Towards rationalization of crystallization screening for small- to medium-sized academic laboratories: the PACT/JCSG+ strategy

Abstract

A crystallization screening process is presented that was developed for a small academic laboratory. Its underlying concept is to combine sparse-matrix screening with systematic screening in a minimum number of crystallization conditions. The sparse-matrix screen is the cherry-picked combination of conditions from the Joint Center for Structural Genomics (JCSG) extended using conditions from other screens. Its aim is to maximize the coverage of crystallization parameter space with no redundancy. The systematic screen, a pH-, anion- and cation-testing (PACT) screen, aims to decouple the components of each condition and to provide information about the protein, even in the absence of crystals, rather than cover a wide crystallization space. This screening strategy is combined with nanolitre-volume dispensing hardware and a small but practical experiment-tracking system. The screens have been tested both at the NKI and in other laboratories and it is concluded that they provide a useful minimal screening strategy.

Structure of fructose bisphosphate aldolase fromEncephalitozoon cuniculi

“…Crystallization of E. cuniculi aldolase with the partial substrate phosphate. E. cuniculi aldolase at 25 mg ml À1 (0.6 mM) in SEC buffer (see above) was initially crystallized in PACT screen (Newman et al, 2005) condition F10 and optimized with the ADDit Additive Screen (Emerald BioSystems). Aldolase stock solutions (0.4 ml) were mixed with reservoir (0.4 ml) and equilibrated against a 80 ml reservoir of precipitant (crystallant) using 96-well Compact Jr plates from Emerald BioSystems.…”

Salvage and storage of infectious disease protein targets in the SSGCID high-throughput crystallization pathway using microfluidics

“…The JCSG+ screen used for the initial trials set up for SSGCID targets arose from data mining conducted by the Joint Center for Structural Genomics, which identified 96 conditions in commercial screens that had been most successful in crystallizing proteins (Newman et al, 2005;Page & Stevens, 2004). 16 of these conditions were taken from Wizard Screens I and II (Emerald BioSystems) in order to compose the MPCS Sweet 16 Screen (Table 1).…”

“…10-15 new targets arrive at the facility each week and are immediately screened for crystallization using traditional sitting-drop vapor diffusion. Following the strategy proposed by Newman et al (2005), all targets are screened against two 96-condition screens, JCSG+ (Emerald BioSystems) and PACT Premier (Molecular Dimensions), giving 192 unique conditions. The experiments are monitored for four weeks Figure 1 Workflow schematic for the salvage study.…”

Structure of triosephosphate isomerase fromCryptosporidium parvum

“…Thawed protein was used to set up four sparse-matrix screens (JCSG+, Cryo and Wizard from Emerald BioStructures, Bainbridge Island, Washington, USA and PACT from Molecular Dimensions, Apopka, Florida, USA) following an extended Newman strategy (Newman et al, 2005). 0.4 ml protein solution was mixed with 0.4 ml well solution and equilibrated against a 100 ml reservoir using 96-well Compact Jr crystallization plates (Emerald BioStructures).…”