Elspeth Garman
Biochemistry Department, Oxford University
South Parks Road, Oxford
OX1 3QU, U.K.
E-mail: [email protected]

HT1: Crystallization

L5: Cryo-cooling: Why and How?

When a crystal has finally been obtained, the next stage in the structure determination pipeline is to prepare it for a diffraction test either at room temperature or 100 K. Often this step is given little thought, and the deleterious effects of various unfavourable crystal handling and cryo-protection protocols are not appreciated. In many laboratories a ‘standard’ route is used, which has worked for other protein crystals in the past. However, entrenched and accepted practices are not necessary the optimum ones for a particular problem, and may not work at all for your particular protein.  The main aim is to avoid the formation of any ice both within the crystal and around it (see Figure below).

This lecture will follow the crystal from growth drop to goniometer, through the identification of a suitable stabilising buffer, a benign cryo-protectant buffer and an effective cooling protocol, emphasising the physical principles which affect the outcome (resolution of diffraction and mosaic spread: with the aim of maximising the former and minimising the latter) [1,2,3]. The practical aspects will be covered during the hands-on session.

Left: Protein crystal in a 1mm diameter mohair fibre loop. Buffer film is opaque with ice due to inadequate concentration of cryoprotectant.
Centre: Same crystal with transparent film – a necessary but not sufficient condition for optimum diffraction.
Right:  200µm sized crystal after optimising the cryo-cooling protocol.


[1] Macromolecular Cryocrystallography. Garman, EF & Schneider, TR.  J.Appl.Cryst. (1997) 30, 211-237.

[2] Cool Data: Quantity and Quality. Garman, EF. Acta Cryst. (1999) D55, 1641-1653.

[3] Practical Macromolecular Cryocrystallography. Pflugrath, JW. Acta Cryst. (2015) F71, 622-642.