- 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 

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

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. 


Brief history and progression of Cryo-EM technology  

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. 


Grand Valley State University

Tertiary protein structure combines the folding of the secondary back bone structure and includes the positions of the side chains of each amino acid. 

Tertiary Protein Structure

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

Tertiary structures of proteins can be further subdivided based on their secondary structures. Proteins can be classified as alpha, beta and alpha/beta and further understood by their topology or how the secondary structure is connected. 
Key secondary classification:
- Beta barrels

Classification based on Secondary Protein Topology

X-ray crystallography is a technique to visualize proteins. This technique utilizes a crystal  of the desired molecule that is exposed to an X-ray beam which then displays a diffraction pattern of the atoms in the molecule. These diffractions are used to to make the 3D image of the protein. 

Drawbacks:
- X-ray only interacts with electrons not the nuclei - the amino acid chains is added to complete the picture
- Lower resolution - possible inaccuracy of the images. 

X-ray Crystallography

NMR stands for nuclear magnetic resonance. this technique allows for visualizing the 3D structure of the protein by observing the nuclei of the proteins resonating in a magnetic field and interacts with the other nearby nuclei to create a 3D image of the protein. 
Drawbacks: 
- Sometimes lower resolution of the X-ray Crystal structures 
- Limited to smaller proteins (<100kD)
- Best when used in combination with X-ray Crystal structures. 

Cryo-EM 

Combinations of the secondary structure is a motif and is another way to classify the tertiary structure of proteins. 
Common motifs in proteins:
- Beta-alpha-beta – alpha helix connecting two parallel strands 
- Beta hairpin – antiparallel strands that are joined by reverse turns
- Alpha alpha – antiparallel alpha helices 
- Greek key – froms an antiparallel Beta sheet 



Protein Motif

Large protein chains that can form two or more clusters. Each cluster is known as a domain. 

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