Electron Microscopy 5

Question 1

List 4 advantages of single particle analysis.

Answer 1

1. Can use heterogenous sample
2. No need to grow crystals
3. Very little sample is required - 3ul
4. Can be automated fairly easily

Question 2

List 3 disadvantages of single particle analysis.

Answer 2

1. Electron dose limitations
2. There can be orientation preferences on the grid - lack of resolution in one dimension
3. Relies on the precision of image alignment

Question 3

What is the name of the new microscope that has improved EM data collection?

Answer 3

Titan Krios

Question 4

What are the advantages of the Titan Krios?

Answer 4

More coherent electron source. 
Less dampening (loss of scattered electrons). 
Highly automated - kept in room alone so unperturbed by environmental changes.

Question 5

Describe the difference between direct electron detectors and older EM cameras.

Answer 5

Previously, electrons hit a scintillator that produced multiple photons, which went on to trigger multiple pixels.
In DEDs there is a cascade of electrons from the scintillator (rather than photons) and one electron tends to trigger one pixel. This increases resolution.

Question 6

Other than increasing resolution, what is the other advantage of DEDs.

Answer 6

The have a quicker read out, that allows motion to be corrected for.

Question 7

How to DEDs detect and correct for motion?

Answer 7

If there has been motion in the sample there will be smearing of the Thon rings. DEDs allow 10 short exposure images to be taken and these can then be averaged to remove the motion.
This improves resolution as it removes blurring.

Question 8

Give an example of how movement can be generated in a sample.

Answer 8

Electron beam hitting the sample can cause it to become charged and this will repel the electrons.

Question 9

What is the name of the newest image processing software?

Answer 9

Relion

Question 10

Describe how Relion works

Answer 10

Make class averages and then assigns these an orientation in Fourier space. 
Alongside the orientation there is a likelihood that it is correct and retains a list of the next most likely orientations. 
This means more than one structure can be generated.

Question 11

What is another method for reducing motion, other than DEDs?

Answer 11

Can replace both the copper grid and carbon film with gold.

Question 12

Why does using Gold reduce motion?

Answer 12

Copper and carbon have different thermal expansion coefficients - so when the beam of electrons heat them up they expand differently. This results in wrinkling of carbon causing movement of the sample.
Replacing with gold means there is no difference in expansion, it also heats up less as it is conductive.

Question 13

What is one disadvantage of using Gold instead of carbon and copper?

Answer 13

There are no Thon rings generated by the Gold, so have to use the ones generated by the sample.

Question 14

Name 2 structures that have been recently been determined by cryo EM to 3.4A.

Answer 14

1. TRPV1 channel

2. Gamma Secretase

Question 15

Briefly describe 2D Electron Crystallography.

Answer 15

Tether proteins to a lipid bilayer and grow 2D crystals. Produces a x,y diffraction pattern (2D).

Question 16

What are the limitations of 2D Electron Crystallography?

Answer 16

1. Hard to get stable 2D crystals
2. Hard to get flat crystals
3. There is charging of the sample, which causes motion (blurring)
4. Missing cone of data (z).

Question 17

Name two 2D Electron crystallography structures and their resolutions.

Answer 17

Bacteriorhodopsin - 3.5A

Aquaporin - 1.9A

Question 18

Describe MicroED (electron diffraction) briefly.

Answer 18

Involves micron or smaller crystals.
The objective lens is switched off so that a diffraction pattern is collected - uses modified crystallography programmes to get structures.

Question 19

What are the limitations of MicroED?

Answer 19

1. Only one microscope in the world that performs this.
2. It is hard to see micro crystals under a light microscope to determine if they have grown. However, it is unknown whether large crystals are easier to grow than micro crystals.

Question 20

Name 1 structure than has been determined using MicroED and its resolution.

Answer 20

Catalase - 3.2A

Single crystal used - tilted in the microscope to get structure