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.

scholarly journals With phases: how two wrongs can sometimes make a right

2010 ◽  
Vol 66 (4) ◽  
pp. 420-425 ◽  
Author(s):  
Pietro Roversi ◽  
Steven Johnson ◽  
Susan M. Lea
Keyword(s):  
Crystal Structures ◽  
Case Studies ◽  
Electron Density ◽  
Heavy Atom ◽  
Direct Methods ◽  
Molecular Replacement ◽  
Asymmetric Unit ◽  
Partial Model ◽  
Density Maps

In isolation, both weak isomorphous/anomalous difference signals from heavy-atom derivatization and phases from partial molecular-replacement solutions for a subset of the asymmetric unit often fall short of producing interpretable electron-density maps. Phases generated from very partial molecular-replacement models (if generated carefully) can be used to reliably locate heavy-atom sites, even if the signal is not sufficiently strong to allow robust finding of the sites using Patterson interpretation or direct methods. Additional advantages are that using molecular-replacement phases to define the heavy-atom substructure avoids the need for subsequent hand determination and/or origin-choice reconciliation and that the partial model can be used to aid the mask determination during solvent flattening. Two case studies are presented in which it was only by combining experimental and molecular-replacement phasing approaches that the crystal structures could be determined.

scholarly journals The role of BamA in the biogenesis of beta-barrel membrane proteins

2014 ◽  
Vol 70 (a1) ◽  
pp. C578-C578
Author(s):  
Nicholas Noinaj ◽  
Adam Kuszak ◽  
Curtis Balusek ◽  
JC Gumbart ◽  
Petra Lukacik ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Outer Membrane ◽  
Crystal Structures ◽  
Outer Membrane Proteins ◽  
Hydrophobic Surface ◽  
Md Simulations ◽  
Structural Features ◽  
Bam Complex ◽  
Beta Barrel

Beta-barrel membrane proteins are essential for nutrient import, signaling, motility, and survival. In Gram-negative bacteria, the beta-barrel assembly machinery (BAM) complex is responsible for the biogenesis of beta-barrel outer membrane proteins (OMPs), with homologous complexes found in mitochondria and chloroplasts. Despite their essential roles, exactly how these OMPs are formed remains unknown. The BAM complex consists of a central and essential component called BamA (an OMP itself) and four lipoproteins called BamB-E. While the structure of the lipoproteins have been reported, the structure of full length BamA has been elusive. Recently though, we described the structure of BamA from two species of bacteria: Neisseria gonorrhoeae and Haemophilus ducreyi. BamA consists of a large periplasmic domain attached to a 16-strand transmembrane beta-barrel domain. Together, our crystal structures and molecule dynamics (MD) simulations revealed several structural features which gave clues to the mechanism by which BamA catalyzes beta-barrel assembly. The first is that the interior cavity is accessible in one BamA structure and conformationally closed in the other. Second, an exterior rim of the beta-barrel has a distinctly narrowed hydrophobic surface, locally destabilizing the outer membrane. Third, the beta-barrel can undergo lateral opening, suggesting a route from the interior cavity in BamA into the outer membrane. And fourth, a surface exposed exit pore positioned above the lateral opening site which may play a role in the biogenesis of extracellular loops. In this presentation, the crystal structures and MD simulations of BamA will be presented along with our work looking at the role of these four structural features in the role of BamA within the BAM complex.

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.

Use of Patterson-based methods automatically to determine the structures of heavy-atom-containing proteins with up to 6000 non-hydrogen atoms in the asymmetric unit

2006 ◽  
Vol 39 (5) ◽  
pp. 728-734 ◽  
Author(s):  
Maria Cristina Burla ◽  
Rocco Caliandro ◽  
Benedetta Carrozzini ◽  
Giovanni Luca Cascarano ◽  
Liberato De Caro ◽  
...  
Keyword(s):  
Crystal Structures ◽  
Protein Data Bank ◽  
Heavy Atom ◽  
Data Bank ◽  
Atomic Resolution ◽  
Asymmetric Unit ◽  
Hydrogen Atoms ◽  
Heavy Atoms ◽  
Density Maps ◽  
Resolution Data

The Patterson superposition methods described by Burlaet al.[J. Appl. Cryst.(2006),39, 527–535], based on the use of the `multiple implication functions', have been enriched by supplementary filtering techniques based on some general (resolution-dependent) features of both the Patterson and the electron density maps. The method has been implemented in a modified version of the programSIR2004and tested using a set of 20 crystal structures selected from the Protein Data Bank, having a number of non-hydrogen atoms in the asymmetric unit larger than 2000, atomic resolution data and some heavy atoms (equal to or heavier than Ca). The new phasing procedure is able to solve most of the test structures, among which there are two proteins with more than 6000 non-hydrogen atoms in the asymmetric unit, so extending by far the complexity today commonly considered as the limit for Patterson-based methods (i.e.about 2000 non-hydrogen atoms).

