Publications by authors named "Cy M Jeffries"

63 Publications

Order from disorder in the sarcomere: FATZ forms a fuzzy but tight complex and phase-separated condensates with α-actinin.

Sci Adv 2021 May 28;7(22). Epub 2021 May 28.

Department of Structural and Computational Biology, Max Perutz Labs, University of Vienna, Campus Vienna Biocenter 5, A-1030 Vienna, Austria.

In sarcomeres, α-actinin cross-links actin filaments and anchors them to the Z-disk. FATZ (filamin-, α-actinin-, and telethonin-binding protein of the Z-disk) proteins interact with α-actinin and other core Z-disk proteins, contributing to myofibril assembly and maintenance. Here, we report the first structure and its cellular validation of α-actinin-2 in complex with a Z-disk partner, FATZ-1, which is best described as a conformational ensemble. We show that FATZ-1 forms a tight fuzzy complex with α-actinin-2 and propose an interaction mechanism via main molecular recognition elements and secondary binding sites. The obtained integrative model reveals a polar architecture of the complex which, in combination with FATZ-1 multivalent scaffold function, might organize interaction partners and stabilize α-actinin-2 preferential orientation in Z-disk. Last, we uncover FATZ-1 ability to phase-separate and form biomolecular condensates with α-actinin-2, raising the question whether FATZ proteins can create an interaction hub for Z-disk proteins through membraneless compartmentalization during myofibrillogenesis.
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http://dx.doi.org/10.1126/sciadv.abg7653DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8163081PMC
May 2021

: expanded functionality and new tools for small-angle scattering data analysis.

J Appl Crystallogr 2021 Feb 1;54(Pt 1):343-355. Epub 2021 Feb 1.

European Molecular Biology Laboratory, Hamburg Site, Notkestrasse 85, Building 25 A, Hamburg, 22607, Germany.

The software suite encompasses a number of programs for the processing, visualization, analysis and modelling of small-angle scattering data, with a focus on the data measured from biological macromolecules. Here, new developments in the package are described. They include , for simulating isotropic 2D scattering patterns; , to perform operations on 2D images and masks; , a method for variance estimation of structural invariants through parametric resampling; , which computes the pair distance distribution function by a direct Fourier transform of the scattering data; , to compute the scattering data from a pair distance distribution function, allowing comparison with the experimental data; a new module in for Bayesian consensus-based concentration-independent molecular weight estimation; , an shape analysis method that optimizes the search model directly against the scattering data; , an application to set up the initial search volume for multiphase modelling of membrane proteins; , to perform quasi-atomistic modelling of liposomes with elliptical shapes; , which models conformational changes in nucleic acid structures through normal mode analysis in torsion angle space; , which reconstructs the shape of an unknown intermediate in an evolving system; and and , for modelling multilamellar and asymmetric lipid vesicles, respectively. In addition, technical updates were deployed to facilitate maintainability of the package, which include porting the graphical interface to Qt5, updating - a plugin to run a subset of tools - to be both Python 2 and 3 compatible, and adding utilities to facilitate mmCIF compatibility in future releases. All these features are implemented in , freely available for academic users at https://www.embl-hamburg.de/biosaxs/software.html.
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http://dx.doi.org/10.1107/S1600576720013412DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7941305PMC
February 2021

Comment on the Optimal Parameters to Derive Intrinsically Disordered Protein Conformational Ensembles from Small-Angle X-ray Scattering Data Using the Ensemble Optimization Method.

J Chem Theory Comput 2021 Apr 16;17(4):2014-2021. Epub 2021 Mar 16.

Centre de Biologie Structurale (CBS), INSERM, CNRS, Université de Montpellier, 29, rue de Navacelles, 34090 Montpellier, France.

The Ensemble Optimization Method (EOM) is a popular approach to describe small-angle X-ray scattering (SAXS) data from highly disordered proteins. The EOM algorithm selects subensembles of coexisting states from large pools of randomized conformations to fit the SAXS data. Based on the unphysical bimodal radius of gyration () distribution of conformations resulting from the EOM analysis, a recent article (Fagerberg et al. 2019, 15 (12), 6968-6983) concluded that this approach inadequately described the SAXS data measured for human Histatin 5 (Hst5), a peptide with antifungal properties. Using extensive experimental and synthetic data, we explored the origin of this observation. We found that the one-bead-per-residue coarse-grained representation with averaged scattering form factors (provided in the EOM as an add-on to represent disordered missing loops or domains) may not be appropriate for EOM analyses of scattering data from short (below 50 residues) proteins/peptides. The method of choice for these proteins is to employ atomistic models (e.g., from molecular dynamics simulations) to sample the protein conformational landscape. As a convenient alternative, we have also improved the coarse-grained approach by introducing amino acid specific form factors in the calculations. We also found that, for small proteins, the search for relatively large subensembles of 20-50 conformers (as implemented in the original EOM version) more adequately describes the conformational space sampled in solution than the procedures optimizing the ensemble size. Our observations have been added as recommendations into the information for EOM users to promote the proper utilization of the program for ensemble-based modeling of SAXS data for all types of disordered systems.
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http://dx.doi.org/10.1021/acs.jctc.1c00014DOI Listing
April 2021

Selection, biophysical and structural analysis of synthetic nanobodies that effectively neutralize SARS-CoV-2.

Nat Commun 2020 11 4;11(1):5588. Epub 2020 Nov 4.

Centre for Structural Systems Biology (CSSB), DESY and European Molecular Biology Laboratory Hamburg, Notkestrasse 85, D-22607, Hamburg, Germany.

