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In situ proteolysis to generate crystals for structure determination: an update.

PloS one (2009-04-09)
Amy Wernimont, Aled Edwards
ABSTRACT

For every 100 purified proteins that enter crystallization trials, an average of 30 form crystals, and among these only 13-15 crystallize in a form that enables structure determination. In 2007, Dong et al reported that the addition of trace amounts of protease to crystallization trials--in situ proteolysis--significantly increased the number of proteins in a given set that produce diffraction quality crystals. 69 proteins that had previously resisted structure determination were subjected to crystallization with in situ proteolysis and ten crystallized in a form that led to structure determination (14.5% success rate). Here we apply in situ proteolysis to over 270 new soluble proteins that had failed in the past to produce crystals suitable for structure determination. These proteins had produced no crystals, crystals that diffracted poorly, or produced twinned and/or unmanageable diffraction data. The new set includes yeast and prokaryotic proteins, enzymes essential to protozoan parasites, and human proteins such as GTPases, chromatin remodeling proteins, and tyrosine kinases. 34 proteins yielded deposited crystal structures of 2.8 A resolution or better, for an overall 12.6% success rate, and at least ten more yielded well-diffracting crystals presently in refinement. The success rate among proteins that had previously crystallized was double that of those that had never before yielded crystals. The overall success rate is similar to that observed in the smaller study, and appears to be higher than any other method reported to rescue stalled protein crystallography projects.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Protease from Bacillus licheniformis, Type VIII, lyophilized powder, 7-15 units/mg solid
Sigma-Aldrich
Papain from papaya latex, lyophilized powder, aseptically filled
Sigma-Aldrich
α-Chymotrypsin from bovine pancreas, (TLCK treated to inactivate residual tryspin activity), Type VII, essentially salt-free, lyophilized powder, ≥40 units/mg protein