Macromolecular Crystallography Part II
|
|
- Phillip Baker
- 6 years ago
- Views:
Transcription
1 Molecular Biology Course 2009 Macromolecular Crystallography Part II Tim Grüne University of Göttingen Dept. of Structural Chemistry November
2 From Experiment to Model Introduction 1/56
3 So Far... From Experiment to Model 2/56
4 The Model The electron density map is the actual result of the X-ray experiment. useless: difficult to interpret. It is per se quite Model: Atom positions Atom types Relationship between atoms: secondary structure, domains, etc... Biologically/ Chemically the model is the final goal of crystallography From Experiment to Model 3/56
5 Visualising a Model ball and stick CPK (space filling) C α trace(smooth) C α trace (coloured by B-factor) ball-and-stick (coloured by B-factor) ribbons The PDB File 4/56
6 Storing Structural Data: the PDB-File Macromolecular Structural Data from crystallography or NMR are stored at the Protein Data Bank (PDB, or the Nucleic Acid Database (NDB, ndbserver.rutgers.edu). The data are stored as PDB-files. Access to the PDB is free. Small molecule data are stored in the Cambridge Data Base (CSD), a commercial product for which a license must be obtained. Small molecule data are stored in CIF-format (which we will not discuss). The PDB-File 5/56
7 The PDB file an Example HEADER LIGASE 28-APR-99 1CLI TITLE AUTHOR REMARK X-RAY CRYSTAL STRUCTURE OF AMINOIMIDAZOLE RIBONUCLEOTIDE C.LI,T.J.KAPPOCK,J.STUBBE,T.M.WEAVER,S.E.EALICK 2 RESOLUTION ANGSTROMS.... CRYST P ATOM 1 N THR A N ATOM 2 CA THR A C ATOM 3 C THR A C... The PDB File 6/56
8 Storing Structural Data: the PDB-File A PDB-file is a simple text file. It contains a header with supplemental information (authors, compound, publication, etc.), the crystallographic space group and unit cell dimensions. The main part of the file are ATOM entries, one per line. An atom entry contains atom type, atom name, residue type it belongs to, and coordinates, occupancy, and B-factor. The PDB-File 7/56
9 Occupancy: An Example of Multiple conformation Initially the model contained only one position for the Tyrosine. But the electron density map suggests that in about half the molecules in the crystal, the side chain of the Tyrosine points in a different direction this can be modelled by setting the occupancies for both orientations of the side chain to 0.5. The PDB-File 8/56
10 Temperature factor of an Atom B factor Even though data are usually collected at 100 K, atoms are not immobile but vibrate thermal motion. The (isotropic) temperature (or B ) factor describes the vibration as a sphere within which the atom oscillates. This is quite a coarse assumption. At high resolution (< 1.6Å), when enough data are available, the vibrations in each of the three directions can be described separately. In that case, 6 parameters are necessary to describe the thermal motions. They are called Anisotropic Displacement Parameters (ADP). A low B-factor indicates a rigid, stable region, while a high B-factor indicates flexibility (e.g. at loops). Later will be explaned why ADP s cannot always be used and the less accurate isotropic B-factor must be used instead. The PDB-File 9/56
11 Illustration of the B factor Isotropic B factors Anisotropic B factors Spherical movement of atoms Ellipsoidal movement of atoms more exact The PDB-File 10/56
12 Data Reliability: The Data to Parameter Ratio Data to Parameter Ratio 11/56
13 Reliability of Data: The Data to Parameter Ratio Measurements are inexact and only approximations. The more often a value is measrued the more trustworthy is becomes: The error estimate becomes better. In macromolecular crystallography we want to determine at least the coordinates for every atom of the structure, i.e., we require 3 data points for every position. The more data were collected for a fixed number of paramersthe more reliable our model can be. We aim at a high data to parameter ratio. Data to Parameter Ratio 12/56
14 Data to Parameter Ratio: Example Estimates Resolution[Å] refined parameters a data/parameters ratio 3.0 x,y,z 0.9:1 2.3 x,y,z; B 1.5:1 1.8 x,y,z; B 3.1:1 1.5 x,y,z; B 5.4:1 1.5 x,y,z; U 11 U 12 U 13 U 23 U 22 U :1 1.1 x,y,z; U 11 U 12 U 13 U 23 U 22 U :1 0.8 x,y,z; U 11 U 12 U 13 U 23 U 22 U 33 16:1 a x,y,z: coordinates; B: isotropic B-value; U ij : anisotropic B-values G. Sheldrick Effectively below 1.8Å, there would not be enough data points to create a reliable model. The data to parameter ratio can be improved by additional (bio ) chemical etc. information. Data to Parameter Ratio 13/56
15 An Example: Data to Parameter Ratio (1/7) Scenario Measure data along a graph Experiment 1: High resolution, 21 data points with errors Experiment 2: Low Resolution, 3 data points with errors measurements Ideal: f(x)=x measurements Ideal: f(x)=x Data to Parameter Ratio 14/56
16 An Example: Data to Parameter Ratio (2/7) Two Models Model 1: g(x) = g 2 x 2 + g 1 x + g 0 Model 2: h(x) = h 3 x 3 + h 1 x + h 0 Both Models contain three parameters, i.e., at least three data points are required for their unambiguous determination. Data to Parameter Ratio 15/56
17 An Example: Data to Parameter Ratio (3/7) Fitting High Resolution Data data x 2 Model x 3 Model x x 0.51 χ 2 = good 0.16x x χ 2 = bad Data to Parameter Ratio 16/56
18 An Example: Data to Parameter Ratio (4/7) Remarks on χ 2 χ 2 is a common error estimator in statistics. χ 2 should be close to 1 for a good model. χ 2 makes a clear distinction between the two models. The reliability of χ 2 depends on a good estimate of the errors of the data points. Data to Parameter Ratio 17/56
19 An Example: Data to Parameter Ratio (5/7) Fitting Low Resolution Data data x 2 Model x 3 Model x x x x Data to Parameter Ratio 18/56
20 An Example: Data to Parameter Ratio (6/7) Problems with Fitting Low Resolution Data: Both Models fit the data perfectly. No error estimates because #data = #parameters. Additional knowledge is required to decide about the correct model. Data to Parameter Ratio 19/56
21 An Example: Data to Parameter Ratio (7/7) Fitting Low Resolution Data Constraints Assuming Constraint: data passes through (0, 0) Model 1: g(x) = g 2 x 2 + g 1 x +g 0 Model 2: h(x) = h 3 x 3 + h 1 x +h data x 2 Model constraint x 3 Model constraint x x χ 2 = x x χ 2 = 14.4 Data to Parameter Ratio 20/56
22 Crystallographic Model Building Model Building 21/56