The coronavirus SARS-CoV-2 is the cause of the ongoing COVID-19 pandemic. Therapeutic neutralizing antibodies constitute a key short-to-medium term approach to tackle COVID-19. However, traditional antibody production is hampered by long development times and costly production. Here, we report the rapid isolation and characterization of nanobodies from a synthetic library, known as sybodies (Sb), that target the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein. Several binders with low nanomolar affinities and efficient neutralization activity were identified of which Sb23 displayed high affinity and neutralized pseudovirus with an IC of 0.6 µg/ml. A cryo-EM structure of the spike bound to Sb23 showed that Sb23 binds competitively in the ACE2 binding site. Furthermore, the cryo-EM reconstruction revealed an unusual conformation of the spike where two RBDs are in the 'up' ACE2-binding conformation. The combined approach represents an alternative, fast workflow to select binders with neutralizing activity against newly emerging viruses.
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http://dx.doi.org/10.1038/s41467-020-19204-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7642358PMC
November 2020

Rapid screening of grown protein crystals via a small-angle X-ray scattering/X-ray powder diffraction synergistic approach.

J Appl Crystallogr 2020 Oct 25;53(Pt 5):1169-1180. Epub 2020 Sep 25.

Institute of Biochemistry, University of Lübeck, Ratzeburger Allee 160, Lübeck 23562, Germany.

Crystallization of recombinant proteins in living cells is an exciting new approach for structural biology that provides an alternative to the time-consuming optimization of protein purification and extensive crystal screening steps. Exploiting the potential of this approach requires a more detailed understanding of the cellular processes involved and versatile screening strategies for crystals in a cell culture. Particularly if the target protein forms crystalline structures of unknown morphology only in a small fraction of cells, their detection by applying standard visualization techniques can be time consuming and difficult owing to the environmental challenges imposed by the living cells. In this study, a high-brilliance and low-background bioSAXS beamline is employed for rapid and sensitive detection of protein microcrystals grown within insect cells. On the basis of the presence of Bragg peaks in the recorded small-angle X-ray scattering profiles, it is possible to assess within seconds whether a cell culture contains microcrystals, even in a small percentage of cells. Since such information cannot be obtained by other established detection methods in this time frame, this screening approach has the potential to overcome one of the bottlenecks of intracellular crystal detection. Moreover, the association of the Bragg peak positions in the scattering curves with the unit-cell composition of the protein crystals raises the possibility of investigating the impact of environmental conditions on the crystal structure of the intracellular protein crystals. This information provides valuable insights helping to further understand the crystallization process.
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http://dx.doi.org/10.1107/S1600576720010687DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7534541PMC
October 2020

Structural basis for DNA recognition and allosteric control of the retinoic acid receptors RAR-RXR.

Nucleic Acids Res 2020 09;48(17):9969-9985

Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Illkirch, France.

Retinoic acid receptors (RARs) as a functional heterodimer with retinoid X receptors (RXRs), bind a diverse series of RA-response elements (RAREs) in regulated genes. Among them, the non-canonical DR0 elements are bound by RXR-RAR with comparable affinities to DR5 elements but DR0 elements do not act transcriptionally as independent RAREs. In this work, we present structural insights for the recognition of DR5 and DR0 elements by RXR-RAR heterodimer using x-ray crystallography, small angle x-ray scattering, and hydrogen/deuterium exchange coupled to mass spectrometry. We solved the crystal structures of RXR-RAR DNA-binding domain in complex with the Rarb2 DR5 and RXR-RXR DNA-binding domain in complex with Hoxb13 DR0. While cooperative binding was observed on DR5, the two molecules bound non-cooperatively on DR0 on opposite sides of the DNA. In addition, our data unveil the structural organization and dynamics of the multi-domain RXR-RAR DNA complexes providing evidence for DNA-dependent allosteric communication between domains. Differential binding modes between DR0 and DR5 were observed leading to differences in conformation and structural dynamics of the multi-domain RXR-RAR DNA complexes. These results reveal that the topological organization of the RAR binding element confer regulatory information by modulating the overall topology and structural dynamics of the RXR-RAR heterodimers.
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http://dx.doi.org/10.1093/nar/gkaa697DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7515732PMC
September 2020

RovC - a novel type of hexameric transcriptional activator promoting type VI secretion gene expression.

PLoS Pathog 2020 09 23;16(9):e1008552. Epub 2020 Sep 23.

Department of Molecular Infection Biology, Helmholtz Centre for Infection Research, Braunschweig, Germany.

Type VI secretion systems (T6SSs) are complex macromolecular injection machines which are widespread in Gram-negative bacteria. They are involved in host-cell interactions and pathogenesis, required to eliminate competing bacteria, or are important for the adaptation to environmental stress conditions. Here we identified regulatory elements controlling the T6SS4 of Yersinia pseudotuberculosis and found a novel type of hexameric transcription factor, RovC. RovC directly interacts with the T6SS4 promoter region and activates T6SS4 transcription alone or in cooperation with the LysR-type regulator RovM. A higher complexity of regulation was achieved by the nutrient-responsive global regulator CsrA, which controls rovC expression on the transcriptional and post-transcriptional level. In summary, our work unveils a central mechanism in which RovC, a novel key activator, orchestrates the expression of the T6SS weapons together with a global regulator to deploy the system in response to the availability of nutrients in the species' native environment.
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http://dx.doi.org/10.1371/journal.ppat.1008552DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7535981PMC
September 2020

Anomeric Selectivity of Trehalose Transferase with Rare l-Sugars.

