Lecture 9 X-ray crystallography Flashcards
Limitation of X-ray crystallography
proteins need to be pure and crystallizable
Main principles of x-ray crystallography
- High energy X-rays (wavelength ~1 Å) are scattered by electrons in atoms
- X-ray scattering by molecules in a crystal results in diffraction patterns
- Diffraction patterns can be computationally transformed to a 3D electron density map.
- The electron density map is then used to re-construct (fit) the corresponding 3D atomic structure
What are structure factors in protein X-ray crystallography
Elementary waves which, when combined, will reconstruct the 3D electron density map
Which factors determine (or have an influence on) the resolving power (max. resolution) of the X-ray diffraction data?
- Intensity X-rays
- Wavelenght X-rays
- Size of the crystals
- Packing of the protein molecules in the crystals
- Protein purity
- Sensitivity of the X-ray detector
Parameters that affect protein crystallization
- protein purity !!!
- protein concentration
- type of buffer & pH
- solution ionic strength & ionic species
- !precipitants: type & concentration!
- additives (divalent ions, cofactors, etc.)
- detergents (for membrane proteins)
- temperature
Precipitants
Molecules use in protein crystallization by making the protein less soluble.
The main principle of these precipitants is that they bind water, and therefore reduce the amount of solvent available to solubilize a protein.
- ammonium sulfate
- methyl pentane diol
- polyethylene glycol
General protein crystallization method
vapour diffusion
1. Protein sample after purification
2. Small beaker containing crystallization cocktail
3. Glass cover slip is added to the beaker
4. Drop of protein is added to glass cover
5. Cocktail is mixed in
6. Glass slide is turned upside down, the vapour is duffesed into the well, concentrations in the drop will increase.
7. At certain point proteins will nucleate, this can be observed under microscope
Why can you not reliably predict the conditions that result in the crystallization of a protein?
- Too many parameters that play a role in crystallization
- We do not understand crystallization well enough
- To predict the crystallization conditions you would need to know the 3D structure of the protein
Preparing crystals for X-ray diffraction
- Crystal is fished from drop using a small fiber loop
- A cryoprotectant (e.g. glycerol) is added to the
crystal solution - Crystal is flash-cooled at 100 Kelvin to prevent
X-ray radiation damage
Crystal soaking
pre-grown protein crystals are soaked in a solution containing the ligand.
Advantage: fast and easy
Co-crystallization
New crystallization experiments are performed
with a mixture of the protein and ligand.
Advantage: allows for conformational changes in the protein upon ligand binding
Model building concrete steps
1: build a model that fits the electron density map
2: calculate structure factors for the model (Fcalc)
3: compare Fobs and Fcalc
4: change model parameters in small steps to minimize R-factor, while keeping the model geometry close to ideal
5. Calculate a new improved electron density map.
B-factors
Atomic B-factors define the uncertainties in the
position of each atom. They are a measure of the
local disorder or flexibility in a crystal structure
high B-factors -> flexible loops at surface
lower B-factors-> interior of a protein (is more ordered than the surface)
Structure factors
represent the reflection amplitudes and phases
Which of the following properties of a protein crystal
structure is/are closely linked to its reliability?
- Upper resolution limit of the diffraction data
- Number of outliers in the Ramachandran plot
- Difference between Rfree and Rwork