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.

Automatic procedure for crystal-structure analysis with the heavy-atom technique*

1971 ◽  
Vol 134 (1-2) ◽  
Author(s):  
Vasantha Pattabhi ◽  
K. Venkatesan
Keyword(s):  
Electron Density ◽  
Least Squares Method ◽  
Heavy Atom ◽  
Site Occupancy ◽  
Crystal Structure Analysis ◽  
Density Maps ◽  
Structure Factors ◽  
Subsequent Calculation ◽  
Density Map ◽  
Electron Density Map

AbstractThis paper deals with a possible method of determining molecular structure directly, using x-ray data. The peaks chosen from the electron-density map calculated using the signs obtained from the contribution of the heavy atom are assigned site-occupancy values which depend upon the peak heights. The occupancy parameter is refined by the least-squares method and the peaks for which the value of the occupancy parameters increases are taken as real atoms in the subsequent calculation of structure factors and electron-density maps. The method has been tested in two hypothetical cases and in a real case.

Removing bias from solvent atoms in electron density maps

2008 ◽  
Vol 41 (4) ◽  
pp. 761-767 ◽  
Author(s):  
Eric N. Brown
Keyword(s):  
Crystal Structures ◽  
Electron Density ◽  
Protein Crystal ◽  
Water Molecules ◽  
Data Set ◽  
Atomic Structures ◽  
Research Article ◽  
Density Maps ◽  
Atom Position

Atomic structures of proteins determinedviaprotein crystallography contain numerous solvent atoms. The experimental data for the determination of a water molecule's O-atom position is often a small contained blob of unidentified electron density. Unfortunately, the nature of crystallographic refinement lets poorly placed solvent atoms bias the future refined positions of all atoms in the crystal structure. This research article presents the technique of omit-maps applied to remove the bias introduced by poorly determined solvent atoms, enabling the identification of incorrectly placed water molecules in partially refined crystal structures. A total of 160 protein crystal structures with 45 912 distinct water molecules were processed using this technique. Most of the water molecules in the deposited structures were well justified. However, a few of the solvent atoms in this test data set changed appreciably in position, displacement parameter or electron density when fitted to the solvent omit-map, raising questions about how much experimental support exists for these solvent atoms.

scholarly journals Determining crystal structures through crowdsourcing and coursework

2016 ◽  
Vol 7 (1) ◽  
Author(s):  
Scott Horowitz ◽  
◽  
Brian Koepnick ◽  
Raoul Martin ◽  
Agnes Tymieniecki ◽  
...  
Keyword(s):  
Crystal Structures ◽  
Electron Density ◽  
Undergraduate Students ◽  
Model Building ◽  
Computer Game ◽  
Real Space ◽  
Amyloid Toxicity ◽  
New Family ◽  
Density Maps ◽  
New Feature

Abstract We show here that computer game players can build high-quality crystal structures. Introduction of a new feature into the computer game Foldit allows players to build and real-space refine structures into electron density maps. To assess the usefulness of this feature, we held a crystallographic model-building competition between trained crystallographers, undergraduate students, Foldit players and automatic model-building algorithms. After removal of disordered residues, a team of Foldit players achieved the most accurate structure. Analysing the target protein of the competition, YPL067C, uncovered a new family of histidine triad proteins apparently involved in the prevention of amyloid toxicity. From this study, we conclude that crystallographers can utilize crowdsourcing to interpret electron density information and to produce structure solutions of the highest quality.

scholarly journals Simulation of electron density maps for two-dimensional crystal structures usingMathematica

2001 ◽  
Vol 34 (5) ◽  
pp. 658-660 ◽  
Author(s):  
Plinio Delatorre ◽  
Walter Filgueira de Azevedo Jr
Keyword(s):  
Crystal Structures ◽  
Electron Density ◽  
Two Dimensional ◽  
Thermal Displacement ◽  
Density Maps ◽  
Cell Dimensions ◽  
Unit Cell Dimensions ◽  
The Relationship ◽  
Dimensional Crystal

The simulations presented here are based on the programMathematicaas a tool to present electron density maps of two-dimensional crystal structures. The models give further insights into the relationship between the thermal displacement parameters and the quality of the electron density maps. Furthermore, users can readily test the effects of several crystallographic parameters on the electron density maps, such as, the number of reflections, the thermal displacement parameters and the unit-cell dimensions.

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.

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