Three-dimensional reconstruction of icosahedral particles from single micrographs in real time at the microscope.
Single particle analysis is a valuable tool in cryo-electron microscopy for determining the structure of biological complexes. However, the conformational state and the preparation of the sample are factors that play a critical role in the ultimate attainable resolution. In some cases extensive analysis at the microscope of a sample under different conditions is required to derive the optimal acquisition conditions. Currently this analysis is limited to raw micrographs, thus conveying only limited information on the structure of the complex. We are developing a computing system that generates a three-dimensional reconstruction from a single micrograph acquired under cryogenic and low dose conditions, and containing particles with icosahedral symmetry. The system provides the microscopist with immediate structural information from a sample while it is in the microscope and during the preliminary acquisition stage. The system is designed to run without user intervention on a multi-processor computing resource and integrates all the processing steps required for the analysis. Tests performed on experimental data sets show that the probability of obtaining a reliable reconstruction from one micrograph is primarily determined by the quality of the sample, with success rates close to 100% when sample conditions are optimal, and decreasing to about 60% when conditions are sub-optimal. The time required to generate a reconstruction depends significantly on the diameter of the particles, and in most instances takes about 1min. The proposed approach can provide valuable three-dimensional information, albeit at low resolution, on conformational states, epitope binding, and stoichiometry of icosahedral multi-protein complexes.