Exposing a sample to an electron microscope does degrade the structural integrity of the sample. It can damage the chemical bonds in larger macromolecules. 

Grand Valley State University

Cryo-electron microscopy is a continually advancing technique that is consistently improving the quality and diversity of protein images. Recently, discoveries have allowed for viewing a protein at the atomic level.

“Cryo” refers to the thin layer of noncrystalline solid water called vitreous ice that is kept at liquid nitrogen temperatures. This solution holds the desired protein to be imaged. EM refers to electron microscopy which is used to create these images. 


Cryo-EM 

Bai, X., McMullan, G., & Scheres, S. H. W. (2015). How cryo-EM is revolutionizing structural biology. Trends in Biochemical Sciences, 40(1), 49–57. https://doi.org/10.1016/j.tibs.2014.10.005

How Cryo-Em is Revolutionizing structural biology

- While NMR provides some insight on the dynamics, it does not determine the atomic structure unless it is a small structure. Cryo-EM is able to create an atomic structure. 
- Both X-ray crystallography and NMR need a near-pure sample. Cryo-EM does not. 
- Crystallization is hampered by a not symmetrical and heterogeneous sample. Cryo-EM structures were successful in creating a structure using the likelihood calculations with an incomplete structure.
- Cryo-EM requires less sample and poses less restriction on sample purity and does not require to be crystallized. 

Cryo-EM: Improvements from NMR and X-ray crystallography 

Downsides to Cryo-EM

- In the beginning of cryo-EM technology, 3D image calculations from 2D images were used in combination with cyro-EM. This approach was successful for proteins with certain structures like helical shapes. 
- For subjects that lack orderly patterns, cryo-EM single-particle analysis which uses a purified solution of macromolecules that may be imaged directly, was used. 3D constructions are calculated from projections of complexes and particles. However, the orientation of the particles are unknown. 
- Gradual progress occurred with a 40 Angstrom (very low resolution structure) to a near 10 Angstrom image around the 2000s. 


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