ACS Catal 2020 Aug 22;10(15):8835-8839. Epub 2020 Jul 22.

Department of Biotechnology, Delft University of Technology, Van der Maasweg 9, 2629 HZ, Delft, The Netherlands.

Retaining LeLoir glycosyltransferases catalyze the formation of glycosidic bonds between nucleotide sugar donors and carbohydrate acceptors. The anomeric selectivity of trehalose transferase from was investigated for both d- and l-glycopyranose acceptors. The enzyme couples a wide range of carbohydrates, yielding trehalose analogues with conversion and enantioselectivity of >98%. The anomeric selectivity inverts from α,α-(1 → 1)-glycosidic bonds for d-glycopyranose acceptors to α,β-(1 → 1)-glycosidic bonds for l-glycopyranose acceptors, while ()-selectivity was retained for both types of sugar acceptors. Comparison of protein crystal structures of trehalose transferase in complex with α,α-trehalose and an unnatural α,β-trehalose analogue highlighted the mechanistic rationale for the observed inversion of anomeric selectivity.
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http://dx.doi.org/10.1021/acscatal.0c02117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7493220PMC
August 2020

Structure of a collagen VI α3 chain VWA domain array: adaptability and functional implications of myopathy causing mutations.

J Biol Chem 2020 09 21;295(36):12755-12771. Epub 2020 Jul 21.

Center for Biochemistry, Medical Faculty, University of Cologne, Cologne, Germany

Collagen VI is a ubiquitous heterotrimeric protein of the extracellular matrix (ECM) that plays an essential role in the proper maintenance of skeletal muscle. Mutations in collagen VI lead to a spectrum of congenital myopathies, from the mild Bethlem myopathy to the severe Ullrich congenital muscular dystrophy. Collagen VI contains only a short triple helix and consists primarily of von Willebrand factor type A (VWA) domains, protein-protein interaction modules found in a range of ECM proteins. Disease-causing mutations occur commonly in the VWA domains, and the second VWA domain of the α3 chain, the N2 domain, harbors several such mutations. Here, we investigate structure-function relationships of the N2 mutations to shed light on their possible myopathy mechanisms. We determined the X-ray crystal structure of N2, combined with monitoring secretion efficiency in cell culture of selected N2 single-domain mutants, finding that mutations located within the central core of the domain severely affect secretion efficiency. In longer α3 chain constructs, spanning N6-N3, small-angle X-ray scattering demonstrates that the tandem VWA array has a modular architecture and samples multiple conformations in solution. Single-particle EM confirmed the presence of multiple conformations. Structural adaptability appears intrinsic to the VWA domain region of collagen VI α3 and has implications for binding interactions and modulating stiffness within the ECM.
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http://dx.doi.org/10.1074/jbc.RA120.014865DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7476709PMC
September 2020

Structure-Based Identification and Functional Characterization of a Lipocalin in the Malaria Parasite Plasmodium falciparum.

Cell Rep 2020 06;31(12):107817

Centre for Structural Systems Biology, 22607 Hamburg, Germany; Bernhard Nocht Institute for Tropical Medicine, 20359 Hamburg, Germany; University of Hamburg, 20146 Hamburg, Germany. Electronic address:

Proteins of the lipocalin family are known to bind small hydrophobic ligands and are involved in various physiological processes ranging from lipid transport to oxidative stress responses. The genome of the malaria parasite Plasmodium falciparum contains a single protein PF3D7_0925900 with a lipocalin signature. Using crystallography and small-angle X-ray scattering, we show that the protein has a tetrameric structure of typical lipocalin monomers; hence we name it P. falciparum lipocalin (PfLCN). We show that PfLCN is expressed in the intraerythrocytic stages of the parasite and localizes to the parasitophorous and food vacuoles. Conditional knockdown of PfLCN impairs parasite development, which can be rescued by treatment with the radical scavenger Trolox or by temporal inhibition of hemoglobin digestion. This suggests a key function of PfLCN in counteracting oxidative stress-induced cell damage during multiplication of parasites within erythrocytes.
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http://dx.doi.org/10.1016/j.celrep.2020.107817DOI Listing
June 2020

Hydration in aqueous osmolyte solutions: the case of TMAO and urea.

Phys Chem Chem Phys 2020 May 14;22(20):11614-11624. Epub 2020 May 14.

European Synchrotron Radiation Facility, 71 Avenue des Martyrs, 38000 Grenoble, France.

The hydration and hydrogen-bond topology of small water solvated molecules such as the naturally occurring organic osmolytes trimethylamine N-oxide (TMAO) and urea are under intense investigation. We aim at furthering the understanding of this complex hydration by combining experimental oxygen K-edge excitation spectra with results from spectra calculated via the Bethe-Salpeter equation based on structures obtained from ab initio molecular dynamics simulations. Comparison of experimental and calculated spectra allows us to extract detailed information about the immediate surrounding of the solute molecules in the solvated state. We quantify and localize the influence of the solute on the hydrogen bond network of the water solvent and find spectroscopic fingerprints of a clear directional asymmetry around TMAO with strong and local kosmotropic influence around TMAO's NO head group and slight chaotropic influence around the hydrophobic methyl groups. The influence of urea on the local water network is qualitatively similar to that of TMAO but weaker in magnitude. The strongest influence of both molecules on the shape of the oxygen K-edge spectra is found in the first hydration shells.
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http://dx.doi.org/10.1039/c9cp06785jDOI Listing
May 2020

Insights into herpesvirus assembly from the structure of the pUL7:pUL51 complex.

