Protein Crystallography Course

SHOOTING

Diffraction

The only requirement for a protein crystal is that it diffracts. Diffraction depends on

Size

The scattering is proportional to the number of unit cells in the crystal.
The number of unit cells is proportional to the volume of the crystal so doubling all dimensions of a cubic crystal will give 8 times the diffraction
Conversely, the number of unit cells in a crystal depends on size of the unit cells, which in turn depends on

the size of the protein
the tightness of the packing of the protein
how many proteins are in the repeating symmetry unit (the asymmetric unit)
the symmetry

Salt diffracts better than protein for the same size crystal because the unit cells are much smaller (spots are further apart).

Order

The scattering depends on how identical each unit cell is. The more identical, the stronger the scattering.
Salt diffracts better than protein because it is more ordered.

In other words, the size of a protein crystal doesn't tell you anything about how well it will diffract.

The First Shot

Find the minimum concentration of glycerol required to cryo-protect your mother liquor.
Put your crystal in the cryoprotectant for a minute or so and mount in a loop.
Collect a large oscillation angle image. If the crystal is salt, the spots will be far apart and might be missed with a small oscillation angle range. Use 5 degrees (most data from protein crystals would be collected with 1 degree oscillations or less). If the crystal is salt then there will be some intense spots at high resolution. Salt crystals have small lattices so you won't see spots at low resolution.

The diffraction quality of a crystal can vary with any combination of:

Strength of diffraction	Quality of diffraction
no diffraction	multiple mosaic crystal
weak diffraction 10 angstroms	mosaic crystal
promising diffraction 3.5-6 angstroms	multiple crystal
good diffraction > 2.8 angstroms	single crystal

Poor Crystals

How can you make a multiple mosaic crystal a single crystal?

Some multiple crystals can be broken apart. Use a glass fibre to gently break up a cluster.
Shoot fresh crystals.Crystals can deteriorate within days of growth.
Mosaic crystals require careful data collection. If the mosaicity does not cause the spots to overlap with one another, data collection is possible.

How can you make weakly diffracting crystals diffract further?

Shoot non-frozen crystals. If you have only ever tried collecting data from flash-cooled crystals, it is a good idea to see how far they will diffract at room temperature. Try mounting them in a capilliary.
Be aware of radiation damage. Some crystals die with exposure to x-rays. This is normally only a problem with non-frozen crystals, but can affect frozen crystals also. If there is weak diffraction on the first image and even less on the second the crystal could be dying almost immediately and you won't see the high resolution limit.
Shoot fresh crystals. Crystals can deteriorate within days of growth.
Treat crystals very gently. Use large loops for freezing.
Keep trying, within reason. Crystal number 30 might be OK. It is unlikely that crystal 300 will be.
Dehydration. Put the crystals in conditions of higher precipitant to shrink the cell. This can produce spectacular improvements in resolution.
Annealing. Freeze crystals in a cryostream (don't shoot), bring them back to room temperature by putting them back in the crystallization solution for a couple of minutes, and re-freeze.

J.M. Harp, D.E. Timm and G.J. Bunick (1998). Macromolecular Crystal Annealing: Overcoming Increased Mosaicity Associated with Cryocrystallography. Acta Cryst. D54, 622 - 628
J. I. Yeh and W. G. J. Hol (1998). A flash-annealing technique to improve diffraction limits and lower mosaicity in crystals of glycerol kinase. Acta Cryst. (1998). D54, 479 - 480

Microgravity. NASA will grow your crystals in space for you
- Biological crystal growth in space
- Microgravity Research Program - Biotechnology
- Experiment Selection Process

If none of the above work, find new conditions with higher precipitant concentrations.
See Enrico Stura's page.

Crystals for cryo-crystallography

Most data is collected at cryo temperatures because mounting crystals in loops is easier than mounting in capillaries and the cryo-temperatures reduce radiation damage.

If your crystals deteriorate within days or weeks of being grown, try "freeze and retrieve" (freezing your crystals in liquid nitrogen and storing them at 100K, then retrieving them for data collection)

It is usually best to grow your crystals in cryoprotectant. See a modified version of Elspeth Garman's protocol for cryoprotecting crystals.