Solution of the structure of the cofactor-binding fragment of CysB: a struggle against non-isomorphism

1999 ◽  
Vol 55 (2) ◽  
pp. 369-378 ◽  
Author(s):  
Koen H. G. Verschueren ◽  
Richard Tyrrell ◽  
Garib N. Murshudov ◽  
Eleanor J. Dodson ◽  
Anthony J. Wilkinson
Keyword(s):  
Electron Density ◽  
Single Molecule ◽  
Model Building ◽  
Heavy Atom ◽  
Data Set ◽  
Crystal Forms ◽  
Cofactor Binding ◽  
Isomorphous Replacement ◽  
Density Maps ◽  
Better Than

The elucidation of the structure of CysB(88–324) by multiple isomorphous replacement (MIR) techniques was seriously delayed by problems encountered at every stage of the analysis. There was extensive non-isomorphism both between different native crystals and between native and heavy-atom-soaked crystals. The heavy-atom substitution was invariably weak and different soaking experiments frequently led to substitution at common sites. These correlated heavy-atom binding sites resulted in an overestimation of the phase information. Missing low-resolution reflections in the native data set, constituting only 2% of the total observations, reduced the power of density modification and phase refinement. Finally, the extensive dimer interface made it difficult to isolate a single molecule in the course of model building into the MIR maps. The power of maximum-likelihood refinement (REFMAC) was exploited in solving the structure by means of iterative cycles of refinement of a partial model, initially comprising only 30% of the protein atoms in the final coordinate set. This technique, which uses experimental phases, can automatically discriminate the correct and incorrect parts of electron-density maps and give properly weighted combined phases which are better than the experimental or calculated ones. This allowed the model to be gradually extended by manual building into improved electron-density maps. A model generated in this way, containing just 50% of the protein atoms, proved good enough to find the transformations needed for multi-crystal averaging between different crystal forms. The averaging regime improved the phasing dramatically such that the complete model could be built. The problems, final solutions and some possible causes for the observed lack of isomorphism are discussed.

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.

scholarly journals Atomic resolution experimental phase information reveals extensive disorder and bound 2-methyl-2,4-pentanediol in Ca2+-calmodulin

2016 ◽  
Vol 72 (1) ◽  
pp. 83-92 ◽  
Author(s):  
Jiusheng Lin ◽  
Henry van den Bedem ◽  
Axel T. Brunger ◽  
Mark A. Wilson
Keyword(s):  
Crystal Structures ◽  
Electron Density ◽  
Full Range ◽  
Binding Pocket ◽  
Data Set ◽  
Conformational Substates ◽  
Density Maps ◽  
Hydrophobic Binding ◽  
Ef Hands ◽  
Critical Regions

Calmodulin (CaM) is the primary calcium signaling protein in eukaryotes and has been extensively studied using various biophysical techniques. Prior crystal structures have noted the presence of ambiguous electron density in both hydrophobic binding pockets of Ca2+-CaM, but no assignment of these features has been made. In addition, Ca2+-CaM samples many conformational substates in the crystal and accurately modeling the full range of this functionally important disorder is challenging. In order to characterize these features in a minimally biased manner, a 1.0 Å resolution single-wavelength anomalous diffraction data set was measured for selenomethionine-substituted Ca2+-CaM. Density-modified electron-density maps enabled the accurate assignment of Ca2+-CaM main-chain and side-chain disorder. These experimental maps also substantiate complex disorder models that were automatically built using low-contour features of model-phased electron density. Furthermore, experimental electron-density maps reveal that 2-methyl-2,4-pentanediol (MPD) is present in the C-terminal domain, mediates a lattice contact between N-terminal domains and may occupy the N-terminal binding pocket. The majority of the crystal structures of target-free Ca2+-CaM have been derived from crystals grown using MPD as a precipitant, and thus MPD is likely to be bound in functionally critical regions of Ca2+-CaM in most of these structures. The adventitious binding of MPD helps to explain differences between the Ca2+-CaM crystal and solution structures and is likely to favor more open conformations of the EF-hands in the crystal.

scholarly journals Automated detection of disulfide bridges in electron density maps using linear discriminant analysis

2005 ◽  
Vol 38 (1) ◽  
pp. 121-125 ◽  
Author(s):  
Thomas R. Ioerger
Keyword(s):  
Discriminant Analysis ◽  
Linear Discriminant Analysis ◽  
Electron Density ◽  
Model Building ◽  
Computational Method ◽  
Training Data ◽  
Decision Threshold ◽  
Disulfide Bridges ◽  
Linear Discriminant ◽  
Density Maps

The ability to recognize disulfide bridges automatically in electron density maps would be useful to both protein crystallographers and automated model-building programs. A computational method is described for recognizing disulfide bridges in uninterpreted maps based on linear discriminant analysis. For each localized spherical region in a map, a vector of rotation-invariant numeric features is calculated that captures various aspects of the local pattern of density. These features values are then input into a linear equation, with coefficients computed to optimize discrimination of a set of training examples (disulfides and non-disulfides), and compared with a decision threshold. The method is shown to be highly accurate at distinguishing disulfides from non-disulfides in both the original training data and in real (experimental) electron density maps of other proteins.

