Computational protocol: The Cation−π Interaction Enables a Halo-TagFluorogenic Probe for Fast No-Wash Live Cell Imaging and Gel-FreeProtein Quantification

Similar protocols

Protocol publication

[…] Halo [15 mg/mL in 20 mM HEPES (pH 7.5)] was purified as described above and crystallized at room temperature using the sitting-drop vapor diffusion method with 0.1 M sodium chloride, 0.1 M HEPES (pH 7.5), and 1.6 M ammonium sulfate as the precipitating solution. The crystals were mounted on loops and soaked in a well solution supplemented with 25% (v/v) ethylene glycol prior to being flash-frozen in liquid nitrogen. The Halo–P9 conjugate [12 mg/mL in 20 mM HEPES (pH 7.5)] was generated as described and crystallized at room temperature using the hanging-drop vapor diffusion method with 0.2 M ammonium acetate, 0.1 M sodium acetate (pH 4.6), and 20% (w/v) PEG 3000 as the precipitating solution. The crystals were mounted on loops and soaked in a well solution supplemented with 21% (v/v) ethylene glycol prior to being flash-frozen in liquid nitrogen.Crystallographic data sets were collected at the Life Sciences Collaborative Access Team and GM/CA Collaborative Access Team beamlines at the Advanced Photon Source and processed using HKL2000. The Halo structure was determined by molecular replacement with PHASER as implemented within the CCP4 software package, using the Rhodococcus rhodochrous haloalkane dehalogenase structure (Protein Data Bank entry 1BN7) as the initial search model. Phases for the Halo–P9 structure were obtained via the same procedure, but with the coordinates of the apo Halo structure used as the search model. Model building and refinement were performed with Coot and Refmac5, respectively. Coordinates and geometric restraints for the P9 ligand were generated in JLigand. Structure validation and Ramachandran analyses were performed using the Molprobity server. Figures were generated with the PyMOL Molecular Graphics software package (Schrödinger LLC).The asymmetric unit of the apo Halo structure is composed of two Halo monomers related by noncrystallographic symmetry. Residues 3–292 are modeled in each chain, and the final model also includes 437 water molecules, five ethylene glycol molecules, and three chloride ions. Molprobity analysis indicates 97.4% of side chains in favorable rotamer conformations (0.6% of side chains are outliers) and a clash score of 0.43 (100th percentile). The Halo–P9 conjugate also contains two monomers in the asymmetric unit, and the final model consists of residues 3–292 in chain A and 3–297 in chain B, 72 water molecules, two ethylene glycol molecules, two P9 ligands, and two chloride ions. Molprobity analysis of this structure shows 96.3% of rotamers in favorable conformations (0.2% outliers) and a clash score of 1.07 (100th percentile). […]

Pipeline specifications

Software tools CCP4, Coot, REFMAC5, MolProbity, PyMOL
Application Protein structure analysis
Chemicals Benzene, Tryptophan