Single-particle cryogenic electron microscopy has recently become a major method for determining the structures of proteins and protein complexes. This has markedly increased the demand for throughput of high-resolution electron microscopes, which are required to produce high-resolution images at high rates. An increase in data-collection throughput can be achieved by using large beam-image shifts combined with off-axis coma correction, enabling the acquisition of multiple images from a large area of the EM grid without moving the microscope stage. Here, the optical properties of the JEOL CRYO ARM 300 electron microscope equipped with a K3 camera were characterized under off-axis illumination conditions. It is shown that efficient coma correction can be achieved for beam-image shifts with an amplitude of at least 10 µm, enabling a routine throughput for data collection of between 6000 and 9000 images per day. Use of the benchmark for the rapid data-collection procedure (with beam-image shifts of up to 7 µm) on apoferritin resulted in a reconstruction at a resolution of 1.7 Å. This demonstrates that the rapid automated acquisition of high-resolution micrographs is possible using a CRYO ARM 300.
Using single particle cryogenic electron microscopy (cryo-EM) high-resolution structures of proteins in different conformations can be reconstructed. Protein function often involves transient functional conformations, which can be resolved using time-resolved cryo-EM (trEM). In trEM, reactions are arrested after a defined delay time by rapid vitrification of protein solution on the EM grid. Despite the increasing interest in trEM among the cryo-EM community, making trEM samples with a time resolution below 100 ms remains challenging. Here we report the design and the realization of a time-resolved cryo-plunger that combines a droplet-based microfluidic mixer with a laser-induced generator of microjets that allows rapid initiation of reaction and rapid plunge-freezing of cryo-EM grids. Using this approach, a time resolution of 5 ms was achieved and the protein density map was reconstructed to a spatial resolution of 2.1 Å. We performed trEM experiments on GroEL:GroES chaperonin complex, these resolved the kinetics of the complex formation and visualized putative short-lived conformations of GroEL-ATP complex.
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