Home About us Contact
|
Home About us Contact | ||
|
|
||
Protein Crystals (protein + crystal)
Terms modified by Protein Crystals Selected AbstractsPrediction of Water,s Mobility and Disorder in Protein Crystals Using Novel Local Hydrophobic DescriptorsMOLECULAR INFORMATICS, Issue 3 2010Yuzhu Pan Abstract The B-factors of crystal structures reflect the atomic fluctuations about their average positions and provide important information about molecular dynamics. Although numerous works have been addressed on theoretical and computational studies of B-factor profile of protein atoms, the methods used for predicting B-factor values of water molecules in protein crystals still remain unexploited. In this article, we describe a new approach that we named local hydrophobic descriptors (LHDs) to characterize the hydrophobic landscapes of protein hydration sites. Using this approach coupled with partial least squares (PLS) regression and least-squares squares support vector machine (LSSVM), we perform a systematic investigation on the linear and nonlinear relationships between the LHDs and water B-factors. Based upon an elaborately selected, large-scale dataset of crystal water molecules, our method predicts B-factor profile with coefficient of determination rpred of 0.554. We demonstrate that (i) the dynamics of water molecules is primarily governed by the local features of hydrophobic potential landscapes, and (ii) the accuracy of predicted B-factor values depends on water packing density. [source] pEffects of UV radiation on the ultrastructure of several red algaePHYCOLOGICAL RESEARCH, Issue 1 2003Frank Poppe SUMMARY The effect of ultraviolet (UV) radiation on the ultrastructure of four red algae, the endemic Antarctic Palmaria decipiens (Reinsch) Ricker and Phycodrys austrogeorgica Skottsberg, the Arctic-cold temperate Palmaria palmata (Linnaeus) O. Kuntze and the cosmopolitan Bangia atropurpurea (Roth) C. Agardh was studied. All four species showed a formation of ,inside-out' vesicles from the chloroplast thylakoids upon exposure to artificial UV-radiation. In P. decipiens, most vesicles were developed after 8 h and in P. palmata, after 48 h of UV exposure. In B. atropurpurea, vesi-culation of thylakoids was observed after 72 h of UV irradiation. In Ph. austrogeorgica, the chloroplast envelope and thylakoid membranes were damaged and the phycobilisomes became detached from the thylakoids after 12 h of UV exposure. Ultraviolet-induced changes in the membrane structure of mitochondria were observed in P. decipiens and P. palmata. However, in P. decipiens they were reversible as was the damage in chloroplast fine structure after 12 h of UV treatment. Protein crystals in Ph. austrogeorgica showed degradation after exposure to UV radiation. Different methods of fixation and embedding macroalgal material are discussed. These findings give insight into the fine structural changes which occur during and after UV exposure and indicate a relationship between the species dependent sensitivity to UV-exposure and the depth distribution of the different species. [source] The plug-based nanovolume Microcapillary Protein Crystallization System (MPCS)ACTA CRYSTALLOGRAPHICA SECTION D, Issue 11 2008Cory J. Gerdts The Microcapillary Protein Crystallization System (MPCS) embodies a new semi-automated plug-based crystallization technology which enables nanolitre-volume screening of crystallization conditions in a plasticware format that allows crystals to be easily removed for traditional cryoprotection and X-ray diffraction data collection. Protein crystals grown in these plastic devices can be directly subjected to in situ X-ray diffraction studies. The MPCS integrates the formulation of crystallization cocktails with the preparation of the crystallization experiments. Within microfluidic Teflon tubing or the microfluidic circuitry of a plastic CrystalCard, ,10,20,nl volume droplets are generated, each representing a microbatch-style crystallization experiment with a different chemical composition. The entire protein sample is utilized in crystallization experiments. Sparse-matrix screening and chemical gradient screening can be combined in one comprehensive `hybrid' crystallization trial. The technology lends itself well to optimization by high-granularity gradient screening using optimization reagents such as precipitation agents, ligands or cryoprotectants. [source] Soaking: the effect of osmotic shock on tetragonal lysozyme crystalsACTA CRYSTALLOGRAPHICA SECTION D, Issue 2 2002F. J. López-Jaramillo Protein crystals crack when they are soaked in a solution with ionic strength sufficiently