In contrast, it remains a challenge to solve high-resolution structure of proteins with a smaller molecular weight, especially those below 100 kDa, using SPA Cryo-EM. Currently, it has become increasingly routine to reconstruct a well-behaved macromolecule with good homogeneity, rigidity, and random orientations in ice and a molecular weight larger than 300 kDa at ~3 Å resolution. New algorithms based on Bayesian statistics have also greatly improved the efficiency of extracting signal from noisy micrographs and heterogeneous datasets 5, 6, 7, 8, 9. Thanks to the significant improvements in the recording speed and detective quantum efficiency of the direct electron detection cameras, more information at both low and high resolutions can be recovered from raw movie stacks, thus improving the reconstruction accuracy 4. Of the various cryo-EM structural determination methods, single-particle analysis (SPA) has drawn the most attention from structural biologists because of its relatively well-established methods for specimen preparation, data collection, image processing, and structural determination 1, 2, 3. With recent technical breakthroughs, cryogenic electron microscopy (cryo-EM) has rapidly become one of the most powerful and efficient technologies to investigate the structures of macromolecules at near-atomic resolution.
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