Combination of methods used in the structure solution of pyruvate:ferredoxin oxidoreductase from two crystal forms

1999 ◽  
Vol 55 (9) ◽  
pp. 1546-1554 ◽  
Author(s):  
E. Chabrière ◽  
A. Volbeda ◽  
J. C. Fontecilla-Camps ◽  
M. Roth ◽  
M.-H. Charon
Keyword(s):  
Electron Density ◽  
Real Space ◽  
Molecular Replacement ◽  
Crystal Forms ◽  
Isomorphous Replacement ◽  
Resolution Electron ◽  
Structure Solution ◽  
Phase Extension ◽  
Pyruvate:Ferredoxin Oxidoreductase ◽  
Combination Of Methods

The structure of the homodimeric 267 kDa pyruvate:ferredoxin oxidoreductase (PFOR) of Desulfovibrio africanus was solved with data from two crystals forms, both containing two monomers per asymmetric unit. Phases were obtained from multiwavelength anomalous dispersion (MAD), solvent flattening (SF), molecular replacement (MR) using a 5 Å resolution electron-density search model, multiple isomorphous replacement (MIR) and, finally, electron-density averaging (DA) procedures. It is shown how the combination of all these techniques was used to overcome problems arising from incompleteness of MAD data and weak phasing power of MIR data. A real-space refinement (RSR) procedure is described to improve MR solutions and obtain very accurate protein envelopes and non-crystallographic symmetry (NCS) transformations from 5 Å resolution phase information. These were crucial for the phase extension to high resolution by DA methods.

EDMA: a computer program for topological analysis of discrete electron densities

2012 ◽  
Vol 45 (3) ◽  
pp. 575-580 ◽  
Author(s):  
Lukáš Palatinus ◽  
Siriyara Jagannatha Prathapa ◽  
Sander van Smaalen
Keyword(s):  
Computer Program ◽  
Electron Density ◽  
Critical Points ◽  
Topological Analysis ◽  
Three Dimensional ◽  
Real Space ◽  
Principal Curvatures ◽  
Electron Densities ◽  
Structure Solution ◽  
Aperiodic Crystals

EDMAis a computer program for topological analysis of discrete electron densities according to Bader's theory of atoms in molecules. It locates critical points of the electron density and calculates their principal curvatures. Furthermore, it partitions the electron density into atomic basins and integrates the volume and charge of these atomic basins.EDMAcan also assign the type of the chemical element to atomic basins based on their integrated charges. The latter feature can be used for interpretation ofab initioelectron densities obtained in the process of structure solution. A particular feature ofEDMAis that it can handle superspace electron densities of aperiodic crystals in arbitrary dimensions.EDMAfirst generates real-space sections at a selected set of phases of the modulation wave, and subsequently analyzes each section as an ordinary three-dimensional electron density. Applications ofEDMAto model electron densities have shown that the relative accuracy of the positions of the critical points, the electron densities at the critical points and the Laplacian is of the order of 10−4or better.

Real-space technique applied to crystal structure determination from powder data

2002 ◽  
Vol 35 (2) ◽  
pp. 182-184 ◽  
Author(s):  
Angela Altomare ◽  
Corrado Cuocci ◽  
Carmelo Giacovazzo ◽  
Antonietta Guagliardi ◽  
Anna Grazia Giuseppina Moliterni ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Powder Diffraction ◽  
Structural Model ◽  
Real Space ◽  
Structure Parameters ◽  
Powder Diffraction Data ◽  
Structure Solution ◽  
Phase Extension ◽  
Space Technique ◽  
New Process

Real-space techniques used for phase extension and refinement in the modern direct procedures forab initiocrystal structure solution of proteins have been optimized for application to powder diffraction data. The new process has been implemented in a modified version ofEXPO[Altomareet al.(1999).J.Appl.Cryst.32, 339–340]. The method is able to supply a structural model that is more complete than that provided by the standardEXPOprogram. The model is then refinedviaa diagonal least-squares procedure, but only when the ratio of the number of observations to the number of structure parameters to be refined is larger than a given threshold.