Elife 2020 05 11;9. Epub 2020 May 11.

Department of Pathology, University of Cambridge, Cambridge, United Kingdom.

Herpesviruses acquire their membrane envelopes in the cytoplasm of infected cells via a molecular mechanism that remains unclear. Herpes simplex virus (HSV)-1 proteins pUL7 and pUL51 form a complex required for efficient virus envelopment. We show that interaction between homologues of pUL7 and pUL51 is conserved across human herpesviruses, as is their association with -Golgi membranes. We characterized the HSV-1 pUL7:pUL51 complex by solution scattering and chemical crosslinking, revealing a 1:2 complex that can form higher-order oligomers in solution, and we solved the crystal structure of the core pUL7:pUL51 heterodimer. While pUL7 adopts a previously-unseen compact fold, the helix-turn-helix conformation of pUL51 resembles the cellular endosomal complex required for transport (ESCRT)-III component CHMP4B and pUL51 forms ESCRT-III-like filaments, suggesting a direct role for pUL51 in promoting membrane scission during virus assembly. Our results provide a structural framework for understanding the role of the conserved pUL7:pUL51 complex in herpesvirus assembly.
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http://dx.doi.org/10.7554/eLife.53789DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7289601PMC
May 2020

Tetrameric Structures of Inorganic CBS-Pyrophosphatases from Various Bacterial Species Revealed by Small-Angle X-ray Scattering in Solution.

Biomolecules 2020 04 7;10(4). Epub 2020 Apr 7.

Shubnikov Institute of Crystallography of Federal Scientific Research Centre "Crystallography and Photonics" of Russian Academy of Sciences, Leninskiy prospect, 59, 119333 Moscow, Russia.

Quaternary structure of CBS-pyrophosphatases (CBS-PPases), which belong to the PPases of family II, plays an important role in their function ensuring cooperative behavior of the enzymes. Despite an intensive research, high resolution structures of the full-length CBS-PPases are not yet available making it difficult to determine the signal transmission path from the regulatory to the active center. In the present work, small-angle X-ray scattering (SAXS) combined with size-exclusion chromatography was applied to determine the solution structures of the full-length wild-type CBS-PPases from three different bacterial species. Previously, in the absence of an experimentally determined full-length CBS-PPase structure, a homodimeric model of the enzyme based on known crystal structures of the CBS domain and family II PPase without this domain has been proposed. Our SAXS analyses demonstrate, for the first time, the existence of stable tetramers in solution for all studied CBS-PPases from different sources. Our findings show that further studies are required to establish the functional properties of these enzymes. This is important not only to enhance our understanding of the relation between CBS-PPases structure and function under normal conditions but also because some human pathogens harbor this class of enzymes.
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http://dx.doi.org/10.3390/biom10040564DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7226116PMC
April 2020

An intrinsically disordered proteins community for ELIXIR.

F1000Res 2019 15;8. Epub 2019 Oct 15.

University of Wisconsin-Madison, Madison, WI, 53706-1544, USA.

Intrinsically disordered proteins (IDPs) and intrinsically disordered regions (IDRs) are now recognised as major determinants in cellular regulation. This white paper presents a roadmap for future e-infrastructure developments in the field of IDP research within the ELIXIR framework. The goal of these developments is to drive the creation of high-quality tools and resources to support the identification, analysis and functional characterisation of IDPs. The roadmap is the result of a workshop titled "An intrinsically disordered protein user community proposal for ELIXIR" held at the University of Padua. The workshop, and further consultation with the members of the wider IDP community, identified the key priority areas for the roadmap including the development of standards for data annotation, storage and dissemination; integration of IDP data into the ELIXIR Core Data Resources; and the creation of benchmarking criteria for IDP-related software. Here, we discuss these areas of priority, how they can be implemented in cooperation with the ELIXIR platforms, and their connections to existing ELIXIR Communities and international consortia. The article provides a preliminary blueprint for an IDP Community in ELIXIR and is an appeal to identify and involve new stakeholders.
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http://dx.doi.org/10.12688/f1000research.20136.1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6880265PMC
January 2020

The dimeric ectodomain of the alkali-sensing insulin receptor-related receptor (ectoIRR) has a droplike shape.

J Biol Chem 2019 11 15;294(47):17790-17798. Epub 2019 Oct 15.

Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow 117997, Russia.

Insulin receptor-related receptor (IRR) is a receptor tyrosine kinase of the insulin receptor family and functions as an extracellular alkali sensor that controls metabolic alkalosis in the regulation of the acid-base balance. In the present work, we sought to analyze structural features of IRR by comparing them with those of the insulin receptor, which is its closest homolog but does not respond to pH changes. Using small-angle X-ray scattering (SAXS) and atomic force microscopy (AFM), we investigated the overall conformation of the recombinant soluble IRR ectodomain (ectoIRR) at neutral and alkaline pH. In contrast to the well-known inverted U-shaped (or λ-shaped) conformation of the insulin receptor, the structural models reconstructed at different pH values revealed that the ectoIRR organization has a "droplike" shape with a shorter distance between the fibronectin domains of the disulfide-linked dimer subunits within ectoIRR. We detected no large-scale pH-dependent conformational changes of ectoIRR in both SAXS and AFM experiments, an observation that agreed well with previous biochemical and functional analyses of IRR. Our findings indicate that ectoIRR's sensing of alkaline conditions involves additional molecular mechanisms, for example engagement of receptor juxtamembrane regions or the surrounding lipid environment.
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http://dx.doi.org/10.1074/jbc.RA119.010390DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6879334PMC
November 2019

SASBDB: Towards an automatically curated and validated repository for biological scattering data.

