Facilities and Methods for the High-Throughput Crystal Structural Analysis of Human Proteins

Abstract

Facilities and methods for the high-throughput crystal structure analysis of human proteins are described as recently established in the Protein Structure Factory, a Berlin-area structural genomics project. Genes encoding human proteins are expressed in either recombinant Escherichia coli or yeast (Saccharomyces cerevisiae or Pichia pastoris). To facilitate and standardize protein purification, the target proteins are produced with various tags for affinity chromatography. For high-throughput crystallization, a robotic station is being set up that has the capacity to handle 960 000 experiments simultaneously. The resulting protein crystals will be subjected to X-ray diffraction experiments at the third-generation synchrotron storage ring BESSY where protein crystallography beamlines are currently under construction. The Protein Structure Factory's strategy for high-throughput production and structure analysis of human proteins is evaluated based on first results.

Discovery, Structure–Activity Relationship Studies, and Crystal Structure of Nonpeptide Inhibitors Bound to the Shank3 PDZ Domain

“…Shank3 PDZ was concentrated to 12 mg mL À1 in the presence of a fivefold molar excess of 36 (racemic mixture). Crystals of the complex were grown at room temperature using the sitting drop vapor diffusion method in a robotics setup [48] by mixing 0.4 mL of protein solution with 0.4 mL precipitant solution (24 % PEG 4000, 14 % 2-propanol, 0.15 m NaOAc). Rod-shaped crystals suitable for X-ray diffraction grew within 1-2 weeks.…”

“…The crystals were cryo-protected for data collection by soaking them for a few seconds in precipitant solution containing 20 % (v/v) PEG 400 and 0.5 mm 36, and were subsequently frozen in liquid nitrogen. Diffraction data to a resolution of 1.83 were collected at 100 K at beamline BL14.1 at the synchrotron-radiation source BESSY, [48] Helmholz-Centrum Berlin, and processed with XDS. [49] Structure determination and refinement.…”

The MX beamlines BL14.1-3 at BESSY II

“…The HZB-MX beamlines (Heinemann et al, 2003;Mueller et al, 2012Mueller et al, , 2015 are designed and built to support all possible MX experiments in a reliable and user friendly way:…”

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Abstract: The Macromolecular Crystallography (MX) group at the Helmholtz-Zentrum Berlin (HZB) is operating three state-of-the-art synchrotron beamlines for MX at BESSY II in Berlin (Heinemann et al, 2003;Mueller et al, 2012Mueller et al, , 2015. The radiation source for all three beamlines BL14.1-3 is a superconducting 7T-wavelength shifter.

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Structural and thermodynamic characterization of the adrenodoxin-like domain of the electron-transfer protein Etp1 from Schizosaccharomyces pombe

“…Crystals of Etp1 fd (516-618) were obtained at 4 °C by the sitting-drop method using a semi-automated dispensing system [25]. The reservoir solution contained 0.04 M HEPES (pH 7.0), 1.8 M Na-citrate, 3% glycerol and was mixed 1:1 with the protein solution, 18.4 mg ml -1 , 20 mM potassium phosphate buffer (pH 7.4) resulting in start droplet volumes of 0.6 μl.…”