GoKawiilCloning, cell culture and protein expression
N-terminally fused mEGFP constructs of TRPM8 were obtained using Gibson assembly to sub-clone TRPM8 genes into a pFastBac1 vector. Mutagenesis was carried out by either PCR with mutagenic primers, or by Gibson assembly, with Q5 high-fidelity polymerase. Construct sequences were verified using Sanger sequencing and whole-plasmid sequencing. Expi293F cells were maintained at 37 °C with 8% ambient CO 2 and grown to a density of 3–3.5 × 106 cells ml−1. To transduce cells for protein overexpression, a baculovirus (generated from two rounds of viral amplification in SF9 cells, according to known protocols) for the N-terminally fused mEGFP Pm or Hs TRPM8 with a human rhinovirus 3C proteolysis site was added dropwise, for a final concentration of 5–10% (vol%/vol%) to the flask while gently mixing the culture. Baculoviruses used in mammalian cell cultures were sequenced routinely by amplifying the coding region using PCR (M13 primer sites), gel extracting the amplicon and subjecting the purified DNA fragments to Sanger sequencing. Virally transduced Expi293F cultures were left at 37 °C with 8% CO 2 for 16 h. To boost expression, the cultures were supplemented with 10 mM sodium butyrate and transferred to an incubator at 30 °C with 5% CO 2 and allowed to incubate for a total of 72 h (post-transduction). Cells were then collected by centrifugation (3,000g, 10 min, 4 °C), washed once with 1× Dulbecco’s phosphate buffered saline (DPBS) pH 7.40 by gently resuspending the cell pellet, and collected by centrifugation (3,000g, 10 min, 4 °C). Cell pellets were flash frozen in liquid nitrogen and kept in a −80 °C freezer until use.
Detergent purification for HDX–MS and cryo-EM samples
All purification steps were carried out at 4 °C or over ice, unless otherwise noted. The avian or human TRPM8 channel was purified with the same general protocol. Briefly, a cell pellet grown from 0.5 l Expi293F cell culture (typically 7–10 g in total) was resuspended into lysis buffer (50 mM HEPES-NaOH pH 7.40, 150 mM NaCl, 50 μg ml−1 DNase I, 10 μg ml−1 RNase, 0.2 mM AEBSF, 50 μg ml−1 soy trypsin inhibitor, 10 μg ml−1 leupeptin, 10 μg ml−1 pepstatin, 1 mM benzamidine-HCl and aprotinin) for a total dilution of 4:1 lysis buffer:cell pellet. The resuspension was adjusted to 0.5% lauryl maltose neopentyl glycol (LMNG)/0.5% glycodiosgenin (GDN), and rotated gently for 1 h at 4 °C. After centrifugation (35,000g, 30 min, 4 °C), the lysate was applied to 1 ml anti-GFP-nanobody-conjugated Sepharose 4B resin (prepared in-house) for 2 h at 4 °C with gentle rotation. The TRPM8-bound resin was washed extensively with wash buffer (20 mM HEPES-NaOH pH 7.40, 150 mM NaCl, 0.05% GDN), then digested in the presence of 10 μg ml−1 PreScission protease (prepared in-house) along with 1 mM dithiothreitol for 2 h. The eluate was concentrated to 0.2 ml and injected onto a Superose 6 Increase 10/300 GL column pre-equilibrated in SEC buffer (20 mM HEPES-NaOH pH 7.40, 150 mM NaCl, 0.005% GDN). For the Pm structures obtained at pH 9, cells were resuspended into lysis buffer containing 50 mM Tris-HCl pH 9.0, 150 mM NaCl, 5 mM CaCl 2 , 50 μg ml−1 DNase I, 10 μg ml−1 RNase, 0.2 mM AEBSF, 50 μg ml−1 soy trypsin inhibitor, 10 μg ml−1 leupeptin, 10 μg ml−1 pepstatin, 1 mM benzamidine-HCl and aprotinin. The resuspended cells were extracted using 0.5% LMNG/0.5% GDN and further purified with anti-GFP nanobody resin. For cryo-EM, the final sample buffer contained 20 mM TRIS pH 9.00, 150 mM NaCl, 5 mM CaCl 2 and 0.0025% GDN.
Cell vesicle purification for cryo-EM
A cell pellet grown from 1.0 l Expi293F cell culture (typically 20–25 g in total) was resuspended into lysis buffer (50 mM HEPES-NaOH pH 7.40, 300 mM KCl) supplemented with 2 mM CaCl 2 , 50 μg ml−1 DNase I, 10 μg ml−1 RNase, 0.2 mM AEBSF, 50 μg ml−1 soy trypsin inhibitor, 10 μg ml−1 leupeptin, 10 μg ml−1 pepstatin, 1 mM benzamidine-HCl, and aprotinin. The mixture was homogenized roughly using a Dounce homogenizer over ice, and the resuspended cells were adjusted to a final volume of 200 ml. The cells were transferred to a metal beaker over ice and lysed using a probe-tip sonicator (2 min total, 5 s on/15 s off, 60% amplitude). The lysate was clarified by centrifugation (12,000g, 15 min, 4 °C) and the supernatant was filtered immediately through a 0.8-μm mixed cellulose acetate filter. The filtrate was then passed through a 10-ml bed of ion exchange resin (Q-Sepharose FastFlow, Cytiva) packed in a gravity column and pre-equilibrated in lysis buffer. The flowthrough was collected and adjusted to 0.5 mM fluorinated fos-choline 8 (Anatrace), which is below its critical micelle concentration of around 3 mM, for a final volume of 240 ml. Vesicles were batch bound to 5 ml anti-GFP nanobody Sepharose 4B resin (prepared in-house) for 2 h at 4 °C with constant rotation. After binding, the resin was collected by centrifugation (500g, 1 min) and transferred to a gravity column. The affinity resin was washed extensively, and the resuspended beads were adjusted to 10 μg ml−1 PreScission protease (prepared in-house) along with 1 mM dithiothreitol. Bound vesicles were eluted by proteolysis for 2 h, and the eluate was collected and concentrated using an Amicon ultra-centrifugal filter (100 kDa, regenerated cellulose) to a final volume of 0.5 ml. After brief centrifugation (10,000g, 2 min, 4 °C), the sample was injected onto a Superose 6 Increase 10/300 column (Cytiva) pre-equilibrated with the same elution buffer. The void volume peak (typically 8.5–9 ml) was pooled and concentrated to 30 μl using a 0.5-ml Amicon ultra-centrifugal filter (100 kDa, regenerated cellulose) and kept on ice. Concentrated vesicle samples were used immediately for or warmed to 37 °C for 5–10 min before cryo-EM sample preparation.