23 Model Building: Getting Started The first steps in building the model consist of finding larger groups of residues with special features. The Secondary Structure Elements of proteins are good starting points. In proteins this is the (C α ) main chain, in nucleic acids the position of the bases. α helices are particularly easy to locate, even at medium to low resolution (2.5 4Å). Model Building 22/56
24 Directionality of α Helices From the main chain (C α chain) one cannot determine the direction, nor which part of the sequence it covers. One gets help from the so-called Christmas tree: the side chains of an α helix point towards the N terminal end of the protein chain. Model Building 23/56
25 β Strands The other secondary structure element of proteins, β strands are also striking but more difficult to build. Especially the direction of the peptide chain can be difficult to find. Model Building 24/56
26 Sequence Docking The secondary structure basically is a Poly-Alanine model with no sequence information. Selenomethionine substituted proteins have become very popular for MAD experiments. The heavy selenium atoms are easy to find in the electron density map and help docking the sequence to the map. Disulphide bridges or metals bound to an active centre can also be helpful. Model Building 25/56
27 Automated Model Building Until a couple of years ago, a crystallographer had to place every residue by hand. At resolution better than, say, 2.5Å building is extremely facilitated by programs like Arp/Warp (A. Perrakis, V. Lamzin), Buccaneer (K. Cowtan), or Resolve (T. Terwilliger), which automatically build large parts of the structure in a couple of hours. Model Building 26/56
28 Manual Model Building Computer programs do not know about biology, certainly not of a specific molecule/structure. Human interaction is therefore required to pay attention to: presence and identification of ligands and/or metal ions (from crystallisation or protein preparation) special interaction for complexes exceptions from standard values used in refinement correct placement of solvent (water) molecules Model Interpretation Model Building 27/56
29 Hydrogen Atoms? X-rays interact with the electron shell of atoms. The strength of interaction is proportional to the total number of electrons. Hydrogen atoms only have one electron. They cannot be detected by X-ray diffraction (unless with very high resolution data < 1Å). During refinement, hydrogens are treated as riding atoms, that is, in a fixed position relative to the groups they belong to (like the carbons of a phenylalanine ring). Instead of completely ignoring hydrogens, this method improves the quality of the model and also aids to keep the correct distances to neighbouring groups. Because of the fixed position, riding atoms do not increase the number of parameters. Model Building 28/56
30 Empty Space? The Solvent Region Arrangement of molecules in the unit cell Electron density map The holes in both pictures are not vacuum. They are filled with solvent, i.e., mostly water molecules. They are disordered, therefore one does not see explicit density in these parts of the crystal. Yet, they still contribute (a little) to the diffraction pattern at low resolution. The treatment of the solvent region in crystallography leaves space for improvement. Model Building 29/56
31 Model Refinement Model Refinement 30/56
32 Refinement & Building Model Building describes the construction of the model, addition and deletion of atoms and ligands. It is mostly done by the crystallographer in front of a computer screen. Model Refinenemt describes the improvement of that model to better match the experimental data ( F meas (hkl) ). It is mostly done by computer programs. The computer program tries small changes of the coordinates and modifcation of the temperature factors to minimise the difference between calculated and measured amplitudes. Model Refinement 31/56
33 Excursus: Crystallographic Theory Given the structure factors F meas (hkl) F meas (hkl) exp iφ(hkl), the electron density at position (x, y, z) is given by the Fourier transformation ρ(x, y, z) = 1 V unit cell h,k,l F meas (hkl) e iφ(hkl) e 2πi(hx+ky+lz) Once a model is known with atom coordinates (x j, y j, z j ), the structure factors can be calculated from the spherical atomic scattering factors f j by F calc (h, k, l) = j f j e 2πi(hx j+ky j +lz j ) (1) The spherical atomic scattering factors f j can be calculated from per atom properties. They are also tabulated (e.g. in the International Tables for Crystallography, Volume C). They include the effect of the temperature factor. Model Refinement 32/56
34 Excursus: Crystallographic Theory There are two sources for the intensities I(hkl): I meas (hkl) = F meas (hkl) 2, which are measured from the X-ray experiment I calc (hkl) = F calc (hkl) 2 calculated from model coordinates. Model refinement minimises the difference between calculated and measured structure factor amplitudes (e.g. with least-squares-methods). Model Refinement 33/56
35 Initial Map Generation Amplitudes F (hkl) initial Map initial Model Phases φ(hkl) For the first map, phases were determined with MAD, or SIR, or Molecular Replacement, etc. These phases are generally of low quality, i.e., they have large errors compared to the real values. Model Refinement 34/56
36 model refinement by program (checks chemical correctness) φ calculate map new model F build model/ match model to map better w.r.t. map! data The model is created/modified based on the map. The map is calculated using the phases from the model. Therefore, the new model is biased against the old model: errors may persist. Model Refinement 35/56
37 Model Building and Refinement (1/2) Creating a model from X-ray data is an iterative process consisting of model building and refinement. Refinement: global improvement of the model with respect to the experimental data. Coordinates of all atoms together with their temperature factors (and sometimes, at very high resolution, even the occupancy), are moved in order to minimise the difference between the measured intensities and the ones calculated from the model. Refinement 36/56
38 Model Building and Refinement (2/2) Model Building: local improvement of the model with respect to the experimental data. Atoms are added, removed, or moved in order to ensure that 1. the model makes sense bio chemically (proximity of atoms, H-bonding, position of solvent molecules, etc.) 2. the model fits the calculated electron density (e.g. check for multiple conformations) Refinement 37/56
