Predicting X-ray diffuse scattering from translation–libration–screw structural ensembles

Identifying the intramolecular motions of proteins and nucleic acids is a major challenge in macromolecular X-ray crystallography. Because Bragg diffraction describes the average positional distribution of crystalline atoms with imperfect precision, the resulting electron density can be compatible with multiple models of motion. Diffuse X-ray scattering can reduce this degeneracy by reporting on correlated atomic displacements. Although recent technological advances are increasing the potential to accurately measure diffuse scattering, computational modeling and validation tools are still needed to quantify the agreement between experimental data and different parameterizations of crystalline disorder. A new tool,phenix.diffuse, addresses this need by employing Guinier's equation to calculate diffuse scattering from Protein Data Bank (PDB)-formatted structural ensembles. As an example case,phenix.diffuseis applied to translation–libration–screw (TLS) refinement, which models rigid-body displacement for segments of the macromolecule. To enable the calculation of diffuse scattering from TLS-refined structures,phenix.tls_as_xyzbuilds multi-model PDB files that sample the underlying T, L and S tensors. In the glycerophosphodiesterase GpdQ, alternative TLS-group partitioning and different motional correlations between groups yield markedly dissimilar diffuse scattering maps with distinct implications for molecular mechanism and allostery. These methods demonstrate how, in principle, X-ray diffuse scattering could extend macromolecular structural refinement, validation and analysis.

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Predicting X-ray Diffuse Scattering from Translation Libration Screw Structural Ensembles

10.1101/012955 ◽

2015 ◽

Author(s):

Andrew Van Benschoten ◽

Pavel Afonine ◽

Thomas Terwilliger ◽

Michael Wall ◽

Colin Jackson ◽

...

Keyword(s):

Diffuse Scattering ◽

Positional Distribution ◽

Data Bank ◽

Bragg Diffraction ◽

Structural Refinement ◽

X Ray ◽

X Ray Crystallography ◽

Atomic Displacements ◽

Technological Advances ◽

Structural Ensembles

Identifying the intramolecular motions of proteins and nucleic acids is a major challenge in macromolecular X-ray crystallography. While Bragg diffraction describes the average positional distribution of crystalline atoms, many different models can fit this distribution equally well. Diffuse X-ray scattering can reduce this degeneracy by directly reporting on correlated atomic displacements. Although recent technological advances are increasing the potential to accurately measure diffuse scattering, computational modeling and validation tools are still needed to quantify the agreement between experimental data and different parameterizations of crystalline disorder. A new tool, phenix.diffuse, addresses this need by employing Guinier’s equation to calculate diffuse scattering from Protein Data Bank (PDB)-formatted structural ensembles. As an example case, phenix.diffuse is applied to Translation-Libration-Screw (TLS) refinement, which models rigid body displacement for segments of the macromolecule. To enable calculation of diffuse scattering from TLS refined structures, phenix.tls_models builds multi-model PDB files that sample the underlying T, L and S tensors. In the glycerophosphodiesterase GpdQ, alternative TLS group partitioning and different motional correlations between groups yield markedly dissimilar diffuse scattering maps with distinct implications for molecular mechanism and allostery. These methods demonstrate how X-ray diffuse scattering can extend macromolecular structural refinement, validation, and analysis.

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Diffuse X-ray Scattering from Correlated Motions in a Protein Crystal

10.1101/805424 ◽

2019 ◽

Author(s):

Steve P. Meisburger ◽

David A. Case ◽

Nozomi Ando

Keyword(s):

Diffuse Scattering ◽

Structural Information ◽

Bragg Diffraction ◽

Protein Crystal ◽

Dynamic Information ◽

X Ray ◽

X Ray Crystallography ◽

X Ray Scattering ◽

Correlated Motions ◽

Hinge Bending

AbstractProtein dynamics are integral to biological function, yet few techniques are sensitive to collective atomic motions. A long-standing goal of X-ray crystallography has been to combine structural information from Bragg diffraction with dynamic information contained in the diffuse scattering background. However, the origin of macromolecular diffuse scattering has been poorly understood, limiting its applicability. We present a detailed diffuse scattering map from triclinic lysozyme that resolves both inter- and intramolecular correlations. These correlations are studied theoretically using both all-atom molecular dynamics and simple vibrational models. Although lattice dynamics reproduce most of the diffuse pattern, protein internal dynamics, which include hinge-bending motions, are needed to explain the short-ranged correlations revealed by Patterson analysis. These insights lay the groundwork for animating crystal structures with biochemically relevant motions.

