Protein Crystallography Course

	Terese Bergfors' Pictorial library of crystallization drop phenomena
	Johan Zeelen's X-tal Protocols crystallization drop phenomena

There are 9 possible things you will see, possibly in combination (photos used with permission as credited).

• Clear drops
• The protein did not reach saturation or did not nucleate.
• Matter
• Fibres, dirt, glass etc
• Should have noted this immediately after setting up the trials
• Precipitate
• If the precipitate is brown and the protein denatured, it won't crystallize
• If the precipitate is white and amorphous and the protein folded, it might still crystallize
• Gels
• Transparent, irregular regions of the drop
• Skin
• process of formation not understood
• crystals can grow before a skin forms but nothing happens afterwards
• Phase Separation
• Hundreds of small round droplets.
• Crystals can grow from phase separation.
• One precipitant has become immisible with another precipitant and phase separated
• May be an oil instead.
• Oils
• Droplets are usually fewer and not as round as compared with phase separation.
• The protein has separated out from the aqueous phase
• Spherulites
• Transparent birefringent clusters
• Can be difficult to distinguish from oils
• Crystals
• Microcrystalline precipitate
• 1D - needles
• 2D - plates
• 3D - boulders

• Immediately set up a screen around the conditions before your protein has a chance to deteriorate.
• If the results are reproducible, sacrifice a drop trying to identify it as salt or protein
• If the results aren't reproducible, try using the crystal for seeding
• Shoot a crystal.

There is no reliable way to tell if your crystals are salt or protein except by collecting an oscillation image (see section on shooting). But you can try:

• Poking the crystal with a very fine glass rod or glass pipette (drawn over a bunsen burner). This is the simplest and probably the best test. If the crystal is protein it should disintegrate very easily.
• Checking the well to see if the precipitant contains crystals.
• Setting up a "no protein" control drop with the buffer (+additives) your protein was in initially to see if crystals grow without protein.
• Running the crystal on a gel and silver staining or doing a western if you have an antibody. A 0.3 x 0.3 x 0.3 crystal has about 15 micrograms of protein in it.
• Using a dye. Hampton's IZIT dye or methylene green will stain protein but not salt.

• It is probably salt. Try to identify the crystal as salt or protein by poking it with a glass fibre.
• If it is protein, it is probably degraded protein. Run a gel for molecular weight determination and to blot for N-terminal sequencing
• Find the original sample used for crystallization or
• Cannibalize a few drops around the drop with the crystal in it or
• Try to reproduce the degradation proteolytically
• Make the construct corresponding to the degradation product
• Set up a screen around the conditions with the construct and see what happens
• If you get nothing, try seeding

If you see a crystal, don't go running down the corridor screaming "EUREKA!!!" until...			.	.
.	you know your crystal isn't salt...	and that it diffracts.

© 1999-2005 Airlie J McCoy, University of Cambridge. All rights reserved.

Last updated: 7 June, 2005