different from the environment in which they grew. It is demonstrated for the case of tetragonal lysozyme that the forces involved and the mechanisms that lead to the formation of cracks are different for hypertonic and hypotonic soaking. Tetragonal lysozyme crystals are very sensitive to hypotonic shocks and, after a certain waiting time, cracks always appear with a characteristic pattern perpendicular to the crystallographic c axis. Conversely, a hypertonic shock is better withstood: cracks do not display any deterministic pattern, are only visible at higher differences in ionic strength and after a certain time a phenomenon of crystal reconstruction occurs and the cracks vanish. At the lattice level, the unit-cell volume expands in hypotonic shock and shrinks under hypertonic conditions. However, the compression of the unit cell is anisotropic: the c axis is compressed to a minimum, beyond which it expands despite the unit-cell volume continuing to shrink. This behaviour is a direct consequence of the positive charge that the crystals bear and the existence of channels along the crystallographic c axis. Both features are responsible for the Gibbs,Donnan effect which limits the free exchange of ions and affects the movement of water inside the channels and bound to the protein. [source] Crystallization and preliminary X-ray diffraction analysis of cytotoxic ribonucleases from bullfrog Rana catesbeianaACTA CRYSTALLOGRAPHICA SECTION D, Issue 11 2001Jyung-Hurng Liu RC-RNases are ribonucleases from Rana catesbeiana oocytes with pyrimidine,guanine sequence specificity. They also possess cell cytotoxicity and lectin activity. Protein crystals of three RC-RNase isozymes, RC-RNase 3, RC-RNase 4 and RC-RNase 6, were grown in various crystal systems under different conditions. Crystals of RC-RNase3 belong to the orthorhombic C2221 space group, with unit-cell parameters a = 66.66, b = 97.38, c = 85.74,Å. Crystals of RC-RNase 4 belong to the trigonal space group P31 or P32, with unit-cell parameters a = b = 32.22, c = 92.12,Å. Crystals of RC-RNase 6 complexed with cytidylyl 2,-5, guanosine belong to the tetragonal space group P41212 or P43212, with unit-cell parameters a = b = 61.80, c = 65.96,Å. [source] Crystallization and preliminary X-ray diffraction studies of AsaP1_E294A and AsaP1_E294Q, two inactive mutants of the toxic zinc metallopeptidase AsaP1 from Aeromonas salmonicida subsp. achromogenesACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 7 2009Xenia Bogdanovi Two mutants of the toxic extracellular zinc endopeptidase AsaP1 (AsaP1_E294Q and AsaP1_E294A) of Aeromonas salmonicida subsp. achromogenes were expressed in Escherichia coli and crystallized by the vapour-diffusion method. Crystals were obtained using several precipitants and different protein concentrations. Protein crystals were found in a monoclinic (C2) as well as an orthorhombic (P212121) space group. The crystals belonging to the monoclinic space group C2 had unit-cell parameters a = 103.4, b = 70.9, c = 54.9,Å, , = 109.3° for AsaP1_E294A, and a = 98.5, b = 74.5, c = 54.7,Å, , = 112.4° for AsaP1_E294Q. The unit-cell parameters of the orthorhombic crystal obtained for AsaP1_E294A were a = 57.9, b = 60.2, c = 183.6,Å. The crystals of the two different mutants diffracted X-rays beyond 2.0,Å resolution. [source] Crystallization and preliminary X-ray diffraction of human interleukin-7 bound to unglycosylated and glycosylated forms of its ,-receptorACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 10 2007Joseph Wickham Jr The interleukin-7 (IL-7) signaling pathway plays an essential role in the development, proliferation and homeostasis of T and B cells in cell-mediated immunity. Understimulation and overstimulation of the IL-7 signaling pathway leads to severe combined immunodeficiency, autoimmune reactions, heart disease and cancers. Stimulation of the IL-7 pathway begins with IL-7 binding to its ,-receptor, IL-7R,. Protein crystals of unglycosylated and glycosylated complexes of human IL-7,IL-7R, extracellular domain (ECD) obtained using a surface entropy-reduction approach diffract to 2.7 and 3.0,Å, respectively. Anomalous dispersion methods will be used to solve the unglycosylated IL-7,IL-7R, ECD complex structure and this unglycosylated structure will then serve as a model in molecular-replacement attempts to solve the structure of the glycosylated IL-7,,-receptor complex. [source] Pressure-induced high-density amorphous ice in protein crystalsJOURNAL OF APPLIED CRYSTALLOGRAPHY, Issue 1 2008Chae Un Kim Crystal cryocooling has been used in X-ray protein crystallography to mitigate radiation