Computational protocol: Kinetic and Structural Requirements for CarbapenemaseActivity in GES-Type β-Lactamases

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Protocol publication

[…] The GES-2 crystals grown under these conditions belonged to space group P21 with cell dimensions a = 42.94 Å, b = 81.47 Å, c = 71.99 Å, β = 101.9°, and diffracted to 1.4 Å resolution. The Matthews coefficient assuming two molecules in the asymmetric unit, is 2.1 Å3/Da (42% solvent content). A complete data set comprising 360 images with an oscillation angle of 0.5° was collected from a single ertapenem-GES-2 crystal on SSRL beamline BL9-2, using a Rayonix MX325 detector with X-rays at 12658 eV (0.97945 Å). The images were processed with XDS and scaled and merged with SCALA. Additional data collection statistics are given in Table . [...] The ertapenem–GES-2 structure was solved by molecular replacement (MR) with the program MOLREP using the apo-GES-2 structure previously reported as the starting model. Prior to the MR calculation, the side chain of residue 170 was truncated to glycine, all water molecules were removed from the apo-GES-2 model, and the atomic displacement parameters were set to a uniform value of 20.0 Å2. The MR solutions for both structures were initially refined using REFMAC, and subsequent rounds of refinement used PHENIX and manual model building with COOT. The final ertapenem–GES-2 structure, refined at 1.4 Å resolution to a Rwork and Rfree of 0.158 and 0.197, respectively, contains 533 residues (A, 268 residues, and B, 265 residues), 486 water molecules, 6 iodide ions, and 2 ertapenem molecules. The atomic coordinates for the crystal structure of the ertapenem-GES-2 complex have been deposited in the Protein Data Bank (PDB accession code 4QU3), along with the observed structure factors. Final refinement statistics are given in Table . […]

Pipeline specifications

Software tools XDS, CCP4, Molrep, PHENIX, Coot
Applications Small-angle scattering, Protein structure analysis
Chemicals Hydrogen