Journal
JOVE-JOURNAL OF VISUALIZED EXPERIMENTS
Volume -, Issue 123, Pages -Publisher
JOURNAL OF VISUALIZED EXPERIMENTS
DOI: 10.3791/55448
Keywords
Biochemistry; Issue 123; structural biology; electron microscopy; electron cryo-microscopy; cryo-EM; TEM; single particle image processing; software development; single particle analysis; Tc toxin
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Funding
- Max Planck Society
- European Council under the European Union's Seventh Framework Programme (FP7) [615984]
- National Institutes of Health [R01 GM60635]
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SPHIRE (SPARX for High-Resolution Electron Microscopy) is a novel open-source, user-friendly software suite for the semi-automated processing of single particle electron cryo-microscopy (cryo-EM) data. The protocol presented here describes in detail how to obtain a near-atomic resolution structure starting from cryo-EM micrograph movies by guiding users through all steps of the single particle structure determination pipeline. These steps are controlled from the new SPHIRE graphical user interface and require minimum user intervention. Using this protocol, a 3.5 angstrom structure of TcdA1, a Tc toxin complex from Photorhabdus luminescens, was derived from only 9500 single particles. This streamlined approach will help novice users without extensive processing experience and a priori structural information, to obtain noise-free and unbiased atomic models of their purified macromolecular complexes in their native state.
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