Computational protocol: The FAD Cofactor of RebC Shifts to an IN Conformation upon Flavin Reduction†,‡

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Protocol publication

[…] Data were collected at beamline 9-2 at the Stanford Synchrotron Radiation Laboratory, integrated in HKL2000, and scaled in Scalepack () without σ cutoff. The unit cell volume of reduced RebC is 602000 Å3, almost twice the volume of substrate-free RebC, because two monomers previously related by a crystallographic 180° rotation shift to be related by a 160° rotation, with consequent alteration of the a, c, and β values of the unit cell (Table ). Data collection statistics are shown in Table . The structure of reduced RebC was solved in CNS (), using rigid-body refinement and the coordinates from PDB ID 2R0G, with reflections flagged as “free” identically to the structure factor file used in refinement of PDB ID 2R0G. Only coordinates for protein atoms were used in rigid-body refinement, with coordinates for water molecules and flavin molecules added later in refinement. Following rigid-body refinement, multiple rounds of refinement, including simulated annealing, were carried out in CNS with alternate rounds of manual adjustment carried out in COOT (). We found no notable differences between the two molecules of RebC in the asymmetric unit, regardless of whether or not noncrystallographic symmetry (NCS) restraints were used. However, to improve the data to parameter ratio, NCS restraints were used throughout refinement. The topology and parameter files for reduced flavin were generated from the FADH2 molecule in the PrnA structure (PDB ID 2ARD) () as well as a higher resolution FAD molecule from cholosterol oxidase (PDB ID 1N4V) (), using XPLO2D (). A composite omit map was used to verify the overall structure, with omit maps also generated for the flavin cofactor and the “melting helix”. There are two protein chains in the asymmetric unit, each containing 529 residues from RebC with 20 amino acids in an N-terminal tag. RebC residues 3−529 from chain A and 2−529 from chain B are included in the final model, with residues 247−250 and 417−423 disordered in chain A and residues 247−250 and 418−421 disordered in chain B. There is also one molecule of reduced flavin in each active site. One sodium atom, positioned near an aspartate, was included in the model. When instead modeled as water, its B-factor was atypically low (5 Å2). More residues are outside of the “most favored” region of a Ramachandran plot than has been observed for other RebC structures (); this is likely a reflection of the lower resolution of these data relative to other RebC structures (Table ). Refinement statistics for the reduced-RebC structure are shown in Table . [...] CAVER () was used to determine if any pathways connected the substrate-binding pocket of RebC to external solvent, with subsequent examination of results in PYMOL (http://pymol.sourceforge.net). Based on the representation of this pathway as spheres with varying radii, the widest pathway identified that leads to solvent in the reduced-RebC structure is too narrow for a putative RebC substrate to traverse. […]

Pipeline specifications

Software tools CNS, Coot, CAVER, PyMOL
Application Protein structure analysis