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A new cryo-EM single-particle ab initio reconstruction method visualizes secondary structure elements in an ATP-fueled AAA+ motor
KTH, School of Technology and Health (STH), Structural Biotechnology.
Lund Univ, Dept Mol Biophys.
Lund Univ, Dept Mol Biophys.
Lund Univ, Dept Biochem.
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2008 (English)In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 375, no 4, 934-947 p.Article in journal (Refereed) Published
Abstract [en]

The generation of ab initio three-dimensional (3D) models is a bottleneck in the studies of large macromolecular assemblies by single-particle cryo-electron microscopy. We describe here a novel method, in which established methods for two-dimensional image processing are combined with newly developed programs for joint rotational 3D alignment of a large number of class averages (RAD) and calculation of 3D volumes from aligned projections (VolRec). We demonstrate the power of the method by reconstructing an similar to 660-kDa ATP-fueled AAA+ motor to 7.5 angstrom resolution, with secondary structure elements identified throughout the structure. We propose the method as a generally applicable automated strategy to obtain 3D reconstructions from unstained single particles imaged in vitreous ice.

Place, publisher, year, edition, pages
2008. Vol. 375, no 4, 934-947 p.
Keyword [en]
electron microscopy; cryo-EM; single particle; reconstruction; alignment
National Category
Chemical Sciences
Identifiers
URN: urn:nbn:se:kth:diva-8111DOI: 10.1016/j.jmb.2007.11.028ISI: 000253098000005OAI: oai:DiVA.org:kth-8111DiVA: diva2:13343
Note
QC 20100719Available from: 2008-03-18 Created: 2008-03-18 Last updated: 2010-10-01Bibliographically approved
In thesis
1. Protein structure dynamics and interplay: by single-particle electron microscopy
Open this publication in new window or tab >>Protein structure dynamics and interplay: by single-particle electron microscopy
2008 (English)Doctoral thesis, comprehensive summary (Other scientific)
Abstract [en]

Single-particle cryo-electron microscopy (cryo-EM) is a method capable of obtaining information about the structural organization and dynamics of large macromolecular assemblies. In the late nineties, the method was suggested to have the potential of generating “atomic resolution” reconstructions of particles above a certain mass. However, visualization of secondary structure elements in cryo-EM reconstructions has so far been achieved mainly for highly symmetrical macromolecular assemblies or by using previously existing X-ray structures to solve the initial alignment problem. A factor that severely limits the resolution for low-symmetry (point group symmetry Cn) particles is the problem of ab initio three-dimensional alignment of cryo-EM projection images of proteins in vitreous ice.

A more general problem in the field of molecular biology is the study of heterogeneous structural properties of particles in preparations of purified macromolecular complexes. If not resolved, structural heterogeneity limits the achievable resolution of a cryo-EM reconstruction and makes correct biological interpretation difficult. If resolved, the heterogeneity instead offers a tremendous biological insight into the dynamic behaviour of a structure, and statistical information about partitioning over subpopulations with distinct structural features within the ensemble of particles may be gained.

This thesis adds to the existing body of methods in the field of single-particle cryo-EM by addressing the problem of ab initio rotational alignment and the problem of resolving structural heterogeneity without using a priori information about the structural variability within large populations of cryo-EM projections of unstained proteins. The thesis aims at making the single-particle cryo-EM method a generally applicable tool for generating subnanometer resolution reconstructions and perform heterogeneity analysis of biological macromolecules.

Place, publisher, year, edition, pages
Stockholm: KTH, 2008. 83 p.
Series
Trita-STH : report, ISSN 1653-3836 ; 2008:1
Keyword
biophysics, biochemistry, molecular
National Category
Physical Chemistry
Identifiers
urn:nbn:se:kth:diva-4669 (URN)978-91-7178-892-4 (ISBN)
Public defence
2008-04-04, Hörsallen plan 4, Novum, Flemingsberg, Huddinge, 13:00
Opponent
Supervisors
Note
QC 20100719Available from: 2008-03-18 Created: 2008-03-18 Last updated: 2010-10-01Bibliographically approved

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