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Forneris F.,University Utrecht | Burnley B.T.,University Utrecht | Burnley B.T.,Research Complex at Harwell | Gros P.,University Utrecht
Acta Crystallographica Section D: Biological Crystallography | Year: 2014

Human factor D (FD) is a self-inhibited thrombin-like serine proteinase that is critical for amplification of the complement immune response. FD is activated by its substrate through interactions outside the active site. The substrate-binding, or 'exosite', region displays a well defined and rigid conformation in FD. In contrast, remarkable flexibility is observed in thrombin and related proteinases, in which Na+ and ligand binding is implied in allosteric regulation of enzymatic activity through protein dynamics. Here, ensemble refinement (ER) of FD and thrombin crystal structures is used to evaluate structure and dynamics simultaneously. A comparison with previously published NMR data for thrombin supports the ER analysis. The R202A FD variant has enhanced activity towards artificial peptides and simultaneously displays active and inactive conformations of the active site. ER revealed pronounced disorder in the exosite loops for this FD variant, reminiscent of thrombin in the absence of the stabilizing Na+ ion. These data indicate that FD exhibits conformational dynamics like thrombin, but unlike in thrombin a mechanism has evolved in FD that locks the unbound native state into an ordered inactive conformation via the self-inhibitory loop. Thus, ensemble refinement of X-ray crystal structures may represent an approach alternative to spectroscopy to explore protein dynamics in atomic detail. © 2014 International Union of Crystallography.

Robinson I.,University College London | Robinson I.,Research Complex at Harwell
Journal of the Physical Society of Japan | Year: 2013

This review examines the physical reasons why nanoparticles differ in structure from the bulk. Certain simple properties of nanoparticles are explained through these structural differences. A powerful method of measuring the three dimensional structure of nanoparticles, Coherent X-ray diffraction (CXD), is introduced. A key experiment is described that uses CXD to study the redistribution of strains on the surface of a Au nanocrystal. Some future perspectives are discussed in conclusion. Copyright © 2013 The Physical Society of Japan.

Clark J.N.,University College London | Huang X.,University College London | Harder R.,Argonne National Laboratory | Robinson I.K.,University College London | Robinson I.K.,Research Complex at Harwell
Nature Communications | Year: 2012

The wave properties of light, particularly its coherence, are responsible for interference effects, which can be exploited in powerful imaging applications. Coherent diffractive imaging relies heavily on coherence and has recently experienced rapid growth. Coherent diffractive imaging recovers an object from its diffraction pattern by computational phasing with the potential of wavelength-limited resolution. Diminished coherence results in reconstructions that suffer from artefacts or fail completely. Here we demonstrate ab initio phasing of partially coherent diffraction patterns in three dimensions, while simultaneously determining the coherence properties of the illuminating wavefield. Both the dramatic improvements in image interpretability and the three-dimensional evaluation of the coherence will have broad implications for quantitative imaging of nanostructures and wavefield characterization with X-rays and electrons. © 2012 Macmillan Publishers Limited. All rights reserved.

Clark J.N.,University College London | Clark J.N.,SLAC | Huang X.,Brookhaven National Laboratory | Harder R.J.,Argonne National Laboratory | And 2 more authors.
Physical Review Letters | Year: 2014

We demonstrate through experiment an example of "mixed state" reconstruction using x-ray ptychography. We demonstrate successful imaging of a vibrating sample that has dynamics that are of one order magnitude faster than the measurement times. We show how increased vibrational amplitude leads to an increased population of illumination modes, a characteristic of partial coherence. Implications of a vibrating sample are explored, with its possible use in manipulating coherent wave field mode shapes and coherence properties. © Published by the American Physical Society.

Wagner A.,Diamond Light Source | Duman R.,Diamond Light Source | Stevens B.,Nottingham Trent University | Ward A.,Research Complex at Harwell
Acta Crystallographica Section D: Biological Crystallography | Year: 2013

X-ray crystallography is the method of choice to deduce atomic resolution structural information from macromolecules. In recent years, significant investments in structural genomics initiatives have been undertaken to automate all steps in X-ray crystallography from protein expression to structure solution. Robotic systems are widely used to prepare crystallization screens and change samples on synchrotron beamlines for macromolecular crystallography. The only remaining manual handling step is the transfer of the crystal from the mother liquor onto the crystal holder. Manual mounting is relatively straightforward for crystals with dimensions of >25 μm; however, this step is nontrivial for smaller crystals. The mounting of microcrystals is becoming increasingly important as advances in microfocus synchrotron beamlines now allow data collection from crystals with dimensions of only a few micrometres. To make optimal usage of these beamlines, new approaches have to be taken to facilitate and automate this last manual handling step. Optical tweezers, which are routinely used for the manipulation of micrometre-sized objects, have successfully been applied to sort and mount macromolecular crystals on newly designed crystal holders. Diffraction data from CPV type 1 polyhedrin microcrystals mounted with laser tweezers are presented.

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