Flashcards in Electron Microscopy 2 Deck (43):
How small can the protein be for EM?
Generally, the minimum size of the protein must be >150kDa (150kDa is pretty small for EM).
How much sample is required for EM?
Does not require much sample (unlike XC) - roughly 2.5ul of material at 0.01-1mg/ml
Why is EM good for visualising MPs?
Can visualise the protein in its native environment.
What is needed to study large multi-protein complexes?
Lots of homogenous material - both compositional and conformational homogeneity.
Which methods be used to test for compositional homogeneity?
Size exclusion chromatography (SEC)
SEC coupled to multi angle light scattering (MALS).
What is key to achieving compositional homogeneity?
What must you balance in sample preparation?
Achieving good signal to noise ratio with preservation of native structure of protein.
What is the major difference between light microscopy and EM?
Light microscopy works in air - EM works in a vacuum.
What is the issue of using a vacuum?
Biological molecules do not like to be contained in a vacuum so must be prepared to withstand it.
When was negative stain developed and what was it for at the time?
In 1959 - developed to study viruses.
What is negative stain made of?
Made of heavy metal salts such as uranium, molybdenum and tungsten. q
What are the steps involved in setting up a negative stain?
Bind the protein to a carbon support and then the heavy metal stain is added which forms a cast around the protein.
What is the benefit of the negative stain in a vacuum?
The protein remains hydrated while in the vacuum.
What are you in fact imaging in negative stain EM?
Actually imaging the heavy metal cast.
How does the negative stain protect the protein?
The metals are radiation hard - are not fired by radiation of inelastically scattered electrons.
Why is this radiation protection beneficial to EM?
Can increase electron dosage without damaging the protein.
How does the negative stain produce a better signal to noise ratio?
They interact with (scatter) electrons very well (better than biological molecules).
What temperature is negative stain EM done at?
What are the advantages of negative stain EM?
Quick to screen samples
High contrast as the heavy metals scatter electrons well.
Able to visualise smaller proteins.
What are the disadvantages of negative stain EM?
Limited in resolution due to grain size - because imaging the cast not the actual protein.
Proteins can be damaged or distorted by the process.
What is different about cryo-EM?
The molecule is encased in ice.
How is the protein cooled?
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.
What should the ice resemble if the process has been done well?
What happens if freezing is not quick enough?
You can end up with contaminants - there can be water particles which distort the image.
What are the advantages of Cryo-EM?
The high resolution is preserved as the protein is in near native/hydrated state.
What are the disadvantages of Cryo-EM?
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.
What are liquid samples applied to?
A copper mesh grid.
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?
Negative stain EM - continuous carbon.
Cryo-EM - holey carbon.
How is holey carbon produced?
Blot the carbon film with buffer using filter paper to produce holes of the desired size.
What are the 2 advantages of using a holey carbon grid?
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.
What is the problem with using a carbon layer?
It is inherently hydrophobic so is hard to get proteins to absorb to a hydrophobic layer.
What is done to resolve this adsorption problem?
The carbon is charged (negatively) - this is known as a plasma treatment.
How is plasma created and how does it interact with carbon?
By ionisation of gas under a low vacuum.
The ions interact with carbon surface to remove contamination and make it hydrophilic.
What does the plasma treatment mean for distribution of sample?
There is a good distribution of proteins in the holes.
What is the issue with holey carbon when it comes to MPs and DNA binding proteins?
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.
How is the issue with holey carbon when looking at MPs and DNA binding proteins resolved?
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.
Why does the extra carbon layer have to be so thin?
So that it does not effect the signal to noise ratio.
What is the advantages of controlling adsorption?
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.
What are the disadvantages of controlling adsorption?
The proteins may fall into preferential orientations
There can be some background noise produced from the carbon film.
What has been used recently to control protein adsorption and why?
Graphene - a single layer of carbon atoms.
It reduces the noise on the image significantly.
How is the sample kept at liquid nitrogen temperature when it is being transferred and in the microscope?
There is a thermos at the end of the sample container than contains liquid nitrogen.
What is low dose imaging?
Using fewer electrons to produce an image