Extended Data Fig. 6: Cryo-EM data processing of GluA2flipQ/TARP γ2 complex at pH 8.0 and pH 5.5.
a, Peak amplitude, entry, and recovery from desensitization measured in pH 7.4 (black), pH 8 (purple), and pH 5.5 (red). Responses were obtained from 200 ms applications of 10 mM L-glutamate. Lines connect values obtained from the same patch. Boxes and whiskers as in Extended Data Fig. 2a. Effect of pH on current peak amplitude (left panel) was revealed by repeated measures ANOVA test F(1.878, 9.392) = 62.36, P < 0.0001, followed post hoc with Tukey’s multiple comparisons test. Similarly, the effect of pH on entry (middle panel) and entry to desensitisation (right panel) was indicated by repeated measures one-way ANOVA F(1.207, 6.036) = 141.6, P < 0.0001, followed post hoc with Tukey’s multiple comparisons test (desensitisation entry), repeated measures ANOVA test F(1.116, 5.582) = 37.06, P = 0.001, followed post hoc with Tukey’s multiple comparisons test (desensitisation recovery). b, Left: 4–20% SDS-PAGE gel of the purified complex. GluA2flipQ migrating at 100 kDa and TARP γ2 at 37 kDa. Right: Representative (from 21,898 micrographs) motion-corrected cryo-EM image at pH 5.5. c, Representative 2D class averages (from 28 class averages containing clear secondary structural features) of particles at pH 5.5. Box size is 180×180 pixels, equivalent to 295.6 × 295.6 Å. d, Summary of particles that were selected sequentially through the image processing pipeline from the initial motion corrected micrographs. Selected particles were those that were contained in well-defined classes in each step. Particles that were selected after 3D classification were used for 3D refinement. e, Consensus maps calculated from focused refinement of the LDB, TMD, and TARP γ2 at pH 8.0 (left) and pH 5.5 (right). The Fourier Shell Correlation curves with estimated resolutions at FSC = 0.143 and angular distributions of the final particles are shown. Note the LBD densities at pH 5.5 are ill-defined compared to pH 8.0, due to conformational heterogeneity. f, Local resolutions of the maps containing the LDB, TMD, and TARP γ2 at pH 8.0 (left) and pH 5.5 (right), calculated using ResMap77. The heatmap reference is displayed at the right with local resolution in Å unit. g, Side chain resolution of the GluA2-TARPγ2 complex determined at pH 5.5; all GluA2 (M1-4) and TARP (TM1-4) are shown.