Cryo-EM grid preparation, screening and data collection
Cryo-EM grids for vesicle samples were prepared using either Quantifoil R1.2/1.3 400 mesh holey carbon grids, Quantifoil R1.2/1.3 300 mesh holy carbon grids coated with ultrathin continuous carbon. Detergent samples were prepared using Quantifoil Au R1.2/1.3 300 mesh holey carbon grids. Grids were glow discharged (15 mA, 30 s) and placed in a Vitrobot Mark IV (FEI Company) set at 4 °C with 100% humidity. For vesicle samples, 2.5 μl concentrated sample was applied directly to grids and, after 5 s of incubation, grids were blotted for 2–5 s and plunge-frozen immediately in liquid ethane cooled in a Dewar of liquid nitrogen. To obtain menthol-bound datasets, vesicles were kept at 4 °C for Pm TRPM8 or ambient temperature (21–24 °C) for the human channel, and a final concentration of 1 mM menthol was added from a stock of 200 mM menthol dissolved in 100% ethanol. After mixing the menthol-treated samples gently at respective temperatures, samples were applied to grids, for a total agonist treatment time of 2–15 min. For detergent samples, 2.5 μl of purified TRPM8 concentrated to 14–16 mg ml−1 was applied directly to grids and, after 10 s of incubation, grids were blotted for 5 s before vitrifying. Grids were transferred and stored under liquid nitrogen until screening or data collection. Grids were screened with a Talos Arctica or Glacios 200 kV cryo-TEM (ThermoFisher Scientific) equipped with a K3 direct detector camera (GATAN), and screening datasets were obtained using SerialEM. Data were collected at the University of California San Francisco (UCSF) Cryo-EM Facility with a Titan Krios cryo-TEM equipped with a K3 camera and Bio Quantum post-column energy filter (counting mode pixel size of 0.8189 Å per pixel after 2× Fourier binning) or at the Janelia Cryo-EM Facility on a Krios equipped with a cold field-emission gun, Selectris X energy filter and Falcon 4i camera (physical pixel size of 0.94). For data collection at UCSF, the zero-loss energy selection slit was set to 10 eV. For vesicle datasets, the target defocus was set at −1.0 to −2.5 µm and, for detergent datasets, the target defocus was set at −0.5 to −2.0 µm.
Cryo-EM data processing and refinement
Dose-weighted, motion-corrected micrographs were obtained using MotionCor2 (ref. 52) and Fourier cropped by a factor of two, or by pre-processing using cryoSPARC53. An initial model of Pm TRPM8 in a membrane was generated in cryoSPARC with a screening dataset obtained on a Glacios cryo-TEM (2,420 micrographs) and refined to approximately 5 Å (440 pixel box size, 0.73 Å per pixel). This initial reconstruction was used to generate a reference volume using relion_image_handler (512 pixel box size, 0.8189 Å per pixel) that was subsequently used in template picking for further processing of datasets obtained on the Krios microscopes. For the vesicle datasets, particles were identified using a combination of Topaz particle picking54 and template picking in cryoSPARC, followed by several rounds of heterogeneous refinement and two-dimensional classification to deplete non-TRPM8 or obvious junk particles. After generating a consensus refinement with cryoSPARC non-uniform refinement, further classification was carried out in either RELION5 (ref. 55) or cryoSPARC. Three-dimensional classification used either a mask capturing only the channel region, or a spherical mask to limit bilayer signal. Further two-dimensional classification of three-dimensional refinements without a circular mask revealed that a range of membrane curvatures are included in reconstructions, indicating that, in our datasets, three-dimensional classes result from a distribution, rather than specific, membrane curvatures (Supplementary Fig. 1c). For the GDN-purified TRPM8 datasets, an initial reference map was generated from each dataset ab initio from particles identified with blob picker in cryoSPARC from a small sub-set of the micrographs. The ab initio model was then subjected to non-uniform refinement without symmetry and used subsequently to generate templates to process the remaining dataset. After several rounds of heterogeneous refinement, a consensus refinement was generated in cryoSPARC for further three-dimensional classification with cryoSPARC. Three-dimensional class averages were subjected to non-uniform refinement using cryoSPARC. Local resolution estimation of refined maps was carried out in cryoSPARC and visualized in UCSF ChimeraX.
... continue reading