Feb 10 Flashcards
What is used to replace Sulfur in AA’s, which AA is more problematic?
Use selenomethionine, it is more easily oxidized than S. So cys is more problematic than met, as cys has disulfide bridging, so Selenomethionine would be even worse
What is the workflow for an experiment using selenomethionine?
Use media with SeMet instead of Methionine.
2. Purify protein, use reducing agents, use MS to determine SeMet incorporation
3. Grow crystals
4. Scan fluorescence to get optimal wavelengths
5. Collect diffraction data from 1 crystal
6. Solve for Se atom positions, estimate phases, and calc electron density maps, fit model to these maps
What 3 wavelengths do crystallographers select for data collection using anomalous scattering, which is the most important?
Peak, inflection, and remote. This is multiwavelength anomalous diffraction (MAD). Peak is most important.
What does multiwavelength Anomalous Diffraction plot look like?
Both start at origin, draw structure factor with protein+heavy atom for both the wavelength where no anomalous scattering (remote wavelength). Then draw structure factor F PH that has a different wavelength (where there is anomalous scattering).
Then we look at getting from end of the vector without anomalous scattering to the one with we need to draw 2 straight lines to get to that point. These are Fr and Fi structure factors.
Then draw a circle without scattering radius, then do same with the scattering radius with a new origin based on the shift depicted by Fr and Fi..
How do we correct for model bias using difference Fourier Techniques?
We draw 2 Fourier maps. One has 2 times the target structure factor minus the model structure factor. The other map is just target-model structure factors. This reduces model bias
What is nonisomorphous phasing models? How get new part?
It is if we add domain to protein, similar structure but diff protein packing so diff space group etc. We use phases of known to estimate structure of new part. Trial and error
We need to split into 2 parts, look at orientation, then position of the molecule, need to match them up. May have multiple copies of the molecule in the same asymmetric unit
What is the orientation search and rotation function?
It is giving correlation between patterson maps with rotation function. You have map of found model, and of your new target. You match the interatomic vectors up,
What is translation search?
It is when you look at correlation between measured structure factor amplitudes and model expected examples. This is an R factor, just to measure how close to predicted value