Software for handling macromolecular envelopes

1999 ◽  
Vol 55 (4) ◽  
pp. 941-944 ◽  
Author(s):  
G. Jacob Kleywegt ◽  
Thomas A. Jones
Keyword(s):  
Electron Density ◽  
Local Density ◽  
Computer Programs ◽  
Real Space ◽  
Atomic Model ◽  
Density Correlation ◽  
Phase Extension ◽  
Crystallographic Symmetry ◽  
Averaging Techniques ◽  
Electron Density Averaging

Macromolecular phase-refinement and phase-extension calculations using real-space electron-density averaging techniques require accurate envelopes (or masks) to define the boundaries of each domain or molecule whose density is to be averaged. An extensive set of tools, implemented in four computer programs (O, MAMA, COMA and MASKIT) are described which can be used to generate such envelopes (either from an atomic model or based on local density-correlation maps), to improve them, to remove overlap owing to crystallographic or non-crystallographic symmetry, to display them and to manipulate them in a variety of manners.

IL MILIONE: a suite of computer programs for crystal structure solution of proteins

2007 ◽  
Vol 40 (3) ◽  
pp. 609-613 ◽  
Author(s):  
Maria C. Burla ◽  
Rocco Caliandro ◽  
Mercedes Camalli ◽  
Benedetta Carrozzini ◽  
Giovanni L. Cascarano ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Electron Density ◽  
Heavy Atom ◽  
Direct Methods ◽  
Computer Programs ◽  
Atomic Resolution ◽  
Protein Crystal ◽  
Anomalous Scattering ◽  
Isomorphous Replacement ◽  
Phase Extension

IL MILIONEis a suite of computer programs devoted to protein crystal structure determination by X-ray crystallography. It may be used in the following key activities. (a)Ab initiophasing,viaPatterson or direct methods. The program may succeed even with structures with up to 6000 non-H atoms in the asymmetric unit, provided that atomic resolution is available, and with data at quasi-atomic resolution (1.4–1.5 Å). (b) Single or multiple isomorphous replacement, single- or multiple-wavelength anomalous diffraction, and single or multiple isomorphous replacement with anomalous scattering techniques. In the first step the program finds the heavy-atom/anomalous scatterer substructure, then automatically uses the above information to phase protein reflections. Phase extension and refinement are performed by electron density modification techniques. (c) Molecular replacement. The orientation and the location of the protein molecules are foundviareciprocal space methods. Phase extension and refinement are performed by electron density modification techniques. All the programs integrated intoIL MILIONEare controlled by means of a user-friendly graphical user interface, which is used to input data and to monitor intermediate and final results by means of real-time updated messages, diagrams and histograms.

Density-modification procedures: improving a calculated map before interpretation

2002 ◽  
Author(s):  
David Blow
Keyword(s):  
Electron Density ◽  
Uniform Density ◽  
Anomalous Scattering ◽  
Molecular Replacement ◽  
Small Proteins ◽  
Isomorphous Replacement ◽  
Structure Factors ◽  
Density Map ◽  
Solvent Molecules ◽  
Electron Density Map

Procedures to determine the phases of the structure factors, by isomorphous replacement, by anomalous scattering, or by molecular replacement, were described in the Chapters 7–9. Using one or more of these methods, phases are generated which allow an electron-density map to be calculated, at a resolution to which the phases are thought to be reliable. In many cases this electron density can be confidently interpreted in terms of atomic positions. But this is not always the case. Quite often, the procedures so far described offer a tantalizing puzzle map, with some features which I think I can interpret, but raising many questions. Before devoting effort to interpreting an unsatisfactory electron-density map, a number of procedures are available, which might make a striking improvement. Perhaps the most important strategy is to seek out more isomorphous and anomalous scattering derivatives. Before doing that, there are other possibilities which may improve an electron-density map without any more experimental data. These methods are known collectively as density modification. The first group of methods exploits features of the electron density which result from the packing of molecules into a crystal. Macromolecular crystals composed of rigid molecules have voids between the molecules filled with disordered solvent, often including the precipitants used in the crystallization process. These solvent regions present featureless density between the structured density of the macromolecules. A high-quality electron-density map will show these featureless regions clearly. In a map of poorer quality, the voids between molecules may be clearly defined, but far from featureless. This provides a method to improve the map. Although some solvent molecules are immobilized on the surface of the macromolecule, those further from the surface are in a disordered liquid-like state which presents a uniform density. Except in very small proteins, the majority of solvent is disordered. If such uniform solvent regions can be recognized, they allow surfaces to be defined which separate solvent regions from protein regions. Two procedures are described below. It has become almost a matter of routine to use one or both of these methods.

