Development of an automated large-scale protein-crystallization and monitoring system for high-throughput protein-structure analyses

Abstract

Protein crystallization remains one of the bottlenecks in crystallographic analysis of macromolecules. An automated large-scale protein-crystallization system named PXS has been developed consisting of the following subsystems, which proceed in parallel under unified control software: dispensing precipitants and protein solutions, sealing crystallization plates, carrying robot, incubators, observation system and image-storage server. A sitting-drop crystallization plate specialized for PXS has also been designed and developed. PXS can set up 7680 drops for vapour diffusion per hour, which includes time for replenishing supplies such as disposable tips and crystallization plates. Images of the crystallization drops are automatically recorded according to a preprogrammed schedule and can be viewed by users remotely using web-based browser software. A number of protein crystals were successfully produced and several protein structures could be determined directly from crystals grown by PXS. In other cases, X-ray quality crystals were obtained by further optimization by manual screening based on the conditions found by PXS.

Conformational change of a unique sequence in a fungal galectin from Agrocybe cylindracea controls glycan ligand‐binding specificity

“…Initial crystallization trials were performed using an automated protein crystallization and monitoring system [7]. After screening, ACG was crystallized by the hanging-drop vapor diffusion method at 20°C in buffer containing 30% PEG 4000, 0.1 M Na citrate (pH 5.6), and 0.2 M ammonium acetate, with or without 5 mM lactose.…”

Structural Basis for Recognition of High Mannose Type Glycoproteins by Mammalian Transport Lectin VIP36

“…Crystallization and X-ray Data Collection-Initial crystallization conditions were screened using the Large Scale Protein Crystallization and Monitoring System (PXS) (33). The crystallization conditions of VIP36 in its Ca 2ϩ -bound form were obtained in a buffer containing 18 mg ml Ϫ1 protein, 15% (w/v) polyethylene glycol 4000, 1.5 M NaCl, and 0.1 M MES (pH 6.5) with incubation at 277 K for 4 days.…”

Structural basis for Arf6-MKLP1 complex formation on the Flemming body responsible for cytokinesis

“…Crystallization conditions were initially searched using a crystallization robot (Hiraki et al, 2006). The final crystallization conditions for the native and Se-Met cMKLP1-Arf6 complexes were 12% w/v PEG 4000, 0.1 M sodium cacodylate, pH 6.5, 50 mM (NH 4 ) 2 SO 4 , 5 mM MgCl 2 , 0.25-1.0% v/v ethyl acetate at 201C.…”