39 Restraints and Constraints The reflection data alone would not be sufficient to create a trustworthy model at worse than, say, 1.5Å. There are too many parameters. Therefore it is necessary to incorporate additional information. The re are two types of auxiliary information: restraints and constraints. Refinement 38/56
40 Restraints and Constraints Constraints reduce the number of parameters. They are expression like Property X must have value Y e.g.: temperature factor is isotropic instead of anisotropic : 4 parameters per atom instead of 9 parameters per atom Restraints increase the number of data. Should be or should be approximately expressions, e.g. distance (N C α ) 1.458Å. Restraints used in refinement encompass bond lengths and bond angles. They are important for macromolecular crystallography, and solving a structure without them would be impossible. Refinement 39/56
41 Traps: Local Minima Refinement programs cannot cross this barrier they would get stuck in the local minimum and could not move the Phenylalanine into the right position. These local minima and the vicious circle make validation of the model necessary. Model Refinement 40/56
42 Refinement: R and R free R and R free 41/56
43 The R Value The difference between calculated and measured amplitudes is a so-called R value R = hkl ( F meas F calc ) hkl ( F meas ) For small molecules, R values between 2% and 5% are normal, for macromolecules, the range is approximately 10% 30%. As a rule of thumb the R value should be about 1/10 of the resolution: a 2.5Å structure should have an R value of 0.25 = 25%. R and R free 42/56
44 Refinement and Overfitting For macromolecular molecules, the data to parameter ratio is not very high at a normal resolution range. Therefore, the R value can be nearly arbitrarily reduced by adding more and more atoms that were not really present in the crystal structure or allowing positions that chemically do not make much sense (stereochemical clashes). This is called overfitting the data. Refinement 43/56
45 Quality Measures (2): The R free -value One measure to reduce overfitting is the R free value. About 5% 10% of the reflections are excluded both from refinement and model building. They remain unconsidered and are like an independent judge : after refinement, the R free value is calculated like the R value, but with the excluded reflections. The two values should not differ too much (model errors) but should also not be too close (model bias). The R free value is common in statistics, but was introduced to crystallography only in the mid 90 s by Axel Brünger. Refinement 44/56
46 Structure Validation Validation 45/56
47 Why Validation? Experimental data never free of errors Scientists never free of prejudice Compared to other technical or physical disciplines, the errors in X-ray experiments are huge. It is easy to create erroneous models non-deliberately. The results - structural models - are often used by non-crystallographers. They must be able to check the quality without knowing too much about crystallography. Validation 46/56
48 Photoactive Yellow Protein: 1989 and This model was published in 1989 (PDB entry 1phy) The correct version: published six years later (PDB entry 2phy) Kleywegt, Acta D(2000), D56 NB: The first structure was published before usage of the R free and other means of validation. It is nowadays very unlikely that such coarse misinterpretations happen. Validation 47/56
49 Caveat: Modelling Models The structure of TBP, the TATA-box binding protein (TBP or TFIIDτ) was published in 1992 (Nikolov et al., Nature 360, pp.40 46). The shape of the molecule suggested that the TATA box sits straight in the groove of the protein. The structure of the complex, published a year later by Kim et al. (Nature 365, pp ) revealed that the DNA was actually heavily bent. Validation 48/56
50 Caveat: What You see is What You get? Another issue with PDB files is that they contain more information than a graphical viewer might be able to display. Many crystallographers include atoms/residues into their structures without experimental support and set their occupancy to zero. While this chemically makes sense, this procedure is error prone for users of the structure. Validation 49/56
51 How to Validate Validation means estimation of the model in comparison with the data. However, since the model was created by refinement against the data, the model is biased. Therefore, there is need for independent factors. All information can be used 1. that did not participate in the creation of the model/ minimisation of the model data difference 2. of which ideal/ average values are known. This means that these information must be the same or similar for all proteins. Validation 50/56
52 The Real Space R factor R and R free are global figures of merit: one number describes the quality of the whole structure. A local figure of merit is the real space R factor or real space correlation coefficient between model and electron density map. It expresses the fit between the electron density and the model. Validation 51/56
53 Dihedral Angles the Ramachandran-Plot The Ramachandran-plot is probably the most famous validation tool. It is based on the two dihedral angles ψ and ϕ. Φ is the angle between the two planes defined by C i 1 N i C α and N i C α i C i. Ψ is the angle between the two planes of N i C α i C i and C α i C i N i+1 Validation 52/56
54 The Ramachandran-Plot The Ramachandran plot shows the φ vs. ψ angles for a structure and the most probable regions derived from the 500 best determined protein structures. β strand α helix left handed α helix Interactive Ramachandran window of the model building program Coot. Everything outside the shaded region is an outlier Validation 53/56
55 The Kleywegt Plot Even more information can be read from the Ramachandran plot, if there are more than one copy of a molecule: the two (or more) copies should be rather similar to each other. If one plots the Ramachandran plot for all molecules into the same diagram and connects corresponding residues, one should NOT obtain a picture like this. Kleywegt, Acta D(2000), D56 Validation 54/56
