Computational protocol: Microseeding – A Powerful Tool for Crystallizing Proteins Complexed with Hydrolyzable Substrates

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Protocol publication

[…] Data processing and structure calculation are described elsewhere (Oswald et al., submitted for publication) but shall be described here briefly. Data sets were collected at beamline BW7A of the EMBL outstation at DESY Hamburg. The dataset was processed with the XDS program package [] and subsequently subjected to twinning analysis utilizing the algorithm of Padilla and Yeates [], which revealed an almost perfect twin. Crystals belonged to space group P21 with cell axis of a= 31.2 Å, b= 212.7 Å and c= 42.8 Å (β = 90.1°) with two protomers in the asymmetric unit. Molecular replacement was performed utilizing the program MOLREP []. As search model, the structure of ChoX/choline (PDB code: 2REG) was depleted of its ligands and water molecules and further reduced to one monomer of ChoX. Further refinement consisted of iterative rounds of restrained refinement, carried out with SHELXL97–2 [] using (h,-k,-l) as twinning operator, and manual rebuilding using COOT []. Initially, water molecules were detected using ARP/wARP [] (threshold of 3.2 sigma) and thereafter their position was manually checked. The model was refined to a RF value of 15.1 and a Rfree value of 21% at a resolution of 1.8 Å. Figures were generated using PyMol []. […]

Pipeline specifications

Software tools XDS, Molrep, SHELX, Coot, ARP/wARP, PyMOL
Applications Small-angle scattering, Protein structure analysis
Chemicals Acetylcholine, Choline