Screening and optimization strategies for macromolecular crystal growth

Abstract

Today the determination of successful crystallization conditions for a particular macromolecule remains a highly empirical process. Sparse-matrix and grid-screening procedures are rapid and economical means to determine preliminary crystallization conditions. During optimization the variable set (pH, precipitant type and precipitant concentration) utilized in these procedures is screened in an attempt to determine appropriate conditions for the nucleation and growth of single crystals suitable for X-ray diffraction analysis. Unfortunately, in many cases this strategy will not produce single crystals suitable for X-ray diffraction analysis. We have explored, in an empirical sense, other tools for use during optimization. First, a new screening protocol is evaluated which employs less classical precipitating agents. Second, a set of 24 electrostatic crosslinking agents are evaluated for their ability to promote crystallization. Third, a panel of more than 30 detergents are evaluated for their ability to prevent sample aggregation and influence crystal growth.

Crystallization, X‐ray studies, and site‐directed cysteine mutagenesis of the DNA‐binding domain of OmpR

“…Initial crystallization conditions were found using the sparse matrix approach in a hanging drop vapor-diffusion experiment (Jancarik & Kim, 1991;Cudney et al, 1994). OmpRc is crystallized at 4 "C from a reservoir buffer containing 18% PEG 8000, 400 mM CaC12, 100 mM cacodylate, 2 mM PME, pH 6.5, using freshly purified protein concentrated with Centricon I O units (Amicon) to 45-60 mg/mL in 20 mM BisTris.HCI, 100 mM KCI, 2 mM PME, pH 6.5.…”

Crystallization of an intact GST-estrogen receptor hormone binding domain fusion protein

“…Initial screening was carried out using the sparse-matrix screening kits from Hampton Research (Crystal screen HI and HII, Jancarik & Kim, 1991;Cudney et al, 1994) 2 gl of the protein, 8 mg ml -a in 20 mM Hepes, 20 mM NaCI, O.05%(w/v) floctylglucoside, pH 8.0, were mixed with 2 gl of the precipitating solution at 293 K. The composition of the crystals was investigated for protein content by using transmission electron microscopy (EM). Negatively stained specimens for EM were prepared as follows.…”

Crystallization and preliminary crystallographic analysis of the complex of the second and third regulatory subunits of human Pol δ

“…Initial crystallization conditions were obtained by screening at 298 K in 96-well plates using the sitting-drop vapor-diffusion method with Crystal Screens I and II (Hampton Research; Jancarik & Kim, 1991;Cudney et al, 1994) and 50% diluted solutions of Crystal Screens I and II. Drops consisting of 1 ml protein solution mixed with 1 ml reservoir solution were equilibrated against 100 ml reservoir solution.…”