Computational protocol: Modulating the DNA polymerase β reaction equilibrium to dissect the reverse reaction

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Protocol publication

[…] Binary complex crystals with nicked DNA were grown as previously described. The time-lapse crystallography was performed as before and is briefly summarized here. Binary pol β/DNA complex crystals were first transferred to a cryosolution containing 15% ethylene glycol, 50 mM imidazole, pH 7.5, 20% PEG3350, 90 mM sodium acetate, 2 mM PNP and 50 mM CaCl2 for 1 h. These ground state (GS) ternary complex crystals were then transferred to a cryosolution containing 200 mM MgCl2 for varying times. All reactions were stopped by freezing the crystals at 100K prior to data collection at the home source, 1.54 Å, or the Advanced Photon Source, 1.0 Å (Argonne National Laboratory). In house data collection was done on a SATURN92 CCD detector system mounted on a MiraMax-007HF rotating anode generator. This allows for anomalous data detection after phasing by molecular replacement. Remote data collection was done at Southeast Regional Collaborative Access Team (SER-CAT) BM-22 beamline at the Advanced Photon Source (Argonne National Laboratory) with the MAR225 area detector. Data were processed and scaled using the HKL2000 software package. Initial models were determined using molecular replacement with the open binary (PDB ID 3ISB) or closed ternary (PDB ID 2FMS) structures of pol β and all Rfree flags were taken from the starting model. Refinement was carried out using PHENIX and model building using Coot,. The metal-ligand coordination restraints were generated by ReadySet (PHENIX) and not utilized until the final rounds of refinement. Partial catalysis models were generated with both the reactant and product species and occupancy refinement was performed. The structural figures were prepared in Pymol (Schrödinger, LLC) and all density maps were generated after performing simulated annealing. Ramachandran analysis determined 100% of non-glycine residues lie in allowed regions and at least 97% in favored regions. […]

Pipeline specifications

Software tools PHENIX, Coot, PyMOL
Application Protein structure analysis
Chemicals Nucleosides, Nucleotides, Oxygen