damage during diffraction data collection. However, cryocooling typically increases crystal mosaicity and often requires a time-consuming search for cryoprotectants. A recently developed high-pressure cryocooling method reduces crystal damage relative to traditional cryocooling procedures and eases or eliminates the need to screen for cryoprotectants. It has been proposed that the formation of high-density amorphous (HDA) ice within the protein crystal is responsible for the excellent diffraction quality of the high-pressure cryocooled crystals. This paper reports X-ray data that confirm the presence of HDA ice in the high-pressure cryocooled protein crystallization solution and protein crystals analyzed at ambient pressure. Diffuse scattering with a spacing characteristic of HDA ice is seen at low temperatures. This scattering then becomes characteristic successively to low-density amorphous, cubic and hexagonal ice phases as the temperature is gradually raised from 80 to 230,K, and seems to be highly correlated with the diffraction quality of crystals. [source] Tracking reflections through cryogenic cooling with topographyJOURNAL OF APPLIED CRYSTALLOGRAPHY, Issue 3 2006Jeffrey J. Lovelace The mosaic structure of a single protein crystal was analyzed by reflection profiling and topography using highly parallel and monochromatic synchrotron radiation. Fine-,-sliced diffraction images (0.002° stills) were collected using a conventional large-area CCD detector in order to calculate reflection profiles. Fine-,-sliced topographic data (0.002°) stills were collected with a digital topography system for three reflections in a region where the Lorentz effect was minimized. At room temperature, several different mosaic domains were clearly visible within the crystal. Without altering the crystal orientation, the crystal was cryogenically frozen (cryocooled) and the experiment was repeated for the same three reflections. Topographs at cryogenic temperatures reveal a significantly increased mosaicity, while the original domain structure is maintained. A model for the observed changes during cryocooling is presented. [source] Radiation damage in protein crystals examined under various conditions by different methodsJOURNAL OF SYNCHROTRON RADIATION, Issue 2 2009Elspeth F. Garman Investigation of radiation damage in protein crystals has progressed in several directions over the past couple of years. There have been improvements in the basic procedures such as calibration of the incident X-ray intensity and calculation of the dose likely to be deposited in a crystal of known size and composition with this intensity. There has been increased emphasis on using additional techniques such as optical, Raman or X-ray spectroscopy to complement X-ray diffraction. Apparent discrepancies between the results of different techniques can be explained by the fact that they are sensitive to different length scales or to changes in the electronic state rather than to movement of atoms. Investigations have been carried out at room temperature as well as cryo-temperatures and, in both cases, with the introduction of potential scavenger molecules. These and other studies are leading to an overall description of the changes which can occur when a protein crystal is irradiated with X-rays at both cryo- and room temperatures. Results from crystallographic and spectroscopic radiation-damage experiments can be reconciled with other studies in the field of radiation physics and chemistry. [source] Feasibility of one-shot-per-crystal structure determination using Laue diffractionACTA CRYSTALLOGRAPHICA SECTION D, Issue 1 2010Sterling Cornaby Crystal size is an important factor in determining the number of diffraction patterns which may be obtained from a protein crystal before severe radiation damage sets in. As crystal dimensions decrease this number is reduced, eventually falling to one, at which point a complete data set must be assembled using data from multiple crystals. When only a single exposure is to be collected from each crystal, the polychromatic Laue technique may be preferable to monochromatic methods owing to its simultaneous recording of a large number of fully recorded reflections per image. To assess the feasibility of solving structures using single Laue images from multiple crystals, data were collected using a `pink' beam at the CHESS D1 station from groups of lysozyme crystals with dimensions of the order of 20,30,µm mounted on MicroMesh grids. Single-shot Laue data were used for structure determination by molecular replacement and correct solutions were obtained even when as few