Protein structure Determination Flashcards
How do protein crystals form?
Network of weak intermolecular interactions containing large channels filled with solvent
How can X-rays be used to determine a protein crystal structure
X-ray lenses don’t exist so diffusion scattering is used. X-rays interact with orbital electrons which re-emits them.
Reflective rays intefere at certain scattering angles
the crystal is rotated to get patterns
How are X-ray scatterings inferred?
Square root of electron density is amplitude
The further out the spot, the higher the resolution
Phase is missing from the data due to no lens
How can the phase problem of X-ray scattering be solved?
Binding heavy metals to the protein which shows dense electron scattering.
e.g replacing Met with seleno-Met
What is a favourable way of adding heavy metals to proteins for X-ray crystallography?
Polyoxometalates (POMs) contain dense metal clusters
These are stable and inert so the buffer doesn’t bind to the heavy metals
How is vapour diffusion done?
Used for growing crystals.
The protein is solubilised with water which then diffuses out and increases protein concentration.
Why are detergents used to isolate membrane proteins?
Prevent aggregation
Water soluble surfactants
Non-polar tails attract grease
Detergent for crystallisation may not work for purification
Forms micelles
What general challenges are the with protein crystallography?
Crystal contacts can be difficult to maintain due to buffer blocking it
Crystallography is unpredictable
Detergent needs to be above the CMC (concentration required to keep protein soluble)
What problems are associated with detergents?
Can disrupt membrane protein stability
Micelles intefere with optical properties
Specific phospholipids in the bilayer may aid its function
What are liposome preparations?
Method of isolating membrane proteins. Useful for compartmentalisation either side of the membrane.
However these are large and unstable
What are lipid nanodiscs
Discs of lipid bilayer that can
They overcome the problems of detergent and liposome preparations by maintaining membrane integrity
Outline the process of cryo EM
- Membrane protein is applied directly to an EM grid
- Blotted and frozen with liquid ethane (-160°C so ice crystals cannot form).
- This results in vitrious ice (not volatile so freezes but isn’t crystallised). Doesn’t evaporate under a vaccuum
- EM grid is put under an electron microscope and high energy electrons are fired
Pros and Cons of cryo-EM
Electrons 10000 times stronger than X-rays so a small sample can be used
Expensive
Radiation can damage proteins so lower doses and frozen samples help
Damaging the protein may reveal movement of the protein
If crystals are in same conditions, may not consider different protein conformations
How is a 3D image made from 2D images
Whole stack of images are superimposed to give different directions of view
Averages of images are used to reduce noise
Put together
What is AlphaFold used for?
Predicting 3D structure from linear amino acid sequence
Neural network that looks for patterns in structures to predict strucutres from amino acid sequence
Doesn’t know chemistry
Can’t predict effect of mutations
Limited use in protein complexes
Makes sense of X-ray/ Cryo-EM data