Protein Sci 2020 01 11;29(1):66-75. Epub 2019 Oct 11.

European Molecular Biology Laboratory, Hamburg Outstation, Hamburg, Germany.

Small-angle scattering (SAS) of X-rays and neutrons is a fundamental tool to study the nanostructural properties, and in particular, biological macromolecules in solution. In structural biology, SAS recently transformed from a specialization into a general technique leading to a dramatic increase in the number of publications reporting structural models. The growing amount of data recorded and published has led to an urgent need for a global SAS repository that includes both primary data and models. In response to this, a small-angle scattering biological data bank (SASBDB) was designed in 2014 and is available for public access at www.sasbdb.org. SASBDB is a comprehensive, free and searchable repository of SAS experimental data and models deposited together with the relevant experimental conditions, sample details and instrument characteristics. SASBDB is rapidly growing, and presently has over 1,000 entries containing more than 1,600 models. We describe here the overall organization and procedures of SASBDB paying most attention to user-relevant information during submission. Perspectives of further developments, in particular, with OneDep system of the Protein Data Bank, and also widening of SASBDB including new types of data/models are discussed.
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http://dx.doi.org/10.1002/pro.3731DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6933840PMC
January 2020

The quaternary structure of insulin glargine and glulisine under formulation conditions.

Biophys Chem 2019 10 11;253:106226. Epub 2019 Jul 11.

Sanofi-Aventis Deutschland GmbH, R&D, Industriepark Höchst, 65926 Frankfurt, Germany.

The quaternary structures of insulin glargine and glulisine under formulation conditions and upon dilution using placebo or water were investigated using synchrotron small-angle X-ray scattering. Our results revealed that insulin glulisine in Apidra® is predominantly hexameric in solution with significant fractions of dodecamers and monomers. Upon dilution with placebo, this equilibrium shifts towards monomers. Insulin glargine in Lantus® and Toujeo® is present in a stable hexamer/dimer equilibrium, which is hardly affected by dilution with water down to 1 mg/ml insulin concentration. The results provide exclusive insight into the quaternary structure and thus the association/dissociation properties of the two insulin analogues in marketed formulations.
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http://dx.doi.org/10.1016/j.bpc.2019.106226DOI Listing
October 2019

Hydration in aqueous solutions of ectoine and hydroxyectoine.

Phys Chem Chem Phys 2018 Nov;20(44):27917-27923

European Synchrotron Radiation Facility, 71 Avenue des Martyrs, 38000 Grenoble, France.

We explore the influence of the two osmolytes ectoine and hydroxyectoine on the structure of pure water and aqueous NaCl solutions using non-resonant X-ray Raman scattering spectroscopy at the oxygen K-edge. Both ectoine and hydroxyectoine are naturally occurring organic osmolytes synthesized by halophilic organisms that live in high-salt and other extreme environments. We find that X-ray spectroscopic data at the oxygen K-edge are consistent with a scenario where both osmolytes affect the hydrogen bonding network of water on a local scale to in effect increase tetrahedral order. This supports the proposed stabilizing mechanism of the osmolytes for proteins: preferential exclusion of the osmolytes from the proteins' surface and preferential hydration of the macromolecules instead of complex alterations to the structure of water on a bulk scale. The effect of NaCl on water, a disruption of hydrogen bonds and tetrahedral order, acts in opposition to the localized water-binding effects of ectoine and hydroxyectoine. For ternary mixtures of osmolyte in the presence of NaCl, the effects seen in the spectra are found to be additive such that the mixed solutes generate a level of oppositional frustration in the water network.
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http://dx.doi.org/10.1039/c8cp05308aDOI Listing
November 2018

Smaller capillaries improve the small-angle X-ray scattering signal and sample consumption for biomacromolecular solutions.

J Synchrotron Radiat 2018 Jul 26;25(Pt 4):1113-1122. Epub 2018 Jun 26.

European Molecular Biology Laboratory (EMBL), Hamburg Outstation c/o DESY, Notkestrasse 85, 22607 Hamburg, Germany.

Radiation damage by intense X-ray beams at modern synchrotron facilities is one of the major complications for biological small-angle X-ray scattering (SAXS) investigations of macromolecules in solution. To limit the damage, samples are typically measured under a laminar flow through a cell (typically a capillary) such that fresh solution is continuously exposed to the beam during measurement. The diameter of the capillary that optimizes the scattering-to-absorption ratio at a given X-ray wavelength can be calculated a priori based on fundamental physical properties. However, these well established scattering and absorption principles do not take into account the radiation susceptibility of the sample or the often very limited amounts of precious biological material available for an experiment. Here it is shown that, for biological solution SAXS, capillaries with smaller diameters than those calculated from simple scattering/absorption criteria allow for a better utilization of the available volumes of radiation-sensitive samples. This is demonstrated by comparing two capillary diameters d (d = 1.7 mm, close to optimal for 10 keV; and d = 0.9 mm, which is nominally sub-optimal) applied to study different protein solutions at various flow rates. The use of the smaller capillaries ultimately allows one to collect higher-quality SAXS data from the limited amounts of purified biological macromolecules.
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http://dx.doi.org/10.1107/S1600577518007907DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6038601PMC
July 2018

Recombinant production of A1S_0222 from Acinetobacter baumannii ATCC 17978 and confirmation of its DNA-(adenine N6)-methyltransferase activity.

