Three-dimensional reconstruction of bovine brain V-ATPase by cryo-electron microscopy and single particle analysis |
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Authors: | Gregorini Marco Wang Jin Xie Xiao-Song Milligan Ronald A Engel Andreas |
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Affiliation: | aMaurice E. Müller Institute for Structural Biology, Biozentrum University of Basel, Klingelbergstrasse 70, 4056 Basel, Switzerland;bUniversity of Texas Southwestern Medical Center at Dallas, Department of Internal Medicine, 6000 Harry Hines Boulevard, Dallas, TX 75390-8591, USA;cThe Scripps Research Institute, Department of Cell Biology, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA |
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Abstract: | Bovine V-ATPase from brain clathrin-coated vesicles was investigated by cryo-electron microscopy and single particle analysis. Our studies revealed great flexibility of the central linker region connecting V1 and V0. As a consequence, the two sub-complexes were processed separately and the resulting volumes were merged computationally. We present the first three-dimensional (3D) map of a V-ATPase obtained from cryo-electron micrographs. The overall resolution was estimated 34 Å by Fourier shell correlation (0.5 cutoff). Our 3D reconstruction shows a large peripheral stalk and a smaller, isolated peripheral density, suggesting a second, less well-resolved peripheral connection. The 3D map reveals new features of the large peripheral stator and of the collar-like density attached to the membrane domain. Our analyses of the membrane domain indicate the presence of six proteolipid subunits. In addition, we could localize the V0 subunit a flanking the large peripheral stalk. |
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Keywords: | Bovine brain V-ATPase Cryo-electron microscopy Single particle analysis 3D reconstruction |
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