use righthandside dials to move residue into density to R128 leave it there quot Back to Unix windowquot gtmerge7atoms on the Unix window and follow prompts molecule name and which residue to merge fromhew X molecule name and which residue to merge tohew 128 Step 5 Contd Back to the Graphics windowquot Click no to deselect moveizone 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 le with just the coordinates for any R residue read it into 0 with odb read and move it into the density at residue 128 Back to Unix windowquot pdbwrite startlpdb 3 to output your new pdb le save to update binary 0 le lysoo 2 stop a to exit from program and save all your work NEVER use QUIT to exit if you want to save work If you nish early explore your unit cell Eg Use crystallographic symmetry to see where all your molecules in the units are On the Unix window gtpdbreadI startlpdb hew2 7 gtmol hew2 obj hew2 z end 3 gtsym7setup follow directions to set up symmetry for your molecule hew2 Molecule name hew2 Cell parameters enter your cell parameters Symmetry P432121 gtsym7sphere follow directions to display symmetry molecules Molecule namehew2 Name for symmetry moleculessym Radius for symmetry50 as an example gtgtgtThis will generate a number of lysozyme molecules related by crystallographic symmetry Back to graphics windowquot Look to see where your molecules are Is it consistent with the space group Can you see how6 symmetry related molecules interact Practical 4 Assignment The structure of lysozyme published in 1965 was the rst enzyme structure ever determined This offered the possibility ofa 39 39 level of 39 quot for its 39 l 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
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