56 Validation Tools also for non-crystallographers Various programs are available to check the quality of a PDB-file, e.g. WhatIF SFcheck ProCheck MolProbity The MolProbity program is available online One can upload a PDF-file or enter a PDB ID-code and various plots. It even checks the flip states of Asn, Gln, His-residue based on possible hydrogen bondings. Validation 55/56
57 Validation: Summary Most of the pretty pictures about proteins represent structures determined by X ray diffraction. But do not be deceived by colours and artistic compositions. Everyone who make use of PDB files / structural data should be aware of possible pitfalls. 1. Read the header information. 2. Consider the resolution and data quality 3. Does the quality and resolution match allow for the details you want to extract? 4. Make use of programs that examine structure and (if available/possible) data Validation 56/56
Protein Crystallography Part II
Molecular Biology Course 2007 Protein Crystallography Part II Tim Grüne University of Göttingen Dept. of Structural Chemistry November 2007 http://shelx.uni-ac.gwdg.de tg@shelx.uni-ac.gwdg.de Overview
More informationMolecular Biology Course 2006 Protein Crystallography Part II
Molecular Biology Course 2006 Protein Crystallography Part II Tim Grüne University of Göttingen Dept. of Structural Chemistry December 2006 http://shelx.uni-ac.gwdg.de tg@shelx.uni-ac.gwdg.de Overview
More informationMacromolecular Crystallography Part II
Molecular Biology Course 2010 Macromolecular Crystallography Part II University of Göttingen Dept. of Structural Chemistry November 2010 http://shelx.uni-ac.gwdg.de tg@shelx.uni-ac.gwdg.de Crystallography
More informationDirect Method. Very few protein diffraction data meet the 2nd condition
Direct Method Two conditions: -atoms in the structure are equal-weighted -resolution of data are higher than the distance between the atoms in the structure Very few protein diffraction data meet the 2nd
More informationProtein Crystallography
Protein Crystallography Part II Tim Grüne Dept. of Structural Chemistry Prof. G. Sheldrick University of Göttingen http://shelx.uni-ac.gwdg.de tg@shelx.uni-ac.gwdg.de Overview The Reciprocal Lattice The
More informationX-ray Crystallography
2009/11/25 [ 1 ] X-ray Crystallography Andrew Torda, wintersemester 2009 / 2010 X-ray numerically most important more than 4/5 structures Goal a set of x, y, z coordinates different properties to NMR History
More informationExperimental Phasing with SHELX C/D/E
WIR SCHAFFEN WISSEN HEUTE FÜR MORGEN Dr. Tim Grüne :: Paul Scherrer Institut :: tim.gruene@psi.ch Experimental Phasing with SHELX C/D/E CCP4 / APS School Chicago 2017 22 nd June 2017 1 - The Phase Problem
More informationProtein crystallography. Garry Taylor
Protein crystallography Garry Taylor X-ray Crystallography - the Basics Grow crystals Collect X-ray data Determine phases Calculate ρ-map Interpret map Refine coordinates Do the biology. Nitrogen at -180
More informationshelxl: Refinement of Macromolecular Structures from Neutron Data
ESS Neutron Protein Crystallography 2013 Aarhus, Denmark shelxl: Refinement of Macromolecular Structures from Neutron Data Tim Grüne University of Göttingen Dept. of Structural Chemistry http://shelx.uni-ac.gwdg.de
More informationExamples of Protein Modeling. Protein Modeling. Primary Structure. Protein Structure Description. Protein Sequence Sources. Importing Sequences to MOE
Examples of Protein Modeling Protein Modeling Visualization Examination of an experimental structure to gain insight about a research question Dynamics To examine the dynamics of protein structures To
More informationMolecular Biology Course 2006 Protein Crystallography Part I
Molecular Biology Course 2006 Protein Crystallography Part I Tim Grüne University of Göttingen Dept. of Structural Chemistry November 2006 http://shelx.uni-ac.gwdg.de tg@shelx.uni-ac.gwdg.de Overview Overview
More informationElectron Density at various resolutions, and fitting a model as accurately as possible.
Section 9, Electron Density Maps 900 Electron Density at various resolutions, and fitting a model as accurately as possible. ρ xyz = (Vol) -1 h k l m hkl F hkl e iφ hkl e-i2π( hx + ky + lz ) Amplitude
More informationTLS and all that. Ethan A Merritt. CCP4 Summer School 2011 (Argonne, IL) Abstract
TLS and all that Ethan A Merritt CCP4 Summer School 2011 (Argonne, IL) Abstract We can never know the position of every atom in a crystal structure perfectly. Each atom has an associated positional uncertainty.
More informationTable 1. Crystallographic data collection, phasing and refinement statistics. Native Hg soaked Mn soaked 1 Mn soaked 2
Table 1. Crystallographic data collection, phasing and refinement statistics Native Hg soaked Mn soaked 1 Mn soaked 2 Data collection Space group P2 1 2 1 2 1 P2 1 2 1 2 1 P2 1 2 1 2 1 P2 1 2 1 2 1 Cell
More informationElectronic Supplementary Information (ESI) for Chem. Commun. Unveiling the three- dimensional structure of the green pigment of nitrite- cured meat
Electronic Supplementary Information (ESI) for Chem. Commun. Unveiling the three- dimensional structure of the green pigment of nitrite- cured meat Jun Yi* and George B. Richter- Addo* Department of Chemistry
More informationResolution and data formats. Andrea Thorn
Resolution and data formats Andrea Thorn RESOLUTION Motivation Courtesy of M. Sawaya Map resolution http://www.bmsc.washington.edu/people/verlinde/experiment.html Data quality indicators Resolution accounts
More informationSummary of Experimental Protein Structure Determination. Key Elements
Programme 8.00-8.20 Summary of last week s lecture and quiz 8.20-9.00 Structure validation 9.00-9.15 Break 9.15-11.00 Exercise: Structure validation tutorial 11.00-11.10 Break 11.10-11.40 Summary & discussion
More informationIntroduction to Comparative Protein Modeling. Chapter 4 Part I
Introduction to Comparative Protein Modeling Chapter 4 Part I 1 Information on Proteins Each modeling study depends on the quality of the known experimental data. Basis of the model Search in the literature
More informationX-ray Crystallography I. James Fraser Macromolecluar Interactions BP204
X-ray Crystallography I James Fraser Macromolecluar Interactions BP204 Key take-aways 1. X-ray crystallography results from an ensemble of Billions and Billions of molecules in the crystal 2. Models in
More informationSHELXC/D/E. Andrea Thorn
SHELXC/D/E Andrea Thorn What is experimental phasing? Experimental phasing is what you do if MR doesn t work. What is experimental phasing? Experimental phasing methods depend on intensity differences.
