Three-dimensional reconstruction of bovine brain V-ATPase by cryo-electron microscopy and single particle analysis.
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 34A 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.
Gregorini
Marco
M
Maurice E. Müller Institute for Structural Biology, Biozentrum University of Basel, Klingelbergstrasse 70, 4056 Basel, Switzerland.
Wang
Jin
J
Xie
Xiao-Song
XS
Milligan
Ronald A
RA
Engel
Andreas
A
D000818:Animals; D001921:Brain / Q000201:enzymology*; D020134:Catalytic Domain; D002417:Cattle; D020285:Cryoelectron Microscopy; D020035:Holoenzymes / Q000737:chemistry; D011487:Protein Conformation; D025262:Vacuolar Proton-Translocating ATPases / Q000648:ultrastructure*
D020035:Holoenzymes; D025262:Vacuolar Proton-Translocating ATPases