Reverse screening.
Major emphasis has been placed in recent years on kits for screening crystallization conditions of macromolecules. Such approaches have undoubtedly speeded up the initial screening and, to a certain extent, helped in reducing the amount of protein required for the initial survey. Factorial screening techniques, either full-factorial or sparse-matrix approaches, have proved successful in the crystallization of many proteins. However, in cases where the amount of protein is limited a systematic approach based on an a priori choice of precipitants may be preferable to an extensive search. The approach described here targets such situations. The approach consists of the determination of the solubility characteristics of the macromolecule under study as a function of precipitant and macromolecule concentrations to define a working range for these parameters. Conditions under which the protein is highly supersaturated, and hence more conducive to nucleation, are established so as to favor the formation of an initial stable nucleus which can be one of the dominant problems that hinders successful crystallization of proteins. Later, changes in solubility as a function of pH and as a result of the introduction of additives are evaluated. In addition, when ligands are available for the formation of macromolecular complexes, screening of different complexes is used as a means to increase the probability of obtaining crystals. Solubility information derived from one, or more, complexes that have been screened can be used for comparison and to aid in the crystallization of other complexes. Cross-seeding between complexes is an intrinsic part of the method and provides an efficient way of obtaining crystals when spontaneous nucleation is hard to achieve. In the example presented here, reverse screening has enabled the production of crystals of several peptide complexes with an anti-malaria antibody.
Department of Molecular Biology, The Scripps Research Institute, La Jolla, California 92037, USA.
E A
EA
Stura
A C
AC
Satterthwait
J C
JC
Calvo
D C
DC
Kaslow
I A
IA
Wilson