scholarly journals Rapid model building of α-helices in electron-density maps

2010 ◽  
Vol 66 (3) ◽  
pp. 268-275 ◽  
Author(s):  
Thomas C. Terwilliger
Keyword(s):  
High Resolution ◽  
Electron Density ◽  
Model Building ◽  
Main Chain ◽  
Rapid Identification ◽  
Side Chains ◽  
Low Resolution ◽  
Density Maps ◽  
Experimental Electron Density

A method for the identification of α-helices in electron-density maps at low resolution followed by interpretation at moderate to high resolution is presented. Rapid identification is achieved at low resolution, where α-helices appear as tubes of density. The positioning and direction of the α-helices is obtained at moderate to high resolution, where the positions of side chains can be seen. The method was tested on a set of 42 experimental electron-density maps at resolutions ranging from 1.5 to 3.8 Å. An average of 63% of the α-helical residues in these proteins were built and an average of 76% of the residues built matched helical residues in the refined models of the proteins. The overall average r.m.s.d. between main-chain atoms in the modeled α-helices and the nearest atom with the same name in the refined models of the proteins was 1.3 Å.

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.

scholarly journals Building brain network in electron density map determined by micro-CT

2014 ◽  
Vol 70 (a1) ◽  
pp. C1752-C1752
Author(s):  
Rino Saiga ◽  
Susumu Takekoshi ◽  
Naoya Nakamura ◽  
Akihisa Takeuchi ◽  
Kentaro Uesugi ◽  
...  
Keyword(s):  
Density Distribution ◽  
Electron Density ◽  
Electron Density Distribution ◽  
Model Building ◽  
Three Dimensional ◽  
Brain Network ◽  
X Ray ◽  
Density Maps ◽  
Density Map ◽  
Electron Density Map

In macromolecular crystallography, an electron density distribution is traced to build a model of the target molecule. We applied this method to model building for electron density maps of a brain network. Human cerebral tissue was stained with heavy atoms [1]. The sample was then analyzed at the BL20XU beamline of SPring-8 to obtain a three-dimensional map of X-ray attenuation coefficients representing the electron density distribution. Skeletonized wire models were built by placing and connecting nodes in the map [2], as shown in the figure below. The model-building procedures were similar to those reported for crystallographic analyses of macromolecular structures, while the neuronal network was automatically traced by using a Sobel filter. Neuronal circuits were then analytically resolved from the skeletonized models. We suggest that X-ray microtomography along with model building in the electron density map has potential as a method for understanding three-dimensional microstructures relevant to biological functions.

High-resolution structural analysis of purple membrane

1983 ◽  
Vol 41 ◽  
pp. 418-421
Author(s):  
R. Henderson ◽  
J.M. Baldwin ◽  
D.A. Agard ◽  
D. Leifer
Keyword(s):  
Single Molecule ◽  
Molecular Layer ◽  
Purple Membrane ◽  
Three Dimensions ◽  
Electron Microscopic ◽  
Crystal Forms ◽  
Protein Molecules ◽  
Single Molecular Layer ◽  
Common Shape ◽  
Better Than

Two crystal forms of the purple membrane from Halobacterium halobium are being investigated. One is the native p3 form which occurs naturally, and the other is an orthorhombic p22121 form which is made artificially with detergent.Both forms diffract to better than 3 Å resolution. They have been studied by electron diffraction at high (3 Å) resolution, and at lower resolution (6.5-7 Å) by electron microscopy. Both crystal forms occur as membrane sheets, containing a single molecular layer of protein molecules with a lipid bilayer filling the space between the protein molecules, the whole having a thickness of about 45-50 Å. The electron microscopic and diffraction analyses have been carried out in three dimensions using specimens tilted at angles of up to 60° to the incident electron beam.The resulting Fourier maps of the two structures enable a common shape for a single molecule of bacteriorhodopsin to be determined, which is free of the ambiguity of molecular boundary normally found in crystal structure analyses at lower resolution than that required to trace the path of the polypeptide chain.

Mapping lipid and detergent molecules at the surface of membrane proteins

2011 ◽  
Vol 39 (3) ◽  
pp. 775-779 ◽  
Author(s):  
Richard J. Cogdell ◽  
Alastair T. Gardiner ◽  
Aleksander W. Roszak ◽  
Sigitas Stončius ◽  
Pavel Kočovský ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Crystal Structures ◽  
Present Article ◽  
Electron Density ◽  
Rhodobacter Sphaeroides ◽  
Binding Sites ◽  
Heavy Atom ◽  
Hydrophobic Surface ◽  
Density Maps ◽  
Blastochloris Viridis

Electron-density maps for the crystal structures of membrane proteins often show features suggesting binding of lipids and/or detergent molecules on the hydrophobic surface, but usually it is difficult to identify the bound molecules. In our studies, heavy-atom-labelled phospholipids and detergents have been used to unequivocally identify these binding sites at the surfaces of test membrane proteins, the reaction centres from Rhodobacter sphaeroides and Blastochloris viridis. The generality of this method is discussed in the present article.

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.

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