Protein Expr Purif 2018 11 22;151:78-85. Epub 2018 Jun 22.

Robert Koch-Institute, Project Group P2, Wernigerode, Germany. Electronic address:

Acinetobacter baumannii appears as an often multidrug-resistant nosocomial pathogen in hospitals worldwide. Its remarkable persistence in the hospital environment is probably due to intrinsic and acquired resistance to disinfectants and antibiotics, tolerance to desiccation stress, capability to form biofilms, and is possibly facilitated by surface-associated motility. Our attempts to elucidate surface-associated motility in A. baumannii revealed a mutant inactivated in a putative DNA-(adenine N6)-methyltransferase, designated A1S_0222 in strain ATCC 17978. We recombinantly produced A1S_0222 as a glutathione S-transferase (GST) fusion protein and purified it to near homogeneity through a combination of GST affinity chromatography, cation exchange chromatography and PD-10 desalting column. Furthermore we demonstrate A1S_0222-dependent adenine methylation at a GAATTC site. We propose the name AamA (Acinetobacteradenine methyltransferase A) in addition to the formal names M.AbaBGORF222P/M.Aba17978ORF8565P. Small angle X-ray scattering (SAXS) revealed that the protein is monomeric and has an extended and likely two-domain shape in solution.
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http://dx.doi.org/10.1016/j.pep.2018.06.009DOI Listing
November 2018

Machine Learning Methods for X-Ray Scattering Data Analysis from Biomacromolecular Solutions.

Biophys J 2018 06;114(11):2485-2492

European Molecular Biology Laboratory, Hamburg, Germany.

Small-angle x-ray scattering (SAXS) of biological macromolecules in solutions is a widely employed method in structural biology. SAXS patterns include information about the overall shape and low-resolution structure of dissolved particles. Here, we describe how to transform experimental SAXS patterns to feature vectors and how a simple k-nearest neighbor approach is able to retrieve information on overall particle shape and maximal diameter (D) as well as molecular mass directly from experimental scattering data. Based on this transformation, we develop a rapid multiclass shape-classification ranging from compact, extended, and flat categories to hollow and random-chain-like objects. This classification may be employed, e.g., as a decision block in automated data analysis pipelines. Further, we map protein structures from the Protein Data Bank into the classification space and, in a second step, use this mapping as a data source to obtain accurate estimates for the structural parameters (D, molecular mass) of the macromolecule under study based on the experimental scattering pattern alone, without inverse Fourier transform for D. All methods presented are implemented in a Fortran binary DATCLASS, part of the ATSAS data analysis suite, available on Linux, Mac, and Windows and free for academic use.
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http://dx.doi.org/10.1016/j.bpj.2018.04.018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6129182PMC
June 2018

Consensus Bayesian assessment of protein molecular mass from solution X-ray scattering data.

Sci Rep 2018 05 8;8(1):7204. Epub 2018 May 8.

European Molecular Biology Laboratory (EMBL) Hamburg Outstation, DESY, Hamburg, Germany.

Molecular mass (MM) is one of the key structural parameters obtained by small-angle X-ray scattering (SAXS) of proteins in solution and is used to assess the sample quality, oligomeric composition and to guide subsequent structural modelling. Concentration-dependent assessment of MM relies on a number of extra quantities (partial specific volume, calibrated intensity, accurate solute concentration) and often yields limited accuracy. Concentration-independent methods forgo these requirements being based on the relationship between structural parameters, scattering invariants and particle volume obtained directly from the data. Using a comparative analysis on 165,982 unique scattering profiles calculated from high-resolution protein structures, the performance of multiple concentration-independent MM determination methods was assessed. A Bayesian inference approach was developed affording an accuracy above that of the individual methods, and reports MM estimates together with a credibility interval. This Bayesian approach can be used in combination with concentration-dependent MM methods to further validate the MM of proteins in solution, or as a reliable stand-alone tool in instances where an accurate concentration estimate is not available.
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http://dx.doi.org/10.1038/s41598-018-25355-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5940760PMC
May 2018

Functional interaction of low-homology FRPs from different cyanobacteria with Synechocystis OCP.

Biochim Biophys Acta Bioenerg 2018 May 7;1859(5):382-393. Epub 2018 Mar 7.

A.N. Bach Institute of Biochemistry, Federal Research Center of Biotechnology of the Russian Academy of Sciences, 119071 Moscow, Russian Federation; M.V. Lomonosov Moscow State University, Department of Biophysics, Faculty of Biology, 119991 Moscow, Russian Federation. Electronic address:

Photosynthesis requires a balance between efficient light harvesting and protection against photodamage. The cyanobacterial photoprotection system uniquely relies on the functioning of the photoactive orange carotenoid protein (OCP) that under intense illumination provides fluorescence quenching of the light-harvesting antenna complexes, phycobilisomes. The recently identified fluorescence recovery protein (FRP) binds to the photoactivated OCP and accelerates its relaxation into the basal form, completing the regulatory circle. The molecular mechanism of FRP functioning is largely controversial. Moreover, since the available knowledge has mainly been gained from studying Synechocystis proteins, the cross-species conservation of the FRP mechanism remains unexplored. Besides phylogenetic analysis, we performed a detailed structural-functional analysis of two selected low-homology FRPs by comparing them with Synechocystis FRP (SynFRP). While adopting similar dimeric conformations in solution and preserving binding preferences of SynFRP towards various OCP variants, the low-homology FRPs demonstrated distinct binding stoichiometries and differentially accentuated features of this functional interaction. By providing clues to understand the FRP mechanism universally, our results also establish foundations for upcoming structural investigations necessary to elucidate the FRP-dependent regulatory mechanism.
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http://dx.doi.org/10.1016/j.bbabio.2018.03.001DOI Listing
May 2018

Sample and Buffer Preparation for SAXS.

