Protein Crystallography Course
Screening around the conditions
Screening around the conditions that give microcrystals is done by varying
- In some cases varying the pH by 0.1 pH unit is enough to stop crystallization
- In the Hampton screens the pH given (that of the buffer) is not the pH of the final solution
- The final protein concentration in the drop can be increased by increasing the ratio of protein to precipitant in the set-up
- If you are crystallizing at 4 degrees you can pre-chill all solutions and work on ice before transferring to the cold room
- Incubators are available from Stratech
- Bigger drops can give bigger crystals because there is more protein in the drops and the rate of equilibration is slower.
- Try sitting drops or sandwich drops
Popular additives are:
- Glycerol, which may stop nucleation and may give you fewer, larger crystals, and has the advantage of doubling as a cryo-protectant. Try anything from 1% to 25% glycerol.
- Ethanol or dioxane, which have the effect of poisoning the crystals and stopping too much nucleation
- Divalent cations like magnesium
- A detergent such as beta-octyl-glucoside
Hampton has three Additive Screens
and three Detergent Screens
each with 24 additives
Molecular Dimensions have a Popular Detergents kit (6 detergents) and an Expanded Detergents kit (58 detergents)
Ulrich Gohlke's Home Page contains a description of the Properties of Detergents.
Finding an appropriate additive can be related to finding conditions in which your protein is monodispersed. Try using different additives to get monodispersed protein (dynamic light scattering), and then set up a screen with that additive in all conditions
Oil on the reservoir
Diffusion can be slowed down by adding a layer of oil on the top of the hanging drop well
The proportion of light silicone oil to heavy paraffin oil must be mixed in different proportions to get an appropriate rate of diffusion
A novel technique to control the rate of vapour diffusion, giving larger protein crystals Chayen, NE, J. Appl. Cryst. 1997, Vol.30, No.Pt2, pp.198-202
Seeding is a way of manipulating your position in phase space
There three different methods of seeding
Enrico Stura has a figure
See Douglas Instrument's paper "Crystallization of a protein by microseeding after establishing its phase diagram"
Macroseeding. Enrico Stura has a figure
and four different ways of using seeding
The seeds are used to grow crystals of the same lattice and symmetry from an identical macromolecule
Heterogeneous seeding (Cross-Seeding).
The seeds are used to grow crystals of the same lattice and symmetry from a different, but usually very similar macromolecule (such as a mutant)
The seeds are used to grow crystals of a different lattice and symmetry from an identical macromolecule
Heterogeneous Epitaxial Seeding.
The seeds are used to grow crystals of a different lattice and symmetry from a different macromolecule e.g. the nucleation of protein crystals on cellulose fibre impurities in the drops
A watched crystal never grows
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© 1999-2005 Airlie J McCoy,
University of Cambridge. All rights reserved.
7 June, 2005