as five crystals were used. [source] Simulation of X-ray frames from macromolecular crystals using a ray-tracing approachACTA CRYSTALLOGRAPHICA SECTION D, Issue 6 2009Kay Diederichs An algorithm is described which simulates a data set obtained from a protein crystal using the rotation method. The diffraction pattern of an ideal crystal is specified by the orientation of the crystal's cell axes with respect to a specified laboratory coordinate system, the distance between the crystal and the detector, the wavelength and the rotation range per frame. However, a realistic simulation of an experiment additionally requires at least a plausible physical model for crystal mosaicity and beam properties. To explore the physical basis of reflection shape and rocking-curve variation, the algorithm simulates the diffraction of a real crystal composed of mosaic blocks which is illuminated with a beam of given divergence and dispersion. Ray tracing for each reflection leads to reflection shapes and rocking curves that appear realistic. A program implementing the algorithm may be used to reproducibly generate data sets that model different physical aspects (imperfections) of the crystal and the experiment. Certain types of systematic errors of the experimental apparatus may also be simulated. Further applications include teaching and characterization of the properties of data-reduction algorithms. [source] Structure determination of a novel protein by sulfur SAD using chromium radiation in combination with a new crystal-mounting methodACTA CRYSTALLOGRAPHICA SECTION D, Issue 8 2005Yu Kitago A novel and easy crystal-mounting technique was developed for the sulfur SAD method using Cr,K, radiation (2.29,Å). Using this technique, the cryo-buffer and cryoloop around the protein crystal can be removed before data collection in order to eliminate their X-ray absorption. The superiority and reproducibility of the data sets with this mounting technique were demonstrated using tetragonal hen egg-white lysozyme crystals. The structure of a novel protein, PH1109, from Pyrococcus horikoshii OT3 was solved using this technique. At the wavelength of Cr,K, radiation, the anomalous signal ,|,F|,/,|F|, of PH1109 is expected to be 1.72% as this protein of 144 residues includes four methionines and two cysteines. Sulfur SAD phasing was performed using SHELXD and SHELXE. In the case of the data set obtained using this novel crystal-mounting technique, 54.9% of all residues were built with side chains automatically by RESOLVE. On the other hand, only 16.0% were built with side chains for the data set collected using the standard cryoloop. These results indicated that this crystal-mounting technique was superior to the standard loop-mounting method for the measurement of small anomalous differences at longer wavelength and yielded better results in sulfur-substructure solution and initial phasing. The present study demonstrates that the sulfur SAD method with a chromium source becomes enhanced and more practical for macromolecular structure determination using the new crystal-mounting technique. [source] A new method for predetermining the diffraction quality of protein crystals: using SOAP as a selection toolACTA CRYSTALLOGRAPHICA SECTION D, Issue 2 2005Robin Leslie Owen A microscope for quantitative analysis of the birefringence properties of samples is introduced. The microscope is used to measure variations in the slow optical axis position (SOAP) across hen egg-white lysozyme, glucose isomerase and fibronectin crystals. By comparing these variations with indicators of diffraction quality, it is shown that the optical properties of a protein crystal provide a non-invasive method of determining crystal diffraction quality before any X-ray data collection is attempted. [source] Accurate rocking-curve measurements on protein crystals grown in a homogeneous magnetic field of 2.4,TACTA CRYSTALLOGRAPHICA SECTION D, Issue 6 2004Daniel Lübbert Differences in mosaicity between lysozyme crystals grown inside and outside a homogeneous magnetic field of 2.4,T and with and without agarose gel were investigated by X-ray diffraction rocking-curve measurements. High angular resolution was achieved using an Si(113) four-reflection Bartels monochromator. The results show that (i) all crystals were highly perfect, (ii) the mosaicities were clearly anisotropic and (iii) the mosaicities varied more strongly within each group of crystals (grown under identical conditions) than the average values across groups. In particular, the effect of the magnetic field on crystal mosaicity was found to be very small. Finally, the