Using Small Angle Solution Scattering Data in Xplor-NIH Structure Calculations

Progress in Nuclear Magnetic Resonance Spectroscopy (Impact Factor: 7.24). 07/2014; 80. DOI: 10.1016/j.pnmrs.2014.03.001


This contribution describes the use of small and wide angle X-ray and small angle neutron scattering for biomolecular structure calculation using the program Xplor-NIH, both with and without NMR data. The current algorithms used for calculating scattering curves are described, and the use of scattering data as a structural restraint is given concrete form as a fragment of an Xplor-NIH structure calculation script. We review five examples of the use of scattering data in structure calculation, including the treatment of single domain proteins, nucleic acids, structure determination of large proteins, and the use of ensemble representations to characterize small and large amplitude motions.

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    • ". The goodness of fit to the experimental scattering curve for each of the 20 individual simulated annealing structures was also measured using the CalcSAXS helper program in the Xplor-NIH (version 2.37) software package [40] [78] and yielded values of χ 2 = 0.81 ± 0.02. e PROCHECK-NMR [90] and MolProbity [91] results include residues (A326–I655) and are of acceptable quality for an NMR structure. "
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    ABSTRACT: Staphylococcus aureus is a medically important bacterial pathogen that during infections acquires iron from human hemoglobin (Hb). It uses two closely related iron regulated surface determinant (Isd) proteins to capture and extract the oxidized form of heme (hemin) from Hb, IsdH and IsdB. Both receptors rapidly extract hemin using a conserved tri-domain unit consisting of two NEAr iron Transporter (NEAT) domains connected by a helical linker domain. To gain insight into the mechanism of extraction we used NMR to investigate the structure and dynamics of the 38.8kDa tri-domain IsdH protein (IsdH(N2N3), A326-D660 with a Y642A mutation that prevents hemin binding). The structure was modeled using long-range paramagnetic relaxation enhancement (PRE) distance restraints, dihedral angle, small angle x-ray scattering, residual dipolar coupling and inter-domain NOE data. The receptor adopts an extended conformation wherein the linker and N3 domains pack against each other via a hydrophobic interface. In contrast, the N2 domain contacts the linker domain via a hydrophilic interface, and based on NMR relaxation data undergoes inter-domain motions enabling it to reorient with respect to the body of the protein. Ensemble calculations were used to estimate the range of N2 domain positions compatible with the PRE data. A comparison of the Hb-free and -bound forms reveals that Hb binding alters the positioning of the N2 domain. We propose that binding occurs through a combination of conformational selection and induced fit mechanisms that may promote hemin release from Hb by altering the position of its F-helix. Copyright © 2015. Published by Elsevier Ltd.
    Full-text · Article · Feb 2015 · Journal of Molecular Biology
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    ABSTRACT: Small angle solution X-ray and neutron scattering recently resurfaced as powerful tools to address an array of biological problems including folding, intrinsic disorder, conformational transitions, macromolecular crowding and self or hetero-assembling of bio-macromolecules. In addition, small angle solution scattering complements crystallography, nuclear magnetic resonance spectroscopy and other structural methods to aid in the structure determinations of multi-domain or multi-component proteins or nucleoprotein assemblies. Neutron scattering with hydrogen/deuterium contrast variation, or X-ray scattering with sucrose contrast variation to a certain extent, is a convenient tool for characterizing the organizations of two-component systems such as a nucleoprotein or a lipid-protein assembly. Time-resolved small and wide-angle solution scattering to study biological processes in real time, and the use of localized heavy-atom labeling and anomalous solution scattering as FRET-like molecular rulers, are amongst promising newer developments. Despite the challenges in data analysis and interpretation, these X-ray/neutron solution scattering based approaches hold great promise for understanding a wide variety of complex processes prevalent in the biological milieu. This article is protected by copyright. All rights reserved.
    No preview · Article · Dec 2014 · Protein Science
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    ABSTRACT: Enzyme I (EI), the first component of the bacterial phosphotransfer signal transduction system, undergoes one of the largest substrate-induced interdomain rearrangements documented to date. Here we characterize the perturbations generated by two small molecules, the natural substrate phosphoenolpyruvate and the inhibitor α-ketoglutarate, on the structure and dynamics of EI using NMR, small-angle X-ray scattering and biochemical techniques. The results indicate unambiguously that the open-to-closed conformational switch of EI is triggered by complete suppression of micro- to millisecond dynamics within the C-terminal domain of EI. Indeed, we show that a ligand-induced transition from a dynamic to a more rigid conformational state of the C-terminal domain stabilizes the interface between the N- and C-terminal domains observed in the structure of the closed state, thereby promoting the resulting conformational switch and autophosphorylation of EI. The mechanisms described here may be common to several other multidomain proteins and allosteric systems.
    Full-text · Article · Jan 2015 · Nature Communications
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