Determination of Protein Structure Flashcards
What is the primary technique for determining 3D structures, ranging from salt to whole virus particles?
X-ray crystallography
What form does the sample need to be in for X-ray crystallography?
Crystals
What proportion of structures in the PDB archive has X-ray diffraction techniques solved?
Over 90%
Give examples of the achievements of crystallography in the last 50 years
The structure of DNA has shown how genetic information is stored, replicated and used
Cro repressor-operator complex shows how proteins can interact with DNA and control the regulation of gene expression
What have elucidating the structures of enzymes shown?
How biochemical processes are catalysed
What have elucidating the structures of virus particles shown?
How they assemble
What have elucidating the structures of proton-translocating ATPase shown?
How the proton gradient is used to make ATP
What can protein crystallography be used for?
Structure-based drug design, development and improvement
What happens when waves impinge on a body?
They are scattered and converted into an image by means of a lens
Using waves for large objects works fine but what are they limited to?
About the wavelength of radiation used
What do we want to resolve about molecules?
The distances between atoms
In Angstroms
What is 1 Angstrom equal to?
E-10 metres
When is the resolution limit reached?
When two point-like objects can not be imaged as two distinct images
What type of waves are needed to resolve atoms?
X-rays
Is visible light far too long or short to resolve atoms?
Far too long
X-rays interact with matter weakly, true or false?
True
What are X-rays scattered by?
Electrons and so heavier atoms with more electrons scatter more
When were X-rays discovered and who by?
8th November 1895
Wilhelm Rontgen
Do X-ray lenses exist?
No
What is a crystal?
A repeating ordered array (LATTICE) of molecules in 3D in which the molecules are identically oriented
How many molecules will a typical protein crystal of 0.3mm in each direction have in each dimension?
E5 molecules in each dimension which is about E15 molecules
What is the total scattering a sum of?
The individual scatterers, or one thousand million million times stronger than the individual molecule
Scattering from a crystal is concentrated in discrete directions determined by what?
The crystal lattice
What does the fourier transform relate?
Te sum of all the reflections to the electron density at all points in the crystal repeating unit
Essentially, what does the fourier transform tell us?
What mixture of sine-waves is required to make up any function
What information about a wave is needed for the fourier transform?
Phase information
What techniques can you use to get phase information of a wave?
Molecular replacement
Multiple isomorphous replacement
Anomalous dispersion
What can we calculate from diffraction data and phases?
Electron density to get an atomic model
What can we calculate from an atomic model?
Electron density and from this diffraction data and phases
What is the iterative process called?
Refinement and rebuilding
What can the difference/omit map be used for?
Finding ligands
What affects resolution?
The maximum scattering angle observed for the data
What is the primary way that stereochemistry of a model can be assessed?
Using a Ramachandran plot
Protein crystals need to be obtained gently, true or false?
True
How are protein crystals generated?
By removing the protein very slowly out of solution
What factors affect protein solubility?
Concentrations of salt/precipitate
pH
Temperature
Name a method for obtaining crytals
Vapour diffusion
Crystals are easy to grow, true or fasle?
False, they need many different conditions testing and optimising
What are the mechanics of X-ray data collection?
The crystal is oscillated about a small angle and the resultant diffraction recorded
