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Cryo-Electron Microscopy and Image Processing Methods for Studying the Ribonucleoprotein Vault

Published online by Cambridge University Press:  02 July 2020

L. B. Kong ,
L. H. Rome  and
P. L. Stewart
L. B. Kong
Affiliation:
Crump Institute for Biological Imaging, Department of Molecular and Medical Pharmacology UCLA School of Medicine, Los Angeles, CA90095
L. H. Rome
Affiliation:
Department of Biological Chemistry UCLA School of Medicine, Los Angeles, CA90095
P. L. Stewart
Affiliation:
Crump Institute for Biological Imaging, Department of Molecular and Medical Pharmacology UCLA School of Medicine, Los Angeles, CA90095
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Extract

Vaults are highly conserved ribonucleoprotein complexes found in thousands of copies per cell in various tissues. The major vault protein, pi 04, accounts for over 70% of the particle mass and has been recently found to be involved in cancer cell multidrug resistance. We have utilized cryoelectron microscopy (cryo-EM), and three-dimensional image reconstruction to generate a 3-D structure of the vault particle. Individual particle images were isolated using the QVIEW software package, which simultaneously performs density exclusion, planar background subtraction, and application of an elliptical mask. The IMAGIC-5 software package was used for the subsequent particle orientation and reconstruction steps (Fig. I).

A symmetry self-search test was used to measure the signal strength for a variety of point groups and the highest correlation was found for the cyclic 8-fold point group. We have utilized the sinogram correlation algorithms in IMAGIC-5 to find the Euler angles of both 1 μm and 2 μm defocus particle images.

Type
Chambers and Channels: Functional Connections in Multiprotein Complexes Studied by Single Chambers and Channels
Information
Microscopy and Microanalysis , Volume 4 , Issue S2: Proceedings: Microscopy & Microanalysis '98, Microscopy Society of America 56th Annual Meeting, Microbeam Analysis Society 32nd Annual Meeting, Atlanta, Georgia July 12-16, 1998 , July 1998 , pp. 976 - 977
Copyright
Copyright © Microscopy Society of America

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References

References:

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5. This work was funded by the National Science Foundation (MCB-9722353). The aid of Dion Baybridge and Amara Siva is gratefully acknowledged.Google Scholar