Protein structure and methods Flashcards
What are 3 protein folding constraints?
Amino acid backbone configurations
Sidechain configurations-rotamers
Energy minimisation-bury hydrophobic residues and satisfy H bonding potential
How are atomic-level structures determined?
Through X-ray crystallography and EM
Why isn’t light microscopy used to ID proteins?
Proteins are too small
What is needed for X-ray crystallography to work?
The proteins need to be crystals
What is the resolution of circular dichroism?
Secondary structure
What is the resolution of small-angle X-ray scattering?
Overall molecular shape
What is the resolution of cryo-EM?
Sub-cellular to nm level
What is the resolution of single-particle analysis?
Atomic-level detail
What does X-ray crystallography require?
An X-ray source, a detector and a sample
What did Kepler (1611) discover?
Predicted regular packing of water particles and determined snowflake symmetry
What id Steno (1669), Haüy (1784) and Bravias (1850) discover?
Described basic properties of crystals
What did Röntgen (1895) discover?
X-Rays
What did WH and WL Bragg discover in 1912?
Determined Bragg’s law to explain diffraction
What did WL Bragg discover in 1912?
Described ZnS model
What did WH and WL Bragg discover in 1913?
Described diamond structure
Bragg’s Law equation
nλ=2dsin(θ)
How much pure protein do you need to crystallise?
~1mg
What is needed for a protein to crystallise?
It must overcome an energy barrier analogous to that for conventional chemical reactions
What does a higher energy barrier mean?
A slower nucleation rate
What does a more supersaturated protein solution mean?
The greater likelihood of a nucleus forming
What are the crystallisation screen components?
Salts
Buffers
Chaotropes/kosmotropes
Physical environmental factors
Why are buffers added and what type of buffer should not be used?
To stop pH changes and phosphate buffers should not be used
What types of chaotropes and kosmotropes are used and why are they used?
Alcohols/poly-ols PEG Glycerol Block-co-polymers They interfere with water
What physical environmental factors should be controlled?
Temperature
Cofactors
Other macromolecules
Gravity
