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    ABSTRACT: 2D crystallography has proven to be an excellent technique to determine the 3D structure of membrane proteins. Compared to 3D crystallography, it has the advantage of visualizing the protein in an environment closer to the native one. However, producing good 2D crystals is still a challenge and little statistical knowledge can be gained from literature. Here, we present a thorough screening of 2D crystallization conditions for a prokaryotic inwardly rectifying potassium channel (>130 different conditions). Key parameters leading to very large and well-organized 2D crystals are discussed. In addition, the problem of formation of multilayers during the growth of 2D crystals is also addressed. An intermediate resolution projection map of KirBac3.1 at 6 Å is presented, which sheds (to our knowledge) new light on the structure of this channel in a lipid environment.
    Biophysical Journal 07/2013; 105(2):398-408. DOI:10.1016/j.bpj.2013.05.054
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    ABSTRACT: X-ray data collection for macromolecular crystallography can lead to highly inhomogeneous distributions of dose within the crystal volume for cases when the crystal is larger than the beam or when the beam is non-uniform (Gaussian-like), particularly when crystal rotation is fully taken into account. Here the spatial distribution of dose is quantitatively modelled in order to compare the effectiveness of two dose-spreading data-collection protocols: helical scanning and translational collection. Their effectiveness in reducing the peak dose per unit diffraction is investigated via simulations for four common crystal shapes (cube, plate, long and short needles) and beams with a wide range of full width half maximum values. By inspection of the chosen metric, it is concluded that the optimum strategy is always to use as flat (top-hat) a beam as possible and to either match the beam size in both dimensions to the crystal, or to perform a helical scan with a beam which is narrow along the rotation axis and matched to the crystal size along the perpendicular axis. For crystal shapes where this is not possible, the reduction in peak dose per unit diffraction achieved through dose spreading is quantified and tabulated as a reference for experimenters.
    Journal of Synchrotron Radiation 01/2013; 20(Pt 1):49-57. DOI:10.1107/S0909049512044706
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    ABSTRACT: Research into radiation damage in macromolecular crystallography has matured over the last few years, resulting in a better understanding of both the processes and timescales involved. In turn this is now allowing practical recommendations for the optimization of crystal dose lifetime to be suggested. Some long-standing questions have been answered by recent investigations, and from these answers new challenges arise and areas of investigation can be proposed. Six papers published in this volume give an indication of some of the current directions of this field and also that of single-particle cryo-microscopy, and the brief summary below places them into the overall framework of ongoing research into macromolecular crystallography radiation damage.
    Journal of Synchrotron Radiation 01/2013; 20(Pt 1):1-6. DOI:10.1107/S0909049512050418
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    ABSTRACT: An extensive radiation chemistry literature would suggest that the addition of certain radical scavengers might mitigate the effects of radiation damage during protein crystallography diffraction data collection. However, attempts to demonstrate and quantify such an amelioration and its dose dependence have not yielded consistent results, either at room temperature (RT) or 100 K. Here the information thus far available is summarized and reasons for this lack of quantitative success are identified. Firstly, several different metrics have been used to monitor and quantify the rate of damage, and, as shown here, these can give results which are in conflict regarding scavenger efficacy. In addition, significant variation in results from data collected from crystals treated in nominally the same way has been observed. Secondly, typical crystallization conditions contain substantial concentrations of chemical species which already interact strongly with some of the X-ray-induced radicals that the added scavengers are intended to intercept. These interactions are probed here by the complementary technique of on-line microspectrophotometry carried out on solutions and crystals held both at 100 K and RT, the latter enabled by the use of a beamline-mounted humidifying device. With the help of computational chemistry, attempts are made to assign some of the characteristic spectral features observed experimentally. A further source of uncertainty undoubtedly lies in the challenge of reliably measuring the parameters necessary for the accurate calculation of the absorbed dose (e.g. crystal size and shape, beam profile) and its distribution within the volume of the crystal (an issue addressed in detail in another article in this issue). While microspectrophotometry reveals that the production of various species can be quenched by the addition of scavengers, it is less clear that this observation can be translated into a significant gain in crystal dose tolerance for macromolecular crystallographers.
    Journal of Synchrotron Radiation 01/2013; 20(Pt 1):23-36. DOI:10.1107/S0909049512046237
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    ABSTRACT: The self-assembly of supramolecular structures that are ordered on the nanometre scale is a key objective in nanotechnology. DNA and peptide nanotechnologies have produced various two- and three-dimensional structures, but protein molecules have been underexploited in this area of research. Here we show that the genetic fusion of subunits from protein assemblies that have matching rotational symmetry generates species that can self-assemble into well-ordered, pre-determined one- and two-dimensional arrays that are stabilized by extensive intermolecular interactions. This new class of supramolecular structure provides a way to manufacture biomaterials with diverse structural and functional properties.