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PDBrenum: a webserver and program providing Protein Data Bank files renumbered according to their UniProt sequences

10.1101/2021.02.14.431128 ◽

2021 ◽

Author(s):

Bulat Faezov ◽

Roland L. Dunbrack

Keyword(s):

Protein Data Bank ◽

Data Bank ◽

Post Translational Modifications ◽

X Ray ◽

X Ray Crystallography ◽

Link Type ◽

Binding Partners ◽

Cryo Electron Microscopy ◽

Comparative Structure ◽

In The Beginning

AbstractThe Protein Data Bank (PDB) was established at Brookhaven National Laboratories in 1971 as an archive for biological macromolecular crystal structures. In the beginning the archive held only seven structures but in early 2021, the database has more than 170,000 structures solved by X-ray crystallography, nuclear magnetic resonance, cryo-electron microscopy, and other methods. Many proteins have been studied under different conditions (e.g., binding partners such as ligands, nucleic acids, or other proteins; mutations and post-translational modifications), thus enabling comparative structure-function studies. However, these studies are made more difficult because authors are allowed by the PDB to number the amino acids in each protein sequence in any manner they wish. This results in the same protein being numbered differently in the available PDB entries. In addition to the coordinates, there are many fields that contain information regarding specific residues in the sequence of each protein in the entry. Here we provide a webserver and Python3 application that fixes the PDB sequence numbering problem by replacing the author numbering with numbering derived from the corresponding UniProt sequences. We obtain this correspondence from the SIFTS database from PDBe. The server and program can take a list of PDB entries and provide renumbered files in mmCIF format and the legacy PDB format for both asymmetric unit files and biological assembly files provided by PDBe. The server can also take a list of UniProt identifiers (“P04637” or “P53_HUMAN”) and return the desired files.AvailabilitySource code is freely available at https://github.com/Faezov/PDBrenum. The webserver is located at: http://dunbrack3.fccc.edu/[email protected] or [email protected].

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Structural and functional characterization of proteins from the fire blight pathogen Erwinia amylovora. A review on the state of the art

Journal of Plant Pathology ◽

10.1007/s42161-020-00682-4 ◽

2020 ◽

Author(s):

Stefano Benini

Keyword(s):

Fire Blight ◽

Erwinia Amylovora ◽

Functional Characterization ◽

Data Bank ◽

Knock Out ◽

X Ray ◽

X Ray Crystallography ◽

Starting Point ◽

Good Starting Point

Abstract Together with genome analysis and knock-out mutants, structural and functional characterization of proteins provide valuable hints on the biology of the organism under investigation. Structural characterization can be achieved by techniques such as X-ray crystallography, NMR, Cryo-EM. The information derived from the structure are a good starting point to comprehend the details of the proteins molecular function for a better understanding of their biological role. This review aims at describing the progress in the structural and functional characterization of proteins from the plant pathogen Erwinia amylovora obtained by structural biology and currently deposited in the Protein Data Bank.

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The critical role of QM/MM X-ray refinement and accurate tautomer/protomer determination in structure-based drug design

Journal of Computer-Aided Molecular Design ◽

10.1007/s10822-020-00354-6 ◽

2020 ◽

Author(s):

Oleg Y. Borbulevych ◽

Roger I. Martin ◽

Lance M. Westerhoff

Keyword(s):

