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J Struct Biol. 2015 Nov;192(2):245-54. doi: 10.1016/j.jsb.2015.09.015. Epub 2015 Sep 28.

A new protocol to accurately determine microtubule lattice seam location.

Author information

1
Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA; Howard Hughes Medical Institute, Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720, USA. Electronic address: ruiz@lbl.gov.
2
Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA; Howard Hughes Medical Institute, Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720, USA. Electronic address: enogales@lbl.gov.

Abstract

Microtubules (MTs) are cylindrical polymers of αβ-tubulin that display pseudo-helical symmetry due to the presence of a lattice seam of heterologous lateral contacts. The structural similarity between α- and β-tubulin makes it difficult to computationally distinguish them in the noisy cryo-EM images, unless a marker protein for the tubulin dimer, such as kinesin motor domain, is present. We have developed a new data processing protocol that can accurately determine αβ-tubulin register and seam location for MT segments. Our strategy can deal with difficult situations, where the marker protein is relatively small or the decoration of marker protein is sparse. Using this new seam-search protocol, combined with movie processing for data from a direct electron detection camera, we were able to determine the cryo-EM structures of MT at 3.5 Å resolution in different functional states. The successful distinction of α- and β-tubulin allowed us to visualize the nucleotide state at the E-site and the configuration of lateral contacts at the seam.

KEYWORDS:

Cryo-EM; Microtubule; Seam

PMID:
26424086
PMCID:
PMC4634897
DOI:
10.1016/j.jsb.2015.09.015
[Indexed for MEDLINE]
Free PMC Article

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