Authors
Xiangan Liu, Ryan H. Rochat, and Wah Chiu
Summary
The 9 Å structure of P-SSP7 was determined by single particle cryo-electron microscopy (cryo-EM) reconstruction without imposing any symmetry. The icosahedral features of the capsid shell of this phage provide a strong signal that greatly facilitates the process of single particle icosahedral orientation determination. Unfortunately, these symmetric features greatly weaken the ease with which the particle’s asymmetric orientation can be determined. Icosahedral reconstructions have been a focus of the field for quite some time as they push the envelope of practically achievable resolution via single particle cryo-EM. While there has always been an interest in symmetry free alignment, the ability to achieve asymmetric reconstructions for particles consisting of a large number of symmetric components had been limited by reconstruction algorithms and the sheer magnitude of data required for the process. As of late, the culmination of new and advanced technology in the field of cryo-EM has permitted researchers to perform asymmetric reconstruction1-7 at subnanometer resolutions. Outlined below is a series of eight steps (also see the online methods and supplementary figure 8 of the related article) that illustrate how Multi-Path Simulated Annealing (MPSA) was used to reconstruct subnanometer resolution phage particle maps without imposing symmetry on the final model.Further details
The protocol was published on Protocol Exchange on 15 June 2010. To see the entire protocol, click on the source link.Advertisement
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