High-Throughput Crystallization Pipeline at the Crystallography Core Facility of the Institut Pasteur

Abstract

The availability of whole-genome sequence data, made possible by significant advances in DNA sequencing technology, led to the emergence of structural genomics projects in the late 1990s. These projects not only significantly increased the number of 3D structures deposited in the Protein Data Bank in the last two decades, but also influenced present crystallographic strategies by introducing automation and high-throughput approaches in the structure-determination pipeline. Today, dedicated crystallization facilities, many of which are open to the general user community, routinely set up and track thousands of crystallization screening trials per day. Here, we review the current methods for high-throughput crystallization and procedures to obtain crystals suitable for X-ray diffraction studies, and we describe the crystallization pipeline implemented in the medium-scale crystallography platform at the Institut Pasteur (Paris) as an example.

Shedding X-ray Light on the Role of Magnesium in the Activity of Mycobacterium tuberculosis Salicylate Synthase (MbtI) for Drug Design

“…The crystallization experiments were performed at 4 °C by the sitting drop vapor diffusion technique in 96-well plates according to established protocols at the Crystallography Core Facility of the Institut Pasteur. 28 The trials were set up with a Mosquito crystal Nanoliter Protein Crystallization Robot (TTP Labtech, Melbourne, UK); the plates were stored in a Rock Imager 1000 (Formulatrix, Bedford, MA, USA) and visually checked through the dedicated image repository, following a specific timetable. The drops were obtained by mixing an equal amount of protein and reservoir solutions to a final volume of 400 nL; the reservoir contained 150 μL of the precipitant mixture.…”

The prefusion structure of herpes simplex virus glycoprotein B

“…The protein was purified on Strep-Tactin affinity resin and by size exclusion chromatography using Superdex 200 16/60 column and 10 mM tris (pH 8) and 50 mM NaCl as running buffer. The protein was concentrated to 6.4 mg/ml and crystallized in 0.1 M tris (pH 8), 18% ethanol at the Institut Pasteur core facility for crystallization (63). They were flash-frozen in liquid nitrogen in cryosolution containing 0.1 M tris (pH 8), 20% ethanol.…”

Extensive flavivirus E trimer breathing accompanies stem zippering of the post‐fusion hairpin

“…Prior to crystallization, the purified protein was concentrated to approximately 0.42 mg/ml and buffer-exchanged to 15 mM Tris-HCl pH 8, 150 mM NaCl. Crystallization conditions were screened in 400 nl sitting drops formed by mixing equal volumes of the protein and reservoir solution in 96-well Greiner plates with a Mosquito robot and monitored by Rock-Imager according to the procedures described by Weber et al [46]. Initial crystals were optimized using a robotized system (Matrix Maker and Mosquito setups).…”