Adv Exp Med Biol 2017 ;1009:11-30

European Molecular Biology Laboratory (EMBL) Hamburg Outstation, DESY, Hamburg, Germany.

In this book chapter, a practical approach for conducting small angle X-ray scattering (SAXS) experiments is given. Our aim is to guide SAXS users through a three-step process of planning, preparing and performing a basic SAXS measurement. The minimal requirements necessary to prepare samples are described specifically for protein and other macromolecular samples in solution. We address the very important aspects in terms of sample characterization using additional techniques as well as the essential role of accurately subtracting background scattering contributions. At the end of the chapter some advice is given for trouble-shooting problems that may occur during the course of the SAXS measurements. Automated pipelines for data processing are described which are useful in allowing users to evaluate the quality of the data 'on the spot' and consequently react to events such as radiation damage, the presence of unwanted sample aggregates or miss-matched buffers.
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http://dx.doi.org/10.1007/978-981-10-6038-0_2DOI Listing
June 2018

Influenza virus Matrix Protein M1 preserves its conformation with pH, changing multimerization state at the priming stage due to electrostatics.

Sci Rep 2017 12 1;7(1):16793. Epub 2017 Dec 1.

Frumkin Institute of Physical Chemistry and Electrochemistry, Russian Academy of Sciences, Moscow, Russia.

Influenza A virus matrix protein M1 plays an essential role in the virus lifecycle, but its functional and structural properties are not entirely defined. Here we employed small-angle X-ray scattering, atomic force microscopy and zeta-potential measurements to characterize the overall structure and association behavior of the full-length M1 at different pH conditions. We demonstrate that the protein consists of a globular N-terminal domain and a flexible C-terminal extension. The globular N-terminal domain of M1 monomers appears preserved in the range of pH from 4.0 to 6.8, while the C-terminal domain remains flexible and the tendency to form multimers changes dramatically. We found that the protein multimerization process is reversible, whereby the binding between M1 molecules starts to break around pH 6. A predicted electrostatic model of M1 self-assembly at different pH revealed a good agreement with zeta-potential measurements, allowing one to assess the role of M1 domains in M1-M1 and M1-lipid interactions. Together with the protein sequence analysis, these results provide insights into the mechanism of M1 scaffold formation and the major role of the flexible and disordered C-terminal domain in this process.
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http://dx.doi.org/10.1038/s41598-017-16986-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5711849PMC
December 2017

Interactions between LHX3- and ISL1-family LIM-homeodomain transcription factors are conserved in Caenorhabditis elegans.

Sci Rep 2017 07 4;7(1):4579. Epub 2017 Jul 4.

School of Life and Environmental Sciences, University of Sydney, NSW, 2006, Australia.

LIM-Homeodomain (LIM-HD) transcription factors are highly conserved in animals where they are thought to act in a transcriptional 'LIM code' that specifies cell types, particularly in the central nervous system. In chick and mammals the interaction between two LIM-HD proteins, LHX3 and Islet1 (ISL1), is essential for the development of motor neurons. Using yeast two-hybrid analysis we showed that the Caenorhabditis elegans orthologs of LHX3 and ISL1, CEH-14 and LIM-7 can physically interact. Structural characterisation of a complex comprising the LIM domains from CEH-14 and a LIM-interaction domain from LIM-7 showed that these nematode proteins assemble to form a structure that closely resembles that of their vertebrate counterparts. However, mutagenic analysis across the interface indicates some differences in the mechanisms of binding. We also demonstrate, using fluorescent reporter constructs, that the two C. elegans proteins are co-expressed in a small subset of neurons. These data show that the propensity for LHX3 and Islet proteins to interact is conserved from C. elegans to mammals, raising the possibility that orthologous cell specific LIM-HD-containing transcription factor complexes play similar roles in the development of neuronal cells across diverse species.
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http://dx.doi.org/10.1038/s41598-017-04587-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5496915PMC
July 2017

The Shigella Virulence Factor IcsA Relieves N-WASP Autoinhibition by Displacing the Verprolin Homology/Cofilin/Acidic (VCA) Domain.

J Biol Chem 2017 Jan 23;292(1):134-145. Epub 2016 Nov 23.

From the Cambridge Institute for Medical Research, Department of Pathology, University of Cambridge, Cambridge Biomedical Campus, Hills Road, Cambridge CB2 0XY, United Kingdom and

