BIOINFORMATICS II BIOL 4550
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This 33 page Class Notes was uploaded by Ezequiel Schaefer on Monday October 19, 2015. The Class Notes belongs to BIOL 4550 at Rensselaer Polytechnic Institute taught by Christopher Bystroff in Fall. Since its upload, it has received 50 views. For similar materials see /class/224828/biol-4550-rensselaer-polytechnic-institute in Biology at Rensselaer Polytechnic Institute.
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Date Created: 10/19/15
Bioinformatics 2 lecture 2 Where do protein structures come from X ray crystallography What does quotresolutionquot mean What is a quottemperature factorquot NMR What is an quotensemblequot Why so many isotopes The Protein Data Bank MOE X ray Crystallography quot1 1quot 3 V229 Electron density map Diffraction pattern The wavelength of X rays is about the size of an atom Mlcromve Visible H Wavelength of Xerays used in crystallography AL54A Frequency cA 2x1018 5391 equot oscillates in an electric eld 06 oscillation is the same frequency as the X rays Atomic nuclei don t oscillate much Electron motion is Slow compared to Xray oscillation 1A atom hydrogen In other word Xrays see electro 39 as if they were st 39 L 39i Diffractometer setup crystal beamstop quot6 X ray beam I I goniostat A protein crystal usually frozen is immersed in a powerful beam of monochromatic X rays An X ray diffractometer All points in a re ection plane scatter in phase Angle from beam to detector is 28 Scattering from any of these points has the same pathlength therefore all e in the re ection plane scatter in phase Note we are now inside the crystal Parallel re ection planes separated by d scatter in phase Bragg s Law Ilk2d sinG The two red lines photon paths differ in length by a Whole number of waves Th eref O repr r6 0 e He SentS a Spot 0 set of BH the det Tag 60 g p t01 ane on onlo D t o o I o I o I o I o o u o Q o I I I to o o I I o o I I 0 I I I I o 0 o o a o I o o a o I II I I a a I o a o o o I I 0 o I o a o I u o a o I o I o o I I O n I I o 0 a o I o I o I u O I C o o a I I o o a o I I a I o o I o D o I I a o I a a n I no o o o I o o I a o o I I o o I o o 0 I I I a o I o o I II a a u I o a o o o o I u I o no o o I I I 00 a o o I I I u o I o a I I o I o I o o o o o o I o o u a I o I o a o n I I a 0 I o o 0 I I I a o I o o o I o I u o I o a 0 u u I o o o n I 0 I o a o n I I o o a o o I s i WWW 14 m f 1434 39 mutants v 3 A 3 3 95 u c l u 1 u all lsll1lsssl u m l The image of the molecule is reconstructed from the Bragg planes Eli Iquot l R 7 7 M E If V llll mi m k x H i i i i i i i i MttxxX i i i H Can you see the image of Bragg 7 E E E Fourier Transformation This is the principle behind J PEG compression HMHHMwimmmwwwwmmWWW Fitting the model to the density 3D electron density map electron density at every point in space Visualized by drawing 3D contours Since we know the amino acid sequence and we know what the amino acids should look like we can quot tquot a model to the density Coordinate re nement Each atom is moved in XY and Z until 1 good stereochemlstry 1s achleved r the Rfact0r 2 there is a good match between the atoms and the density Each atom is assigned a B factor or quottemperaturefactorquot to better t the density high B density pro le 4 parameters are re ned for each atom 10W B density pro le Re ned coordinates are deposited in the Protein Data Bank WWWI CSbOI g How to View a PDB le In Netscape go to WWWrcsborg make a bookmark Search for the protein With PDB code quot1CA2quot Download it Use the quotjotquot editor or quotViquot if you prefer to View the le quotjotquot and quotViquot are UniX commands Find the lines on the following slide as they are discussed What39s in a PDB le HEADER CMPND REMARK lines contain reference information including quality measures HET FORMUL Any attached ligands are de ned here OHELIX SHEET TURN The locations of secondary structures in the chain are de ned here OATOM lines contain information about the atoms more later HETATM hetero atoms Lines are like ATOM lines but the atom names and group names are de ned in the HETNAM lines There is no direct information about What atoms are bonded to What This is determined by distances or atom names No direct information about the formal or partial charges on atoms Partial charges may be calculated using quantum mechanics Anatomy of a PDB le the ATOM line 0616 1613 2082 100 6881 1 8DFR 152 ATOM N VAL 1 6 ke word ATOM 7 1 1 atom number 67 80 footnotes and labels 61 66 Temperature factor 13 16 atom name 55 60 Occupancy factor 17 altloc indicator 47 54 Z coordinate 18 20 residue name 39 46 Y coordinate 22 chain identi er o tional 23 26 residue number 27 insertion code optional 31 38 X coordinate 28 30 not used Usually but not always residues are numbered sequentially 123 etc Often the numbering starts from a number other than 1 Coordinates are in orthogonal angstroms by convention May be converted to crystallographic coordinates using CRYST lines Exploring carbonic anhydrase a crystal structure Try these Rasmol commands cartoons color structure restrict sheet restrict helix cartoons restrict not helix and not sheet cartoons How much of each secondary structure iS lere rotate translate scale Exploring carbonic anhydrase a crystal structure More Rasmol commands COL select all cartoons off select protein wireframe Ca