J. Appl. Cryst.の発刊に際して
10 Mar 1970-Vol. 12, Iss: 1, pp 1-1
About: The article was published on 1970-03-10 and is currently open access. It has received 8159 citations till now.
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TL;DR: The electron-density map after density modification, resulting from the phasing of a single-wavelength data set and based purely on the anomalous deltaf" contribution, was clearly interpretable; an almost complete model of the protein was built by wARP without human intervention in all four cases.
Abstract: Glucose isomerase from Streptomyces rubiginosus was crystallized in two forms: I222, with one molecule of 44 kDa in the asymmetric unit, and P2(1)2(1)2, with two unique molecules. The I222 structure is known, but the P2(1)2(1)2 form has not been solved before. X-ray diffraction data for the P2(1)2(1)2 form were collected at a wavelength of 1.54 A and data for the I222 form were collected at three different wavelengths: 1.34, 1.07 and 0.98 A. The amount of anomalous signal from one Mn and eight S atoms in these data sets varies from 1.24% to as low as 0.56%. The dual-space direct-methods program SHELXD, run against the Bijvoet differences, gave a clear solution of all anomalous scatterers for all data sets. The Mn positions only were used for SAD phasing of all four data sets. The electron-density map after density modification, resulting from the phasing of a single-wavelength data set and based purely on the anomalous deltaf" contribution, was clearly interpretable; an almost complete model of the protein was built by wARP without human intervention in all four cases. As far as is known, this is the first time that an anomalous signal as low as 0.6% has successfully been used to determine the structure of a macromolecule.
30 citations
Cites methods from "J. Appl. Cryst.の発刊に際して"
...As an example of using the anomalous signal of a metal, the structure of rusticyanin (Harvey et al., 1998) has been solved at 2.1 AÊ from a signal of about 1% from one Cu atom in combination with the direct-methods approach using OASIS (Hao et al., 2000)....
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TL;DR: A structure-based biophysical screening approach has identified for the first time a synthetic small molecule, 2-phenylamino-4-methyl-5-acetylthiazole, that binds to the active site of the β-Ketoacyl-ACP synthase.
Abstract: Fatty-acid synthesis in bacteria is of great interest as a target for the discovery of antibacterial compounds. The addition of a new acetyl moiety to the growing fatty-acid chain, an essential step in this process, is catalyzed by β-ketoacyl-ACP synthase (KAS). It is inhibited by natural antibiotics such as cerulenin and thiolactomycin; however, these lack the requirements for optimal drug development. Structure-based biophysical screening revealed a novel synthetic small molecule, 2-phenylamino-4-methyl-5-acetylthiazole, that binds to Escherichia coli KAS I with a binding constant of 25 µM as determined by fluorescence titration. A 1.35 A crystal structure of its complex with its target reveals noncovalent interactions with the active-site Cys163 and hydrophobic residues of the fatty-acid binding pocket. The active site is accessible through an open conformation of the Phe392 side chain and no conformational changes are induced at the active site upon ligand binding. This represents a novel binding mode that differs from thiolactomycin or cerulenin interaction. The structural information on the protein–ligand interaction offers strategies for further optimization of this low-molecular-weight compound.
30 citations
Cites background or methods from "J. Appl. Cryst.の発刊に際して"
...E. coli KAS I For the E. coli KAS I protein, structural information is available for the apoprotein and for several protein–inhibitor complexes (Olsen et al., 1999, 2001; Price et al., 2001)....
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...The diffraction images were processed using XDS and scaled with XSCALE (Kabsch, 1993)....
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TL;DR: The structural determination procedures of a gap junction channel at 3.5 Å resolution are described, including the preparation of crystals, intensity data collection, data processing, phasing and structural refinement.
