Computational protocol: Structural Effect of the Asp345a Insertion in Penicillin-BindingProtein 2 from Penicillin-Resistant Strains of Neisseria gonorrhoeae

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Protocol publication

[…] Crystals were cryoprotected in mother liquor, flash-frozen in liquid nitrogen, and diffraction data were collected at SER-CAT beamline ID22 at the Advanced Photon Source at the Argonne National Laboratory (Argonne, IL) in 1° oscillations with an exposure time of 3 s per frame and a crystal–detector distance of 200 mm. Data were processed with HKL2000. The structure was determined by molecular replacement using the CCP4 program PHASER with the TPase domain of wild-type PBP2 as the search model [molecule A of Protein Data Bank (PDB) entry 3EQU]. The structure was determined with two molecules in the asymmetric unit and then refined by alternating rounds of REFMAC refinement and manual building using O. Residues 542–545 of molecule A and residues 504–511 and 543–545 of molecule B could be not be modeled because of weak or absent electron density. Alternative conformations were modeled for the side chain of Ser551 in molecule A. A non-native alanine at the N-terminus resulting from the construct was modeled as Ala236 in both molecules of the asymmetric unit.The stereochemistry of the final model was assessed by PROCHECK. The φ and ψ angles for Leu447 in both molecules of the asymmetric unit occupy the disallowed region of the Ramachandran plot. Both of these residues, however, exhibit excellent electron density. These residues are present on the α9−α10 loop; its conformation is influenced by the absence of the N-terminal domain of PBP2 in the truncated construct (see ). Asp346a and Thr347 of molecule B both occupy the generously allowed region of the Ramachandran plot, but these findings can be explained by the weak electron density at the Asp insertion site. […]

Pipeline specifications

Software tools CCP4, PROCHECK
Application Protein structure analysis
Organisms Neisseria gonorrhoeae
Chemicals Hydrogen, Penicillins