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Cryo-electron microscopy at atomic resolution

Wednesday, September 24, 2014 — Poster Session IV

10:00 a.m. –12:00 p.m.

FAES Academic Center



* FARE Award Winner


  • D. Matthies
  • A. Bartesaghi
  • S. Banerjee
  • A. Merk
  • S. Subramaniam


High-resolution structures of proteins and protein complexes are currently determined using either X-ray crystallography or NMR spectroscopy, and in selected instances, from cryo-electron microscopy of ordered protein assemblies. Here we show that emerging methods in single-particle cryo-EM now allow structure determination at near-atomic resolution, even for smaller protein complexes with low symmetry. We solved the structure of the ~465 kDa Escherichia coli β-galactosidase at ~3.2 Å resolution using single-particle cryo-EM. The majority of the side-chains, the termini, and the geometry of the active sites, including a catalytic Mg2+ ion, can be clearly discerned in the density map. The cryo-EM structure closely matches the crystal structure, although there are significant local differences throughout the protein, with clear evidence for conformational changes resulting from crystal contacts. Inspection of the map reveals systematically weaker densities for negatively charged residues. We show that these exhibit more pronounced effects of radiation damage as determined by comparison of density maps obtained using electron doses ranging from 10 to 30 electrons/Å2. In addition to establishing the feasibility of determining structures, at near-atomic resolution, of protein complexes (<500 kDa) with low symmetry, our analyses also provide a measure of the effects of radiation damage in high-resolution cryo-EM.

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