Bioinformatics 2: Lecture 1 - Protein Structure Determination Methods, Study notes of Biology

Download Bioinformatics 2: Lecture 1 - Protein Structure Determination Methods and more Study notes Biology in PDF only on Docsity! 1 Bioinformatics 2 -- lecture 1 Where do protein structures come from? X-ray crystallography What does "resolution" mean? What is a "temperature factor"? NMR What is an "ensemble"? Why so many isotopes? The PDB Rasmol here do protein structures co e fro ? X-ray crystallography hat does "resolution" mean? hat is a "temperature factor"? N R hat is an "ensemble"? hy so many isotopes? The P B Ras ol Web site for the course www.bioinfo.rpi.edu/~bystrc/courses/biol4550/ From my homepage (www.bioinfo.rpi.edu/~bystrc) click on "Teaching" then "Bioinformatics 2" 2 What is covered in this course. •Protein structure determination methods •Coordinates, rotation, superposition •Protein classification •Homology modeling •Molecular mechanics, dynamics •Force fields and molecular simulations •Molecular surfaces •Secondary and tertiary structure prediction •Ligand docking •Protein design, ligand design Rasmol MAGE InsightII What you need to know.. ...to take this course: Unix basics (see "unixetc.pdf" on the web site) chemistry, organic and physical biology, biochemistry basic matrix algebra Bioinformatics I: Sequence analysis (very helpful) 5 Diffractometer setup X-ray source X -r ay d et ec to r goniostat beamstop x-ray beam diffracted x-rays crystal A protein crystal (usually frozen) is immersed in a powerful beam of monochromatic X-rays. An X-ray diffractometer 6 All points in a reflection plane scatter in phase same angle, θ, with beam and scattered Xray Scattering from any of these points has the same pathlength, therefore, all e- in the reflection plane scatter in phase. θ θ 2θ detectorso ur ce Note: we are now inside the crystal Parallel reflection planes separated by d scatter in phase d θ nλ=2d sinθBragg’s Law: The two red lines (photon paths) differ in length by a whole number of waves. 7 Therefore, one spot on the detector represents a set of Bragg planes. d The image of the molecule is reconstructed from the Bragg planes. 10 How to view a PDB file In a UNIX window, type "netscape" Go to www.rcsb.org (make a bookmark!) Search for the protein with PDB code "1CA2" Download it. Use the "jot" editor (or "vi" if you prefer) to view the file. ("jot" and "vi" are Unix commands) What's in a PDB file •Header lines contain author and method information, including quality measures. Any attached ligands are defined here. •"ATOM" lines contain information about the positions of atoms, their names and numbers, what chain and what "type" (i.e. amino acid) they belong to, and possibly other qualities (temperature factor, occupancy). •No direct information about what atoms are bonded to what. (determined by distances) •No direct information about the formal or partial charges on atoms. (pKa's are determined experimentally by titration. Partial charges may be calculated using quantum mechanics.) 11 In class exercise: PDB and Rasmol A molecular display program. You can download the latest version (2.7.2.1 if possible) for your computer from: http://www.bernstein-plus-sons.com/software/rasmol/ In a UNIX window, type “rasmol” Open the downloaded PDB file. (or you can start by typing "rasmol file", where file is a PDB file.) 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 shift + rotate translate scale How much of each secondary structure is there? 12 Exploring carbonic anhydrase, a crystal structure More Rasmol commands : select all cartoons off select protein wireframe 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? Cα Cα Nuclear Magnetic Resonance NMR Isotopes that have nuclear spin = 1/2 1H, 13C, 15N and 31P ..can adopt two orientations in a magnetic field (H). At equilibrium slightly more spins are aligned with the field than against it. up down Flipping from up to down absorbs radio waves of a frequency that depends on its precession rate, which depends on its environment. H 15 NOESY NOESY spectra tell us which 1H are physically close in space, causing the Nuclear Oberhauser Effect (NOE). 1H with NOEs show absorbsion at one H's frequency when pulsed with radio waves at the frquency of the other. NOE pairs are constrained to a defined distance. H H H H H H H Distance geometry is the problem of solving for the atomic positions that satisfy the constraint distances and the stereochemistry, simultaneously. Molecular dynamics is used to refine the solution(s). NMR result: an ensemble of structures Some regions have fewer constraints than others, due to fewer observed NOEs 16 Other types of experiments Additional information about the conformation may be gained by • H/D-exchange Deuterium (2H) is invisible to NMR. Disappearing 1H's tell us which ones are exposed to solvent. Especially amide NH's. • 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 PDB >80% 16% Coordinates Cartesian. One set per molecule. internal, converted to ensemble of Cartesian Requires pure protein, large perfect crystals pure protein, high conc., no aggregation MW limits >500kD sometimes possible <30kD Advantages more precise. Temperature factors. fast, does not require crystals. H/D-Exchange Disadvantages ambiguous sidechains, difficult and time consuming, limited to well- ordered molecules. imprecise, limited to smaller macromolecules 17 Download an NMR structure From the PDB, find and download the structure with PDB code "1I2V" (defensin). Name it " 1I2V.pdb" Open Rasmol with this structure: "rasmol 1I2V.pdb" From the menu: Display-->backbone Identify the disordered regions: set picking ident Click to get the sequence numbers. Levinthal’s Paradox How many degrees of freedom are there in a polypeptide chain, length 100 amino acids? 100 * 3 [alpha, beta, or loop] = 300 How many conformations are possible of the backbone? 3300 !!! A protein can search maybe 108 conformations per second. Can a protein try all possible states? C C C C C N N O OH H ψφ FOLDING