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J Struct Biol. 2014 Apr;186(1):8-18. doi: 10.1016/j.jsb.2014.02.016. Epub 2014 Mar 5.

Single particle analysis integrated with microscopy: a high-throughput approach for reconstructing icosahedral particles.

Author information

1
Departments of Chemistry & Biochemistry, and Molecular Biology, University of California, San Diego, La Jolla, CA 92093-0378.
2
Department of Microbiology, Immunology & Molecular Genetics, University of California, Los Angeles, Los Angeles, CA 90095-7364.
#
Contributed equally

Abstract

In cryo-electron microscopy and single particle analysis, data acquisition and image processing are generally carried out in sequential steps and computation of a three-dimensional reconstruction only begins once all the micrographs have been acquired. We are developing an integrated system for processing images of icosahedral particles during microscopy to provide reconstructed density maps in real-time at the highest possible resolution. The system is designed as a combination of pipelines to run in parallel on a computer cluster and analyzes micrographs as they are acquired, handling automatically all the processing steps from defocus estimation and particle picking to origin/orientation determination. An ab initio model is determined independently from the first micrographs collected, and new models are generated as more particles become available. As a proof of concept, we simulated data acquisition sessions using three sets of micrographs of good to excellent quality that were previously recorded from different icosahedral viruses. Results show that the processing of single micrographs can keep pace with an acquisition rate of about two images per minute. The reconstructed density map improves steadily during the image acquisition phase and its quality at the end of data collection is only moderately inferior to that obtained by expert users who processed semi-automatically all the micrographs after the acquisition. The current prototype demonstrates the advantages of integrating three-dimensional image processing with microscopy, which include an ability to monitor acquisition in terms of the final structure and to predict how much data and microscope resources are needed to achieve a desired resolution.

KEYWORDS:

3D reconstruction; Automation; Cryo-electron microscopy; Icosahedral particles; Image processing; Single particle analysis

PMID:
24613762
PMCID:
PMC4070310
DOI:
10.1016/j.jsb.2014.02.016
[Indexed for MEDLINE]
Free PMC Article

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