More informationCCP4 Diamond 2014 SHELXC/D/E. Andrea Thorn
CCP4 Diamond 2014 SHELXC/D/E Andrea Thorn SHELXC/D/E workflow SHELXC: α calculation, file preparation SHELXD: Marker atom search = substructure search SHELXE: density modification Maps and coordinate files
More informationAnisotropy in macromolecular crystal structures. Andrea Thorn July 19 th, 2012
Anisotropy in macromolecular crystal structures Andrea Thorn July 19 th, 2012 Motivation Courtesy of M. Sawaya Motivation Crystal structures are inherently anisotropic. X-ray diffraction reflects this
More informationJoana Pereira Lamzin Group EMBL Hamburg, Germany. Small molecules How to identify and build them (with ARP/wARP)
Joana Pereira Lamzin Group EMBL Hamburg, Germany Small molecules How to identify and build them (with ARP/wARP) The task at hand To find ligand density and build it! Fitting a ligand We have: electron
More informationNitrogenase MoFe protein from Clostridium pasteurianum at 1.08 Å resolution: comparison with the Azotobacter vinelandii MoFe protein
Acta Cryst. (2015). D71, 274-282, doi:10.1107/s1399004714025243 Supporting information Volume 71 (2015) Supporting information for article: Nitrogenase MoFe protein from Clostridium pasteurianum at 1.08
More informationWhy do We Trust X-ray Crystallography?
Why do We Trust X-ray Crystallography? Andrew D Bond All chemists know that X-ray crystallography is the gold standard characterisation technique: an X-ray crystal structure provides definitive proof of
More informationX-ray Crystallography. Kalyan Das
X-ray Crystallography Kalyan Das Electromagnetic Spectrum NMR 10 um - 10 mm 700 to 10 4 nm 400 to 700 nm 10 to 400 nm 10-1 to 10 nm 10-4 to 10-1 nm X-ray radiation was discovered by Roentgen in 1895. X-rays
More informationPDBe TUTORIAL. PDBePISA (Protein Interfaces, Surfaces and Assemblies)
PDBe TUTORIAL PDBePISA (Protein Interfaces, Surfaces and Assemblies) http://pdbe.org/pisa/ This tutorial introduces the PDBePISA (PISA for short) service, which is a webbased interactive tool offered by
More informationModel and data. An X-ray structure solution requires a model.
Model and data An X-ray structure solution requires a model. This model has to be consistent with: The findings of Chemistry Reflection positions and intensities Structure refinement = Model fitting by
More informationHandout 12 Structure refinement. Completing the structure and evaluating how good your data and model agree
Handout 1 Structure refinement Completing the structure and evaluating how good your data and model agree Why you should refine a structure We have considered how atoms are located by Patterson, direct
More information7.91 Amy Keating. Solving structures using X-ray crystallography & NMR spectroscopy
7.91 Amy Keating Solving structures using X-ray crystallography & NMR spectroscopy How are X-ray crystal structures determined? 1. Grow crystals - structure determination by X-ray crystallography relies
More informationDictionary of ligands
Dictionary of ligands Some of the web and other resources Small molecules DrugBank: http://www.drugbank.ca/ ZINC: http://zinc.docking.org/index.shtml PRODRUG: http://www.compbio.dundee.ac.uk/web_servers/prodrg_down.html
More informationCrystal lattice Real Space. Reflections Reciprocal Space. I. Solving Phases II. Model Building for CHEM 645. Purified Protein. Build model.
I. Solving Phases II. Model Building for CHEM 645 Purified Protein Solve Phase Build model and refine Crystal lattice Real Space Reflections Reciprocal Space ρ (x, y, z) pronounced rho F hkl 2 I F (h,
More informationRietveld Structure Refinement of Protein Powder Diffraction Data using GSAS
Rietveld Structure Refinement of Protein Powder Diffraction Data using GSAS Jon Wright ESRF, Grenoble, France Plan This is a users perspective Cover the protein specific aspects (assuming knowledge of
More informationIgE binds asymmetrically to its B cell receptor CD23
Supplementary Information IgE binds asymmetrically to its B cell receptor CD23 Balvinder Dhaliwal 1*, Marie O. Y. Pang 2, Anthony H. Keeble 2,3, Louisa K. James 2,4, Hannah J. Gould 2, James M. McDonnell
More informationSUPPLEMENTARY INFORMATION
SUPPLEMENTARY INFORMATION doi:10.1038/nature11539 Supplementary Figure 1 Schematic representation of plant (A) and mammalian (B) P 2B -ATPase domain organization. Actuator (A-), nucleotide binding (N-),
More informationMolecular Modeling lecture 2
Molecular Modeling 2018 -- lecture 2 Topics 1. Secondary structure 3. Sequence similarity and homology 2. Secondary structure prediction 4. Where do protein structures come from? X-ray crystallography
More informationGet familiar with PDBsum and the PDB Extract atomic coordinates from protein data files Compute bond angles and dihedral angles
CS483 Assignment #2 Due date: Mar. 1 at the start of class. Protein Geometry Bedbug spit? Just say NO! Purpose of this assignment Get familiar with PDBsum and the PDB Extract atomic coordinates from protein
More informationPreparing a PDB File
Figure 1: Schematic view of the ligand-binding domain from the vitamin D receptor (PDB file 1IE9). The crystallographic waters are shown as small spheres and the bound ligand is shown as a CPK model. HO
More informationPymol Practial Guide
Pymol Practial Guide Pymol is a powerful visualizor very convenient to work with protein molecules. Its interface may seem complex at first, but you will see that with a little practice is simple and powerful
More informationSupporting Information. Synthesis of Aspartame by Thermolysin : An X-ray Structural Study
Supporting Information Synthesis of Aspartame by Thermolysin : An X-ray Structural Study Gabriel Birrane, Balaji Bhyravbhatla, and Manuel A. Navia METHODS Crystallization. Thermolysin (TLN) from Calbiochem
