Extended Data Fig. 1: Functional characterization of THIK1EM and cryo-EM analysis in MSP1E1 nanodiscs.
a, Exemplar Two Electrode Voltage Clamp (TEVC) recordings of K2P13.1(THIK-1) (grey) and THIK1EM (orange) in Xenopus oocytes. 0 mV trace is indicated. Inset shows protocol. b, Representative TEVC current-voltage responses for K2P13.1(THIK-1) (black) and THIK1EM (orange). Inset shows protocol. c, Average currents I/I<FL> at 0 mV where I<FL> = average currents for full length K2P13.1 (THIK-1). n.s. p > 0.05. Statistical analysis was performed using Kruskal-Wallis test (nonparametric ANOVA) followed by Dunn’s multiple comparisons test. Error bars are S.E.M. d, Exemplar SEC (Superose 6 Increase 10/300 GL) for THIK1EM:MSP1E1 nanodiscs. e, peak fraction SDS-PAGE from ‘d’. More than three purifications were carried out for consistency. f, electromicrograph (~105,000x magnification) from 14,254 movies, and g, 2D class averages. h, Workflow for cryo-EM data processing for the THIK1EM:MSP1E1 nanodisc complex in cryoSPARC-3.286. Red arrow indicates the class of particles extracted without Fourier cropping after the initial cleanup. These were further subjected to heterogeneous, non-uniform, and local refinement jobs that resulted in the final map at 2.65 Å (red box). i, Particle distribution plot and j, gold-standard Fourier Shell Correlation (FSC) curves for the THIK1EM:MSP1E1 nanodisc complex.