spatial distribution of mosaic blocks inside a protein crystal was visualized with a novel diffraction technique using a high spatial resolution two-dimensional CCD detector. [source] Analysis of the influence of coupled diffusion on transport in protein crystal growth for different gravity levelsACTA CRYSTALLOGRAPHICA SECTION D, Issue 10-1 2002D. Castagnolo Diffusion has a central role in protein crystal growth both in microgravity conditions and on ground. Recently several reports have been focused on the importance to use the generalized Fick's equations in n -component systems where crystals grow. In these equations the total flux of each component is produced by the own concentration gradient (main flow) and by the concentration gradient of the other components (cross-flow) present in the system. However in literature the latter effect is often neglected, and the so-called pseudo-binary approximation is used. Lin et al. (1995) proposed a mathematical model to evaluate the concentration profile of the species present around a growing protein crystal. Although the model is reliable, it suffers of the pseudo-binary approximation (neglecting cross term diffusion coefficients and using binary diffusion coefficients), probably because of the lack of multicomponent diffusion data. The present model is based on the experimental set-up proposed by Lin et al. (1995). Nevertheless we have included the coupled diffusion effects, according to the correct description of the matter transport through the generalized Fick's equations. The crystal growth rate is calculated for different gravity levels. The model has been applied to the ternary lysozyme-NaCl-water and quaternary lysozyme-poly(ethylene glycol) (PEG)-NaCl-water systems using recent diffusion data. [source] Synchrotron white-beam X-ray topography of ribonuclease S crystalsACTA CRYSTALLOGRAPHICA SECTION D, Issue 4 2002W. M. Vetter With careful experimental design, indexed synchrotron white-beam X-ray topographs of ribonuclease S crystals at ambient temperature could be recorded with a definition and contrast comparable to that of monochromatic beam topographs of other proteins reported in the literature. By excluding wavelengths longer than 1,Å from the white beam with a filter, a radiation dose equivalent to that required to record about 18 topographs could be tolerated without appreciable radiation damage to the samples. Bragg angles of 0.5° or less were required to select low-index harmonically pure reflections with high intensities and extinction lengths only several times the sample's thickness. The resulting X-ray topographs in some cases showed topographic detail and in others showed the even featureless background that has been considered characteristic of a protein crystal of low mosaicity. The ribonuclease S crystals were well ordered single crystals of a quality comparable to other protein crystals that have been studied by X-ray topography. [source] How to get the magic triangle and the MAD triangle into your protein crystalACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 10 2009Tobias Beck The magic triangle 5-amino-2,4,6-triiodoisophthalic acid (I3C) and the MAD triangle 5-amino-2,4,6-tribromoisophthalic acid (B3C) are two representatives of a novel class of compounds that combine heavy atoms for experimental phasing with functional groups for protein interactions. These compounds are readily available and provide easy access to experimental phasing. The preparation of stock solutions and the incorporation of the compounds into protein crystals are discussed. As an example of incorporation via cocrystallization, the incorporation of B3C into bovine trypsin, resulting in a single site with high occupancy, is described. [source] Elucidation of the mechanism and end products of glutaraldehyde crosslinking reaction by X-ray structure analysisBIOTECHNOLOGY & BIOENGINEERING, Issue 3 2007Yariv Wine Abstract Glutaraldehyde has been used for several decades as an effective crosslinking agent for many applications including sample fixation for microscopy, enzyme and cell immobilization, and stabilization of protein crystals. Despite of its common use as a crosslinking agent, the mechanism and chemistry involved in glutaraldehyde crosslinking reaction is not yet fully understood. Here we describe feasibility study and results obtained from a new approach to investigate the process of protein crystals stabilization by glutaraldehyde crosslinking. It involves exposure of a model protein crystal (Lysozyme) to glutaraldehyde in alkaline or acidic pH for different incubation periods and reaction arrest by medium