Lec 22 Flashcards
What is the advantage of CRYO EM
Don’t need crystals, can just use the liquid
What is the general scheme of EM
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
In cryo em and x ray diffraction what is the difference
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
What is the diffence in how x rays scatter and electrons scatter
X rays scatter off electrons
Electrons scatter off of the electrostatic potential (both the protons and the electrons)
What is the size limitation by each methods NMR, x ray crystallography, CRYO EM, light microscopy
NMR (<50 kDa) so small
X ray crystallography (1-500 kDa) small
Cryo em (>50 kDa) bigger better cant do small
Light microscopy
What is sub tomogram averaging
Can look at proteins within the same sample
Can look at the proteins in vivo and get a good resolution image of that protein
What is the difference between an electron microscope and a light microscope
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
How do we measure resolution for EM NMR and XRD (x ray diffraction)
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
Which would give you better resolution: a protien that is flexible of more rigid
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
In each other the techniques (NMR , XRD, EM) what do we want the protein to be
Not flexible, want it rigid to get the best resolution
XRD vs cryo em
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
What is unique about cryo em
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)
What is the main challenge with cryo em
Get low contrast images
What is negative staining in EM
Instead of the blurry image from EM, when doing the staining, you get a contour of the protein
How do you get a negative stain of a image
And what is the benefit
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
In negative staining what are we actually imaging
Not imaging protein, the imprint of the protein through the stain
Why do we use uranium formate or uranium acetate in the negative staining of a protein
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
What are the cons of negative staining
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
What are the pros of negative staining
Only need tiny amounts of protein
Easy to do and cheaper than cryo EM
Good for quick quality control to check quality of proteins
How can negative stain help with protein purification
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
When doing negative stating what is importantly to check for
The buffer because there may be artifacts due to the buffer
This is why a control is important
What else can negative staining tell us
The oligeomeric shape of the protein
Gives an idea of where other proteins (like antibodies) bind to your protein
How do you do vitrification of a sample
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
