CryoEM and NMR

Question 1

Why can EM provide high resolution structures?

Answer 1

• Electrons have very short wavelength
• Electrons can be focussed using a shaped magnetic field

Question 2

Under what conditions must a CryoEM structure be obtained?

Answer 2

In a vacuum at Liquid nitrogen temperatures

Question 3

How are the issues of taking images in a vacuum overcome for cryoEM?

Answer 3

Plunge freeze sample and take images at liquid nitrogen temperature, protein keeps native conformation

Question 4

How does cryoEM produce a 3D structure?

Answer 4

Images are obtained at different orientations and combine to produce 3D structure

Question 5

How is the problem of the electron beam destroying the sample overcome?

Answer 5

• Use a low dose electron beam
• Combine thousands of equivalent particle images to build up signal:noise

Question 6

How was the coronavirus spike protein structure determined?

Answer 6

by CryoEM

Question 7

How can normal EM be used to help produce a protein structure?

Answer 7

It can be used to quickly determine if the purified protein sample will provide a meaningful structure

Question 8

How is a high resolution structure obtained using CryoEM?

Answer 8

The sequence can be fitted to the CryoEM atom density

Question 9

What are the advantges of CryoEM?

Answer 9

• Does not require a crystal
• Protein structure can be found in multiple conformations
• Samples not dehydrated or stained so structure remains close to native structure

Question 10

What are the disadvantages of CryoEM?

Answer 10

• Specimen preparation can be difficult to ensure optimum particle distribution
• Lack of contrast requires advanced computer processing to produce image
• Very expensive and limited access to equipment

Question 11

How can atomic nuclei communicate with each other?

Answer 11

• Through bonds (chemical coupling)
• Through space (Nuclear Overhauser Effect)

Question 12

How are NMR structures determined?

Answer 12

By calculating structures which are compatible with pairwise distance constraints found from NMR experiments

Question 13

Where do pairwise distance constraints mostly arise from?

Answer 13

¹H–¹H NOE interactions

Question 14

What might proteins be labelled with to produce NMR structures?

Answer 14

¹⁵N or ¹³C

Question 15

Why might proteins be labelled with radioactive isotopes for NMR structure determination?

Answer 15

Protein Structure can be determined from protein within a cell

Question 16

How can NMR be used to find areas of protein which are well characterised?

Answer 16

• Overlay multiple structures which conform to distance constraints
• The more overlap, the better characterised that portion of protein is

Question 17

Why do NMR and crystal structures not match 100%?

Answer 17

Due to packing of proteins in a crystal lattice

Question 18

Why does X-ray Crystallography not give electron density for some parts of the protein?

Answer 18

In some parts of the protein, the protein is very disordered

Question 19

What does a negative NOE value for N–H indicate about a region of protein?

Answer 19

That portion of protein structure is more disordered and is free to rotate in solution

Question 20

What are the advantages of using NMR spec to determine protein structure?

Answer 20

• Structure can be determined directly from proteins in solution
• Can provide good resolution
• Useful information for dynamics

Question 21

What are the disadvantages of using NMR spec to determine protein structure?

Answer 21

• Structures are very complicated and difficult to interpret
• Requires relatively large amount of pure sample