exchange with crystallization medium to remove unbound glutaraldehyde. The crystals were subsequently incubated in diluted buffer affecting dissolution of un-crosslinked crystals. Samples from the resulting solution were subjected to protein composition analysis by gel electrophoresis and mass spectroscopy while crosslinked, dissolution resistant crystals were subjected to high resolution X-ray structural analysis. Data from gel electrophoresis indicated that the crosslinking process starts at specific preferable crosslinking site by lysozyme dimer formation, for both acidic and alkaline pH values. These dimer formations were followed by trimer and tetramer formations leading eventually to dissolution resistant crystals. The crosslinking initiation site and the end products obtained from glutaraldehyde crosslinking in both pH ranges resulted from reactions between lysine residues of neighboring protein molecules and the polymeric form of glutaraldehyde. Reaction rate was much faster at alkaline pH. Different reaction end products, indicating different reaction mechanisms, were identified for crosslinking taking place under alkaline or acidic conditions. Biotechnol. Bioeng. 2007;98:711,718. © 2007 Wiley Periodicals, Inc. [source] On slow protein crystal nucleation: cluster-cluster aggregation on diffusional encountersCRYSTAL RESEARCH AND TECHNOLOGY, Issue 1 2009C. N. NanevArticle first published online: 7 NOV 200 Abstract With a view to experimental results on protein crystal nucleation the effects of cluster coalescence are probed semi-quantitatively. The steric association restriction, which stems from the patchy surface of the protein molecules, explains both experimentally measured low crystal nucleation rate and coalescence limitations for crystalline clusters of protein molecules. The conclusion is that due to its action, and the impact of rotational diffusion, the coalescence of critical (and/or supercritical) clusters should be rejected as a conceivable alternative for explaining the slow nucleation of protein crystals. Besides, the analysis of cluster-cluster aggregation on diffusional encounters may be of more general interest; it may be helpful by considering the coalescence of structured bio-nano-particles. (© 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim) [source] Gold nanoparticles induce protein crystallizationCRYSTAL RESEARCH AND TECHNOLOGY, Issue 6 2008F. Hodzhaoglu Abstract Nucleation of protein crystals by gold nanoparticles was observed. Lysozyme and ferritin were used as model proteins. The effect was established with uncoated gold nanoparticles and with gold nanoparticles coated by 16-mercaptodecanoic acid. (© 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim) [source] Application of mean-separation-works method to protein crystal nucleationCRYSTAL RESEARCH AND TECHNOLOGY, Issue 3 2008Christo N. Nanev Abstract Using mean-separation-works method of Stranski and Kaischew calculations of nucleus form and energy barrier for its formation are performed for globular protein crystals. This is done on the basis of a simple model suggested for crystal nucleation of such proteins. The prerequisite for the model is the fact that strict selection of definite sticky patches on protein molecule surface is obligatory for forming crystal lattices. The calculation results are in agreement with experimental data. (© 2008 WILEY -VCH Verlag GmbH & Co. KGaA, Weinheim) [source] Protein crystal nucleation: Recent notionsCRYSTAL RESEARCH AND TECHNOLOGY, Issue 1 2007Christo N. Nanev Abstract The nucleation of protein crystals is reconsidered taking into account the specificity of the protein molecules. In contrast to the homogeneous surface properties of small molecules, the protein molecule surface is highly inhomogeneous. Over their surfaces proteins exhibit high anisotropic distribution of patches, which are able to form crystalline bonds, the crystallization patch representing only a small fraction of the total surface of the protein molecule. Therefore, an appropriate spatial orientation of the colliding protein molecules is required in order to create a crystalline cluster. This scenario decreases considerably the success ratio of the attempt frequency for crystal nucleation. On the other hand a heterogeneous nucleation of (protein) crystals may be accelerated due to the arrival on some support of under-critical clusters that are formed in bulk solution; when arriving there they may acquire the property of critical nuclei. Thus, a plausible explanation of important peculiarities of protein crystal nucleation, as inferred from the experimental