Ammonium citrate

H₁R-T4L was expressed in yeast Pichia pastoris. Ligand binding assays were performed as described in Methods. Pichia pastoris membranes were solubilized using 1% (w/v) n-dodecyl-β-D-maltopyranoside and 0.2% (w/v) cholesteryl hemisuccinate, and purified by immobilized metal ion affinity chromatography (IMAC). After IMAC, the C-terminal GFP was cleaved by Tobacco Etch virus (TEV) protease. Then the sample mixture was passed through IMAC to remove the cleaved His-tagged GFP and TEV protease. Receptor crystallization was performed by lipidic cubic phase (LCP) method. The protein-LCP mixture contained 40% (w/w) receptor solution, 54% (w/w) monoolein, and 6% (w/w) cholesterol. Crystals were grown in 40-50 nl protein-laden LCP boluses overlaid by 0.8 μl of precipitant solution (26-30% (v/v) PEG400, 300 mM ammonium phosphate, 10 mM MgCl₂, 100 mM Na-citrate pH 4.5 and 1 mM doxepin) at 20 °C. Crystals were harvested directly from LCP matrix and flash frozen in liquid nitrogen. X-ray diffraction data were collected at 100 K with a beam size of 10 × 10 microns on the microfocus beamline I24 at the Diamond Light Source (UK). Data collection, processing, structure solution and refinement are described in Methods.

Except where indicated, we grew all strains (M. smegmatis, M. smegmatis-pBP10, Mtb H37Rv and Mtb H37Rv-pBP10 at 37 °C in Middlebrook 7H9 medium (Becton Dickinson) with 0.05% Tween-80 and albumin, dextrose, catalase (Middlebrook ADC Enrichment, BBL Microbiology) with or without 30 μg ml⁻¹ kanamycin or on Middlebrook 7H10 (Becton Dickinson) medium with oleic acid plus albumin, dextrose, catalase (Middlebrook ADC Enrichment, BBL Microbiology) with or without 30 μg ml⁻¹ kanamycin. We grew strains to an optical densityof ~1 at A₆₀₀ (OD₆₀₀) and stored them in 15% glycerol at −80 °C. For in vitro log-phase experiments, we maintained replicate rolling cultures in log phase by subculturing every 1–3 d for ~40 generations in the absence of antibiotics. We recorded each dilution to calculate total bacterial numbers. We determined the percentage of mycobacteria carrying plasmid as the number of CFUs on 7H10 agar with kanamycin over the number of CFUs on 7H10 agar without kanamycin. We compared different media conditions to achieve a variety of growth rates, including 7H9 medium diluted with sterile water, with 0.05% Tween to minimize clumping. For the starvation experiments, we grew Mtb in minimal medium consisting of 3.33 mM L-asparagine, 5.74 mM KH₂PO₄, 10.6 mM Na₂HPO₄, 40.6 μM MgSO₄·7H₂0, 4.50 μM CaCl₂, 0.619 μM ZnSO₄ and 50 mg/L ferric ammonium citrate. We measured log-phase growth rates at the initiation of growth (high plasmid frequency) and again at the end (low plasmid frequency) to assess the fitness cost of plasmid carriage. We grew hypoxic cultures in 7H9 medium in spinner flasks with 2% oxygen flow-through as previously described32 (link). Before plating, we brought cultures to their original volumes to account for evaporation from the constant air flow. For in vitro stationary phase and starvation experiments with Mtb, we grew rolling cultures for ~20 d without subculturing. For the hypoxic experiments, we maintained Mtb in 7H9 medium in 96-well plates placed inside airtight bags with a Gaspak EZ Anaerobe Container System Sachet (Becton Dickinson) and a Gaspak Dry Anaerobic Indicator Strip (Becton Dickinson).