Computational protocol: Molecular basis for allosteric specificity regulation in class Ia ribonucleotide reductase from Escherichia coli

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Protocol publication

[…] Diffraction data were collected at the Advanced Photon Source at Argonne National Laboratory. The CDP/dATP data set was collected on beamline 24ID-C at 100 K on an ADSC Q315 detector. The UDP/dATP, ADP/dGTP, and GDP/TTP data sets were collected on beamline 24ID-C at 100 K on a Pilatus 6M detector. All data were processed using HKL2000 () ().All four α4β4 complex structures soaked with substrates and effectors were solved to the full extent of the data resolution using the previously published 3.95-Å resolution structure () with all nucleotides removed. Rfree test sets were chosen to contain the same reflections across all data sets. For all structures, initial refinement was carried out in CNS 1.3 () and later in Phenix () with model building performed in COOT (). Refinement consisted of rigid body, simulated annealing, positional and individual B-factor refinement with no sigma cutoff. Loose non-crystallographic symmetry restraints were used throughout refinement. Simulated annealing composite omit maps generated in CNS 1.3 and Phenix were used to verify the models. All figures were made using the PyMOL Molecular Graphics System, version 1.5.0.4 (Schrödinger, LLC). Final refinement statistics are shown in . In addition to the substrate/specificity effector pairs bound in each structure, all structures have dATP or its hydrolysis product dADP in the allosteric activity site. In some cases, the high concentration of nucleotide used (10 mM) has resulted in more than one nucleotide molecule bound near this allosteric site. […]

Pipeline specifications

Software tools CNS, Coot, PyMOL
Databases DADP
Application Protein structure analysis
Organisms Escherichia coli
Chemicals Adenosine Diphosphate, Cytidine Diphosphate, Deoxyribonucleotides, Guanosine Diphosphate, Hydrogen, Purines, Pyrimidines, Ribonucleotides, Uridine Diphosphate