Abstract: Intercellular signalling is an essential characteristic of multicellular organisms. Gap junctions, which consist of arrays of intercellular channels, permit the exchange of ions and small molecules between adjacent cells. Here, the structural determination of a gap junction channel composed of connexin 26 (Cx26) at 3.5 A resolution is described. During each step of the purification process, the protein was examined using electron microscopy and/or dynamic light scattering. Dehydration of the crystals improved the resolution limits. Phase refinement using multi-crystal averaging in conjunction with noncrystallographic symmetry averaging based on strictly determined noncrystallographic symmetry operators resulted in an electron-density map for model building. The amino-acid sequence of a protomer structure consisting of the amino-terminal helix, four transmembrane helices and two extracellular loops was assigned to the electron-density map. The amino-acid assignment was confirmed using six selenomethionine (SeMet) sites in the difference Fourier map of the SeMet derivative and three intramolecular disulfide bonds in the anomalous difference Fourier map of the native crystal.
30 citations
Cites methods from "J. Appl. Cryst.の発刊に際して"
...The data set collected at SLS was integrated and scaled with XDS and XSCALE (Kabsch, 1993)....
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TL;DR: A study of the structure of micells of the surfactant polysorbate 80 in aqueous dimethyl sulfoxide solutions shows that the micelles change from core–shell cylinder micelle to core– shell discus micellers between concentrations of 20 and 30% dimethyl sulphoxide.
Abstract: The structures of micelles of the surfactant polysorbate 80 (Tween 80) in 0–50% aqueous dimethyl sulfoxide (DMSO) solutions (pH 7.2, ionic strength 2.44 mM) were investigated by means of small-angle X-ray scattering. At DMSO concentrations of 0–20%, core–shell cylinder micelles formed, and at 30–50% DMSO, core–shell discus micelles formed, that is, changing the hydrophobicity of the DMSO solvent mixture changed the micelles from core–shell cylinder micelles to core–shell discus micelles.
30 citations
Cites background or methods from "J. Appl. Cryst.の発刊に際して"
...3 of Aizawa (2009)....
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...The sample preparation method is the same as that described by Aizawa (2009)....
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...Because polysorbate 80 forms core–shell cylinder micelles in 100% water (Aizawa, 2009), we attempted to fit the SAXS data obtained at DMSO concentrations between 0 and 50% to the core–shell cylinder model for dilute particle solutions....
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...…solution and for a reference solution, and intensities were calibrated and transformed into scattering cross sections {SCS(q), where q is the scattering vector [q = (4 / )sin , where is the wavelength of the X-rays and 2 is the scattering angle]} based on the method presented by Aizawa (2009)....
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...…core–shell cylinder model for dilute particle solutions and the least-squares fit calculation methods for the model and the SAXS data are detailed by Aizawa (2009). n is the number density of particles; R and core are the radius of the circular base of the core cylinder and the scattering length…...
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TL;DR: The effects of commonly encountered impurities on various membrane-protein crystallization regimes are investigated and it is found that the lipidic cubic phase crystallization methodology is the most robust, tolerating protein contamination levels of up to 50%, with little effect on crystal quality.
Abstract: When starting a protein-crystallization project, scientists are faced with several unknowns. Amongst them are these questions: (i) is the purity of the starting material sufficient? and (ii) which type of crystallization experiment is the most promising to conduct? The difficulty in purifying active membrane-protein samples for crystallization trials and the high costs associated with producing such samples require an extremely pragmatic approach. Additionally, practical guidelines are needed to increase the efficiency of membrane-protein crystallization. In order to address these conundrums, the effects of commonly encountered impurities on various membrane-protein crystallization regimes have been investigated and it was found that the lipidic cubic phase (LCP) based crystallization methodology is more robust than crystallization in detergent environments using vapor diffusion or microbatch approaches in its ability to tolerate contamination in the forms of protein, lipid or other general membrane components. LCP-based crystallizations produced crystals of the photosynthetic reaction center (RC) of Rhodobacter sphaeroides from samples with substantial levels of residual impurities. Crystals were obtained with protein contamination levels of up to 50% and the addition of lipid material and membrane fragments to pure samples of RC had little effect on the number or on the quality of crystals obtained in LCP-based crystallization screens. If generally applicable, this tolerance for impurities may avoid the need for samples of ultrahigh purity when undertaking initial crystallization screening trials to determine preliminary crystallization conditions that can be optimized for a given target protein.
