Extracellular cap domain is an essential component of the TRPV1 gating mechanism.

Construct The full-length 13-lined ground squirrel TRPV1 (GenBank KU877439.1 , residues 1–840) 5 used in cryo-EM studies was cloned into a pEG BacMam vector 35 , followed by thrombin cleavage site (LVPRG), enhanced green fluorescent protein (eGFP) for monitoring during expression and a C-terminal streptavidin affinity tag (WSHPQFEK) for purification. Expression and purification sqTRPV1 construct was expressed and purified as previously described for mTRPV3 (refs. 22 , 23 ), with minor modifications. Bacmids and baculoviruses were produced using a standard method 35 . Briefly, baculovirus was made in Sf9 cells for ~72 h (Thermo Fisher Scientific, mycoplasma test negative, GIBCO #12659017) and was added to the suspension adapted HEK 293 cells lacking N-acetyl-glucosaminyltransferase I (GnTI − , mycoplasma test negative, ATCC #CRL-3022) that were maintained in Freestyle 293 media (Gibco-Life Technologies #12338-018) supplemented with 2% FBS at 37 °C and 5% CO 2 . Twenty-four hours after transduction, 10 mM sodium butyrate was added to enhance protein expression, and the temperature was reduced to 30 °C. Seventy-two hours after transduction, the cells were harvested by centrifugation at 5471 × g for 15 min using a Sorvall Evolution RC centrifuge (Thermo Fisher Scientific), washed in phosphate-buffered saline (pH 8.0), and pelleted by centrifugation at 3202 × g for 10 min using an Eppendorf 5810 centrifuge. The cell pellet was resuspended in ice-cold lysis buffer, containing 20 mM Tris (pH 8.0), 150 mM NaCl, 0.8 μM aprotinin, 4.3 μM leupeptin, 2 μM pepstatin A, 1 mM phenylmethylsulfonyl fluoride and 1 mM β-mercaptoethanol (βME). Cells were subsequently lysed using a Misonix Sonicator with a preset programme (six cycles of 15 s “on” at the amplitude of 8 followed by 15 s “off”; this programme was repeated three times for optimal cell lysis) under constant stirring on ice. Unbroken cells and cell debris were pelleted using an Eppendorf 5810 centrifuge at 3202 × g and 4 °C for 10 min. The supernatant was subjected to ultracentrifugation in a Beckman Coulter ultracentrifuge using a Beckman Coulter Type 45Ti rotor at 186,000 × g and 4 °C for 1 h to pellet the membranes. The membrane pellet was mechanically homogenized and solubilized in the lysis buffer supplemented with 20 mM n-Dodecyl β- d -maltoside under stirring at 4 °C for 2 h. Insoluble material was removed by ultracentrifugation for 40 min in a Beckman Coulter Type 45Ti rotor at 186,000 × g , and the supernatant was added to strep resin and rotated for 14–16 h at 4 °C. Next, the resin was washed with ten column volumes of buffer containing 20 mM Tris (pH 8.0), 150 mM NaCl, 1 mM βME and 0.01% (w/v) glyco-diosgenin (GDN), and the protein was eluted with the same buffer supplemented with 2.5 mM d -desthiobiotin. The eluted protein was concentrated using a 100 kDa NMWL centrifugal filter (MilliporeSigma™ Amicon™) to 0.5 mg ml −1 . The sample was then digested with thrombin (1:200 mass ratio of thrombin to eluted protein) for 1 h at 22 °C, and centrifuged in a Sorvall MTX 150 Micro-Ultracentrifuge (Thermo Fisher Scientific) using a S100AT4 rotor for 30 min at 66,000 × g and 4 °C before injecting into a size-exclusion chromatography (SEC) column. The protein was further purified using a Superose™ 6 10/300 GL SEC column attached to an AKTA FPLC (GE Healthcare) and equilibrated in 150 mM NaCl, 20 mM Tris, 1 mM βME and 0.01% GDN (pH 8.0). The tetrameric peak fractions were pooled and concentrated using 100 kDa NMWL centrifugal filter (MilliporeSigma™ Amicon™) to 2.4–3.8 mg ml −1 .

Cryo-EM sample preparation and data collection

Au/Au grids were prepared as described in the literature 36 . Briefly, grids were prepared by first coating C-flat (Protochips, Inc., Morrisville, NC) CF-1.2/1.3–2Au mesh holey carbon grids with ~60 nm gold, using an Edwards Auto 306 evaporator. Subsequently, an Ar/O 2 plasma treatment (4 min, 50 watts, 35.0 sccm Ar, 11.5 sccm O 2 ) was used to remove the carbon with a Gatan Solarus (model 950) Advanced Plasma Cleaning System (Gatan, Pleasanton, CA, USA). The grids were again plasma treated (H 2 /O 2 , 25 s, 10 watts, 6.4 sccm H 2 , 27.5 sccm O 2 ) prior to sample application to make their surfaces hydrophilic. RTX and capsaicin were dissolved in dimethyl sulfoxide (DMSO) to a concentration of 10 mM and 50 mM, respectively, and further diluted in buffer containing 20 mM Tris (pH 8.0), 150 mM NaCl, 1 mM βME and 0.01% GDN to a concentration of 1.5 mM. Ligands were added to protein 30 min before grid preparation at the final concentration of 50 μM (RTX) and 300 μM (capsaicin). Images of frozen-hydrated particles of sqTRPV1 (3.8 mg ml −1 ) in apo state were collected on a Titan Krios transmission electron microscope (TEM; Thermo Fisher Scientific) operating at 300 kV, and equipped with a post-column GIF Quantum energy filter and a Gatan K3 Summit direct electron detection (DED) camera (Gatan, Pleasanton, CA, USA) using Leginon. A total number of 11,191 micrographs were collected in counting mode with an image pixel size of 1.06 Å and a defocus range of −0.8 to −2.5 µm, and energy filter slit of 30 eV. The total dose of ~65 e − Å −2 was attained by using a dose rate of ~30 e − pixel −1 s −1 across 50 frames for 2.5 s total exposure time. Images for frozen-hydrated particles of sqTRPV1 (2.4 mg ml −1 ) in the presence of capsaicin were collected on a Titan Krios TEM (Thermo Fisher Scientific) operating at 300 kV, and equipped with a post-column GIF Quantum energy filter and a Gatan K3 Summit DED camera (Gatan, Pleasanton, CA, USA) using Leginon. A total number of 9453 micrographs were collected in counting mode with an image pixel size of 0.83 Å and a defocus range of −1.0 to −2.5 µm. The total dose of ~58.5 e − Å −2 was attained by using a dose rate of ~16 e − pixel −1 s −1 across 50 frames for 2.5 s total exposure time. Images of frozen-hydrated particles of sqTRPV1 in the presence of RTX were collected on a Polara 300 kV TEM (FEI) with a Schottky field emission gun, cartridge loading system and a Gatan K3 Summit DED camera (Gatan, Pleasanton, CA, USA) using Leginon. A total number of 3253 micrographs were collected in counting mode with an image pixel size of 0.95 Å and a defocus range of −1.5 to −3.5 µm. The total dose of ~70.8 e − Å −2 was attained by using a dose rate of ~16 e − pixel −1 s −1 across 40 frames for 4 s of total exposure time.

