Computational protocol: The structure of hydrogenase-2 from Escherichia coli: implications for H2-driven proton pumping

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Protocol publication

[…] Diffraction data for the as-isolated enzyme were collected at beamline P14, PETRA III (Hamburg) at a wavelength of 0.976 Å using a Pilatus 6M hybrid pixel array detector, whereas data for the reduced enzyme were collected at beamline I03 (Diamond Light Source, U.K.) at a wavelength of 0.9794 Å using a Pilatus 6M detector. All data sets were collected at 100 K using a helical line scan to minimize photo-reduction during exposure to X-rays. All data reduction was performed using DIALS [] and Aimless []. Initial phase estimates for the as-isolated enzyme were generated using Phaser [] with pdb file 5A4M as a search model. Initial electron density maps were fragmented and difficult to interpret, manual addition of [4Fe4S] clusters in the proximal position and resetting the occupancy of the other metal centers using COOT [] followed by 50 cycles of all atom refinement in REFMAC5 [] greatly improved the electron density allowing automated model building using BUCCANEER []. Subsequent rounds of modeling and refinement were performed manually using COOT and REFMAC5, using TLS restraints and isotropic B-factors. Phase estimates for the reduced structures were calculated by rigid-body positional refinement of the as-isolated Hyd-2 model into the unit cell of the reduced structures using REFMAC5 followed by iterative rounds of manual rebuilding (COOT) and restrained refinement (REFMAC5). Structure superpositions were performed using LSQKAB [], and analysis of protein interfaces was carried out using PISA []. The occupancy of the SD oxygen atom at Cys546 was estimated by deleting the carbonyl oxygen atom of nearby residue Arg428 (an atom present at 100% occupancy) and the SD oxygen atom of Cys546. Electron density maps were recalculated for the structure with the missing atoms followed by comparison of the relative heights of the Fo–Fc map peaks for the missing O atoms. […]

Pipeline specifications

Software tools CCP4, Coot, REFMAC5, Buccaneer
Application Protein structure analysis
Organisms Escherichia coli
Chemicals Hydrogen