Protein structure and methods

Question 1

What are 3 protein folding constraints?

Answer 1

Amino acid backbone configurations
Sidechain configurations-rotamers
Energy minimisation-bury hydrophobic residues and satisfy H bonding potential

Question 2

How are atomic-level structures determined?

Answer 2

Through X-ray crystallography and EM

Question 3

Why isn’t light microscopy used to ID proteins?

Answer 3

Proteins are too small

Question 4

What is needed for X-ray crystallography to work?

Answer 4

The proteins need to be crystals

Question 5

What is the resolution of circular dichroism?

Answer 5

Secondary structure

Question 6

What is the resolution of small-angle X-ray scattering?

Answer 6

Overall molecular shape

Question 7

What is the resolution of cryo-EM?

Answer 7

Sub-cellular to nm level

Question 8

What is the resolution of single-particle analysis?

Answer 8

Atomic-level detail

Question 9

What does X-ray crystallography require?

Answer 9

An X-ray source, a detector and a sample

Question 10

What did Kepler (1611) discover?

Answer 10

Predicted regular packing of water particles and determined snowflake symmetry

Question 11

What id Steno (1669), Haüy (1784) and Bravias (1850) discover?

Answer 11

Described basic properties of crystals

Question 12

What did Röntgen (1895) discover?

Answer 12

X-Rays

Question 13

What did WH and WL Bragg discover in 1912?

Answer 13

Determined Bragg’s law to explain diffraction

Question 14

What did WL Bragg discover in 1912?

Answer 14

Described ZnS model

Question 15

What did WH and WL Bragg discover in 1913?

Answer 15

Described diamond structure

Question 16

Bragg’s Law equation

Answer 16

nλ=2dsin(θ)

Question 17

How much pure protein do you need to crystallise?

Answer 17

~1mg

Question 18

What is needed for a protein to crystallise?

Answer 18

It must overcome an energy barrier analogous to that for conventional chemical reactions

Question 19

What does a higher energy barrier mean?

Answer 19

A slower nucleation rate

Question 20

What does a more supersaturated protein solution mean?

Answer 20

The greater likelihood of a nucleus forming

Question 21

What are the crystallisation screen components?

Answer 21

Salts
Buffers
Chaotropes/kosmotropes
Physical environmental factors

Question 22

Why are buffers added and what type of buffer should not be used?

Answer 22

To stop pH changes and phosphate buffers should not be used

Question 23

What types of chaotropes and kosmotropes are used and why are they used?

Answer 23

Alcohols/poly-ols
PEG
Glycerol
Block-co-polymers
They interfere with water

Question 24

What physical environmental factors should be controlled?

Answer 24

Temperature
Cofactors
Other macromolecules
Gravity

Question 25

How are crystals destroyed by X-rays?

Answer 25

Primary damage-Covalent bond absorbs the X-ray and is broken

Secondary damage- Free radicals diffuse through the crystal

Question 26

How long does it take for diffraction to stop due to radiation damage?

Answer 26

<2 minutes

Question 27

How is phased X-ray diffraction data visualised?

Answer 27

As an e- density map

Question 28

How is the e- density map interpreted?

Answer 28

By building an atomic model