data, is suggested. (© 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim) [source] Growth of large protein crystals by a large-scale hanging-drop methodJOURNAL OF APPLIED CRYSTALLOGRAPHY, Issue 4 2010Keisuke Kakinouchi A method for growing large protein crystals is described. In this method, a cut pipette tip is used to hang large-scale droplets (maximum volume 200,µl) consisting of protein and precipitating agents. A crystal grows at the vapor,liquid interface; thereafter the grown crystal can be retrieved by droplet,droplet contact both for repeated macroseeding and for mounting crystals in a capillary. Crystallization experiments with peroxiredoxin of Aeropyrum pernix K1 (thioredoxin peroxidase, ApTPx) and hen egg white lysozyme demonstrated that this large-scale hanging-drop method could produce a large-volume crystal very effectively. A neutron diffraction experiment confirmed that an ApTPx crystal (6.2,mm3) obtained by this method diffracted to beyond 3.5,Å resolution. [source] Autolabo: an automated system for ligand-soaking experiments with protein crystalsJOURNAL OF APPLIED CRYSTALLOGRAPHY, Issue 4 2010Michihiro Sugahara Ligand soaking of protein crystals is important for the preparation of heavy-atom derivative crystals for experimental phasing as well as for large-scale ligand screening in pharmaceutical developments. To facilitate laborious large-scale ligand screening, to reduce the risk of human contact with hazardous ligand reagents and to increase the success rate of the soaking experiments, a protein crystallization robot `Autolabo' has been developed and implemented in the high-throughput crystallization-to-structure pipeline at RIKEN SPring-8 Center. The main functions of this robotic system are the production of protein crystals for experiments, the ligand soaking of these crystals and the observation of soaked crystals. The separate eight-channel dispensers of Autolabo eliminate the cross-contamination of reagents which should be strictly avoided in the ligand-soaking experiment. Furthermore, the automated approach reduces physical damage to crystals during experiments when compared with the conventional manual approach, and thereby has the potential to yield better quality diffraction data. Autolabo's performance as a ligand-soaking system was evaluated with a crystallization experiment on ten proteins from different sources and a heavy-atom derivatization experiment on three proteins using a versatile cryoprotectant containing heavy-atom reagents as ligands. The crystallization test confirmed reliable crystal reproduction in a single condition and the capability for crystallization with nucleants to improve crystal quality. Finally, Autolabo reproducibly derivatized the test protein crystals with sufficient diffraction quality for experimental phasing and model building, indicating a high potentiality of this automated approach in ligand-soaking experiments. [source] HATODAS II , heavy-atom database system with potentiality scoringJOURNAL OF APPLIED CRYSTALLOGRAPHY, Issue 3 2009Michihiro Sugahara HATODAS II is the second version of HATODAS (the Heavy-Atom Database System), which suggests potential heavy-atom reagents for the derivatization of protein crystals. The present expanded database contains 3103 heavy-atom binding sites, which is four times more than the previous version. HATODAS II has three new criteria to evaluate the feasibility of the search results: (1) potentiality scoring for the predicted heavy-atom reagents, (2) exclusion of the disordered amino acid residues based on the secondary structure prediction and (3) consideration of the solvent accessibility of amino acid residues from a homology model. In the point mutation option, HATODAS II suggests possible mutation sites into reactive amino acid residues such as Met, Cys and His, on the basis of multiple sequence alignments of homologous proteins. These new features allow the user to make a well informed decision as to the possible heavy-atom derivatization experiments of protein crystals. [source] Instrument-independent specification of the diffraction geometry and polarization state of the incident X-ray beamJOURNAL OF APPLIED CRYSTALLOGRAPHY, Issue 1 2009Marc Schiltz This work augments the proposal of Schwarzenbach & Flack [J. Appl. Cryst. (1989), 22, 601,605], who have advocated the use of a diffractometer-independent definition of the azimuthal angle , to specify the diffraction geometry of a Bragg reflection. It is here proposed that one additional angle ,, which is also based on a diffractometer-independent definition, is needed to encode the direction of linear