30 citations
Cites methods from "J. Appl. Cryst.の発刊に際して"
...While the actual format has changed (Cherezov & Caffrey, 2006; Cherezov et al., 2004; Nollert, 2002), this methodology has been key to the crystallization and subsequent structure determination of a number of membrane proteins, most prominently that of the -adrenenergic receptor (Cherezov et al.,…...
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References
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TL;DR: The goals of the PDB are described, the systems in place for data deposition and access, how to obtain further information and plans for the future development of the resource are described.
Abstract: The Protein Data Bank (PDB; http://www.rcsb.org/pdb/ ) is the single worldwide archive of structural data of biological macromolecules. This paper describes the goals of the PDB, the systems in place for data deposition and access, how to obtain further information, and near-term plans for the future development of the resource.
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TL;DR: New features added to the refinement program SHELXL since 2008 are described and explained.
Abstract: The improvements in the crystal structure refinement program SHELXL have been closely coupled with the development and increasing importance of the CIF (Crystallographic Information Framework) format for validating and archiving crystal structures. An important simplification is that now only one file in CIF format (for convenience, referred to simply as `a CIF') containing embedded reflection data and SHELXL instructions is needed for a complete structure archive; the program SHREDCIF can be used to extract the .hkl and .ins files required for further refinement with SHELXL. Recent developments in SHELXL facilitate refinement against neutron diffraction data, the treatment of H atoms, the determination of absolute structure, the input of partial structure factors and the refinement of twinned and disordered structures. SHELXL is available free to academics for the Windows, Linux and Mac OS X operating systems, and is particularly suitable for multiple-core processors.
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TL;DR: CCP4mg is a project that aims to provide a general-purpose tool for structural biologists, providing tools for X-ray structure solution, structure comparison and analysis, and publication-quality graphics.
Abstract: CCP4mg is a project that aims to provide a general-purpose tool for structural biologists, providing tools for X-ray structure solution, structure comparison and analysis, and publication-quality graphics. The map-fitting tools are available as a stand-alone package, distributed as `Coot'.
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TL;DR: The PHENIX software for macromolecular structure determination is described and its uses and benefits are described.
Abstract: Macromolecular X-ray crystallography is routinely applied to understand biological processes at a molecular level. However, significant time and effort are still required to solve and complete many of these structures because of the need for manual interpretation of complex numerical data using many software packages and the repeated use of interactive three-dimensional graphics. PHENIX has been developed to provide a comprehensive system for macromolecular crystallographic structure solution with an emphasis on the automation of all procedures. This has relied on the development of algorithms that minimize or eliminate subjective input, the development of algorithms that automate procedures that are traditionally performed by hand and, finally, the development of a framework that allows a tight integration between the algorithms.
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TL;DR: A description is given of Phaser-2.1: software for phasing macromolecular crystal structures by molecular replacement and single-wavelength anomalous dispersion phasing.
Abstract: Phaser is a program for phasing macromolecular crystal structures by both molecular replacement and experimental phasing methods. The novel phasing algorithms implemented in Phaser have been developed using maximum likelihood and multivariate statistics. For molecular replacement, the new algorithms have proved to be significantly better than traditional methods in discriminating correct solutions from noise, and for single-wavelength anomalous dispersion experimental phasing, the new algorithms, which account for correlations between F+ and F−, give better phases (lower mean phase error with respect to the phases given by the refined structure) than those that use mean F and anomalous differences ΔF. One of the design concepts of Phaser was that it be capable of a high degree of automation. To this end, Phaser (written in C++) can be called directly from Python, although it can also be called using traditional CCP4 keyword-style input. Phaser is a platform for future development of improved phasing methods and their release, including source code, to the crystallographic community.
17,755 citations