Electron Microscopy

Question 1

What size range is X-ray crystallography useful?
What size range is Cryo-EM useful?

Answer 1

• 1-100 angstroms
• 100-1,000,000 angstroms.

Question 2

What are the pros of NMR?

Answer 2

1) great for dynamic and kinetic information
2) No need to crystalize
3) high resolution
4) accurate and detailed model (not including hydrogens)

Question 3

What are the cons of NMR?

Answer 3

1) proteins must be labelled by expensive heavy isotopes
2) High sample purity and homogeneity is required
3) Difficult for computational structure determination
4) Can’t study membrane proteins easily
5) Limited to smaller proteins (<50kD)
6) Requires lots of protein.

Question 4

What are the pros of single particle cryo-EM?

Answer 4

1) Great for dynamics
2) No need to crystalize
3) medium to high resolution
4) Models need not be as accurate
5) Short and easy sample preparation
6) Shows structure in native state
7) Sample need not be completely pure

Question 5

What are the cons of single particle cryo-EM?

Answer 5

1) Lower resolution than XRD and NMR
2) Generally only used for samples of higher molecular weights
3) Very expensive EM techniques and costly EM equipment
4) Preferred orientation is only shown

Question 6

What are the measures used to assess the quality of the model attained using XRD, NMR and Cryo-EM?

Answer 6

XRD: R-factor
NMR: RMSD (root mean square deviation)
Cryo-EM: FSC

Question 7

What are pros of using negative EM staining?

Answer 7

1) Requires very low concentration of protein
2) Easier and cheaper than cryo-EM
3) Quick quality control

Question 8

What a