polarization for those experiments where this quantity is of importance. This definition is then extended to the cases of partially and/or elliptically polarized X-ray beams, and the use of three normalized Stokes parameters, P1, P2 and P3, together with ,, is advocated in order to characterize exhaustively the polarization state of the incident beam. The conventions proposed here present a general, unambiguous and economical means of encoding the information about the diffraction geometry, without the need to record any further information about the instrument, crystal orientation matrix and goniometer angles. Data-processing software using these definitions to analyse polarization-dependent phenomena becomes instrument-independent and completely general. These methods have been implemented in the macromolecular phasing program SHARP for exploiting the polarization anisotropy of anomalous scattering in protein crystals. [source] Early stages of protein crystallization as revealed by emerging optical waveguide technologyJOURNAL OF APPLIED CRYSTALLOGRAPHY, Issue 3 2008Attia Boudjemline A highly sensitive method for studying the onset of protein crystallization in real time using an optical-waveguide-based technique is reported. Dual polarization interferometry uses light from sensing and reference waveguides to produce an interference pattern, which when the sensing waveguide is immersed in a protein solution supplies information on the thickness and density of any protein adlayer on the sensing waveguide's surface. This technique provides evidence that crystallization proceeds via large protein aggregates but, more strikingly, shows dramatic light loss from the sensing waveguide at a very early stage during crystallization. The technique proves relatively insensitive to the crystallization of small molecules or poorly formed protein crystals and affords a method of distinguishing crystal formation from the formation of other protein aggregates or salt crystals. The experimental setup currently necessitates crystallization using the batch method, and precipitant mixing at high supersaturation is known to introduce a greater variability compared with methods such as vapour diffusion or dialysis, but first results promise to bridge the paucity of real-time methods available to distinguish the onset of protein crystallization from other forms of aggregation. [source] Pressure-induced high-density amorphous ice in protein crystalsJOURNAL OF APPLIED CRYSTALLOGRAPHY, Issue 1 2008Chae Un Kim Crystal cryocooling has been used in X-ray protein crystallography to mitigate radiation damage during diffraction data collection. However, cryocooling typically increases crystal mosaicity and often requires a time-consuming search for cryoprotectants. A recently developed high-pressure cryocooling method reduces crystal damage relative to traditional cryocooling procedures and eases or eliminates the need to screen for cryoprotectants. It has been proposed that the formation of high-density amorphous (HDA) ice within the protein crystal is responsible for the excellent diffraction quality of the high-pressure cryocooled crystals. This paper reports X-ray data that confirm the presence of HDA ice in the high-pressure cryocooled protein crystallization solution and protein crystals analyzed at ambient pressure. Diffuse scattering with a spacing characteristic of HDA ice is seen at low temperatures. This scattering then becomes characteristic successively to low-density amorphous, cubic and hexagonal ice phases as the temperature is gradually raised from 80 to 230,K, and seems to be highly correlated with the diffraction quality of crystals. [source] A parallel program using SHELXD for quick heavy-atom partial structural solution on high-performance computersJOURNAL OF APPLIED CRYSTALLOGRAPHY, Issue 2 2007Zheng-Qing Fu A parallel algorithm has been designed for SHELXD to solve the heavy-atom partial structures of protein crystals quickly. Based on this algorithm, a program has been developed to run on high-performance multiple-CPU Linux PCs, workstations or clusters. Tests on the 32-CPU Linux cluster at SER-CAT, APS, Argonne National Laboratory, show that the parallelization dramatically speeds up the process by a factor of roughly the number of CPUs applied, leading to reliable and instant heavy-atom sites solution, which provides the practical opportunity to employ heavy-atom search as an alternative tool for anomalous scattering data quality evaluation during single/multiple-wavelength anomalous diffraction (SAD/MAD) data collection at synchrotron beamlines. [source] | ||||||||||||||||