Page 70 - Macromolecular Crystallography

CHAPTER 4

First analysis of macromolecular

crystals

Sherin S. Abdel-Meguid, David Jeruzalmi, and
Mark R. Sanderson

4.1 Macromolecular cryocrystallography or a removal of water from its exterior. The objective
of this procedure is to enable the water in and around
The past decade and a half have witnessed an almost
a crystal to form stabilizing, vitreous ice upon
complete revolution in the way that macromolec-
shock-cooling, rather than crystalline ice that would
ular diffraction data are recorded. The promise of
damage the lattice. Cryoprotection is commonly
diffraction data measurements essentially free of the
accomplished by one of three methods: (1) slow
effects of radiation damage has driven a change
equilibration of crystals in a cryostabilization buffer;
from older methods requiring crystalline samples
(2) quick passage of the crystal through a cryostabi-
to be mounted in thin glass capillaries for measure-
lization buffer (‘quick dip’ method); or (3) transfer
ments at ambient temperatures (or so) to newer,
of the sample into water-immiscible hydrocarbons.
experimental schemes that enable measurements
These are discussed in detail below. The slow equili-
at cryogenic temperatures from crystals mounted
bration approach allows the cryostabilization buffer,
in free-standing films. Preparation of macromole-
containing the antifreeze agent, to thoroughly per-
cular crystalline samples for measurements at cryo-
meate the crystal. Formation of crystalline ice in
genic temperatures can be considered to have three
and around the crystal is thus suppressed during
separable stages. These are: cryoprotection, shock-
shock cooling. However, some samples do not tol-
cooling, and cryogenic transfer to the X-ray diffrac-
erate exposure to antifreeze agents for the time
tion camera. Each of these will be described in
required for a full equilibration. In such cases, the
detail below. However, as in any experimental tech-
quick-dip method or a transfer to a water immisci-
nique, numerous variations to solve specific prob-
ble hydrocarbon is indicated (Hope, 1988; Kwong
lems have been, and continue to be, reported and
and Liu, 1999; Riboldi-Tunnicliffe and Hilgenfeld,
the interested reader is urged to consult issues of the
1999). These methods suppress formation of dam-
Journal of Applied Crystallography and Acta Crystallo-
aging crystalline ice around the crystal by either
graphica, section D. The theoretical and experimen-
coating the surface with antifreeze-containing buffer
tal aspects of macromolecular cryocrystallography
or an immiscible hydrocarbon. Formation of crys-
have been reviewed in Garman and Schneider, 1997
talline ice around the crystal is suppressed, while
and Rodgers, 1997.
the water within the crystal (thought to be kinetically
less prone to form crystalline ice) remains in a native
4.2 Cryoprotection of macromolecular
crystals state. The goal of all of these approaches is to define a
reproducible handling procedure that enables shock
Cryoprotection of crystalline samples involves cooling of a macromolecular crystal with its X-ray
either the introduction of antifreeze agents into and diffractive properties (as observed at ambient or sub-
around the crystal (which contain 40–90% solvent) zerotemperatures)preservedintact(orinsomecases
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