5. Seeing atoms

2016 ◽  
pp. 94-106
Author(s):  
A. M. Glazer
Keyword(s):  
Synchrotron Radiation ◽  
Electron Density ◽  
Molecular Replacement ◽  
Phase Information ◽  
Phase Determination ◽  
Isomorphous Replacement ◽  
Multiple Wavelength ◽  
Density Maps ◽  
A New Technique ◽  
Difference Fourier

It is clear that knowledge of the relative phases is essential if we wish to find the atoms in a crystal. So what do we do if we do not have phase information? ‘Seeing atoms’ describes the phase problem and the different methods of phase determination used by crystallographers: a difference Fourier map; the Patterson method; electron density maps; multiple isomorphous replacement; multiple-wavelength anomalous dispersion using synchrotron radiation, which is often used in macromolecular crystallography; molecular replacement, commonly used in protein crystallography; the Sayre equation, a mathematical relationship that enables probable values for the phases of some diffracted beams to be found; and a new technique called charge flipping.

Derivative manipulation in the structure solution of the integral membrane LH2 complex

1999 ◽  
Vol 55 (8) ◽  
pp. 1428-1431 ◽  
Author(s):  
S. M. Prince ◽  
G. McDermott ◽  
A. A. Freer ◽  
M. Z. Papiz ◽  
A. M. Lawless ◽  
...  
Keyword(s):  
Electron Density ◽  
Light Harvesting ◽  
Heavy Atom ◽  
Light Harvesting Complex ◽  
Data Comparison ◽  
Rhodopseudomonas Acidophila ◽  
Isomorphous Replacement ◽  
Density Maps ◽  
Structure Solution

The structure of the peripheral light-harvesting complex from Rhodopseudomonas acidophila strain 10050 was determined by multiple isomorphous replacement methods. The derivatization of the crystals was augmented by the addition of a backsoaking stage. The soak/backsoaked data comparison had greater isomorphism and showed simpler Patterson maps than the standard native/soak comparison. Amplitudes from the derivatized then backsoaked crystals and from the derivatized crystals were compared in order to extract a subset of heavy-atom sites. Using this information, the full array of sites were found from a derivative/native comparison, eventually leading to excellent electron-density maps.

The 4.5 Å Resolution Structure of a Bacterial Serine Protease from Streptomyces Griseus

1974 ◽  
Vol 52 (3) ◽  
pp. 208-220 ◽  
Author(s):  
P. W. Codding ◽  
L. T. J. Delbaere ◽  
K. Hayakawa ◽  
W. L. B. Hutcheon ◽  
M. N. G. James ◽  
...  
Keyword(s):  
Electron Density ◽  
Streptomyces Griseus ◽  
Protein Mass ◽  
Isomorphous Replacement ◽  
Resolution Electron ◽  
Cell Dimensions ◽  
Metal Derivatives ◽  
Density Map ◽  
Unit Cell Dimensions ◽  
Electron Density Map

Three crystalline modifications of the bacterial serine peptidase Streptomyces griseus Protease B have been grown. A 4.5 Å resolution electron density map of one form has been computed from the multiple isomorphous replacement (MIR) phases deduced from two heavy metal derivatives plus the anomalous dispersion effects of one of the derivatives. The crystalline modification used was grown from 0.7–1.0 M KH2PO4 at pH 4.2. These crystals have space group P21212 and unit cell dimensions of a = 44.15 (5) Å, b = 108.72 (10) Å, and c = 37.28 (5) Å. The crystal asymmetric unit contains a protein mass of approximately 19 000 daltons. The electron density map, mean figure of merit 0.80, clearly shows the molecular boundary; relatively long stretches of extended chain are discernable. The active site has been identified from a difference electron density map computed using the MIR protein phases and the amplitude differences between a crystal of the enzyme inhibited at the active serine in solution by p-iodobenzenesulfonyl fluoride (PIPSYL) and those from a crystal of the native enzyme. In addition to showing the site of the PIPSYL binding, there is an apparent conformational change in which the histidine side chain moves away from the serine residue by approximately 4.3 Å.

