Microloop

GLIC wt in 100 mM NaCl, 10 mM Tris-HCl (pH 7.4), and 0.5 mM DDM was concentrated to between 9–10 mg/ml with an Amicon Ultra 50 KDa cutoff concentrator (EMD Millipore, Billerica, MA). Prior to crystallization setup, the protein was supplemented with 50 μM DHA (from 300 mM DHA stock in ethanol) and 0.5 mg/ml E.coli polar extract (Avanti Polar Lipids) and incubated on ice for 1 hr. The protein was crystallized at 4°C by sitting drop vapor diffusion in Cryschem plates (Hampton Research, Aliso Viejo, CA) with a 1:1 mixture (1 μl each) of protein and reservoir solution (225 mM ammonium sulfate, 50 mM sodium acetate, pH 3.9–4.2 and 9–12% PEG4000). Crystals typically formed within one week and typically took 2–3 weeks to reach full size. The crystals were cryoprotected by adding 6 μL reservoir solution supplemented with 30% ethyleneglycol to the drop, and directly frozen in liquid nitrogen using appropriately sized microloops (MiTeGen, Ithaca, NY) or cryoloops (Hampton Research).

X-ray data were collected using the macromolecular crystallography beamline F1 at the Cornell High Energy Synchrotron Source (CHESS), which provided a 12.693 keV X-ray beam collimated to 0.1 mm diameter. Room-temperature data collection was performed using the plastic capillary sheathing method^{48 (link)}. Crystals were harvested using low-scatter kapton loops (MicroLoops, MiTeGen), taking care to minimize the amount of solvent surrounding the crystal, and placed within 2 mm diameter, 25 μm wall poly(ethylene terephthalate) capillaries (MicroRT, MiTeGen) with 10 μL reservoir solution in the tip. During X-ray exposure, images were recorded every 0.1^∘ using a pixel-array detector (Pilatus3 6M, Dectris) while rotating the sample at 1^∘ s⁻¹. A dose rate of 1.3 kGy s⁻¹ was estimated assuming a flux of 2.5 × 10¹⁰ photons s⁻¹ and a mass energy-absorption coefficient⁴⁹ of μ_en ∕ ρ = 2.0 cm² g⁻¹. After 50 s of exposure (~65 kGy), the sample was refreshed by translating to a new spot or replacing the crystal. A background dataset was collected for each crystal by translating the sample out of the beam along the spindle axis and collecting 1 s exposures while rotating at 1^∘ s⁻¹.

The diffraction patterns were measured at 270 K on a Rigaku FRX rotating anode X-ray generator at the copper wavelength (Kα, λ = 1.54 Å). The source is equipped with Osmic Varimax HF optics and a Rigaku© HyPix6000 detector on a 2θ arm of a Rigaku partial chi AFC11 goniometer. The samples were mounted in MicroLoops from MiTeGen on a goniometer head under the cryostream nitrogen flux. The diffraction patterns correspond to a 360° rotation along the phi axis (perpendicular to the direct beam with omega and chi axes at the 0 position) with an exposure time of 360 s. Data were integrated with CrysalisPro (Rigaku Oxford Diffraction, Ltd., Yarnton, Oxfordshire, England). After performing the first X-ray diffraction experiments, we used the remaining sample of Hfq11 filaments, resuspended the filaments in H₂O, and centrifuged the filaments to discard the water. The procedure was repeated multiple times to record a second set of diffraction data.

Purified desalted NTSR1-LF-T4L and NTSR1-ELF-T4L were adjusted to 100 μM Tris (2-carboxyethyl) phosphine hydrochloride (TCEP) and 350 μM NTS_8–13 and concentrated to an estimated 30 mg ml⁻¹ using a 100,000 MWCO concentrator (Amicon Ultra, Millipore). After the addition of NTS_8–13 to 1.5 mM and centrifugation (TLA 120.1 rotor, 128,000g, 30 min, 4 °C, Beckman), the sample was mixed with 1.5 parts by weight of a mix of monoolein with cholesterol (10:1) using the two-syringe method44 (link). The resulting lipidic cubic phase45 (link) mix was dispensed in 65–75 nl drops onto Laminex plates (Molecular Dimensions) and overlaid with 750 nl (NTSR1-LF-T4L) or 875 nl (NTSR1-ELF-T4L) precipitant solution using a Mosquito LCP robot (TTP Labtech). Crystals of NTSR1-LF-T4L grew at 20 °C after 3 days in precipitant solution consisting of 19.8–23.4% (v/v) PEG 400, 80 mM Hepes pH 7.0–7.4, 2 mM TCEP and 50 mM lithium citrate. Crystals of NTSR1-ELF-T4L grew in precipitant solution consisting of 16–24% (v/v) PEG 400, 75 mM Hepes pH 7.0–8.0, 1.7 mM TCEP, 32 mM lithium citrate and 0.9 mM NTS_8–13. Crystals were harvested directly from LCP using micro-loops (MiTeGen) and immediately flash frozen in liquid nitrogen without adding extra cryoprotectant.