Drug Design ◽

Critical Role ◽

Pharmaceutical Research ◽

Energy Functional ◽

Structural Refinement ◽

Structure Based Drug Design ◽

X Ray ◽

X Ray Crystallography ◽

Refinement Method ◽

The Impact

Abstract Conventional protein:ligand crystallographic refinement uses stereochemistry restraints coupled with a rudimentary energy functional to ensure the correct geometry of the model of the macromolecule—along with any bound ligand(s)—within the context of the experimental, X-ray density. These methods generally lack explicit terms for electrostatics, polarization, dispersion, hydrogen bonds, and other key interactions, and instead they use pre-determined parameters (e.g. bond lengths, angles, and torsions) to drive structural refinement. In order to address this deficiency and obtain a more complete and ultimately more accurate structure, we have developed an automated approach for macromolecular refinement based on a two layer, QM/MM (ONIOM) scheme as implemented within our DivCon Discovery Suite and "plugged in" to two mainstream crystallographic packages: PHENIX and BUSTER. This implementation is able to use one or more region layer(s), which is(are) characterized using linear-scaling, semi-empirical quantum mechanics, followed by a system layer which includes the balance of the model and which is described using a molecular mechanics functional. In this work, we applied our Phenix/DivCon refinement method—coupled with our XModeScore method for experimental tautomer/protomer state determination—to the characterization of structure sets relevant to structure-based drug design (SBDD). We then use these newly refined structures to show the impact of QM/MM X-ray refined structure on our understanding of function by exploring the influence of these improved structures on protein:ligand binding affinity prediction (and we likewise show how we use post-refinement scoring outliers to inform subsequent X-ray crystallographic efforts). Through this endeavor, we demonstrate a computational chemistry ↔ structural biology (X-ray crystallography) "feedback loop" which has utility in industrial and academic pharmaceutical research as well as other allied fields.

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Measuring and modeling diffuse scattering in protein X-ray crystallography

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1524048113 ◽

2016 ◽

Vol 113 (15) ◽

pp. 4069-4074 ◽

Cited By ~ 25

Author(s):

Andrew H. Van Benschoten ◽

Lin Liu ◽

Ana Gonzalez ◽

Aaron S. Brewster ◽

Nicholas K. Sauter ◽

...

Keyword(s):

Diffuse Scattering ◽

Structure Refinement ◽

Normal Modes ◽

Coarse Grained ◽

X Ray Diffraction ◽

Diffuse Intensity ◽

X Ray ◽

Protein Motions ◽

X Ray Crystallography ◽

Standard Data

X-ray diffraction has the potential to provide rich information about the structural dynamics of macromolecules. To realize this potential, both Bragg scattering, which is currently used to derive macromolecular structures, and diffuse scattering, which reports on correlations in charge density variations, must be measured. Until now, measurement of diffuse scattering from protein crystals has been scarce because of the extra effort of collecting diffuse data. Here, we present 3D measurements of diffuse intensity collected from crystals of the enzymes cyclophilin A and trypsin. The measurements were obtained from the same X-ray diffraction images as the Bragg data, using best practices for standard data collection. To model the underlying dynamics in a practical way that could be used during structure refinement, we tested translation–libration–screw (TLS), liquid-like motions (LLM), and coarse-grained normal-modes (NM) models of protein motions. The LLM model provides a global picture of motions and was refined against the diffuse data, whereas the TLS and NM models provide more detailed and distinct descriptions of atom displacements, and only used information from the Bragg data. Whereas different TLS groupings yielded similar Bragg intensities, they yielded different diffuse intensities, none of which agreed well with the data. In contrast, both the LLM and NM models agreed substantially with the diffuse data. These results demonstrate a realistic path to increase the number of diffuse datasets available to the wider biosciences community and indicate that dynamics-inspired NM structural models can simultaneously agree with both Bragg and diffuse scattering.

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Average structures of the disordered β-phase of Pigment Red 170: a single-crystal X-ray diffraction study

Acta Crystallographica Section B Structural Science Crystal Engineering and Materials ◽

10.1107/s2052520614000407 ◽

2014 ◽

Vol 70 (2) ◽

pp. 283-295 ◽

Cited By ~ 4

Author(s):

Rangana Warshamanage ◽

Anthony Linden ◽

Martin U. Schmidt ◽

Hans-Beat Bürgi

Keyword(s):

Single Crystal ◽

Diffuse Scattering ◽

Bragg Diffraction ◽

X Ray Diffraction ◽

Group Setting ◽

X Ray ◽

Space Groups ◽

Average Structure ◽

Β Phase ◽

Layer Stacking

The β-phase of the industrially important Pigment Red 170 (β-P.R. 170) has a structure with severe layer stacking disorder. The single-crystal X-ray diffraction pattern consists of a difficult-to-disentangle mix of Bragg diffraction superimposed on rods of diffuse scattering which impede the estimation of accurate Bragg intensities. Two average monoclinic structure models with the same unit-cell dimensions, but different extents of disorder in the layers and different space groups seem plausible, one with the non-conventional space group settingB21/g(No. 14,Z′ = 2) and one inP21/a(No. 14,Z′ = 4). Disordered molecules related by a translation of 0.158bare present in all layers of theB21/gmodel and in every second layer of theP21/amodel. Layer-to-layer contacts are practically the same in both models. According to order–disorder theory, both models are valid superposition structures. Structure-factor calculations show that the pattern of strong and weak Bragg reflections is very similar for the two models.Rfactors indicate that theB21/gmodel is the most economic representation of the average structure. However, given the limitations in data processing, theP21/amodel should not be discarded and further insight sought from a detailed analysis of the experimental diffuse scattering. The difficulties encountered in this analysis raise the question of whether or not the concept of an average structure is applicable in practice to β-P.R. 170.

