Lecture 8 - RH

Question 1

What are the ways of examining 3D structures of proteins?

Answer 1

X-ray crystallography

Nuclear Magnetic Resonance Imaging

CryoEM, AFM

Solution scattering (laser, X-ray and neutron)

Circular Dichroism (CD)

Homology modeling and ligand fitting

Question 2

What limits NMR?

Answer 2

Size of the molecule must be small

Question 3

How does solution scattering work?

Answer 3

Laser, X-ray and neutron beam are shined through a protein structure and the scattering pattern can be measured

Question 4

What is Circular Dichroism useful for?

Answer 4

Finding the secondary structures in a solution

Question 5

How does X-ray crystallography work?

Answer 5

Electron clouds weakly diffract X-rays in a way that is proportional to the number or density of electrons. Position of atoms are determined by interpretting the diffraction pattern.

Question 6

What is required for crystallisation to occur?

Answer 6

Supersaturation

Question 7

How are crystals generated in the lab?

Answer 7

Crystals form when protein solution is slowly dehydrated (water is removed) under controlled conditions that favour the production of ordered crystal lattices rather than disordered aggregates

Question 8

How are crystals of high throughput produced?

Answer 8

Via the use of multi-well plates

Question 9

What kind of crystals are used for X-ray crystallography?

Answer 9

Large single crystals

Rotate plane polarised light (Rainbow structures should show)

Have few if any growth defects

Question 10

What are some common growth defects with crystals?

Answer 10

Twins are formed when 2 crystal lattices intersect making it difficult to collect X-ray data relating to a single lattice

Cusps or clefts in crystals occur when protein solution is depleted locally in a droplet

Crystalline aggregates are when multiple crystals form as clusters

Question 11

What are the 7 crystal classes found in nature?

Answer 11

Cubic

Tetragonal

Orthorhombic

Hexagonal

Triclinic

Monoclinic

Rhombohedral

Question 12

Are lattices completely occupied by molecules?

Answer 12

Yes, solvent occupies the gaps between proteins.

Question 13

How are crystals prepared for X-ray diffraction experiments?

Answer 13

Single crystals are isolated from drops and mounted into a capillary or 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 N2 or He

Question 14

What is the usual composition of protein crystals?

Answer 14

60% protein and 40% water