Traditional direct phasing procedures

2013 ◽  
Author(s):  
Carmelo Giacovazzo
Keyword(s):  
Crystal Structure ◽  
Least Squares ◽  
Fourier Transforms ◽  
Direct Methods ◽  
Real Space ◽  
Reciprocal Space ◽  
Interatomic Distances ◽  
Patterson Function ◽  
Structure Solution ◽  
Phase Extension

Which phasing methods can be included in the category direct methods, and which require a different appellation? Originally, direct phasing was associated with those approaches which were able to derive phases directly from the diffraction moduli, without passing through deconvolution of the Patterson function. Since a Patterson map provides interatomic distances, and therefore lies in ‘direct space’, direct methods were also referred to as reciprocal space methods, and Patterson techniques as real-space methods. Historically, direct methods use 3-,4-, . . . , n-phase invariants and 1-2-, . . . phase seminvariants via the tangent formula or its modified algorithms. Since the 1950s, about a half a century of scientific effort has fallen under the above definition. Such approaches are classified here as traditional direct methods. Today, the situation is more ambiguous, because: (i) modern direct methods programs involve steps operating both in reciprocal space and in direct space, the latter mainly devoted to phase extension and refinement (see Chapter 8); (ii) in the past decade, new phasing methods for crystal structure solution (see Chapter 9) have been developed, based on the properties of Fourier transforms, which again work both in direct and in reciprocal space. Should they therefore be considered to be outside the direct methods category or not? Our choice is as follows. Direct methods are all of the approaches which allow us to derive phases from diffraction amplitudes, without passing through a Patterson function deconvolution. Thus, we also include in this category, charge flipping and VLD (vive la difference), here classified as non-traditional direct methods; their description is postponed to Chapter 9. In accordance with the above assumptions, in this chapter we will shortly illustrate traditional direct phasing procedures, with particular reference to those which are current and in regular use today: mainly the tangent procedures (see Section 6.2) and the cosine least squares technique, which is the basic tool of the shake and Bake method (see Section 6.4).

The crystal structure of myoglobin II. Finback whale myoglobin

1956 ◽  
Vol 237 (1209) ◽  
pp. 255-276 ◽  
Keyword(s):  
Real Space ◽  
Unit Cell ◽  
Paramagnetic Resonance ◽  
Crystal Forms ◽  
Replacement Method ◽  
Isomorphous Replacement ◽  
Cell Dimensions ◽  
The Mean ◽  
Absolute Magnitudes ◽  
Polypeptide Chains

Myoglobin of Balaenoptera physalus (finback whale) normally forms orthorhombic crystals with space group P2 1 2 1 2 and cell dimensions a = 97·4, b = 39·8, c = 42·5 Å; the unit cell contains four molecules. The intensities of the reflexions in the three principal zones have been used to compute Patterson projections, which exhibit rod-like features parallel to z . These rods are 10 Å apart in approximately hexagonal packing and have nodes at 5 Å intervals; they are taken to be the vector equivalents of a set of quasi-parallel polypeptide chains with the same orientation in real space, the mean chain direction being about the same in all four molecules in the cell. Further evidence for this conclusion is derived from the radial distribution of intensities, which is anisotropic in the sense that there is a marked preponderance of 10 Å reflexions in the [001] zone; and from the absolute magnitudes of the reflexions, which are compatible with the hypothesis that 25 to 50% of the molecule is made up of parallel polypeptide chains. If the chains were only approximately parallel they might account for a larger proportion of the molecule. The changes in the low-order reflexions produced by variation in the electron density of the suspension medium have been used to find the position of the molecules in the unit cell; confirmation of the results is found in the Patterson projections. The orientation of the haem group relative to the crystal axes is derived from measurements of paramagnetic resonance (Bennett & Ingram 1956 a ) and of optical dichroism. It is concluded that the plane of the haem group is inclined at 41° to the mean chain direction. Unambiguous information about the structure of the molecule will only be obtained by the isomorphous replacement method; meanwhile the above results, taken in conjunction with others derived from different crystal forms of myoglobin, have been used to discuss some plausible models. The most favoured is 42 Å long (in the chain direction) and has a cross-section of 25 x 35 Å. A molecule having these dimensions might consist of two layers of; polypeptide chains, each layer containing three chains. This arrangement is compatible with one of two favoured Fourier projections derived by applying the inequality relations of Cochran (1952) to the [001] zone.

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