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

Abstract

The MPCS Plug Maker is a microcapillary‐based protein‐crystallization system for generating diffraction‐ready crystals from nanovolumes of protein. Crystallization screening using the Plug Maker was used as a salvage pathway for proteins that failed to crystallize during the initial observation period using the traditional sitting‐drop vapor‐diffusion method. Furthermore, the CrystalCards used to store the crystallization experiments set up by the Plug Maker are shown be a viable container for long‐term storage of protein crystals without a discernable loss of diffraction quality with time. Use of the Plug Maker with SSGCID proteins is demonstrated to be an effective crystal‐salvage and storage method.

Chapter 1. Practical Approaches for In Situ X-ray Crystallography: from High-throughput Screening to Serial Data Collection

“…For in situ data collection, the capillary containing the droplets was cut and sealed, and fixed on a magnetic base. The commercial CrystalCard device, 75,76 by Protein BioSolutions, works on the same principle. The crystals produced can be harvested or measured in situ, either directly inside the chip or by coupling with a capillary.…”

Inhibitor-bound complexes of dihydrofolate reductase-thymidylate synthase fromBabesia bovis

“…0.4 ml protein solution was mixed with 0.4 ml reservoir solution and equilibrated against 100 ml reservoir solution using 96-well Compact Jr plates from Emerald BioSystems. The protein solution consisted of 11 mg ml À1 BbDHFR-TS in SEC buffer and crystal conditions were optimized using the Microcapillary Protein Crystallization System (MPCS) from Emerald BioSystems (Gerdts et al, 2008(Gerdts et al, , 2010Christensen et al, 2011). The BbDHFR-TS crystal used to solve the apo structure (PDB entry 3i3r) was obtained from a crystal card running a microfluidic gradient focused at 20%(w/v) polyethylene glycol (PEG) 8000 and 100 mM N-cyclohexyl-2-aminoethanesulfonic acid (CHES) pH 9.5.…”