Lecture 14 - Protein Structural Analysis

Question 1

What are the pros and cons of NMR, and how does it work?

Answer 1

Pros: can obtain very high resolution, can view in solution, can show dynamic regions
Cons: limited to <40kDa, no single unique solution
Apply electromagnetic field, can cause nuclei to flip spin, energy absorbed recorded and absorption profile created

Question 2

What are the pros/cons of X-ray crystallography and how does it work?

Answer 2

Pros: can obtain highest resolution, can use for very large proteins
Cons: need to be able to crystallize protein, may introduce artifacts/deformation, can’t do membrane proteins, not in native environment
Expose crystal to X-rays (many images from many angles), collect diffraction patterns, then use math to create electron density map and slot in amino acids according to known aa sequence

Question 3

What are the pros/cons of TEM and how does it work?

Answer 3

Pros: can be used for very large structures, can be in native conditions
Cons: medium resolution
Record projection images, take many images at different orientations, images aligned and averaged and merged to get 3D structure
Can use NMR for smaller segments and dock within overall TEM envelope