Protein Crystallography Course
The only requirement for a protein crystal is that it diffracts.
Diffraction depends on
In other words, the size of a protein crystal doesn't
tell you anything about how well it will diffract.
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)
Salt diffracts better than protein for the same size
crystal because the unit cells are much smaller (spots are further apart).
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
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
||multiple mosaic crystal
|weak diffraction 10 angstroms
|promising diffraction 3.5-6 angstroms
|good diffraction > 2.8 angstroms
How can you make a multiple mosaic crystal a single crystal?
How can you make weakly diffracting crystals diffract
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.
If none of the above work, find new conditions with higher precipitant concentrations.
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.
Put the crystals in conditions of higher precipitant to shrink the cell. This can produce
spectacular improvements in resolution.
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,
- 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
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.
After all the effort it takes to get crystals,
it can be very tempting to collect poor data from poor crystals in the
hope that any problems can be sorted out in data processing or even at
structure solution. It is much more effective to spend the time sorting
out the problems with the crystals instead.
© 1999-2005 Airlie J McCoy,
University of Cambridge. All rights reserved.
24 December, 2006