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Abstract: Photo-excitation can drive strongly-correlated electron insulators into competing conducting phases, resulting in giant and ultrafast changes of their electronic and magnetic properties. The underlying non-equilibrium dynamics involve many degrees of freedom at once, whereby sufficiently short optical pulses can trigger the corresponding collective modes of the solid along temporally coherent pathways. Their characteristic frequencies of these modes range between the few GHz of acoustic vibrations to the tens or even hundreds of THz for purely electronic excitations. Virtually all experiments to date have used 100-fs or longer pulses, detecting only comparatively slow lattice dynamics. Here, we use sub-10-fs optical pulses to study the photo-induced insulator-metal transition in the magneto-resistive manganite Pr0.7Ca0.3MnO3. At room temperature, we find that the time-dependent pathway toward the metallic phase is accompanied by coherent 31-THz oscillations of the optical reflectivity, significantly faster than all lattice vibrations. These high-frequency oscillations are suggestive of coherent orbital waves, crystal-field excitations triggered here by Impulsive Stimulated Raman Scattering. Orbital waves are likely to be initially localized to the small polarons of this room-temperature manganite, coupling to other degrees of freedom at longer times and seeding the coalescence of photo-domains into a metallic phase.
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Aug 2007
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Abstract: When using coherent x-rays to perform lensless imaging, it is the complex wave field exiting the sample or, in the case of the Bragg geometry, the deformed electron density distribution of a crystal, that is being sought. For most samples, to some extent, the image will be complex, containing both an amplitude and phase variation across the sample. We have developed versions of the hybrid input–output (HIO) and error reduction (ER) algorithms that are very robust for the inversion to complex objects from three-dimensional (3D) coherent x-ray diffraction (CXD) data measured around a Bragg spot of a small crystal. The development and behavior of these algorithms will be discussed in the context of inverting a 3D CXD pattern measured around a (111) Bragg spot of a silver nanocube.
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Mar 2010
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Abstract: The metal cofactor determines the thermal stability in cupredoxins, but how the redox state of copper modulates their melting points remains unknown. The metal coordination environment is highly conserved in cyanobacterial plastocyanins. However, the oxidised form is more stable than the reduced one in thermophilic Phormidium, but the opposite occurs in mesophilic Synechocystis. We have performed neutral amino-acid substitutions at loops of Phormidium plastocyanin far from the copper site. Notably, mutation P49G/G50P confers a redox-dependent thermal stability similar to that of the mesophilic plastocyanin. Moreover, X-ray absorption spectroscopy reveals that P49G/G50P mutation makes the electron density distribution at the oxidised copper site shift towards that of Synechocystis plastocyanin.
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Jun 2010
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Abstract: The PILATUS 1M detector, developed at the Paul Scherrer Institut, is a single-photon-counting hybrid pixel detector designed for macromolecular crystallography. With more than 1 million pixels covering an area of 243 × 210 mm, it is the largest such device constructed to date. The detector features a narrow point spread function, very fast readout and a complete absence of electronic noise. Unfortunately, this prototype detector has numerous defective pixels and sporadic errors in counting that complicate its operation. With appropriate experimental design, it was largely possible to work around these problems and successfully demonstrate the application of this technology to structure determination. Conventional coarse [phi]-sliced data were collected on thaumatin and a refined electron density map was produced that showed the features expected of a map at 1.6 Å resolution. The results were compared with the performance of a reference charge-coupled device detector: the pixel detector is superior in speed, but showed higher R-factors because of the counting errors. Complete fine [phi]-sliced data sets recorded in the continuous-rotation mode showed the predicted advantages of this data collection strategy and demonstrated the expected reduction of R-factors at high resolution. A new readout chip has been tested and shown to be free from the defects of its predecessor; a PILATUS 6M detector incorporating this new technology is under construction.
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Aug 2006
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Abstract: X-ray crystallography is currently the most successful method for determining the three-dimensional structure of membrane proteins. Nevertheless, growing the crystals required for this technique presents one of the major bottlenecks in this area of structural biology. This is especially true for the alpha-helical type membrane proteins that are of particular interest due to their medical relevance. To address this problem we have undertaken a detailed analysis of the crystallization conditions from 121 alpha-helical membrane protein structures deposited in the Protein Data Bank. This information has been analyzed so that the success of different parameters can be easily compared for different membrane protein families. Concurrent with this analysis, we also present the new sparse matrix crystallization screen MemGold.
