Electron Microscopy 2

Question 1

How small can the protein be for EM?

Answer 1

Generally, the minimum size of the protein must be >150kDa (150kDa is pretty small for EM).

Question 2

How much sample is required for EM?

Answer 2

Does not require much sample (unlike XC) - roughly 2.5ul of material at 0.01-1mg/ml

Question 3

Why is EM good for visualising MPs?

Answer 3

Can visualise the protein in its native environment.

Question 4

What is needed to study large multi-protein complexes?

Answer 4

Lots of homogenous material - both compositional and conformational homogeneity.

Question 5

Which methods be used to test for compositional homogeneity?

Answer 5

SDS PAGE
Size exclusion chromatography (SEC)
SEC coupled to multi angle light scattering (MALS).

Question 6

What is key to achieving compositional homogeneity?

Answer 6

Purity.

Question 7

What must you balance in sample preparation?

Answer 7

Achieving good signal to noise ratio with preservation of native structure of protein.

Question 8

What is the major difference between light microscopy and EM?

Answer 8

Light microscopy works in air - EM works in a vacuum.

Question 9

What is the issue of using a vacuum?

Answer 9

Biological molecules do not like to be contained in a vacuum so must be prepared to withstand it.

Question 10

When was negative stain developed and what was it for at the time?

Answer 10

In 1959 - developed to study viruses.

Question 11

What is negative stain made of?

Answer 11

Made of heavy metal salts such as uranium, molybdenum and tungsten. q

Question 12

What are the steps involved in setting up a negative stain?

Answer 12

Bind the protein to a carbon support and then the heavy metal stain is added which forms a cast around the protein.

Question 13

What is the benefit of the negative stain in a vacuum?

Answer 13

The protein remains hydrated while in the vacuum.

Question 14

What are you in fact imaging in negative stain EM?

Answer 14

Actually imaging the heavy metal cast.

Question 15

How does the negative stain protect the protein?

Answer 15

The metals are radiation hard - are not fired by radiation of inelastically scattered electrons.

Question 16

Why is this radiation protection beneficial to EM?

Answer 16

Can increase electron dosage without damaging the protein.

Question 17

How does the negative stain produce a better signal to noise ratio?

Answer 17

They interact with (scatter) electrons very well (better than biological molecules).

Question 18

What temperature is negative stain EM done at?

Answer 18

Room temperature.

Question 19

What are the advantages of negative stain EM?

4

Answer 19

Quick to screen samples
High contrast as the heavy metals scatter electrons well.
Radiation hard
Able to visualise smaller proteins.

Question 20

What are the disadvantages of negative stain EM?

2

Answer 20

Limited in resolution due to grain size - because imaging the cast not the actual protein.
Proteins can be damaged or distorted by the process.

Question 21

What is different about cryo-EM?

Answer 21

The molecule is encased in ice.

Question 22

How is the protein cooled?

Answer 22

There is a vessel containing liquid ethane that has been cooled to liquid nitrogen temperature - the protein is plunged into vessel and cools very rapidly.

Question 23

What should the ice resemble if the process has been done well?

Answer 23

Glass

Question 24

What happens if freezing is not quick enough?

Answer 24

You can end up with contaminants - there can be water particles which distort the image.

Question 25

What are the advantages of Cryo-EM? | 1

Answer 25

The high resolution is preserved as the protein is in near native/hydrated state.

Question 26

What are the disadvantages of Cryo-EM? | 4

Answer 26

Radiation damage can occur - so cannot use a high electron dosage on the sample. Low signal to noise ratio Slow to screen samples - each time a protein is frozen everything involved must be at liquid nitrogen temperature. Easy to contaminate.

Question 27

What are liquid samples applied to?

Answer 27

A copper mesh grid.

Question 28

What are the two things this copper mesh grid can be coated with and which one is used for Negative stain EM and which one is used for Cryo-EM?

Answer 28

Negative stain EM - continuous carbon. | Cryo-EM - holey carbon.

Question 29

How is holey carbon produced?

Answer 29

Blot the carbon film with buffer using filter paper to produce holes of the desired size.

Question 30

What are the 2 advantages of using a holey carbon grid?

Answer 30

They eliminate any absorption and scattering of the electron beam by the carbon film, which generates noise and obstructs the signal. The holes also allow for pockets of solvent to form - within these pockets the specimen remains fully hydrated even when it has been frozen.

Question 31

What is the problem with using a carbon layer?

Answer 31

It is inherently hydrophobic so is hard to get proteins to absorb to a hydrophobic layer.

Question 32

What is done to resolve this adsorption problem?

Answer 32

The carbon is charged (negatively) - this is known as a plasma treatment.

Question 33

How is plasma created and how does it interact with carbon?

Answer 33

By ionisation of gas under a low vacuum. | The ions interact with carbon surface to remove contamination and make it hydrophilic.

Question 34

What does the plasma treatment mean for distribution of sample?

Answer 34

There is a good distribution of proteins in the holes.

Question 35

What is the issue with holey carbon when it comes to MPs and DNA binding proteins?

Answer 35

They will preferentially land on the carbon rather than in the holes - this is because DNA binding proteins are positively charged and are attracted to the negatively charged carbon and MPs are avoiding the pockets of solvent in the holes as they are hydrophobic.

Question 36

How is the issue with holey carbon when looking at MPs and DNA binding proteins resolved?

Answer 36

A second very thin layer of carbon is placed on top of the original carbon film - this forms wells were holes are and reduced preference for where the protein lands.

Question 37

Why does the extra carbon layer have to be so thin?

Answer 37

So that it does not effect the signal to noise ratio.

Question 38

What is the advantages of controlling adsorption? | 3

Answer 38

Allows protein adsorption to the grid - increases the local protein concentration when preferentially falling in holes. Buffer may be exchanged on the grid after the protein has been bound - allows for detergent (MP) and sucrose to be removed. CTF parameters estimation.

Question 39

What are the disadvantages of controlling adsorption? | 2

Answer 39

The proteins may fall into preferential orientations | There can be some background noise produced from the carbon film.

Question 40

What has been used recently to control protein adsorption and why?

Answer 40

Graphene - a single layer of carbon atoms. | It reduces the noise on the image significantly.

Question 41

How is the sample kept at liquid nitrogen temperature when it is being transferred and in the microscope?

Answer 41

There is a thermos at the end of the sample container than contains liquid nitrogen.

Question 42

What is low dose imaging?

Answer 42

Using fewer electrons to produce an image

Question 43

Why is low dose imaging good?

Answer 43

It minimises the radiation damage that can occur from inelastically scattered electrons.