More informationNMR, X-ray Diffraction, Protein Structure, and RasMol
NMR, X-ray Diffraction, Protein Structure, and RasMol Introduction So far we have been mostly concerned with the proteins themselves. The techniques (NMR or X-ray diffraction) used to determine a structure
More information4. Constraints and Hydrogen Atoms
4. Constraints and ydrogen Atoms 4.1 Constraints versus restraints In crystal structure refinement, there is an important distinction between a constraint and a restraint. A constraint is an exact mathematical
More informationLikelihood and SAD phasing in Phaser. R J Read, Department of Haematology Cambridge Institute for Medical Research
Likelihood and SAD phasing in Phaser R J Read, Department of Haematology Cambridge Institute for Medical Research Concept of likelihood Likelihood with dice 4 6 8 10 Roll a seven. Which die?? p(4)=p(6)=0
More information11/6/2013. Refinement. Fourier Methods. Fourier Methods. Difference Map. Difference Map Find H s. Difference Map No C 1
Refinement Fourier Methods find heavy atom or some F s phases using direct methods locate new atoms, improve phases continue until all atoms found in more or less correct position starting point of refinement
More informationScattering by two Electrons
Scattering by two Electrons p = -r k in k in p r e 2 q k in /λ θ θ k out /λ S q = r k out p + q = r (k out - k in ) e 1 Phase difference of wave 2 with respect to wave 1: 2π λ (k out - k in ) r= 2π S r
More informationProtein Structure and Visualisation. Introduction to PDB and PyMOL
Protein Structure and Visualisation Introduction to PDB and PyMOL 1 Feedback Persons http://www.bio-evaluering.dk/ 2 Program 8.00-8.15 Quiz results 8.15-8.50 Introduction to PDB & PyMOL 8.50-9.00 Break
More informationTools for Cryo-EM Map Fitting. Paul Emsley MRC Laboratory of Molecular Biology
Tools for Cryo-EM Map Fitting Paul Emsley MRC Laboratory of Molecular Biology April 2017 Cryo-EM model-building typically need to move more atoms that one does for crystallography the maps are lower resolution
More informationStructure factors again
Structure factors again Remember 1D, structure factor for order h F h = F h exp[iα h ] = I 01 ρ(x)exp[2πihx]dx Where x is fractional position along unit cell distance (repeating distance, origin arbitrary)
More informationGarib N Murshudov MRC-LMB, Cambridge
Garib N Murshudov MRC-LMB, Cambridge Contents Introduction AceDRG: two functions Validation of entries in the DB and derived data Generation of new ligand description Jligand for link description Conclusions
More informationViewing and Analyzing Proteins, Ligands and their Complexes 2
2 Viewing and Analyzing Proteins, Ligands and their Complexes 2 Overview Viewing the accessible surface Analyzing the properties of proteins containing thousands of atoms is best accomplished by representing
More information1. Protein Data Bank (PDB) 1. Protein Data Bank (PDB)
Protein structure databases; visualization; and classifications 1. Introduction to Protein Data Bank (PDB) 2. Free graphic software for 3D structure visualization 3. Hierarchical classification of protein
More informationFull wwpdb X-ray Structure Validation Report i
Full wwpdb X-ray Structure Validation Report i Mar 10, 2018 01:44 am GMT PDB ID : 1MWP Title : N-TERMINAL DOMAIN OF THE AMYLOID PRECURSOR PROTEIN Authors : Rossjohn, J.; Cappai, R.; Feil, S.C.; Henry,
More informationSmall Molecule Crystallography Lab Department of Chemistry and Biochemistry University of Oklahoma 101 Stephenson Parkway Norman, OK
Small Molecule Crystallography Lab Department of Chemistry and Biochemistry University of Oklahoma 101 Stephenson Parkway Norman, OK 73019-5251 Sample: KP-XI-cinnamyl-chiral alcohol Lab ID: 12040 User:
More informationUltra-high resolution structures in validation
Ultra-high resolution structures in validation (and not only...) Mariusz Jaskolski Department of Crystallography,, A. Mickiewicz University Center for Biocrystallographic Research, Polish Academy of Sciences,
More informationAnomalous dispersion
Selenomethionine MAD Selenomethionine is the amino acid methionine with the Sulfur replaced by a Selenium. Selenium is a heavy atom that also has the propery of "anomalous scatter" at some wavelengths,
More informationCALIFORNIA INSTITUTE OF TECHNOLOGY BECKMAN INSTITUTE X-RAY CRYSTALLOGRAPHY LABORATORY
APPENDIX F Crystallographic Data for TBA Tb(DO2A)(F-DPA) CALIFORNIA INSTITUTE OF TECHNOLOGY BECKMAN INSTITUTE X-RAY CRYSTALLOGRAPHY LABORATORY Date 11 January 2010 Crystal Structure Analysis of: MLC23
More informationAutomated Protein Model Building with ARP/wARP
Joana Pereira Lamzin Group EMBL Hamburg, Germany Automated Protein Model Building with ARP/wARP (and nucleic acids too) Electron density map: the ultimate result of a crystallography experiment How would
More informationSupplementary Information
1 Supplementary Information Figure S1 The V=0.5 Harker section of an anomalous difference Patterson map calculated using diffraction data from the NNQQNY crystal at 1.3 Å resolution. The position of the
More informationManipulating Ligands Using Coot. Paul Emsley May 2013
Manipulating Ligands Using Coot Paul Emsley May 2013 Ligand and Density... Ligand and Density... Ligand and Density... Protein-ligand complex models are often a result of subjective interpretation Scoring
More informationIntroduction to" Protein Structure
Introduction to" Protein Structure Function, evolution & experimental methods Thomas Blicher, Center for Biological Sequence Analysis Learning Objectives Outline the basic levels of protein structure.
