Membrane Proteins - Per Bullough Flashcards
What is the lattice of spots showing in Xray diffraction?
The magnified image of the protein crystal lattice
What does Braggs Law tell us
On a crystal surface, the angle of incidence will reflect with the same angle of scattering
What is the problem with X-ray?
No good lens can be created as wavelengths are too short = PHASE PROBLEM
Means we cannot tell relative positions of atom in molecule
How are crystallised proteins formed and prepared for X-ray Crystallography
Protein removed from membrane using detergent
Vapour diffusion by either hanging drop or sitting drop -> supersaturate protein solution until it precipitates out. Use a reservoir around the drop with higher concentration of precipitant solution. Causes water vapour on drop to evaporate leaving only crystallised protein.
Point where crystals form = supersaturation point
What does Braggs law explain?
If wavelengths are fired at a correct angle, where they are only one wavelength apart, their peaks and troughs will align causing reinforcement = very strong signal
What do the characteristics of diffraction spots tell us
Closer to outside = higher resolution information
Darker spot = more intense, so area of higher electron density
Regular arrangement shows it is a crystal, so crystallisation was successful
In general we are told: density of atoms and the density arrangement
What 2 methods can we use to solve the phase problem?
- Molecular replacement –> using a known, related crystal model’s phase information. Optimise orientations until they match
If no similar structure is available then use:
2. Heavy metal atom labelling –> isomorphous replacement. Bind heavy metal (U, Lb, Pl) to crystal and compare diffraction patterns. Use information of where heavy metal binds as anchor points
How and why do we find the correct detergent for a protein?
All slightly denature protein over time. Need it harsh to remove but not too harsh to denature soon
Need to find balance between one that’s best for crystallisation and also for purification -> most common is: DDM and 12M
How does the CMC come into this?
Conc at which micelles self-associate. Need to keep concentration above this to maintain protein solubility
What is good about detergent belts?
Mimic natural environment of protein better than micelles. Can improve crystallisation
How can crystal contacts be increased? why do this?
Proteins can only form contacts between parts not covered in detergent. Smaller proteins have bigger spaces. Bigger spaces make it harder to crystallize
Improved using Fab or Fv antibody fragments to help link proteins
Process of CryoEM:
1) Apply detergent-solubilised protein onto EM grid and blotted to leave a thin film of solution (by removing excess fluid).
2) Rapidly freeze sample by plunging into liquid ethane.
3)Transfer to electron microscope and fire electrons at the sample in a vaccuum.
What is an EM grid?
metal grid with film of carbon with many holes used to hold protein samples for CryoEM.
Why is rapid freezing essential in CryoEM?
Rapidly freezing the proteins in solution prevents the additional crystallisation of the water, ensuring vitrified ICE is formed (non-volatile)
Why is it essential that a sample used in electron microscopy is non-volatile?
So the solution doesn’t spontaneously form a gas, and interfere with the micrograph.
What are the advantages of electron microscopy of protein samples over Xray diffraction?
- Lenses can be used, and-so phase information can be deduced.
- The samples don’t need to be arranged in a lattice with random orientations preferred, crystallisation conditions therefore don’t need to be found.
- Higher Resolution - electrons moving at a shorter wavelength than the Xrays
- Scattering is 10,000 x greater than with X-rays, so much smaller samples need to be prepared (cost saving)
- Protein samples aren’t required to be in set orientations, unlike in X-ray diffraction, requiring manual rotation of crystal to give different orientations of the protein.
- Can be used to identify multiple protein conformations.
What are similarities and differences between X-ray and CryoEM?
Similarities:
- Multiple copies of protein needed
- Extraction of protein from membrane using detergent
- Want different orientations
-
Differences:
- CryoEM can use lens
- Needs smaller samples
- Uses EM images vs diffraction pattern
- Lower cost
- Higher resolution
- sample arranged in different orientations already
- Making 3D models: Single particle averaging vs ???
Does X-ray crystallography show any advantages over CryoEM?
Better at achieving very high resolution in atomic detail for smaller proteins (under a few hundred kDa)
CryoEM better at bigger proteins with more disordered assemblies
What are the issues with electron microscopy in the identification of protein structure?
Radiation damage caused by the electron ray, causes the protein domains to get damage and move, lowering the resolution of the image.
How has the issue of protein damage by electron beam been circumvented in electron microscopy?
- Sensitivity of electron detectors increased, and-so lower dose of electrons needed to capture data.
- Motion correction - techniques compensate for the movement of sample by tracking molecules
- Lower temperatures reduce molecule movement.
How are 3D models / images constructed from electron micrographs?
- Single particle averaging is used to remove background noise and identify the 2D orientations -> These are then aligned by orientation and classified by similarity, averages from each class are converted in a 3D shape according to their Euler angles -> 3D map created is a summation of contributions from each projection image.
What is Alpha Fold?
A program that uses a computational learning algorithm to predict 3D protein structures using 10^5 known structures from the Protein Data Bank (PDB).
The program produces a prediction with area colour coded based on confidence calculations.
How does Alpha Fold work?
It uses the sequence information to search related sequences, analysing amino acids changes and will try to see if there’s a correlation between one amino acid changing another, if so, they’re co-evolving and likely close in 3D space.
What are the limitations of Alpha fold?
- If the protein has few related sequences it’ll struggle to produce a structure.
- Can only estimate the structures of naturally evolving sequences, not as effective with mutations.
- Doesn’t necessarily do sensible chemistry (and-so don’t make sense)
- Limited use for large protein complexes
