L6: X-Ray Crystallography

Question 1

Determining a protein structure by X-ray crystallography, step by step

Answer 1

1. Grow high quality protein crystals
2. Collect X-ray diffraction images
3. Calculate electron density maps, build and refine crystal structure
4. Display and interpret the protein’s 3-D structure

Question 2

Which crystals are required for structure determination?

Answer 2

Crystals suitable for X-ray crystallographic analysis need to be:
• Large single crystals
• Rotate plane polarised light
• Have few if any growth defects

Question 3

How are structures determined using X-ray crystallography?

Answer 3

Based on the simple property that electrons around each atom will scatter x-rays, proportional to the number or density of electrons.
Positions of individual atoms are determined by the diffraction of X-rays by many identical molecules in an ordered array like a crystal

Question 4

Preparing crystals for X-ray diffraction experiments

Answer 4

• Single crystals are isolated from drops and mounted into a capillary or “more commonly” a nylon loop
• Capillaries are used for X-ray diffraction at ambient temperatures
• Nylon loops are used for X-ray diffraction at low temperatures by cooling with liquid N2 or He

Question 5

Which cryoprotectant is used in x-ray crystallography?

Answer 5

ethyleneglycol - reduces freezing point so that it doesn’t form water crystals

Question 6

What is the rough distance between individuals amino acids in a protein chain?

Answer 6

~3.4 A

Question 7

SAXS

Answer 7

Small-angle x-ray scattering:
• X-ray scattering by proteins in solution occurs at small
angles compared to X-ray diffraction (reflections at
high angles)
• Scattering is proportional to that of a single particle
averaged over all orientations yielding low resolution
(estimated between 1-10 nm) information
• A scattering (SAXS) pattern contains information about the size, shape and internal structure of the particle

Question 8

CryoEM

Answer 8

• Vitrified water (amorphous ice) containing a single layer of
biomacromolecules is imaged by transmission electron
microscopy (TEM) at low temperatures
• To enhance contrast, many single particles need to be imaged and averaged by 2D matching and clustering algorithms
• Imaging of randomly orientated molecules allows 3D maps and reconstruction of proteins and biological assemblies at high resolution (starting to rival some crystal structures