Computational protocol: Structural and Biochemical Studies of a Moderately Thermophilic Exonuclease I from Methylocaldum szegediense

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Protocol publication

[…] For crystallization studies the MszExo I gene was synthesized with an N-terminal cleavable strep tag. The preparation of MszExo I protein for crystallization is described in supporting information (). Apo MszExo I crystals were grown by mixing the protein at a concentration of 8 mg/ml with 20% PEG6K, 0.1 M Tris-HCl pH8, 0.2 M calcium chloride at a protein to precipitant ratio of 1:1. Crystals appeared within 3 days, and grew to a suitable size for data collection after 10–14 days. MszExo I crystals were cryo-protected by dipping them into a solution consisting of the components used for crystallization supplemented with 20% glycerol for 2–5 sec followed by quick flash freezing in liquid nitrogen and storage for data collection.Diffraction data were collected at beamline I02 of Diamond Light Source, United Kingdom. Data was processed using the XIA2 pipeline. The apo MszExo I structure was solved by molecular replacement (using PHASER) with E. coli Exo I structure as the starting model (PDB ID: 1FXX). COOT visualization software was used for model building and REFMAC5 for refinement. Selected data collection and refinement statistics are given in . […]

Pipeline specifications

Software tools xia2, Coot, REFMAC5
Application Protein structure analysis
Organisms Escherichia coli, Bacteria
Chemicals Magnesium