Electron microscopy of thin-sectioned three-dimensional crystals of SecA protein from Escherichia coli: Structure in projection at 40 Å resolution

Arthur J. Weaver, Alasdair W. McDowall, Donald B. Oliver, Johann Deisenhofer

Research output: Contribution to journalArticle

15 Citations (Scopus)

Abstract

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 × 0.1 × 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 crosslinked 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 Å 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 C2221 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.

Original languageEnglish (US)
Pages (from-to)87-96
Number of pages10
JournalJournal of Structural Biology
Volume109
Issue number2
DOIs
StatePublished - 1992

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Escherichia coli Proteins
Electron Microscopy
Epoxy Resins
Ammonium Sulfate
Glutaral
Citric Acid
X-Ray Diffraction
Electrodes
Proteins
Cell Count
Escherichia coli
Peptides
Membranes

ASJC Scopus subject areas

  • Structural Biology

Cite this

Electron microscopy of thin-sectioned three-dimensional crystals of SecA protein from Escherichia coli : Structure in projection at 40 Å resolution. / Weaver, Arthur J.; McDowall, Alasdair W.; Oliver, Donald B.; Deisenhofer, Johann.

In: Journal of Structural Biology, Vol. 109, No. 2, 1992, p. 87-96.

Research output: Contribution to journalArticle

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