spacefill 80 set picking distance now pick atoms left mouse button What is the average distance between bonded atoms atoms separated by two bonds three bonds What is the distance between neighboring alpha carbons Rasmol Viewing temperature factors Temperature factors also called Bfactors are the results from crystallography that measure the disorder of each atom Find the Bfactors in the le 1ca2pdb What is the minimum and maximum B View lca2 in Rasmol Color the atoms by temperature either by selecting the menu item colours gtTemperature or by typing at the prompt color temperature You can select atoms based on their B factors using for example select temperature gt 2000 Note you must use lOOXB to select Mean square displacement ltu2gt is proportional to B ltu2gt B87I52 NMR Nuclear Magnetic Resonance Isotopes that have nuclear spin 2 12 1H 13C 15N and 31F can adopt two orientations in a magnetic eld H At equilibrium slightly more spins are aligned With the eld than against it down 4 up Flipping from up to down absorbs radio waves of a frequency that depends on its precession rate Which depends on its environment Steps in Protein NMR Overview I Grow protein in 13C andor 15N enriched media 2 Purify and concentrate protein 3 Collect NMR spectra 23 or 4 dimensions 4 Assign the peaks TOCSYCOSY 5 Assign distance constraints NOESY 6 Solve the distance geometry problem Collecting NMR data Pulse sequence de nes Which atoms are broadcasting Absorption spectrum indicates Which atoms are receiving Short pulses quotringquot through a spin system gt TOCSY used to assign peaks to atoms Long pulses resonate through space gt N OESY used to assign distances between atoms D A Assigning resonances A TOCSY experiment nds cross talk between 1H in a quotspin systemquot Characteristic sets of resonances allow the easy identi cation of amino acids A COSY experiment nds cross peaks between 1H that are separated by 2 or 3 bonds Chemical shifts for ILE NH 819 H OH 423 3H 190 yCH3 097094 yCHZ 148119 H 6CH3 089 This 1H is not part of the spin system Isoleuc1ne sp1n system TOCSYCOSY for 116 Isoleuclne Ila I A3MPTB3X A AA 39 SCH 0393 IY yCH3 095 T TOCSY peaks red diamond 7H COSY peaks blue circle 1 48 SH 130 00 H3N Clt H D uH 7 423 H 3 CH3 9H2 CH3 NOESY nonlocal neighbor 1H NOESY spectra tell us Which 1H are physically close in space causing the Nuclear Oberhauser Effect NOE 1H With NOEs show absorbsion at one H39s frequency When pulsed With radio waves at the frquency of the other NOE pairs are constrained to a de ned distance Distance geometry is the problem of solving for the 4 atomic positions that satisfy the constraint distances and the stereochemistry simultaneously 39 Molecular dynamics is used to re ne the solutions NMR result an ensemble of Some regions have fewer constraints than others due to fewer observed NOEs Other types of experiments Additional information about the conformation may be gained by 0 HD exchange Deuterium 2H is invisible to NMR Disappearing 1H39s tell us Which ones are exposed to solvent Especially amide NH39s 0 Temperature sensitivity of resonances Chemical shift oh 1H changes With T less if H bonded NSQSY Direct coupling of 15N to 1H through a single bond Xray versus NMR of structures in the gt80 16 PDB Coordinates Cartesian One set per internal converted to molecule ensemble of Cartesian Requires pure protein large perfect pure protein high conc no crystals aggregation MW limits gt500kD sometimes possible lt30kD Advantages more precise Temperature fast does not require factors crystals HD Exchange Disadvantages ambiguous sidechains imprecise limited to difficult and time smaller macromolecules consuming limited to well ordered molecules Download an NMR structure From the PDB nd and download the structure With PDB code quot112Vquot defensin Name it quot 112Vpdbquot Open Rasmol With this structure quotrasmol 112Vpdbquot From the menu Displaygtbackb0ne Identify the disordered regions set picking ident Click to get the sequence numbers Frequently asked questions With PDB les What do I do about Missing sidechain atoms If needed add the atoms and energy minimize them Use model building tools Name the atoms correctly Missing backbone atoms If small model using a loop search and energy minimization Large missing sections cannot be cured Multiple occupancy occ lt 100 Remove all but one of the multiple copies using a text edtor Solvent atoms HOH usually In general don t display them Useful only When doing all atom molecular dynamics Frequently asked questions With PDB les What do I do about Ligands Useful for docking studies Should be unselected before drawing surfaces May be saved as a separate model Multiple chains Unless the molecule is a multimer use only one chain Use a text editor to remove all but one Avoid this problem by downloading the PDB Biological Unit le Multiple NMR models Display all models Find the disodered parts Then edit the le keeping only one model and removing the disordered parts 52 CHE MI CAL 39 COMPUTING GROUP INC Introduction to the Industrial Strength molecular modeling program MOE Start moe on SGI use moe ngVisual 0X3l Familiarize yourself With the mouse Help gtMouse Familiarize yourself With the graphical user interface GUI Help gtTutorials gtGetting Started click on GUI Run rst part of MoeTour Help gtTutorials gtGetting Started
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