Cryo-EM

Cryo-electron microscopy (cryo-EM) is a technique used to image biomolecules such as proteins and nucleic acids at near-atomic resolutions. In cryo-EM, a sample is rapidly frozen in a thin layer of amorphous ice which preserves its near-native hydrated state. The frozen sample is then imaged under an electron cryo-microscope, allowing visualization of its structure. Key advantages of cryo-EM over other structural biology techniques are its ability to image macro-molecular complexes without the need for crystallization and its single particle analysis approach which facilitates structural determination even from heterogeneous samples.

Sample Preparation for Cryo-EM

The first step in Cryo Electron Microscopy involves sample preparation where the biomolecule of interest is purified and concentrated to a suitable concentration range, typically 0.1-10 mg/ml. The sample is then applied to a support grid coated with a thin perforated polymer layer and rapidly frozen by being plunged into liquid ethane or propane cooled by liquid nitrogen. This process of vitrification leads to formation of amorphous glassy ice which immobilizes the sample molecules in their near-native hydrated state. The frozen sample grid is then loaded into the electron cryo-microscope for imaging under low temperatures close to liquid nitrogen to prevent sample degradation by beam irradiation.

Data Acquisition using Cryo-EM

In cryo-EM, the vitrified biological sample is imaged using an electron cryo-microscope operating at liquid nitrogen temperatures. An electrons industry overview beam passes through the thin frozen sample layer on the grid, interacting with the specimen. An imaging detector then records the scattered electrons which contain structural information about the sample. Hundreds to thousands of microscope images or "micrographs" are acquired at different sample orientations. The resolution obtainable depends on various cryo-EM hardware parameters like detector pixel size, voltage of the electron beam and spherical aberration of the microscope lenses. State-of-the-art cryo-EMs can now achieve resolutions better than 3Å routinely.

Image Processing Workflow in Cryo-EM

The large datasets acquired in cryo-EM need extensive computational analysis to reconstruct the 3D structure of the sample. Individual sample particles are identified and extracted from the micrographs using various computational tools. Reference-free 2D classification is performed to separate heterogeneous populations and assess quality. Then 3D classification refines the initial structure while sorting particles into structurally distinct groups. Selected particles are then used in 3D auto-refinement which iteratively optimizes particle alignments and the reconstructed 3D density map. Post-processing further improves map resolution and contrasts. Computational routines are now highly automated, making cryo-EM accessible to non-expert users. Breakthroughs in direct electron detectors, super-resolution microscopy, and cryo-focused ion beam milling are further boosting cryo-EM resolutions.

Applications of Cryo-EM

Since coming of age in the last decade, cryo-EM has revolutionized structural biology by enabling determination of structures which were challenging using other techniques. It has enabled de novo structure determination of large protein complexes and assemblies such as ribosomes, viruses and membrane proteins at near-atomic resolutions. Cryo-EM structures have significantly enhanced our understanding of fundamental cellular processes like transcription, translation and signaling. It has also enabled mechanistic insights into pathogens like SARS-CoV-2. Recent advances now make cryo-EM accessible for solving challenging smaller protein complexes below 100kDa in size. Its ability to analyze heterogeneous and dynamic systems has potential applications in biotechnology and drug development. With further hardware and software improvements, cryo-EM is projected to become the dominant technique for high resolution structural biology in the coming years.

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