    Nature Nanotechnology 07/2011; 6(9):558-62. DOI:10.1038/nnano.2011.122
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    ABSTRACT: The potential in macromolecular crystallography for using multiple crystals to collect X-ray diffraction data simultaneously from assemblies of up to seven crystals is explored. The basic features of the algorithms used to extract data and their practical implementation are described. The procedure could be useful both in relation to diffraction data obtained from intergrown crystals and to alleviate the problem of rapid diffraction decay arising from the effects of radiation damage.
    Acta Crystallographica Section D Biological Crystallography 07/2011; 67(Pt 7):608-18. DOI:10.1107/S0907444911015617
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    ABSTRACT: Most signaling pathways in cells involve numerous phosphorylation reactions. Some of the rules for kinase-substrate specificity are known, but a complete description of all substrates is missing. Research published in Science Signaling addresses the process of mitosis and asks how the relevant kinases recognize substrate sequence motifs and, in the cellular context, what substrates are phosphorylated and where. The results increase our molecular understanding of how individual events are coordinated during the process of cell division and show the importance of both sequence epitopes for kinase specificity and the notion of a sense of place through localization in subcellular compartments.
    Science Signaling 06/2011; 4(179):pe31. DOI:10.1126/scisignal.2002234
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    ABSTRACT: The signaling state of the photoactive yellow protein (PYP) photoreceptor is transiently developed via isomerization of its blue-light-absorbing chromophore. The associated structural rearrangements have large amplitude but, due to its transient nature and chemical exchange reactions that complicate NMR detection, its accurate three-dimensional structure in solution has been elusive. Here we report on direct structural observation of the transient signaling state by combining double electron electron resonance spectroscopy (DEER), NMR, and time-resolved pump-probe X-ray solution scattering (TR-SAXS/WAXS). Measurement of distance distributions for doubly spin-labeled photoreceptor constructs using DEER spectroscopy suggests that the signaling state is well ordered and shows that interspin-label distances change reversibly up to 19 Å upon illumination. The SAXS/WAXS difference signal for the signaling state relative to the ground state indicates the transient formation of an ordered and rearranged conformation, which has an increased radius of gyration, an increased maximum dimension, and a reduced excluded volume. Dynamical annealing calculations using the DEER derived long-range distance restraints in combination with short-range distance information from (1)H-(15)N HSQC perturbation spectroscopy give strong indication for a rearrangement that places part of the N-terminal domain in contact with the exposed chromophore binding cleft while the terminal residues extend away from the core. Time-resolved global structural information from pump-probe TR-SAXS/WAXS data supports this conformation and allows subsequent structural refinement that includes the combined energy terms from DEER, NMR, and SAXS/WAXS together. The resulting ensemble simultaneously satisfies all restraints, and the inclusion of TR-SAXS/WAXS effectively reduces the uncertainty arising from the possible spin-label orientations. The observations are essentially compatible with reduced folding of the I(2)' state (also referred to as the 'pB' state) that is widely reported, but indicates it to be relatively ordered and rearranged. Furthermore, there is direct evidence for the repositioning of the N-terminal region in the I(2)' state, which is structurally modeled by dynamical annealing and refinement calculations.
    Journal of the American Chemical Society 06/2011; 133(24):9395-404. DOI:10.1021/ja200617t
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    ABSTRACT: The rate of radiation damage to macromolecular crystals at both room temperature and 100 K has previously been shown to be reduced by the use of certain radical scavengers. Here the effects of sodium nitrate, an electron scavenger, are investigated at 100 K. For sodium nitrate at a concentration of 0.5 M in chicken egg-white lysozyme crystals, the dose tolerance is increased by a factor of two as judged from the global damage parameters, and no specific structural damage to the disulfide bonds is seen until the dose is greatly in excess (more than a factor of five) of the value at which damage appears in electron density maps derived from a scavenger-free crystal. In the electron density maps, ordered nitrate ions adjacent to the disulfide bonds are seen to lose an O atom, and appear to protect the disulfide bonds. In addition, results reinforcing previous reports on the effectiveness of ascorbate are presented. The mechanisms of action of both scavengers in the crystalline environment are elucidated.
    Journal of Synchrotron Radiation 05/2011; 18(Pt 3):346-57. DOI:10.1107/S0909049511007163
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    ABSTRACT: Radiation damage in macromolecular crystallography has become a mainstream concern over the last ten years. The current status of research into this area is briefly assessed, and the ten new papers published in this issue are set into the context of previous work in the field. Some novel and exciting developments emerging over the last two years are also summarized.
    Journal of Synchrotron Radiation 05/2011; 18(Pt 3):313-7. DOI:10.1107/S0909049511013859
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