Computational protocol: Exploring local solvation environments of a heme protein using the spectroscopic reporter 4-cyano-l-phenylalanine††Electronic supplementary information (ESI) available. See DOI: 10.1039/c8ra02000k

Similar protocols

Protocol publication

[…] As-purified untagged Cs H-NOX I5pCNF protein was buffer exchanged into 20 mM TEA (pH 7.5) using a PD10 desalting column and concentrated to 25 mg mL–1 using a 5000 kDa membrane spin concentrator at 4000 rpm. The protein was crystallized via sitting drop vapor diffusion by mixing 1 μL of protein with 1 μL of precipitant solution (Hampton Index Condition 80 : 0.2 M ammonium acetate, 0.1 M HEPES, pH 7.5, 25% PEG 3350) and equilibrating the drop against 99 μL of precipitant solution. Red crystals formed within 4 days, were mounted on loops, and then flash frozen in liquid nitrogen for storage.X-ray diffraction data were collected at the Advanced Photon Source (APS) at Argonne National Laboratory (ANL) on NE-CAT 24-ID-E beamline (λ = 0.979180 Å). Crystals diffracted to 1.85 Å and data were processed in HKL2000 () in space group P21. The protein crystallized with two protein monomers in the asymmetric unit. The protein component (heme and water molecules removed) of the WT structure (PDB ID ; 1U55) with I5 mutated to an alanine was used for molecular replacement in PHASER. The resulting structure was refined in Phenix against 1.85 Å data with alternate cycles of positional and atomic displacement parameter refinement with manual refitting in Coot. Following a round of refinement with alanine modeled at site 5, pCNF was modeled into the difference density at this site (Fig. S1). The final model contains 2 chains with 185 residues in chain A and 187 residues in chain B. Two heme molecules and 197 water molecules were also modeled to produce the final refinement parameters of Rwork of 19.8% and Rfree of 25.8%. […]

Pipeline specifications

Software tools PHENIX, Coot
Applications Small-angle scattering, Protein structure analysis
Organisms Caldanaerobacter subterraneus, Escherichia coli, Dipturus trachyderma
Chemicals Amino Acids, Heme, Hydrogen, Superoxides