Lec 22

Question 1

What is the advantage of CRYO EM

Answer 1

Don’t need crystals, can just use the liquid

Question 2

What is the general scheme of EM

Answer 2

Take a sample of proteins put it on a grid with holes

Embed the sample in the grid through vitrification

Plunge it into liquid Ethane

Get 2D projection images of the sample

Put them together to get a 3D structure

Question 3

In cryo em and x ray diffraction what is the difference

Answer 3

In x ray , you are measuring the diffraction from the sample from and x ray beam in Fourier space phase it lost

You rely on constructive interference because it’s an array of the same repeating units

In EM you measure the scattering of the electron beam from the sample, in real space (since not diffraction) so no phase is lost

No repeating units

Question 4

What is the diffence in how x rays scatter and electrons scatter

Answer 4

X rays scatter off electrons

Electrons scatter off of the electrostatic potential (both the protons and the electrons)

Question 5

What is the size limitation by each methods NMR, x ray crystallography, CRYO EM, light microscopy

Answer 5

NMR (small

X ray crystallography (100kDa to a mega da)

Cryo em (bigger)

Light microscopy

Question 6

What is sub tomogram averaging

Answer 6

Can look at proteins within the same sample

Can look at the proteins in vivo and get a good resolution image of that protein

Question 7

What is the difference between an electron microscope and a light microscope

Answer 7

In light microscope you have a light source, lenses that can focus the beam to make if bigger or smaller

Limited to a wavelength of 400nm though

In electron microscopy you have an electron gun which excites the electron and speed them up to a specific charge

The path of the electrons gets manipulated by an electromagnets, so there are no lenses, magnet cause magnification of the beam

Question 8

How do we measure resolution for EM NMR and XRD (x ray diffraction)

Answer 8

In EM we use the Fourier shell correlation

In NMR we use the root mean squared deviation of our model, if <1 it’s a good structure

In XRD we look at the diffraction of the sample (further away spots better resolution since that’s the smallest spacing you can measure in the crystal) which is determined by the crystal quality

Question 9

Which would give you better resolution: a protien that is flexible of more rigid

Answer 9

A more rigid

Since averaging the samples

in nmr and x ray crystallography, if the protein is moving it’s hard to get a good resolved image of that part

Question 10

In each other the techniques (NMR , XRD, EM) what do we want the protein to be

Answer 10

Not flexible, want it rigid to get the best resolution

Question 11

XRD vs cryo em

Answer 11

X-ray beam vs electron beam

Protein crystal vs a vitrified/frozen protein sample

Diffraction pattern (in Fourier space) vs a 2D projection

Need to know the phases to get back to real space vs getting the orientation (angles) of the particles

Electron density map vs a 3D reconstruction

Protein atomic model vs atomic model

Question 12

What is unique about cryo em

Answer 12

All happens in real space not Fourier space

Can separate out each and every particle in our sample (don’t need very pure samples likes in NMR and XRD)

Question 13

What is the main challenge with cryo em

Answer 13

Get low contrast images

Question 14

What is negative staining in EM

Answer 14

Instead of the blurry image from EM, when doing the staining, you get a contour of the protein

Question 15

How do you get a negative stain of a image

And what is the benefit

Answer 15

Benefit is You can have very low concentration of sample

Apply the protein to the EM grid

Apply the stain (uranium acetate, any uranium compund)

Blot to take away excess liquid then apply more stain, blot

Fixed the protein into the grid using uranium acetate or uranium formate

Question 16

In negative staining what are we actually imaging

Answer 16

Not imaging protein, the imprint of the protein through the stain

Question 17

Why do we use uranium formate or uranium acetate in the negative staining of a protein

Answer 17

They can cover the protein like an envelopeand when they do, they scatter the electrons very strong

This gives a very nice contrast to the background

Question 18

What are the cons of negative staining

Answer 18

The uranium is not easy to use

The protein particles are distorted during the staining

When staining there is no water (so no hydration shell) which can change the shape of the particle leading to artifacts

Artifacts can also show up if the stain is uneven

If using detergents to solubilize the membrane protien, the detergents can make artifacts

The resolution is limited to 15-20 A because your limited to the size of the uranium acetate actetate/formate grain

Question 19

What are the pros of negative staining

Answer 19

Only need tiny amounts of protein

Easy to do and cheaper than cryo EM

Good for quick quality control to check quality of proteins

Question 20

How can negative stain help with protein purification

Answer 20

In SEC,

it can tell you what part of the peak to collect because you don’t know just from the peak if the protein is folded/misfolded

When you do the stain, you can tell if the proteins are active/folded or misfolded

You don’t know the proportion of active protein in the solution for activity assays until you do the staining

Question 21

When doing negative stating what is importantly to check for

Answer 21

The buffer because there may be artifacts due to the buffer

This is why a control is important

Question 22

What else can negative staining tell us

Answer 22

The oligeomeric shape of the protein

Gives an idea of where other proteins (like antibodies) bind to your protein

Question 23

How do you do vitrification of a sample

Answer 23

Start with liquid nitrogen to Cool down the machine

apply gas ethane which slowly condense to form liquid ethane

Then the EM grid gets submerged into liquid ethane

Apply the sample onto the grid

Since want very thin slice of the sample we blot away excess liquid

Take the grid with the sample and liquid Ethan and cool it in the liquid nitrogen