Image processing

All processing was completed in RELION 37 and/or cryoSPARC 38 (Supplementary Table 1 ). The initial drift and beam-induced motions were corrected using MotionCor2 (ref. 39 ) algorithm implemented in RELION, and contrast transfer function (CTF) estimation was performed using Gctf 40 . Following CTF estimation, micrographs were manually inspected and those with outliers in defocus values, ice thickness and astigmatism, as well as micrographs with lower predicted CTF-correlated resolution were excluded from the rest of the processing pipeline (individually assessed for each parameter relative to overall distribution; no set threshold). Initial set of particles was manually picked in RELION and further classified into 100 2D classes to create templates for the template-based picking in RELION that were used for the final round of picking. Picked particles were further 2D- and 3D-classified in iterative classification using RELION and/or cryoSPARC. The reported resolutions and local resolution predictions of the final maps were calculated in RELION and cryoSPARC, with the resolution range estimated by the gold standard Fourier shell correlation 0.143 criterion 41 . EM density visualization was done in UCSF Chimera 42 and UCSF ChimeraX 43 . Model building To build models of TRPV1 in Coot 44 , we used the previously published cryo-EM structures of TRPV1 as guides. The models were tested for overfitting by shifting their coordinates by 0.5 Å (using shake) in Phenix, refining each shaken model against a corresponding unfiltered half map, and generating densities from the resulting models in Chimera. Structures were visualized, and figures were prepared in UCSF Chimera, UCSF ChimeraX and Pymol 45 . Patch-clamp electrophysiology sqTRPV1-pMO 5 or sqTRPV1 d606–628-pMO constructs were co-transfected with pcDNA3-GFP into HEK293T Δ PIEZO1 (ref. 46 ) using Lipofectamine 3000 (Thermo Fisher), according to the manufacturer’s instructions. The following day, cells were plated onto glass coverslips coated with Matrigel Matrix (BD Bioscience) and analysed by voltage-clamp electrophysiology within 24 h after plating. Bath solution contained (in mM): 140 KCl, 10 HEPES, 1 MgCl 2 , 10 glucose, pH 7.3 (pH adjusted with KOH) and the pipette solution contained (in mM): 140 NaCl, 5 KCl, 10 HEPES, 1 EGTA, 1 MgCl 2 , pH 7.4 (pH adjusted with NaOH). To form the gigaohm seal, positive (+10 mmHg) pressure was maintained inside the electrode while approaching the cell, and released upon touching the cell membrane. Spontaneous single-channel events were recorded in the cell-attached mode at room temperature at indicated voltages. Single-channel openings are shown as upward deflections, representing outward current. Currents were sampled at 25 kHz and filtered at 1 kHz. Single-channel data was analysed using Clampfit 10.8. Mean single-channel amplitudes were calculated by fitting the current-amplitude histograms with Gaussian curves. The unitary conductance for sqTRPV1 was calculated by fitting the slope of the current–voltage relationship to the linear equation. NPo and mean open time values were calculated from 10 s recordings using Clampfit. RTX (Alomone, 2 mM stock in ethanol) was applied by perfusion. Oocyte electrophysiology Defolliculated Xenopus laevis oocytes, stages V–VI ( Xenopus 1), were injected with sqTRPV1 mRNA (10 ng) and assayed 48–72 h later by two-electrode voltage clamp in calcium-free ND96, containing (in mM): 3 KCl, 96 NaCl, 2 MgCl 2 and 10 HEPES pH 7.4 (with NaOH), supplemented with 200 nM or 2 µM capsaicin. Currents were evoked by 200-ms voltage steps from −100 to 50 mV, in 10 mV increments, from a holding potential of 0 mV, and digitized at 2 kHz. The reversal potential was estimated from current–voltage plots. Capsaicin (Sigma-Aldrich, 1 mM stock in DMSO) was applied by perfusion. For temperature sensitivity experiments, currents were recorded in a gap-free mode at a holding potential of −80 mV. Temperature was controlled using the SC-20 in-line heater (Warner) and monitored by a thermistor placed in the chamber near the oocyte. Reporting summary Further information on research design is available in the Nature Research Reporting Summary linked to this article.

Supplementary information Supplementary Information Reporting Summary