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1) Product Images from "Vanilloid-dependent TRPV1 opening trajectory from cryoEM ensemble analysis"
Article Title: Vanilloid-dependent TRPV1 opening trajectory from cryoEM ensemble analysis
Journal: Nature Communications
Figure Legend Snippet: RTx-dependent long-range conformational changes in TRPV1. a Cylinder representation of TRPV1 in turquoise (one subunit) and gray (the rest of the channel). The approximate distances from the RTx binding site (reference residue Y511) to subdomains are shown. b The cryo-EM densities (surface) and respective models (sticks) depicting close-up views of the vanilloid binding sites in TRPV1 C, RTx (skyblue), thresholding 0.19, TRPV1 IO, RTx (yellow), thresholding 0.04, and TRPV1 O, RTx (pink), thresholding 0.033. c The cryo-EM densities (surface) and respective models (sticks) depicting close-up views of the selectivity filter in TRPV1 C,RTx (skyblue), thresholding 0.19, TRPV1 IC,RTx (green), thresholding 0.04, TRPV1 IO,RTx (yellow), thresholding 0.1, and TRPV1 O,RTx (pink), thresholding 0.033. d – e Close-up view of the overlays of TRPV1 C, RTx (skyblue), TRPV1 IO, RTx (yellow), and TRPV1 O, RTx (pink) regarding the cytoplasmic domain, and S6 gate e , respectively.
Techniques Used: Binding Assay, Cryo-EM Sample Prep
Figure Legend Snippet: PH-S5-S6 triad hydrogen bond network in TRPV1 for RTx gating. a The cryo-EM maps (surface) and respective models (sticks) depicting the tripartite hydrogen bond network of PH-S5-S6 in TRPV1 C, RTx (skyblue), thresholding 0.15, TRPV1 IC, RTx (yellow), thresholding 0.045, TRPV1 IO, RTx (gold), thresholding 0.1, and TRPV1 O, RTx (pink), thresholding 0.04. The black dotted-lines indicate hydrogen bonds. The red dotted-lines indicate distance measurements between atoms where hydrogen bonds are broken. b – e TRPV1 Y584F and T641A reduce large cation permeabilty (YO-PRO-1, M.W. 376 Da) in the presence of RTx. Representative inside-out current traces of TRPV1 WT b , TRPV1 Y584F c , and TRPV1 T641A d . Current traces for basal, RTx (200 nM) activation (red trace) and intracellular application of 10 μM YO-PRO-1 (blue trace). e Summary of current inhibition by YO-PRO-1 (10 µM) of TRPV1 WT, TRPV1 Y584F and TRPV1 T641A after application of a saturating concentration of RTx (200 nM). Data are presented as mean ± s.e.m.; P
Techniques Used: Cryo-EM Sample Prep, Activation Assay, Inhibition, Concentration Assay
Figure Legend Snippet: RTx-mediated TRPV1 gating mechanism. In the unstimulated apo state, the channel is closed both at the selectivity filter and S6 gate. 1 Initially, RTx binds with no significant conformational changes. 2 RTx binding induces S6 gate dilation. 3 The M644 sidechain flips outward, and the CD moves towards the channel core. 4 Finally, rearrangement of the PL, PH, TJ results in further dilation of the S6 gate.
Techniques Used: Binding Assay
Figure Legend Snippet: Thermal titration cryo-EM experiment and the effect of RTx on TRPV1 heat sensitivity. a A representative macroscopic current time-course (top panel) recorded from a HEK-293T cell expressing rat TRPV1 in response to the temperature ramp (10–50 °C) at a membrane potential of −60 mV and then followed by a saturating concentration of RTx (50 nM) and 20 µM ruthenium red (RR). The dashed line indicates zero current. The recorded temperature is shown in the middle panel. The Arrhenius plot for the temperature activation was shown in the bottom panel. Fitted Q 10 values for high (blue line) and low (red line) temperature ranges are shown. b A representative time-course recording for RTx-bound TRPV1 temperature sensitivity. First the channel was challenged by 10 nM RTx for ~20 s followed by a temperature ramp (10–48 °C), then a saturating concentration of RTx (50 nM) was introduced, and finally RR (20 µM) was applied to completely block the channel. The dashed line indicates zero current. The recorded temperature is shown in the middle panel and the Arrhenius plot for the temperature activation is shown in the bottom panel. Fitted Q 10 values for high and low temperature (T) ranges are shown. c Q 10 values as a function of I/I 50nM RTx for low and high temperature ranges. Each experiment was conducted as shown in a and b . The low T range Q 10 value is steady at 1.7, while the high T range Q 10 rapidly collapses from ~38 to ~3. Each pair of high and low temperature sensitivity data points represents independent time-course recordings from individual cells ( n = 17 cells). Source data are provided as a Source Data file. d Representative micrographs of TRPV1 recorded in the presence of 50 μM RTx at 4 °C, 25 °C and 48 °C, respectively. Cryo-EM maps of RTx-TRPV1 determined at 4 °C (class I, class II, and class III), 25 °C (class A and class B), and 48 °C (class α). Note the differences between central pore sizes amongst different classes at 4 °C. The classes not found in each dataset are shown as transparent. The pie charts depict particle distributions among classes for each dataset along with representative micrographs. Each pie chart represents an average value for four independent data processes (Supplementary Fig. 2a , b ).
Techniques Used: Titration, Cryo-EM Sample Prep, Expressing, Concentration Assay, Activation Assay, Blocking Assay
Figure Legend Snippet: Pore comparison across TRPV1 C, RTx , TRPV1 IC, RTx , TRPV1 IO, RTx , and TRPV1 O, RTx . The cryo-EM densities (grey surface) and respective models (cartoon) depicting bottom-up views of the S6 gate (top), top-down views of the selectivity filter (middle), top-down views of the monomeric outer pore (bottom), and local estimated resolutions for TRPV1 C, RTx a , blue, thresholding 0.12); TRPV1 IC, RTx b , cyan, thresholding 0.035); TRPV1 IO, RTx c , orange, thresholding 0.09); TRPV1 O, RTx,4 °C d , green, thresholding 0.1); TRPV1 O, RTx,25 °C e , brown, thresholding 0.08); and TRPV1 O, RTx,48 °C f , red, thresholding 0.033).
Techniques Used: Cryo-EM Sample Prep
Figure Legend Snippet: RTx-dependent conformational trajectory of TRPV1. a Comparison of the pore domain structures, only two subunits are shown for clarity, with the S6 gate (S6b), selectivity filter (SF), pore loop (PL) and pore helix (PH) as indicated. The pore profiles are shown as surfaces (gray). The red arrows indicate direction of movement. b Comparison of TRPV1 C,RTx (gray) and TRPV1 IC,RTx (green) structures (left) and close-up view of TRPV1 C,RTx and TRPV1 IC,RTx pore region (right). c The cryo-EM densities and the models for M644 in TRPV1 IC,RTx (green) and TRPV1 IO,RTx (gold). The cryo-EM map thresholdings are 0.03, and 0.04, respectively. d Comparison of TRPV1 IO, RTx (gold) and TRPV1 O, RTx (pink) outer pore region. Representative residues showing large motions are shown as sticks. TJ, turret junction. Phospholipids are shown as sticks and cryo-EM densities, with thresholding at 0.035 and 0.029, respectively.
Techniques Used: Cryo-EM Sample Prep