Practical macromolecular cryocrystallography

Abstract

Cryocrystallography is an indispensable technique that is routinely used for single-crystal X-ray diffraction data collection at temperatures near 100 K, where radiation damage is mitigated. Modern procedures and tools to cryoprotect and rapidly cool macromolecular crystals with a significant solvent fraction to below the glass-transition phase of water are reviewed. Reagents and methods to help prevent the stresses that damage crystals when flash-cooling are described. A method of using isopentane to assess whether cryogenic temperatures have been preserved when dismounting screened crystals is also presented.

Keywords: annealing; automounter; cryocrystallography; cryoprotectant; crystal mounting; flash-cooling; high pressure.

Figure 1

Notable temperatures in cryocrystallography. A vertical temperature scale is colored with four major regions marked: (i) dehydration, where crystals are damaged by drying, (ii) iceification, where crystals are damaged by ice formation when freezing, (iii) vitrification, where ice does not form when suitable cryoprotectants and adequate speed in flash-cooling are used, and (iv) data collection, where diffraction experiments are typically performed. Abbreviations used: m.p., melting point; f.p., freezing point; b.p., boiling point; STP, standard temperature and pressure. Glycerol solution temperature points are from Lane (1925 ▶). Ethylene glycol solution temperature points are from Cordray et al. (1996 ▶). Other temperature points are referred to in the text.

Figure 2

Accessories for cryocrystallography. Clockwise from lower left: gloves; shallow Dewar with automounter puck and clamping tongs holding a cryovial; purple foam Dewar holding a small glass Dewar with clear plastic cover; cryovials, cryomounting pin; safety glasses; curved magnetic wand; cryotongs.

Figure 3

Hand tools for cryocrystallography. From the top: forceps for grasping mounting pins; cryovial forceps with O-ring to maintain closure; self-closing forceps to hold vials and mounts without finger pressure; cryovial locking tongs; cryovials and cryobases with pins and loops; cryotongs; straight magnetic wand; curved magnetic wand. Items are from Hampton Research, MiTeGen and Molecular Dimensions

Figure 4

Close-up of five cryomounts. Left to right: Hampton Research ALS-style with steel pin; SPINE with steel pin; Hampton Research CryoCap copper with ledge; MiTeGen copper RT; Rigaku RFID mounting pin.

Figure 5

A typical X-ray diffractometer setup with a cryosystem. At the center is a crystal mounted on a pin magnetically held on a goniometer head. Clockwise from lower right corner: X-ray source collimator pointing towards the crystal with X-ray beamstop; black microscope; microscope display of the crystal in the cross-hairs mounted in a loop; X-ray detector; cryosystem nozzle pointing at the crystal; orange X-ray shutter indicator lamp. Note that the cryonozzle is pointing obliquely at the sample position so that the cryogenic gas impinges neither on the goniometer head nor on the X-ray detector, nor does it obscure the view of the crystal through the microscope.

Figure 6

Flow diagram to cryoprotect a crystal. There are two major requirements for a good cryoprotectant solution: (i) it must be vitrified when flash-cooled (left side of the flowchart) and (ii) the crystal must tolerate the cryoprotecting treatment and diffract well when treated with the cryoprotectant solution (right side). Y, yes; N, no. Good technique is also required. Also, a test of crystal diffraction at room temperature is sometimes warranted as noted by the box at the upper right and the circle-enclosed RT symbols.

Figure 7

ACTOR from Rigaku. An automated sample-mounting robot with Dewar, robot with end effector, motorized goniometer head and software (photograph courtesy of Rigaku Americas Corp.).

Figure 8

Three views of a crystal mounted at room temperature in a MiTeGen mount with a thin-walled poly(ethylene terephthalate) capillary. Left, full view with a MiTeGen RT base and dual-diameter copper pin. Note the liquid in the top of capillary. Middle, crystal, loop, pin, copper. Right, close-up of the crystal in the loop.