Computational protocol: Characterizationof a Cross-Linked Protein–NucleicAcid Substrate Radical in the Reaction Catalyzed by RlmN

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Protocol publication

[…] Crystals of RlmN C118A (10 mg/mL in 10 mM HEPES, pH 7.5) were obtained in a Coy anaerobic chamber using the hanging-drop vapor-diffusion method with 7.5% (w/v) PEG 6000, 0.1 M HEPES, pH 7.5, 5% (v/v) 2-methyl-2,4-pentanediol as the precipitant and 0.25 M LiCl as the well solution. Crystals appeared within 1 week at room temperature and were mounted on rayon loops for data collection. Samples were flash-frozen by direct plunge into liquid nitrogen after cryoprotection in precipitating solution supplemented with 30% (v/v) PEG 400. To determine the structure of C118A RlmN with SAM, crystals were soaked in a 5 mM solution of SAM in mother liquor for 30 min at room temperature and harvested as described above. All crystallographic data sets were collected at the Life Sciences Collaborative Access Team beamlines at the Advanced Photon Source and processed using the HKL2000 software package. The structure was solved by molecular replacement with PHASER using the structure of wt Ec RlmN (PDB accession code 3RF9) as the search model. Refinement and model building were performed with REFMAC5 and Coot. In each structure, two RlmN molecules were found in the asymmetric unit. In the C118A RlmN+SAM structure, the final model consists of residues 17–348 in chain A, residues 17–375 in chain B, two [4Fe-4S] clusters, and two molecules of SAM. In the C118A RlmN structure, the final model consists of residues 17–352 and residues 361–374 in chains A and B (residues 353–360 are disordered in both chains), two [4Fe–4S] clusters, and 231 water molecules. Ramachandran plots generated with Molprobity indicate 100% of residues in allowed and additionally allowed regions. Structural superpositions were performed using the secondary-structure matching method. Electron density maps were calculated with FFT. Figures were generated with the PyMOL Molecular Graphics System (Schrödinger, LLC). reports all data collection and refinement statistics. […]

Pipeline specifications

Software tools REFMAC5, Coot, MolProbity, PyMOL
Application Protein structure analysis
Organisms Dipturus trachyderma
Chemicals Adenine, Carbon, S-Adenosylmethionine