TB3 - Map Interpretation & Model Building Flashcards
Why can we identify proline and not methionine in maps?
Prolines change the backbone conformation; methionine is highly flexible so hard to find.
Compare initial and experimental data in XRC and cryo-EM.
XRC: initial = crystal; experimental = structure factor amplitudes
Cryo-EM: initial = protein model; experimental = electron density maps
Why does XRC refinement flip between real and reciprocal space?
It is often the case to alternate between real and reciprocal space as often refinement programs won’t allow shifts that have too large an energy barrier, even if the structure is stuck in a local minimum. It therefore needs to be manually shifted into the correct region of electron density whilst still observing the previous knowledge.
What is the atomic displacement parameter?
(Also known as the temperature or B factor), this describes the degree to which the electron density is spread out. It gives an idea of the true static or dynamic mobility of the atom.
What are anisotropic displacement factors?
These are a much more realistic description of atomic motion as simple B factors assume the motion available to an atom is spherical, however atoms aren’t equally free to move in all directions due to restraints of covalent bonds. ATFs require far more parameters and high resolution due to these large numbers of parameters per atom.
What is TLS refinement?
The vibrational motions of each of the chosen groups are defined using:
• Translation (linear motion)
• Libration (rocking around a central pivot)
• Screw rotation (describes translation/rotation correlation)
What is occupancy?
A measure of the fraction of molecules at which atom j occupies the position in the model. This won’t always be 1 – if soaking heavy atoms, there’s no guarantee of completely saturated binding, in which case you need to change the occupancy value to refine the model.
What is anisotropy?
Means having a different value when measured in different directions. This comes from the data and also from vibration along torsion angles, creating displacement.
What is bulk solvent correction?
It is harder to model positions of atoms the further away you get from the surface of the protein because they become more random. Thus, a model is applied assuming a distribution of water within the solvent regions.
What is the R-value?
the overall fit between the data and the model
What is Rfree?
measures how well the model predicts a subset of measured intensities. It is calculated from a small subset of randomly chosen intensities which are then not used in refinement. They are used in cross-validation which entails assessing the agreement between calculated and observed data.
What are the two types of maps generally used for improving XRC models after refinement?
- FO – FC (difference map - shows where the experimental density and atomic model disagree)
- 2 FO – FC (electron density map – used to build the model into)
What two methods are used in cryo-EM for scoring electron density maps against molecule models?
Cross-correlation scoring and gradient driven minimization
Pros and cons of cross-correlation scoring.
- Accurate – matches the shape of the experimental map with the map calculated from the model
- Very slow – new map calculated for each change
- Not used during refinement but useful in validation
Pros and cons of gradient driven minimization
- Atom-centered – optimizes positions of atoms in the map
- Very fast
- Better at high resolution than low resolution
- Very dependent on geometry restraints
