Lecture 8 DA

Question 1

What is the most common experimental means for obtaining 3D structures of proteins?

Answer 1

X-ray crystallography.

Question 2

How does X-ray crystallography work?

Answer 2

Electron clouds of proteins scatter and weakly diffract the X-rays, proportional to the density of electrons.

Question 3

How are atom positions determined using X-ray crystallography?

Answer 3

By interpreting the diffraction using an ordered array of the molecule, such as a crystal.

Question 4

When doing X-ray crystallography, how many times is the crystal hit with X-rays?

Answer 4

Several hundred times, done as the crystal rotates.

Question 5

How are electron density maps generated from diffraction patterns?

Answer 5

Algorithms are used, building a 3D structure.

Question 6

What is the metastable region of crystallisation?

Answer 6

Spontaneous nucleation theoretically shouldnt occur, but is a condition where the crystal grows very uniformly.

Question 7

What happens at the labile region of crystallisation? What kind of solution is needed?

Answer 7

Protein more likely to interact with itself than the solvent, so crystallises. A supersaturated solution.

Question 8

What method is used to grow protein crystals?

Answer 8

Crystallisation is controlled using vapour diffusion.

Question 9

How do crystals form?

Answer 9

When a protein solution is slowly dehydrated under controlled conditions that favour ordered lattice growth, rather than disorderred aggregates - precipitates.

Question 10

Describe the process of crystallisation by vapour diffusion (3).

Answer 10

• A well is used, with an inverted coverslip containing a suspended droplet of protein solution.
• The well beneath the coverslip is filled with a concentrated precipitant solution reservoir.
• Water from the protein solution droplet will diffuse downwards to the reservoir in the well, via equilibrium laws.

Question 11

What is commonly used as a precipitant solution in vapour diffusion?

Answer 11

Polyethylene glycol.

Question 12

How can multiple wells (ie. a matrix of wells) used in vapour diffusion be used to set parameters? Why is this useful?

Answer 12

Can be used to set certain parameters, such as a precipitant concentration gradient horizontally, as well as, for example, a pH gradient vertically.
As only a droplet of protein solution is needed, allows one to effectively determine the best conditions for protein crystallisation at once.

Question 13

How are quasicrystals structured?

Answer 13

Their interior is disordered, but their exterior is ordered.

Question 14

Where is the barrier to nucleation?

Answer 14

Between the labile and metastable region.

Question 15

What kind of crystals are suitable for X-ray crystallography (3)?

Answer 15

They need to be:

• Large single crystals
• able to rotate plane polarised light
• Have few, if any, defects

Question 16

What are twin crystals? Are they suitable for analysis?

Answer 16

They are formed when two crystal lattices intersect.

Makes it difficult to collect x-ray data relating to a single lattice, algorithms can be used.

Question 17

How do cusps/clefts occur in crystals during growth?

Answer 17

When the solution around a crystal is depleted locally, due to crystallising too fast, causes defects.

Question 18

What are crystalline aggregates?

Answer 18

When multiple crystals form as clusters.

Question 19

How are proteins in crystals arranged in crystals (aside from being an ordered lattice)?

Answer 19

Arranged to fill as much space as possible, but gaps always remain, filled by solvent.

Question 20

What are single crystals mounted on for analysis?

Answer 20

A capillary, or nylon loop.

Question 21

What is the difference between a capillary mount, vs a nylon loop?

Answer 21

Capillary - for diffraction at ambient temperatures.

Nylon loops - for diffraction at low temperatures.

Question 22

What kind of coolant is used for nylon loops and capillary mounts?

Answer 22

Capillary - none

Nylon loop - liquid N2/He

Question 23

When rapid cooling is necessary for analysis, what is the crystal mounted on beforehand, and what does it form after cooling?

Answer 23

A cryoprotectant that forms glass upon rapid cooling.

Question 24

What normally happens to protein crystals during cooling? What purpose do cryoprotectants have regarding this?

Answer 24

Microscopic ice crystals form during rapid cooling, forming ice rings that interfere with diffraction.
Seen as diffuse rings on the diffraction pattern.
Cryoprotectants prevents these crystals from forming.

Question 25

What percentage of protein crystals is water by volume?

Answer 25

40-60%

Question 26

What kind of device can be used to produce high brilliance x-rays?

Answer 26

Synchrotron.

Question 27

How are diffraction patterns converted to a 3D structure? Why is it beneficial to know the protein sequence beforehand?

Answer 27

Positions between the dots are measured in A/nm.
Algorithms are used.
Protein sequence is normally known beforehand.
Residues are fit to the electron densities from the data.

Question 28

Missing electron densities are often found. What can they be attributed to?

Answer 28

Typically water molecules.

Question 29

What kind of information does a PDB file give, the storage type of x-ray crystallography?

Answer 29

Atom positions are recorded using their z, y, and z coordinates, as well as occupancy n, and temperature B.
Collectively, gives the 3D structure.