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      Three-dimensional structure of basal body triplet revealed by electron cryo-tomography

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          Abstract

          Basal bodies and centrioles play central roles in microtubule (MT)-organizing centres within many eukaryotes. They share a barrel-shaped cylindrical structure composed of nine MT triplet blades. Here, we report the structure of the basal body triplet at 33 Å resolution obtained by electron cryo-tomography and 3D subtomogram averaging. By fitting the atomic structure of tubulin into the EM density, we built a pseudo-atomic model of the tubulin protofilaments at the core of the triplet. The 3D density map reveals additional densities that represent non-tubulin proteins attached to the triplet, including a large inner circular structure in the basal body lumen, which functions as a scaffold to stabilize the entire basal body barrel. We found clear longitudinal structural variations along the basal body, suggesting a sequential and coordinated assembly mechanism. We propose a model in which δ-tubulin and other components participate in the assembly of the basal body.

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          Most cited references54

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          Improved methods for building protein models in electron density maps and the location of errors in these models.

          Map interpretation remains a critical step in solving the structure of a macromolecule. Errors introduced at this early stage may persist throughout crystallographic refinement and result in an incorrect structure. The normally quoted crystallographic residual is often a poor description for the quality of the model. Strategies and tools are described that help to alleviate this problem. These simplify the model-building process, quantify the goodness of fit of the model on a per-residue basis and locate possible errors in peptide and side-chain conformations.
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            Proteomic characterization of the human centrosome by protein correlation profiling.

            The centrosome is the major microtubule-organizing centre of animal cells and through its influence on the cytoskeleton is involved in cell shape, polarity and motility. It also has a crucial function in cell division because it determines the poles of the mitotic spindle that segregates duplicated chromosomes between dividing cells. Despite the importance of this organelle to cell biology and more than 100 years of study, many aspects of its function remain enigmatic and its structure and composition are still largely unknown. We performed a mass-spectrometry-based proteomic analysis of human centrosomes in the interphase of the cell cycle by quantitatively profiling hundreds of proteins across several centrifugation fractions. True centrosomal proteins were revealed by both correlation with already known centrosomal proteins and in vivo localization. We identified and validated 23 novel components and identified 41 likely candidates as well as the vast majority of the known centrosomal proteins in a large background of nonspecific proteins. Protein correlation profiling permits the analysis of any multiprotein complex that can be enriched by fractionation but not purified to homogeneity.
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              Centrioles, centrosomes, and cilia in health and disease.

              Centrioles are barrel-shaped structures that are essential for the formation of centrosomes, cilia, and flagella. Here we review recent advances in our understanding of the function and biogenesis of these organelles, and we emphasize their connection to human disease. Deregulation of centrosome numbers has long been proposed to contribute to genome instability and tumor formation, whereas mutations in centrosomal proteins have recently been genetically linked to microcephaly and dwarfism. Finally, structural or functional centriole aberrations contribute to ciliopathies, a variety of complex diseases that stem from the absence or dysfunction of cilia.
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                Author and article information

                Journal
                EMBO J
                The EMBO Journal
                Nature Publishing Group
                0261-4189
                1460-2075
                01 February 2012
                13 December 2011
                13 December 2011
                : 31
                : 3
                : 552-562
                Affiliations
                [1 ]simpleThe Howard Hughes Medical Institute, University of California , San Francisco, CA, USA
                [2 ]simpleCentro Nacional de Biotecnologia—CSIC, Campus Universidad Autonoma , Madrid, Spain
                [3 ]simpleDepartment of Biochemistry and Biophysics, University of California , San Francisco, CA, USA
                Author notes
                [a ]Department of Biochemistry and Biophysics, Howard Hughes Medical Institute, University of California at San Francisco, 600 16th Street, Room S412D, San Francisco, CA 94158-2517, USA. Tel.: +1 415 476 2521; Fax: +1 415 476 1902; E-mail: agard@ 123456msg.ucsf.edu
                Article
                emboj2011460
                10.1038/emboj.2011.460
                3273388
                22157822
                6bac348a-2ecf-487a-ac17-ebbf72ba827e
                Copyright © 2012, European Molecular Biology Organization

                This is an open-access article distributed under the terms of the Creative Commons Attribution Noncommercial No Derivative Works 3.0 Unported License, which permits distribution and reproduction in any medium, provided the original author and source are credited. This license does not permit commercial exploitation or the creation of derivative works without specific permission.

                History
                : 14 August 2011
                : 15 November 2011
                Categories
                Article

                Molecular biology
                centriole,chlamydomonas reinhardtii,basal body,three-dimensional reconstruction,electron cryo-tomography

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