Lesson 12
Structural Biophysics
In the last lesson, we learned how to express and purify proteins. Once you have the recombinant protein of interest, what do you do with it? This lesson focuses on structural biophysics or structural biology, which aims to elucidate the structure (architecture or arrangement of atoms in 3-dimmensional space) of the protein. There are some techniques, such as small-angle scattering, which can give low-resolution information. There are also several ways to elucidate the high resolution atomic structure, such as X-ray crystallography, cryo-EM, and NMR (which will be discussed in lesson 14, and skipped for now).
Small-Angle Scattering
Small-angle scattering (SAS) techniques include small-angle neutron scattering (SANS) and small-angle X-ray scattering (SAXS). These are considered low-resolution techniques because they have a resolution of 1-2 nm (a carbon - carbon single bond is 0.154 nm and a hydrogen bond is ~0.2 nm). You can think of low-resolution as more "blob-like" structures where you cannot resolve individual atoms. Watch the video on this Small Angle Scattering Biological Database page to learn more about SAS techniques.
X-ray Crystallography
Historically, one of the most common ways to determine a protein structure is through X-ray crystallography. The very first protein structure (of myoglobin) was solved by X-ray crystallography in 1958.
To perform X-ray crystallography you must first obtain protein crystals. Protein crystals contain millions of protein molecules arranged in a repetitive pattern called a unit cell. When X-ray beams are passed through the crystal, the repetitive nature of the protein molecules allows for the X-ray beam to scatter in a manner indicative of the protein structure. Watch this video on obtaining protein crystals (there are several other videos in this series if you are interested).
Using specialized software, a mesh framework is constructed from the X-ray diffraction data representing the electron density (shown in blue, right). The amino acids of the protein sequence of your protein are then placed into this mesh to obtain the protein structure.
If you have interest, read this article for more detailed information about protein crystallography.
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Cryo-EM
Cryo-electron microscopy is increasing becoming a popular technique to study the structure of proteins, especially those that are large or dynamic (which can be hard to study with other biophysical techniques).
In cyro-EM, the protein sample is applied onto a mesh-like grid and quickly flash frozen at very cold temperatures. Using a very powerful microscope, pictures are taken at many angles and computer software is used to "stitch" these photos together and determine the overall protein structure. Recent advances in both microscope power, hardware, and software have allowed for an increase in the resolution of protein structures determined by cryo-EM, and large protein structures have been determined which were previously impossible. This advancement has been called the "cryo-EM revolution". Read this Nature News article about recent advances in cryo-EM and watch this brief video from UCSF describing cyro-EM and comparing it to X-ray crystallography.
Assignment: I would like you to choose two proteins which have a determined structure and present them to the group. Be sure to include the function/importance of the protein, how the structure was solved, and what the structure looks like. Feel free to use any of the resources we have learned throughout the tutorial including the PDB and Uniprot, look up papers, or use resources such as PDB-101 which has short articles about protein structures. Add your slides to the google slides shared with you so that I can share with the group during our meeting.
"Protein-RNA complex and electron density" bysc63 is licensed under CC BY-NC 2.0