Cryo-EM

Question 1

What are we imaging with cryo-EM?

Answer 1

Single particles. Large proteins or macromolecule complexes

Question 2

Why are the samples frozen in amorphous ice for cryo-EM?

Answer 2

Prevents the sample from drying out or being damaged by the electron beam

Question 3

Why does the ice need to be amorphous? What would happen if the ice formed crystals?

Answer 3

Ice crystals would diffract the electron beam and interfere with your image. Amorphous ice doesn’t diffract

Question 4

How do we focus the image with cryo-EM?

Answer 4

Magnets

Question 5

What happens to the orientation of the proteins when they are frozen?

Answer 5

They all get frozen in whatever orientation they were in and we get an image of every single one

Question 6

What sort of image do we get with cryo-EM?

Answer 6

Electron density map

Question 7

How do we prepare a sample for cryo-EM?

Answer 7

Protein solution is placed in a tiny metal mesh and the excess solution is removed. Then the solution is rapidly frozen by dunking it into liquid ethane that is cooled by liquid nitrogen. The cooling causes rapid freezing and the proteins are frozen in different orientations

Question 8

How do we reconstruct the image with cryo-EM using 2D images of the protein?

Answer 8

Computers combine thousands of the 2D images of the protein frozen in different orientations into a 3D image

Question 9

What is different about the image reconstructed from cryo-EM data vs the image from x-ray crystallography?

Answer 9

The image in cryo-EM is an actual photo of the molecule, so it isn’t a computer generated image

Question 10

What two traits of a protein will produce better cryo-EM images?

Answer 10

Internal symmetry and larger proteins

Question 11

What are the 2 recent developments that have allowed cryo-EM to become very useful?

Answer 11

Camera technology has now become faster and have cameras that detect electrons, and computer algorithms that allow more accurate image reconstruction

Question 12

Does cryo-EM provide any information on protein movements?

Answer 12

Yes

Question 13

Does cryo-EM have good resolution?

Answer 13

Yes, approaching the resolution of x-ray crystallography as technology gets better

Question 14

What is the smallest protein structure so far seen with cryo-EM?

Answer 14

Hemoglobin, which is 64 kDa (but does have internal symmetry)

Question 15

What are the pros of cryo-EM?

Answer 15

No crystal, shows an actual image of the protein surface and shape, resolution is getting better

Question 16

What are the cons of cryo-EM?

Answer 16

Lower resolution than x-ray crystallography, lower size limit, equipment is expensive