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.
Citations
More filters
TL;DR: In this paper, a simple, fast and affordable method for transient protein expression in mammalian cells is reported, which combines several features necessary for the production of suitable samples for structural biology, in particular protein crystallography, namely high protein yield, straightforward purification, selenomethionine incorporation and control of N-linked glycosylation.
Abstract: Most proteins for structural biology studies are produced by high-level expression in Escherichia coli. However, prokaryotic based expression systems fail to generate correctly folded functional forms of many proteins and hence a variety of eukaryotic based expression systems have been developed. Of these, yeast and baculovirus-infected insect cells currently represent the expression systems of choice for structural biologists. Here, protocols for a simple, fast and affordable method for transient protein expression in mammalian cells are reported. The results demonstrate that it combines several features necessary for the production of suitable samples for structural biology, in particular protein crystallography, namely high protein yield, straightforward purification, selenomethionine incorporation and control of N-linked glycosylation. The system is suitable for use in conventional laboratories or can be implemented in a medium- or high-throughput pipeline.
702 citations
Cites background or methods from "J. Appl. Cryst.の発刊に際して"
...These timescales mean that compared with other eukaryotic expression systems the mammalian transient transfection method is significantly faster than the baculovirus-infected insect-cell system or generation of yeast stable integrants (timescale considerations discussed in Mancia et al., 2004;…...
[...]
...This is sufficient for screening a large number of crystallization conditions using nanolitre robotic systems (Walter et al., 2003, 2005)....
[...]
TL;DR: The fully automated pipeline, BALBES, integrates a redesigned hierarchical database of protein structures with their domains and multimeric organization, and solves molecular-replacement problems using only input X-ray and sequence data.
Abstract: The number of macromolecular structures solved and deposited in the Protein Data Bank (PDB) is higher than 40 000. Using this information in macromolecular crystallography (MX) should in principle increase the efficiency of MX structure solution. This paper describes a molecular-replacement pipeline, BALBES, that makes extensive use of this repository. It uses a reorganized database taken from the PDB with multimeric as well as domain organization. A system manager written in Python controls the workflow of the process. Testing the current version of the pipeline using entries from the PDB has shown that this approach has huge potential and that around 75% of structures can be solved automatically without user intervention.
700 citations
Cites background from "J. Appl. Cryst.の発刊に際して"
...Although the input is sufficiently simple, the output files contain all the process information, including the results of the analysis of the data by SFCHECK, REFMAC and MOLREP....
[...]
...The system uses currently available programs including MOLREP (Lebedev et al., 2008), REFMAC (Murshudov et al., 1997) and SFCHECK (Vaguine et al., 1999)....
[...]
...All of these approaches are built around one or more of the popular molecular-replacement programs AMoRe (Navaza, 1987), MOLREP (Vagin & Teplyakov, 1997; Lebedev et al., 2008) and Phaser (Storoni et al., 2004)....
[...]
Journal Article•
TL;DR: In this paper, a Bayesian approach to estimating map quality is developed and used in the PHENIX AutoSol wizard to make decisions during automated structure solution, and the skewness of electron density is found to be the best indicator of actual map quality.
Abstract: Ten measures of experimental electron-density-map quality are examined and the skewness of electron density is found to be the best indicator of actual map quality. A Bayesian approach to estimating map quality is developed and used in the PHENIX AutoSol wizard to make decisions during automated structure solution.
691 citations
TL;DR: Algorithms are presented that allowed a web-server implementation of PDB_REDO, and the first user results are discussed.
681 citations
Cites methods from "J. Appl. Cryst.の発刊に際して"
...Automated pipelines are available for data reduction (e.g. Otwinowski & Minor, 1997; Vonrhein et al., 2011; Krug et al., 2012; Monaco et al., 2013; Winter et al., 2013), experimental phasing (e.g. Panjikar et al., 2005; Terwilliger et al., 2009; Pannu et al., 2011), molecular replacement (e.g.…...
[...]
TL;DR: A description is given of a maximum-likelihood approach to absolute structure determinations of biologically active molecules and how this approach can be applied to solve the mystery of why polyene-like structures occur in nature.
Abstract: A new probabilistic approach is introduced for the determination of the absolute structure of a compound which is known to be enantiopure based on Bijvoet-pair intensity differences. The new method provides relative probabilities for different models of the chiral composition of the structure. The outcome of this type of analysis can also be cast in the form of a new value, along with associated standard uncertainty, that resembles the value of the well known Flack x parameter. The standard uncertainty we obtain is often about half of the standard uncertainty in the value of the Flack x parameter. The proposed formalism is suited in particular to absolute configuration determination from diffraction data of biologically active (pharmaceutical) compounds where the strongest resonant scattering signal often comes from oxygen. It is shown that a reliable absolute configuration assignment in such cases can be made on the basis of Cu Kα data, and in some cases even with carefully measured Mo Kα data.
675 citations
Cites methods from "J. Appl. Cryst.の発刊に際して"
...…K sealed-tube X-ray generator with graphite monochromator at 100 K. (4) Measured on a Bruker AXS SMART 6000 system with a Siemens Cu K rotatinganode tube and focusing multilayer optics at 100 K. (a) Data integrated using EvalCCD (Duisenberg et al., 2003) and scaled using SADABS (Sheldrick, 1996)....
[...]
...(2) Measured on a Nonius KappaCCD system with a Nonius Mo K rotating-anode X-ray generator at 100 K. (3) Measured on a Bruker AXS SMART 6000 system with a Cu K sealed-tube X-ray generator with graphite monochromator at 100 K. (4) Measured on a Bruker AXS SMART 6000 system with a Siemens Cu K rotatinganode tube and focusing multilayer optics at 100 K. (a) Data integrated using EvalCCD (Duisenberg et al., 2003) and scaled using SADABS (Sheldrick, 1996)....
[...]
References
More filters
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.
34,239 citations
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.
28,425 citations
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'.
27,505 citations
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.
18,531 citations
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