X-ray crystallography Flashcards
What are the features of proteins suited to analysis by EM, X-ray crystallography, or NMR?
EM - large, multimeric complexes
X-ray - proteins <100 kDa, protein/DNA complexes
NMR - proteins <40 kDa
Name the two enzymes from Ideonella sakaiensis that degrade polyesters
PETase: degrades polymers into soluble oligomers
MHETase: deacetylates oligomers and breaks into monomers
Give an overview of crystal structure determination process
- Protein purification
- Crystallisation
- Diffraction
- Fourier transform
- Electron density mapping
- Atomic modelling
- Refinement of phases
Steps 4-7 repeated iteratively, improving the model each time
Define a crystal
A lattice array of regularly repeating identical blocks
Why are protein crystals, rather than single proteins, used for x-ray crystallography?
to amplify the signal - a single protein does not diffract strongly enough
Why is cold gas used during x-ray diffraction?
To preserve the crystal from being destroyed by x-rays
Why is a goniometer used during x-ray diffraction?
To collect diffraction images from multiple angles
What is the purpose of CCD detector?
To convert diffracted x-ray beams into electric signals
Define x-rays
Highly penetrating, high energy, short wavelength electromagnetic waves
What is the typical wavelength of x-rays? What range is used for crystallography experiments?
0.5 to 4 A; 0.6-1.8 A is the commonly used range
What scatters the X-rays?
Electrons of an atom they encounter
What three features determine the intensity of a reflection spot?
1) The number/density of electrons around the atom
2) Scattering angle (theta)
3) Displacement of the atom from its mean position (B factor)
Name two sources of x-ray waves
Rotating anode generator
Synchrotron
Name three properties that describe a wave
Amplitude, wavelength and phase
Give a formula for Bragg’s Law
nl = 2d sin(t)
