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      X-ray refinement significantly underestimates the level of microscopic heterogeneity in biomolecular crystals

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      1 , a , 2 , b , 1
      Nature Communications
      Nature Pub. Group

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          Abstract

          Biomolecular X-ray structures typically provide a static, time- and ensemble-averaged view of molecular ensembles in crystals. In the absence of rigid-body motions and lattice defects, B-factors are thought to accurately reflect the structural heterogeneity of such ensembles. In order to study the effects of averaging on B-factors, we employ molecular dynamics simulations to controllably manipulate microscopic heterogeneity of a crystal containing 216 copies of villin headpiece. Using average structure factors derived from simulation, we analyse how well this heterogeneity is captured by high-resolution molecular-replacement-based model refinement. We find that both isotropic and anisotropic refined B-factors often significantly deviate from their actual values known from simulation: even at high 1.0 Å resolution and R free of 5.9%, B-factors of some well-resolved atoms underestimate their actual values even sixfold. Our results suggest that conformational averaging and inadequate treatment of correlated motion considerably influence estimation of microscopic heterogeneity via B-factors, and invite caution in their interpretation.

          Abstract

          The structural heterogeneity of a biomolecular crystal structure is typically captured using atomic B-factors, determined during structure refinement. Here, the authors use molecular dynamics to show that this strategy is flawed, and that crystallographic B-factors underestimate structural heterogeneity.

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          Most cited references55

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          Biomolecular simulation: a computational microscope for molecular biology.

          Molecular dynamics simulations capture the behavior of biological macromolecules in full atomic detail, but their computational demands, combined with the challenge of appropriately modeling the relevant physics, have historically restricted their length and accuracy. Dramatic recent improvements in achievable simulation speed and the underlying physical models have enabled atomic-level simulations on timescales as long as milliseconds that capture key biochemical processes such as protein folding, drug binding, membrane transport, and the conformational changes critical to protein function. Such simulation may serve as a computational microscope, revealing biomolecular mechanisms at spatial and temporal scales that are difficult to observe experimentally. We describe the rapidly evolving state of the art for atomic-level biomolecular simulation, illustrate the types of biological discoveries that can now be made through simulation, and discuss challenges motivating continued innovation in this field.
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            Prediction of chain flexibility in proteins

            Naturwissenschaften, 72(4), 212-213
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              The catalytic pathway of horseradish peroxidase at high resolution.

              A molecular description of oxygen and peroxide activation in biological systems is difficult, because electrons liberated during X-ray data collection reduce the active centres of redox enzymes catalysing these reactions. Here we describe an effective strategy to obtain crystal structures for high-valency redox intermediates and present a three-dimensional movie of the X-ray-driven catalytic reduction of a bound dioxygen species in horseradish peroxidase (HRP). We also describe separate experiments in which high-resolution structures could be obtained for all five oxidation states of HRP, showing such structures with preserved redox states for the first time.
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                Author and article information

                Journal
                Nat Commun
                Nat Commun
                Nature Communications
                Nature Pub. Group
                2041-1723
                07 February 2014
                : 5
                : 3220
                Affiliations
                [1 ]Department of Structural and Computational Biology, Max F. Perutz Laboratories, University of Vienna , Campus Vienna Biocenter 5, A-1030 Vienna, Austria
                [2 ]Biophysical Structural Chemistry, Leiden University , PO Box 9502, 2300 RA Leiden, The Netherlands
                Author notes
                Article
                ncomms4220
                10.1038/ncomms4220
                3926004
                24504120
                7a5e96a1-6cd5-44f4-8ec8-f0f257373320
                Copyright © 2014, Nature Publishing Group, a division of Macmillan Publishers Limited. All Rights Reserved.

                This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. To view a copy of this licence visit http://creativecommons.org/licenses/by/3.0/.

                History
                : 17 July 2013
                : 07 January 2014
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