More informationwwpdb X-ray Structure Validation Summary Report
wwpdb X-ray Structure Validation Summary Report io Jan 31, 2016 06:45 PM GMT PDB ID : 1CBS Title : CRYSTAL STRUCTURE OF CELLULAR RETINOIC-ACID-BINDING PROTEINS I AND II IN COMPLEX WITH ALL-TRANS-RETINOIC
More informationVisualization of Macromolecular Structures
Visualization of Macromolecular Structures Present by: Qihang Li orig. author: O Donoghue, et al. Structural biology is rapidly accumulating a wealth of detailed information. Over 60,000 high-resolution
More informationBioinformatics. Macromolecular structure
Bioinformatics Macromolecular structure Contents Determination of protein structure Structure databases Secondary structure elements (SSE) Tertiary structure Structure analysis Structure alignment Domain
More informationScientific Integrity: A crystallographic perspective
Scientific Integrity: A crystallographic perspective Ian Bruno - Director, Strategic Partnerships The Cambridge Crystallographic Data Centre @ijbruno @ccdc_cambridge Scientific Integrity: Can We Rely on
More informationFull wwpdb X-ray Structure Validation Report i
Full wwpdb X-ray Structure Validation Report i Mar 8, 2018 08:34 pm GMT PDB ID : 1RUT Title : Complex of LMO4 LIM domains 1 and 2 with the ldb1 LID domain Authors : Deane, J.E.; Ryan, D.P.; Maher, M.J.;
More informationFull wwpdb X-ray Structure Validation Report i
Full wwpdb X-ray Structure Validation Report i Mar 13, 2018 04:03 pm GMT PDB ID : 5NMJ Title : Chicken GRIFIN (crystallisation ph: 6.5) Authors : Ruiz, F.M.; Romero, A. Deposited on : 2017-04-06 Resolution
More informationExperimental phasing in Crank2
Experimental phasing in Crank2 Pavol Skubak and Navraj Pannu Biophysical Structural Chemistry, Leiden University, The Netherlands http://www.bfsc.leidenuniv.nl/software/crank/ X-ray structure solution
More informationCharge density refinement at ultra high resolution with MoPro software. Christian Jelsch CNRS Université de Lorraine
Charge density refinement at ultra high resolution with MoPro software Christian Jelsch CNRS Université de Lorraine Laboratoire de Cristallographie & Résonance Magnétique & Modélisations (CRM2) Nancy,
More informationEnsemble refinement of protein crystal structures in PHENIX. Tom Burnley Piet Gros
Ensemble refinement of protein crystal structures in PHENIX Tom Burnley Piet Gros Incomplete modelling of disorder contributes to R factor gap Only ~5% of residues in the PDB are modelled with more than
More informationAPPENDIX E. Crystallographic Data for TBA Eu(DO2A)(DPA) Temperature Dependence
APPENDIX E Crystallographic Data for TBA Eu(DO2A)(DPA) Temperature Dependence Temperature Designation CCDC Page 100 K MLC18 761599 E2 200 K MLC17 762705 E17 300 K MLC19 763335 E31 E2 CALIFORNIA INSTITUTE
More informationSmall Molecule Crystallography Lab Department of Chemistry and Biochemistry University of Oklahoma 101 Stephenson Parkway Norman, OK
Small Molecule Crystallography Lab Department of Chemistry and Biochemistry University of Oklahoma 101 Stephenson Parkway Norman, OK 73019-5251 Sample: KP-XI-furan-enzymatic alcohol Lab ID: 12042 User:
More informationDetermination of the Substructure
Monday, June 15 th, 2009 Determination of the Substructure EMBO / MAX-INF2 Practical Course http://shelx.uni-ac.gwdg.de Overview Substructure Definition and Motivation Extracting Substructure Data from
More informationFull wwpdb X-ray Structure Validation Report i
Full wwpdb X-ray Structure Validation Report i Jan 14, 2019 11:10 AM EST PDB ID : 6GYW Title : Crystal structure of DacA from Staphylococcus aureus Authors : Tosi, T.; Freemont, P.S.; Grundling, A. Deposited
More informationHandout 13 Interpreting your results. What to make of your atomic coordinates, bond distances and angles
Handout 13 Interpreting your results What to make of your atomic coordinates, bond distances and angles 1 What to make of the outcome of your refinement There are several ways of judging whether the outcome
More informationFull wwpdb X-ray Structure Validation Report i
Full wwpdb X-ray Structure Validation Report i Mar 14, 2018 02:00 pm GMT PDB ID : 3RRQ Title : Crystal structure of the extracellular domain of human PD-1 Authors : Lazar-Molnar, E.; Ramagopal, U.A.; Nathenson,
More informationFull wwpdb X-ray Structure Validation Report i
Full wwpdb X-ray Structure Validation Report i Jan 17, 2019 09:42 AM EST PDB ID : 6D3Z Title : Protease SFTI complex Authors : Law, R.H.P.; Wu, G. Deposited on : 2018-04-17 Resolution : 2.00 Å(reported)
More informationTable S1. Overview of used PDZK1 constructs and their binding affinities to peptides. Related to figure 1.
Table S1. Overview of used PDZK1 constructs and their binding affinities to peptides. Related to figure 1. PDZK1 constru cts Amino acids MW [kda] KD [μm] PEPT2-CT- FITC KD [μm] NHE3-CT- FITC KD [μm] PDZK1-CT-
More information1 Introduction. Abstract
High-resolution Structure Refinement George M. Sheldrick Institut für Anorganische Chemie der Universität Göttingen Tammannstraße 4, D-37077 Göttingen, Germany gsheldr@shelx.uni-ac.gwdg.de Abstract The
More informationIntroduction Molecular Structure Script Console External resources Advanced topics. JMol tutorial. Giovanni Morelli.
