Computational protocol: Aminoglycoside resistance profile and structural architecture of the aminoglycoside acetyltransferase AAC(6')-Im

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Protocol publication

[…] AAC(6’)-Im was crystallized as previously described . Three different crystal forms were initially obtained, and data collection from form III (an AAC(6’)-Im-kanamycin complex in space group P65) has been described . This data was reprocessed to 1.95 Å resolution using XDS and AIMLESS and data are presented in Table S1. A data set to 1.7 Å resolution was collected from a form II apo-AAC(6’)-Im crystal at SSRL beamline BL12-2 using X-rays at 12658 eV (0.9795 Å). A total of 950 fine phi-sliced (0.2º rotation) images with a 0.2 sec exposure were measured using a Pilatus 6M PAD detector running in shutterless mode. The images were processed using XDS , and scaled and merged with AIMLESS . Data collection statistics are also given in Table S1.The AAC(6’)-Im-kanamycin A structure was solved as described and refined using PHENIX , giving a final model comprising 1507 protein atoms, one kanamycin ligand and 83 water molecules, with final Rwork and Rfree values of 17.05% and 19.43% respectively. The apo-AAC(6’)-Im structure was solved by molecular replacement using the refined AAC(6’)-Im-kanamycin A structure, with the kanamycin A and water molecules removed. Two independent molecules were found in the asymmetric unit and subsequently refined using PHENIX . The final Rwork and Rfree values were 18.46% and 22.93% respectively (see Table S1 for final statistics for both structures). The atomic coordinates and the structure factors for apo AAC(6')-Im and the AAC(6')-Im-kanamycin A complex were deposited to the Protein Data Bank with PDB codes 6BFF and 6BFH, respectively. […]

Pipeline specifications

Software tools XDS, CCP4, PHENIX
Applications Small-angle scattering, Protein structure analysis
Chemicals Acetyl Coenzyme A, Aminoglycosides, Kanamycin