Electron microscopy of thin-sectioned three-dimensional crystals of SecA protein from Escherichia coli: structure in projection at 40 A resolution.
SecA is a single-chain, membrane-associated polypeptide (102 kDa) which functions as an essential component of the protein export machinery of Escherichia coli. SecA has been crystallized from ammonium sulfate as small, three-dimensional bipyramidal crystals (0.1 x 0.1 x 0.05 mm). These crystals did not demonstrate detectable diffraction of X-rays from rotating anode sources. For study by electron microscopy, individual crystals were cross-linked in glutaraldehyde and OsO4 solutions, dehydrated, embedded in epoxy resin, and sectioned normal to crystallographic axial directions inferred from the external morphology of the crystals. Fourier transformation of processed images of untilted thin sections stained with uranyl acetate and lead citrate show reflections extending to 31 A resolution. Diffraction data and reconstructed images of the projected density of the unit cell contents indicate that the bipyramidal SecA crystals belong to orthorhombic space group C222(1) with unit cell dimensions a = 414 A, b = 381 A, and c = 243 A. Filtered images and density maps of mutually orthogonal projections of the unit cell contents are consistent with a three-dimensional model in which the asymmetric unit contains eight SecA monomers. The large unit cell dimensions and packing of protein monomers suggest that SecA is crystallizing as an oligomer of either dimers or tetramers.
Weaver
A J
AJ
Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas 75235.
McDowall
A W
AW
Oliver
D B
DB
Deisenhofer
J
J
D000251:Adenosine Triphosphatases / Q000648:ultrastructure*; D001426:Bacterial Proteins / Q000648:ultrastructure*; D004926:Escherichia coli / Q000737:chemistry*; D029968:Escherichia coli Proteins; D026901:Membrane Transport Proteins; D008854:Microscopy, Electron; D011487:Protein Conformation; D000069816:SEC Translocation Channels; D000081416:SecA Proteins
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D026901:Membrane Transport Proteins; D000069816:SEC Translocation Channels; D000251:Adenosine Triphosphatases; D000081416:SecA Proteins