Shigella flexneri is a bacterial pathogen that invades cells of the gastrointestinal tract, causing severe dysentery. Shigella mediates intracellular motility and spreading via actin comet tail formation. This process is dependent on the surface-exposed, membrane-embedded virulence factor IcsA, which recruits the host actin regulator N-WASP. Although it is clear that Shigella requires N-WASP for this process, the molecular details of this interaction and the mechanism of N-WASP activation remain poorly understood. Here, we show that co-expression of full-length IcsA and the Shigella membrane protease IcsP yields highly pure IcsA passenger domain (residues 53-758). We show that IcsA is monomeric and describe the solution structure of the passenger domain obtained by small-angle X-ray scattering (SAXS) analysis. The SAXS-derived models suggest that IcsA has an elongated shape but, unlike most other autotransporter proteins, possesses a central kink revealing a distinctly curved structure. Pull-down experiments show direct binding of the IcsA passenger domain to both the WASP homology 1 (WH1) domain and the GTPase binding domain (GBD) of N-WASP and no binding to the verprolin homology/cofilin/acidic (VCA) region. Using fluorescence polarization experiments, we demonstrate that IcsA binding to the GBD region displaces the VCA peptide and that this effect is synergistically enhanced upon IcsA binding to the WH1 region. Additionally, domain mapping of the IcsA interaction interface reveals that different regions of IcsA bind to the WH1 and GBD domains of N-WASP. Taken together, our data support a model where IcsA and N-WASP form a tight complex releasing the N-WASP VCA domain to recruit the host cell machinery for actin tail formation.
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http://dx.doi.org/10.1074/jbc.M116.758003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5217673PMC
January 2017

Preparing monodisperse macromolecular samples for successful biological small-angle X-ray and neutron-scattering experiments.

Nat Protoc 2016 Nov 6;11(11):2122-2153. Epub 2016 Oct 6.

European Molecular Biology Laboratory (EMBL) Hamburg Outstation, DESY, Hamburg, Germany.

Small-angle X-ray scattering (SAXS) and small-angle neutron scattering (SANS) are techniques used to extract structural parameters and determine the overall structures and shapes of biological macromolecules, complexes and assemblies in solution. The scattering intensities measured from a sample contain contributions from all atoms within the illuminated sample volume, including the solvent and buffer components, as well as the macromolecules of interest. To obtain structural information, it is essential to prepare an exactly matched solvent blank so that background scattering contributions can be accurately subtracted from the sample scattering to obtain the net scattering from the macromolecules in the sample. In addition, sample heterogeneity caused by contaminants, aggregates, mismatched solvents, radiation damage or other factors can severely influence and complicate data analysis, so it is essential that the samples be pure and monodisperse for the duration of the experiment. This protocol outlines the basic physics of SAXS and SANS, and it reveals how the underlying conceptual principles of the techniques ultimately 'translate' into practical laboratory guidance for the production of samples of sufficiently high quality for scattering experiments. The procedure describes how to prepare and characterize protein and nucleic acid samples for both SAXS and SANS using gel electrophoresis, size-exclusion chromatography (SEC) and light scattering. Also included are procedures that are specific to X-rays (in-line SEC-SAXS) and neutrons, specifically preparing samples for contrast matching or variation experiments and deuterium labeling of proteins.
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http://dx.doi.org/10.1038/nprot.2016.113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5402874PMC
November 2016

X-Ray Solution Scattering Study of Four Escherichia coli Enzymes Involved in Stationary-Phase Metabolism.

PLoS One 2016 26;11(5):e0156105. Epub 2016 May 26.

EMBL, Hamburg Outstation, c/o DESY, Hamburg, Germany.

The structural analyses of four metabolic enzymes that maintain and regulate the stationary growth phase of Escherichia coli have been performed primarily drawing on the results obtained from solution small angle X-ray scattering (SAXS) and other structural techniques. The proteins are (i) class I fructose-1,6-bisphosphate aldolase (FbaB); (ii) inorganic pyrophosphatase (PPase); (iii) 5-keto-4-deoxyuronate isomerase (KduI); and (iv) glutamate decarboxylase (GadA). The enzyme FbaB, that until now had an unknown structure, is predicted to fold into a TIM-barrel motif that form globular protomers which SAXS experiments show associate into decameric assemblies. In agreement with previously reported crystal structures, PPase forms hexamers in solution that are similar to the previously reported X-ray crystal structure. Both KduI and GadA that are responsible for carbohydrate (pectin) metabolism and acid stress responses, respectively, form polydisperse mixtures consisting of different oligomeric states. Overall the SAXS experiments yield additional insights into shape and organization of these metabolic enzymes and further demonstrate the utility of hybrid methods, i.e., solution SAXS combined with X-ray crystallography, bioinformatics and predictive 3D-structural modeling, as tools to enrich structural studies. The results highlight the structural complexity that the protein components of metabolic networks may adopt which cannot be fully captured using individual structural biology techniques.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0156105PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4881948PMC
July 2017

Domain-swap polymerization drives the self-assembly of the bacterial flagellar motor.

Nat Struct Mol Biol 2016 Mar 8;23(3):197-203. Epub 2016 Feb 8.

Structural and Computational Biology Division, Victor Chang Cardiac Research Institute, Darlinghurst, New South Wales, Australia.

Large protein complexes assemble spontaneously, yet their subunits do not prematurely form unwanted aggregates. This paradox is epitomized in the bacterial flagellar motor, a sophisticated rotary motor and sensory switch consisting of hundreds of subunits. Here we demonstrate that Escherichia coli FliG, one of the earliest-assembling flagellar motor proteins, forms ordered ring structures via domain-swap polymerization, which in other proteins has been associated with uncontrolled and deleterious protein aggregation. Solution structural data, in combination with in vivo biochemical cross-linking experiments and evolutionary covariance analysis, revealed that FliG exists predominantly as a monomer in solution but only as domain-swapped polymers in assembled flagellar motors. We propose a general structural and thermodynamic model for self-assembly, in which a structural template controls assembly and shapes polymer formation into rings.
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http://dx.doi.org/10.1038/nsmb.3172DOI Listing
March 2016