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Mar 2008
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Abstract: The biological role of manganese (Mn2+) has been a long-standing puzzle, since at low concentrations it activates several polymerases whilst at higher concentrations it inhibits. Viral RNA polymerases possess a common architecture, reminiscent of a closed right hand. The RNA-dependent RNA polymerase (RdRp) of bacteriophage {phi}6 is one of the best understood examples of this important class of polymerases. We have probed the role of Mn2+ by biochemical, biophysical and structural analyses of the wild-type enzyme and of a mutant form with an altered Mn2+-binding site (E491 to Q). The E491Q mutant has much reduced affinity for Mn2+, reduced RNA binding and a compromised elongation rate. Loss of Mn2+ binding structurally stabilizes the enzyme. These data and a re-examination of the structures of other viral RNA polymerases clarify the role of manganese in the activation of polymerization: Mn2+ coordination of a catalytic aspartate is necessary to allow the active site to properly engage with the triphosphates of the incoming NTPs. The structural flexibility caused by Mn2+ is also important for the enzyme dynamics, explaining the requirement for manganese throughout RNA polymerization.
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Oct 2008
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Abstract: New World arenaviruses, which cause severe hemorrhagic fever, rely upon their envelope glycoproteins for attachment and fusion into their host cell. Here we present the crystal structure of the Machupo virus GP1 attachment glycoprotein, which is responsible for high-affinity binding at the cell surface to the transferrin receptor. This first structure of an arenavirus glycoprotein shows that GP1 consists of a novel alpha/beta fold. This provides a blueprint of the New World arenavirus attachment glycoproteins and reveals a new architecture of viral attachment, using a protein fold of unknown origins.
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Jan 2009
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Abstract: We measured coherent x-ray diffraction (CXD) on zeolite microcrystals in order to gain information on internal density distribution and to learn more about the strain developed during the synthesis and attachment process on the substrate. From the distortion and asymmetry of the diffraction pattern on the (020) Bragg peak, the strain field distribution is estimated. We inverted the diffraction patterns from a less strained crystal to obtain the three-dimensional image of the shape and internal strain fields using the error reduction and hybrid input–output phase retrieval algorithms. We also show a few examples of characteristic distortion modes relevant to CXD of zeolites.
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Mar 2010
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Abstract: The cryocooling of protein crystals to temperatures of around 100 K drastically reduces X-ray-induced radiation damage. The majority of macromolecular data collection is therefore performed at 100 K, yielding diffraction data of higher resolution and allowing structure determination from much smaller crystals. However, at third-generation synchrotron sources radiation damage at 100 K still limits the useful data obtainable from a crystal. For data collection at 15 K, realised by the use of an open-flow helium cryostat, a further reduction of radiation damage is expected. However, no systematic studies have been undertaken so far. In this present study, a total of 54 data sets have been collected from holoferritin and insulin crystals at 15 and 90 K in order to identify the effect of the lower data-collection temperature on the radiation damage. It is shown that data collection at 15 K has only a small positive effect for insulin crystals, whereas for holoferritin crystals radiation damage is reduced by 23% compared with data collection at 90 K.
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Mar 2007
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Abstract: Eukaryotic membrane proteins are often difficult to produce in large quantities, which is a significant obstacle for further structural and biochemical investigation. Based on the analysis of 43 eukaryotic membrane proteins, we present a cost-effective high-throughput approach for rapidly screening membrane proteins that can be overproduced to levels of >1 mg per liter in Saccharomyces cerevisiae. We find that 70% of the well expressed membrane proteins tested in this system are stable, targeted to the correct organelle, and monodisperse in either Fos-choline 12 (FC-12) or n-dodecyl-?-d-maltoside. We illustrate the advantage of such an approach, with the purification of monodisperse human and yeast nucleotide-sugar transporters to unprecedented levels. We estimate that our approach should be able to provide milligram quantities for at least one-quarter of all membrane proteins from both yeast and higher eukaryotic organisms.
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Aug 2007
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