Lecture 7 - Cryo EM

Question 1

Describe the Aberrations in electron microscopy

Answer 1

Spherical aberration: Lens-dependent, causes the wave to bend

Chromatic aberration: Wavelength-dependent, focal point can change

Astigmatic aberration: Lens dependent,
instead of a round beam, an oval-shaped beam formed

Question 2

Describe the different electron interactions

Answer 2

Not scattered
Backscattered
Transmitted Inelastically
Transmitted Elastically

Question 3

What is a CTF?

Answer 3

A contrast transfer function is the set of conditions that have been applied to manipulate an image

Question 4

What is low and high defocus?

Answer 4

Low defocus: Low contrast, High resolution

High defocus: High contrast, Low resolution

Question 5

What is the Pipeline for Single Particle Analysis (SPA)?

Answer 5

1. Particle Picking
2. Classification
3. Alignment and Averaging
   Reiterate

Question 6

What is the process for creating 3D models?

Answer 6

1. Get an initial model at low resolution from Alphafold e.g..
2. Project this initial model at known angles.
3. Compare experimental images to these projections.
4. Assign angles for 3D reconstruction.
   Reiterate

Question 7

What is the sample preparation for negative stain EM?

Answer 7

Add a drop of solution containing heavy elements to the sample

Once a particle is coated, remove the excess solution

Particle appears light on a dark barkground

Question 8

What is the sample preparation for Cryo-EM?

Answer 8

Add uL of sample to liquid ethane at -160C

Blot sample, this will give nL of the sample

Thin layer of ice formed

Question 9

Features of Negative Stain EM?

Answer 9

Simple Prep
High contrast
Distortion seen
Dehydration
Low resolution

Question 10

Features of Cryo-EM

Answer 10

Complex Prep
Low contrast
3D structure preserved
Hydrated state seen
High resolution