Resolving macromolecular structures from electron cryo-tomography data using sub-tomogram averaging in RELION
| Autor: | Bharat, TAM, Scheres, SHW |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2016 |
| Předmět: |
0301 basic medicine
Electron Microscope Tomography Hepatitis B virus Computer science Macromolecular Substances Image processing Electron Saccharomyces cerevisiae General Biochemistry Genetics and Molecular Biology Article 03 medical and health sciences Software Capsid Image Processing Computer-Assisted Cryopreservation Contrast transfer function business.industry 030104 developmental biology Macromolecular Complexes Tomography business Algorithm Ribosomes |
| Popis: | Electron cryo-tomography (cryo-ET) is a technique that is used to produce three-dimensional pictures (tomograms) of complex objects like asymmetric viruses, cellular organelles or whole cells from a series of tilted electron cryo-microscopy (cryo-EM) images. Averaging of macromolecular complexes found within tomograms is known as sub-tomogram averaging, and this technique allows structure determination of macromolecular complexes in situ. Sub-tomogram averaging is also gaining in popularity for the calculation of initial models for single-particle analysis. We describe herein, a protocol for sub-tomogram averaging from cryo-ET data using the RELION software (http://www2.mrc-lmb.cam.ac.uk/relion). RELION was originally developed for cryo-EM single-particle analysis and the sub-tomogram averaging approach presented in this protocol has been implemented in the existing workflow for single-particle analysis so that users may conveniently tap into existing capabilities of the RELION software. We describe how to calculate three-dimensional models for the contrast transfer function (CTF), which describe the transfer of information in the imaging process, and we illustrate the results of classification and subtomogram averaging refinement for cryo-ET data of purified hepatitis B capsid particles and S. cerevisiae 80S ribosomes. Using the steps described in this protocol, along with the troubleshooting and optimisation guidelines, high-resolution maps can be obtained where secondary structure elements are resolved. |
| Databáze: | OpenAIRE |
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