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  Unraveling the structure of membrane proteins in situ by transfer function corrected cryo-electron tomography

Eibauer, M., Hoffmann, C., Plitzko, J. M., Baumeister, W., Nickell, S., & Engelhardt, H. (2012). Unraveling the structure of membrane proteins in situ by transfer function corrected cryo-electron tomography. JOURNAL OF STRUCTURAL BIOLOGY, 180(3), 488-496. doi:10.1016/j.jsb.2012.09.008.

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 Creators:
Eibauer, Matthias1, Author              
Hoffmann, Christian1, Author              
Plitzko, Jürgen M.1, Author              
Baumeister, Wolfgang1, Author              
Nickell, Stephan1, Author              
Engelhardt, Harald1, Author              
Affiliations:
1Baumeister, Wolfgang / Molecular Structural Biology, Max Planck Institute of Biochemistry, Max Planck Society, ou_1565142              

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Free keywords: PARTICLE ELECTRON CRYOMICROSCOPY; MYCOBACTERIAL OUTER-MEMBRANE; MOLECULAR ARCHITECTURE; ENVELOPE PROTEIN; 26S PROTEASOME; CELL-WALL; MICROSCOPY; SMEGMATIS; RESOLUTION; COMPLEXCryo-electron microscopy; 3D electron microscopy; Contrast transfer function (CTF); Modulation transfer function (MTF); Subtomogram averaging; Porin; Outer membrane protein; Mycobacterium smegmatis;
 Abstract: Cryo-electron tomography in combination with subtomogram averaging allows to investigate the structure of protein assemblies in their natural environment in a close to live state. To make full use of the structural information contained in tomograms it is necessary to analyze the contrast transfer function (CTF) of projections and to restore the phases of higher spatial frequencies. CTF correction is however hampered by the difficulty of determining the actual defocus values from tilt series data, which is due to the low signal-to-noise ratio of electron micrographs. In this study, an extended acquisition scheme is introduced that enables an independent CTF determination. Two high-dose images are recorded along the tilt axis on both sides of each projection, which allow an accurate determination of the defocus values of these images. These values are used to calculate the CTF for each image of the tilt series. We applied this scheme to the mycobacterial outer membrane protein MspA reconstituted in lipid vesicles and tested several variants of Cif estimation in combination with subtomogram averaging and correction of the modulation transfer function (MTF). The 3D electron density map of MspA was compared with a structure previously determined by X-ray crystallography. We were able to demonstrate that structural information up to a resolution of 16.8 angstrom can be recovered using our CTF correction approach, whereas the uncorrected 3D map had a resolution of only 26.2 angstrom. (C) 2012 Elsevier Inc. All rights reserved.

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Language(s): eng - English
 Dates: 2012-12
 Publication Status: Published in print
 Pages: 9
 Publishing info: -
 Table of Contents: -
 Rev. Type: Peer
 Identifiers: ISI: 000311471000011
DOI: 10.1016/j.jsb.2012.09.008
 Degree: -

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Title: JOURNAL OF STRUCTURAL BIOLOGY
Source Genre: Journal
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Publ. Info: 525 B ST, STE 1900, SAN DIEGO, CA 92101-4495 USA : ACADEMIC PRESS INC ELSEVIER SCIENCE
Pages: - Volume / Issue: 180 (3) Sequence Number: - Start / End Page: 488 - 496 Identifier: ISSN: 1047-8477