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      Gctf: Real-time CTF determination and correction

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          Abstract

          Accurate estimation of the contrast transfer function (CTF) is critical for a near-atomic resolution cryo electron microscopy (cryoEM) reconstruction. Here, a GPU-accelerated computer program, Gctf, for accurate and robust, real-time CTF determination is presented. The main target of Gctf is to maximize the cross-correlation of a simulated CTF with the logarithmic amplitude spectra (LAS) of observed micrographs after background subtraction. Novel approaches in Gctf improve both speed and accuracy. In addition to GPU acceleration (e.g. 10–50×), a fast ‘1-dimensional search plus 2-dimensional refinement (1S2R)’ procedure further speeds up Gctf. Based on the global CTF determination, the local defocus for each particle and for single frames of movies is accurately refined, which improves CTF parameters of all particles for subsequent image processing. Novel diagnosis method using equiphase averaging (EPA) and self-consistency verification procedures have also been implemented in the program for practical use, especially for aims of near-atomic reconstruction. Gctf is an independent program and the outputs can be easily imported into other cryoEM software such as Relion (Scheres, 2012) and Frealign (Grigorieff, 2007). The results from several representative datasets are shown and discussed in this paper.

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          How cryo-EM is revolutionizing structural biology.

          For many years, structure determination of biological macromolecules by cryo-electron microscopy (cryo-EM) was limited to large complexes or low-resolution models. With recent advances in electron detection and image processing, the resolution by cryo-EM is now beginning to rival X-ray crystallography. A new generation of electron detectors record images with unprecedented quality, while new image-processing tools correct for sample movements and classify images according to different structural states. Combined, these advances yield density maps with sufficient detail to deduce the atomic structure for a range of specimens. Here, we review the recent advances and illustrate the exciting new opportunities that they offer to structural biology research.
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            Ribosome. The structure of the human mitochondrial ribosome.

            The highly divergent ribosomes of human mitochondria (mitoribosomes) synthesize 13 essential proteins of oxidative phosphorylation complexes. We have determined the structure of the intact mitoribosome to 3.5 angstrom resolution by means of single-particle electron cryogenic microscopy. It reveals 80 extensively interconnected proteins, 36 of which are specific to mitochondria, and three ribosomal RNA molecules. The head domain of the small subunit, particularly the messenger (mRNA) channel, is highly remodeled. Many intersubunit bridges are specific to the mitoribosome, which adopts conformations involving ratcheting or rolling of the small subunit that are distinct from those seen in bacteria or eukaryotes. An intrinsic guanosine triphosphatase mediates a contact between the head and central protuberance. The structure provides a reference for analysis of mutations that cause severe pathologies and for future drug design. Copyright © 2015, American Association for the Advancement of Science.
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              Structure of the TRPA1 ion channel suggests regulatory mechanisms

              The TRPA1 ion channel (a.k.a the ‘wasabi receptor’) is a detector of noxious chemical agents encountered in our environment or produced endogenously during tissue injury or drug metabolism. These include a broad class of electrophiles that activate the channel through covalent protein modification. TRPA1 antagonists hold potential for treating neurogenic inflammatory conditions provoked or exacerbated by irritant exposure. Despite compelling reasons to understand TRPA1 function, structural mechanisms underlying channel regulation remain obscure. Here, we use single-particle electron cryo-microscopy to determine the structure of full-length human TRPA1 to ~4Å resolution in the presence of pharmacophores, including a potent antagonist. A number of unexpected features are revealed, including an extensive coiled-coil assembly domain stabilized by polyphosphate co-factors and a highly integrated nexus that converges on an unpredicted TRP-like allosteric domain. These findings provide novel insights into mechanisms of TRPA1 regulation, and establish a blueprint for structure-based design of analgesic and anti-inflammatory agents.
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                Author and article information

                Contributors
                Journal
                J Struct Biol
                J. Struct. Biol
                Journal of Structural Biology
                Academic Press
                1047-8477
                1095-8657
                1 January 2016
                January 2016
                : 193
                : 1
                : 1-12
                Affiliations
                Medical Research Council Laboratory of Molecular Biology, Division of Structural Studies, Francis Crick Avenue, Cambridge CB2 0QH, UK
                Article
                S1047-8477(15)30100-3
                10.1016/j.jsb.2015.11.003
                4711343
                26592709
                440f006d-7493-4618-8480-a778ab7ef0a2
                © 2015 MRC Laboratory of Molecular Biology. Published by Elsevier Inc.

                This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

                Categories
                Article

                Biophysics
                1d (2d, 3d), one(two, three) dimensional,cryoem, cryo-electron microscopy,ccc, cross-correlation coefficient,ccd, charge coupled device,ccf, cross-correlation function,ctf, contrast transfer function,dqe, detective quantum efficiency,epa, equiphase averaging,fft, fast fourier transform,gpu, graphic processing unit,hav, hepatitis a virus,las, logarithmic amplitude spectra,nfs, network file system,snr, signal to noise ratio,ssd, solid state disk,contrast transfer function,cryo-electron microscopy,gpu program,ctf determination

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