Rapid experimental SAD phasing and hot-spot identification with halogenated fragments

4-Bromopyrazole and 4-iodopyrazole bind to many small molecule binding hot spots in target proteins. This promiscuous binding enables the use of these compounds for experimental phase determination by single-wavelength anomalous dispersion (SAD). The low cost and safety of the compounds make them excellent choices for addition to the protein crystallographer’s toolkit.


S1.1. Expression, purification, and crystallization of HIV-1 RT
HIV-1 RT construct RT52A (crystallization optimized mutant) was expressed/purified/crystallized as described previously (Bauman et al., 2008;Bauman et al., 2013). A plasmid encoding RT52A was transformed into BL21-CodonPlus®-RIL (Stratagene, La Jolla, CA) competent cells and grown on LB-agar plates containing 35 mg/liter streptomycin and 0.1% (w/v) glucose. A single colony was picked and grown overnight in LB + 35 mg/liter streptomycin and 0.5% glucose at 37°C with shaking. The overnight culture was diluted 100-fold into a culture of 4 liters and incubated at 37°C with shaking. At an OD600 of 0.9, the cells were induced with 1 mM IPTG and incubated for three hours prior to pelleting and storage at -80°C.

S1.3. Crystallization of proteinase K
Proteinase K was purchased from Sigma-Aldrich (St. Louis, MO) and crystallized as described previously (Beck et al., 2010). 20 mg/ml proteinase K was mixed with an equal volume of 100 mM Tris pH 7.2 and 1.28 M ammonium sulfate.
The RT compound/cryo soaking solutions were prepared with crystallization well solution with the addition of 80 mM L-arginine, 5% (v/v) ethylene glycol, and 20% (v/v) DMSO (containing compound). 80 mM L-arginine was included to improve the solubility of the ligands (Bauman et al., 2013). Crystals of RT52A-rilpivirine were harvested three months after crystals formed. The crystals were placed in compound/cryo soaking drops for ten minutes before flash freezing in liquid nitrogen. volume of 10% and concentration of 10 mM) for 2 hours before flash freezing in liquid nitrogen.
Proteinase K ligand soaks were performed by addition of 500 mM iodopyrazole directly to well solution with the addition of 30% (v/v) glycerol for cryoprotection. Crystals were soaked in the ligand solution containing cryoprotectant for ten minutes before flash freezing in liquid nitrogen.

S1.5. Data collection and processing
X-ray diffraction data collection was performed at the Cornell High Energy Synchrotron Source (CHESS) F1 beamline, the National Synchrotron Light Source (NSLS) beamline X25, and the CABM Macromolecular X-ray Crystallography Facility. The diffraction data were indexed, processed, scaled, and merged using HKL2000 (Otwinowski et al., 1997).

S1.6. DFT calculations
The geometry was fully optimized for each compound in its singlet ground state using M06 functional (Zhao et al., 2008) as implemented in Spartan'14 v1.1.0 (Spartan'14, Wavefunction Inc, Irvine, CA) with the 6-311+G** basis set, which in the valence space is of triple-zeta quality and of double-zeta-quality polarization functions (Glukhovstev et al., 1995;Yao et al., 2007;Ribieto et al., 2012;Shallangwa et al.,2014). The structures were verified to be at a minimum energy without any imaginary frequencies. Single point energy calculations were computed using the 6-311++G** basis set with a diffuse function on all the atoms including the hydrogens using the optimized structures. Electrostatic potential energy maps were generated at a 0.002 (arbitrary unit) isosurface. The electrostatic surface potential (ESP) provides a charge density distribution which gives a visual indication of probable interactions of a point-like charged species with organic molecules (Naray-Szabo et al., 1995;Mircescu et al., 2011).

Figure S1
Calculated electrostatic potential surfaces for 4-bromopyrazole, 4-iodopyrazole, and related analogs. Full table of derivatives tested for binding to RT-rilpivirine with density functional theory calculations of the electrostatic potential energy surface.