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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.