Gen 19th, 2017 1 2 Create and edit Display and view Mesurament and labelling Surface and Orbitals 3 4 from Database Protein Enzyme Crystal Structure and Unit Cell 5 Symmetry Animation General information
More informationFull wwpdb X-ray Structure Validation Report i
Full wwpdb X-ray Structure Validation Report i Jan 28, 2019 11:10 AM EST PDB ID : 6A5H Title : The structure of [4+2] and [6+4] cyclase in the biosynthetic pathway of unidentified natural product Authors
More informationWeb-based Auto-Rickshaw for validation of the X-ray experiment at the synchrotron beamline
Web-based Auto-Rickshaw for validation of the X-ray experiment at the synchrotron beamline Auto-Rickshaw http://www.embl-hamburg.de/auto-rickshaw A platform for automated crystal structure determination
More informationFull wwpdb X-ray Structure Validation Report i
Full wwpdb X-ray Structure Validation Report i Mar 8, 2018 10:24 pm GMT PDB ID : 1A30 Title : HIV-1 PROTEASE COMPLEXED WITH A TRIPEPTIDE INHIBITOR Authors : Louis, J.M.; Dyda, F.; Nashed, N.T.; Kimmel,
More informationX-Ray structure analysis
X-Ray structure analysis Kay Diederichs kay.diederichs@uni-konstanz.de Analysis of what? Proteins ( /ˈproʊˌtiːnz/ or /ˈproʊti.ɨnz/) are biochemical compounds consisting of one or more polypeptides typically
More informationSOLVE and RESOLVE: automated structure solution, density modification and model building
Journal of Synchrotron Radiation ISSN 0909-0495 SOLVE and RESOLVE: automated structure solution, density modification and model building Thomas Terwilliger Copyright International Union of Crystallography
More informationIntroduction to Structure Preparation and Visualization
Introduction to Structure Preparation and Visualization Created with: Release 2018-4 Prerequisites: Release 2018-2 or higher Access to the internet Categories: Molecular Visualization, Structure-Based
More informationCOMP 598 Advanced Computational Biology Methods & Research. Introduction. Jérôme Waldispühl School of Computer Science McGill University
COMP 598 Advanced Computational Biology Methods & Research Introduction Jérôme Waldispühl School of Computer Science McGill University General informations (1) Office hours: by appointment Office: TR3018
More informationSmall-Angle Scattering Atomic Structure Based Modeling
Small-Angle Scattering Atomic Structure Based Modeling Alejandro Panjkovich EMBL Hamburg 07.12.2017 A. Panjkovich (EMBL) BioSAS atomic modeling 07.12.2017 1 / 49 From the forest to the particle accelerator
More informationDirect Methods and Many Site Se-Met MAD Problems using BnP. W. Furey
Direct Methods and Many Site Se-Met MAD Problems using BnP W. Furey Classical Direct Methods Main method for small molecule structure determination Highly automated (almost totally black box ) Solves structures
More informationProtein Struktur (optional, flexible)
Protein Struktur (optional, flexible) 22/10/2009 [ 1 ] Andrew Torda, Wintersemester 2009 / 2010, AST nur für Informatiker, Mathematiker,.. 26 kt, 3 ov 2009 Proteins - who cares? 22/10/2009 [ 2 ] Most important
More informationFull wwpdb X-ray Structure Validation Report i
Full wwpdb X-ray Structure Validation Report i Feb 17, 2018 01:16 am GMT PDB ID : 1IFT Title : RICIN A-CHAIN (RECOMBINANT) Authors : Weston, S.A.; Tucker, A.D.; Thatcher, D.R.; Derbyshire, D.J.; Pauptit,
More informationPrinciples of Physical Biochemistry
Principles of Physical Biochemistry Kensal E. van Hold e W. Curtis Johnso n P. Shing Ho Preface x i PART 1 MACROMOLECULAR STRUCTURE AND DYNAMICS 1 1 Biological Macromolecules 2 1.1 General Principles
More informationPhase problem: Determining an initial phase angle α hkl for each recorded reflection. 1 ρ(x,y,z) = F hkl cos 2π (hx+ky+ lz - α hkl ) V h k l
Phase problem: Determining an initial phase angle α hkl for each recorded reflection 1 ρ(x,y,z) = F hkl cos 2π (hx+ky+ lz - α hkl ) V h k l Methods: Heavy atom methods (isomorphous replacement Hg, Pt)
More informationSUPPLEMENTARY INFORMATION
Supplementary Results DNA binding property of the SRA domain was examined by an electrophoresis mobility shift assay (EMSA) using synthesized 12-bp oligonucleotide duplexes containing unmodified, hemi-methylated,
More informationBasics of protein structure
Today: 1. Projects a. Requirements: i. Critical review of one paper ii. At least one computational result b. Noon, Dec. 3 rd written report and oral presentation are due; submit via email to bphys101@fas.harvard.edu
More informationX- ray crystallography. CS/CME/Biophys/BMI 279 Nov. 12, 2015 Ron Dror
X- ray crystallography CS/CME/Biophys/BMI 279 Nov. 12, 2015 Ron Dror 1 Outline Overview of x-ray crystallography Crystals Electron density Diffraction patterns The computational problem: determining structure
More informationSupporting Information
Submitted to Cryst. Growth Des. Version 1 of August 22, 2007 Supporting Information Engineering Hydrogen-Bonded Molecular Crystals Built from 1,3,5-Substituted Derivatives of Benzene: 6,6',6''-(1,3,5-Phenylene)tris-1,3,5-triazine-2,4-diamines
More informationModeling Biological Systems Opportunities for Computer Scientists
Modeling Biological Systems Opportunities for Computer Scientists Filip Jagodzinski RBO Tutorial Series 25 June 2007 Computer Science Robotics & Biology Laboratory Protein: πρώτα, "prota, of Primary Importance
More informationMacromolecular Phasing with shelxc/d/e
Sunday, June 13 th, 2010 CCP4 Workshop APS Chicago, June 2010 http://shelx.uni-ac.gwdg.de Overview Substructure Definition and Motivation Extracting Substructure Data from measured Data Substructure Solution
More informationFull wwpdb X-ray Structure Validation Report i
Full wwpdb X-ray Structure Validation Report i Mar 8, 2018 06:13 pm GMT PDB ID : 5G5C Title : Structure of the Pyrococcus furiosus Esterase Pf2001 with space group C2221 Authors : Varejao, N.; Reverter,
More information1.b What are current best practices for selecting an initial target ligand atomic model(s) for structure refinement from X-ray diffraction data?!
1.b What are current best practices for selecting an initial target ligand atomic model(s) for structure refinement from X-ray diffraction data?! Visual analysis: Identification of ligand density from
More information