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New developments in crystallography: exploring its technology, methods and scope in the molecular biosciences

Bioscience Reports ◽

10.1042/bsr20170204 ◽

2017 ◽

Vol 37 (4) ◽

Cited By ~ 7

Author(s):

John R. Helliwell

Keyword(s):

Crystal Structure ◽

Data Bank ◽

Crystal Structure Analysis ◽

Public Understanding ◽

Biological Macromolecules ◽

Hydrogen Atoms ◽

X Ray ◽

The Public ◽

X Ray Crystallography ◽

Chemical Catalysis

Since the Protein Data Bank (PDB) was founded in 1971, there are now over 120,000 depositions, the majority of which are from X-ray crystallography and 90% of those made use of synchrotron beamlines. At the Cambridge Structure Database (CSD), founded in 1965, there are more than 800,000 ‘small molecule’ crystal structure depositions and a very large number of those are relevant in the biosciences as ligands or cofactors. The technology for crystal structure analysis is still developing rapidly both at synchrotrons and in home labs. Determination of the details of the hydrogen atoms in biological macromolecules is well served using neutrons as probe. Large multi-macromolecular complexes cause major challenges to crystallization; electrons as probes offer unique advantages here. Methods developments naturally accompany technology change, mainly incremental but some, such as the tuneability, intensity and collimation of synchrotron radiation, have effected radical changes in capability of biological crystallography. In the past few years, the X-ray laser has taken X-ray crystallography measurement times into the femtosecond range. In terms of applications many new discoveries have been made in the molecular biosciences. The scope of crystallographic techniques is indeed very wide. As examples, new insights into chemical catalysis of enzymes and relating ligand bound structures to thermodynamics have been gained but predictive power is seen as not yet achieved. Metal complexes are also an emerging theme for biomedicine applications. Our studies of coloration of live and cooked lobsters proved to be an unexpected favourite with the public and schoolchildren. More generally, public understanding of the biosciences and crystallography’s role within the field have been greatly enhanced by the United Nations International Year of Crystallography coordinated by the International Union of Crystallography. This topical review describes each of these areas along with illustrative results to document the scope of each methodology.

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xMDFF: molecular dynamics flexible fitting of low-resolution X-ray structures

Acta Crystallographica Section D Biological Crystallography ◽

10.1107/s1399004714013856 ◽

2014 ◽

Vol 70 (9) ◽

pp. 2344-2355 ◽

Cited By ~ 33

Author(s):

Ryan McGreevy ◽

Abhishek Singharoy ◽

Qufei Li ◽

Jingfen Zhang ◽

Dong Xu ◽

...

Keyword(s):

Molecular Dynamics ◽

Large Scale ◽

Protein Structures ◽

Data Bank ◽

Real Space ◽

Low Resolution ◽

X Ray ◽

X Ray Crystallography ◽

Atomic Structures ◽

Electron Density Map

X-ray crystallography remains the most dominant method for solving atomic structures. However, for relatively large systems, the availability of only medium-to-low-resolution diffraction data often limits the determination of all-atom details. A new molecular dynamics flexible fitting (MDFF)-based approach, xMDFF, for determining structures from such low-resolution crystallographic data is reported. xMDFF employs a real-space refinement scheme that flexibly fits atomic models into an iteratively updating electron-density map. It addresses significant large-scale deformations of the initial model to fit the low-resolution density, as tested with synthetic low-resolution maps of D-ribose-binding protein. xMDFF has been successfully applied to re-refine six low-resolution protein structures of varying sizes that had already been submitted to the Protein Data Bank. Finally,viasystematic refinement of a series of data from 3.6 to 7 Å resolution, xMDFF refinements together with electrophysiology experiments were used to validate the first all-atom structure of the voltage-sensing protein Ci-VSP.

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