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.

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.

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.

Phase perturbation as a means of electron density map quality evaluation

2008 ◽  
Vol 41 (1) ◽  
pp. 31-37 ◽  
Author(s):  
Olga Kirillova
Keyword(s):  
Electron Density ◽  
Data Sets ◽  
Molecular Replacement ◽  
Data Set ◽  
Density Maps ◽  
Phase Perturbation ◽  
Replacement Solution ◽  
Electron Density Map ◽  
Trial Phase

This paper describes a new means for evaluating the quality of crystallographic electron density maps. It has been found that a better data set possesses greater robustness against perturbations applied to the phases. Thus it allows recognition of a more precise phase set and provides a way to select the best or reject the worst from several noisy data sets derived from the same crystal structure. The results indicate that calculation of the correlations by the procedure described here can be useful in ranking electron density maps in this aspect of quality. The method suggested has potential use for selecting a better molecular replacement solution, as well as for evaluating trial phase sets inab initiophasing procedures.

scholarly journals Photoinduced isomerization sampling of retinal in bacteriorhodopsin

2021 ◽  
Author(s):  
Zhong Ren
Keyword(s):  
Electron Density ◽  
Binding Pocket ◽  
Proton Pumping ◽  
Time Resolved ◽  
Numerical Resolution ◽  
Light Sensing ◽  
Molecular Events ◽  
Density Maps ◽  
Light Effects ◽  
Protein Pocket

Abstract Photoisomerization of retinoids inside a confined protein pocket represents a critical chemical event in many important biological processes from animal vision, non-visual light effects, to bacterial light sensing and harvesting. Light driven proton pumping in bacteriorhodopsin entails exquisite electronic and conformational reconfigurations during its photocycle. However, it has been a major challenge to delineate transient molecular events preceding and following the photoisomerization of the retinal from noisy electron density maps when varying populations of intermediates coexist and evolve as a function of time. Here I report several distinct early photoproducts deconvoluted from the recently observed mixtures in time-resolved serial crystallography. This deconvolution substantially improves the quality of the electron density maps hence demonstrates that the all-trans retinal undergoes extensive isomerization sampling before it proceeds to the productive 13-cis configuration. Upon light absorption, the chromophore attempts to perform trans-to-cis isomerization at every double bond coupled with the stalled anti-to-syn rotations at multiple single bonds along its polyene chain. Such isomerization sampling pushes all seven transmembrane helices to bend outward, resulting in a transient expansion of the retinal binding pocket, and later, a contraction due to recoiling. These ultrafast responses observed at the atomic resolution support that the productive photoreaction in bacteriorhodopsin is initiated by light-induced charge separation in the prosthetic chromophore yet governed by stereoselectivity of its protein pocket. The method of a numerical resolution of concurrent events from mixed observations is also generally applicable.

scholarly journals Photoinduced isomerization sampling of retinal in bacteriorhodopsin

2021 ◽  
Author(s):  
Zhong Ren
Keyword(s):  
Electron Density ◽  
Binding Pocket ◽  
Proton Pumping ◽  
Time Resolved ◽  
Numerical Resolution ◽  
Light Sensing ◽  
Molecular Events ◽  
Density Maps ◽  
Light Effects ◽  
Protein Pocket

Photoisomerization of retinoids inside a confined protein pocket represents a critical chemical event in many important biological processes from animal vision, non-visual light effects, to bacterial light sensing and harvesting. Light driven proton pumping in bacteriorhodopsin entails exquisite electronic and conformational reconfigurations during its photocycle. However, it has been a major challenge to delineate transient molecular events preceding and following the photoisomerization of the retinal from noisy electron density maps when varying populations of intermediates coexist and evolve as a function of time. Here I report several distinct early photoproducts deconvoluted from the recently observed mixtures in time-resolved serial crystallography. This deconvolution substantially improves the quality of the electron density maps hence demonstrates that the all-trans retinal undergoes extensive isomerization sampling before it proceeds to the productive 13-cis configuration. Upon light absorption, the chromophore attempts to perform trans-to-cis isomerization at every double bond coupled with the stalled anti-to-syn rotations at multiple single bonds along its polyene chain. Such isomerization sampling pushes all seven transmembrane helices to bend outward, resulting in a transient expansion of the retinal binding pocket, and later, a contraction due to recoiling. These ultrafast responses observed at the atomic resolution support that the productive photoreaction in bacteriorhodopsin is initiated by light-induced charge separation in the prosthetic chromophore yet governed by stereoselectivity of its protein pocket. The method of a numerical resolution of concurrent events from mixed observations is also generally applicable.

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.

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.

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.

scholarly journals DNA conformational transitions inferred from re-evaluation ofm|Fo| −D|Fc| electron-density maps

2017 ◽  
Vol 73 (7) ◽  
pp. 600-608 ◽  
Author(s):  
Tomoko Sunami ◽  
Toshiyuki Chatake ◽  
Hidetoshi Kono
Keyword(s):  
Transcriptional Regulation ◽  
Crystal Structures ◽  
Electron Density ◽  
Conformational Transitions ◽  
Conformational Flexibility ◽  
Dna Packaging ◽  
Biological Processes ◽  
Density Maps ◽  
Conformational Variations ◽  
Better Than

Conformational flexibility of DNA plays important roles in biological processes such as transcriptional regulation and DNA packagingetc. To understand the mechanisms of these processes, it is important to analyse when, where and how DNA shows conformational variations. Recent analyses have indicated that conventional refinement methods do not always provide accurate models of crystallographic heterogeneities and that some information on polymorphism has been overlooked in previous crystallographic studies. In the present study, them|Fo| −D|Fc| electron-density maps of double-helical DNA crystal structures were calculated at a resolution equal to or better than 1.5 Å and potential conformational transitions were found in 27% of DNA phosphates. Detailed analyses of them|Fo| −D|Fc| peaks indicated that some of these unassigned densities correspond to ZI ↔ ZII or A/B → BI conformational transitions. A relationship was also found between ZI/ZII transitions and metal coordination in Z-DNA from the detected peaks. The present study highlights that frequent transitions of phosphate backbones occur even in crystals and that some of these transitions are affected by the local molecular environment.

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