Jan 17 Flashcards
What do you get from XRD pattern? What don’t you get?
Get intensities and positions of points, do not get the phases
What holds protein crystals together?
There is water present in crystal, there are weak intermolecular forces so the crystal is fragile
Is it possible for 2 different crystal types to arise from the same growth conditions and mother liquor?
Yes
What is crystal mosaicity?
It is that protein crystals are made of smaller blocks that are roughly aligned. Because of this, x rays will diffract slighly differently depending on orientation of smaller blocks. This is the mosaic spread of a crystal
Do protein crystals contain water, do they need it?
They need it, if they are dry, they do not diffract. The water molecules are ordered
How do we know that proteins in solution are the same as in a crystal?
They still have function while in crystal. Crosslinking also supports crystal structures, can do NMR and overlap structure with one from PXRD, they are generally similar
Describe hanging and sitting drop crystallization.
Hanging has some protein and precipitant in the drop. If [precipitant] is higher in the well below drop, then water leaves the drop so protein and precipitant concentrations go up, then protein conc. goes down as precipitant stays same as crystals form
Sitting is good as detergents if needed for MB protein in drop reduce surface tension, so sitting more practical. These can be modified during experiement (like adding more precipitant to well etc.)
When crystallization is too fast, what do you get?
Amorphous precipitation or you get really small crystals
What happens if protein and precipitant concentrations stay high and it stays in nucleation part of phase diagram?
This is bad as all protein in sample going towards making new crystals and not growing existing ones.
What are the 2 types of seeding, explain the process.
Macroseeding is using tiny crystal, put in solution with protein and precipitant conc. so in growth conditions. Usually need to etch the surface of crystal as surface is poisoned, need to give surface for crystal to grow on.
Microseeding uses a crushed crystal that is diluted to get a few small nuclei for seeding.
How is microdialysis done?
Have a “button” with a small well with the protein solution. It is kept in the well with MB. This button is placed in precipitant solution, the precipitant goes into the well (diffusion). Easy to make changes to precipitant solutions.
