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1 Supplementary Materials for Structure of a eukaryotic voltage-gated sodium channel at near-atomic resolution Huaizong Shen, Qiang Zhou, Xiaojing Pan, Zhangqiang Li, Jianping Wu, Nieng Yan* This PDF file includes: Figs. S1 to S10 Table S1 *Corresponding author. nyan@tsinghua.edu.cn Published 9 February 2017 on Science First Release DOI: /science.aal4326

2 Shen et al Supplementary Figures and Legends 2

3 Shen et al 3

4 Shen et al 4

5 Figure S1 Sequence Alignment of Na v PaS with the human and two closely related insect Na v channels. The sequences are aligned using ClustalW with minor manual adjustment. Secondary structural elements of Na v PaS are indicated above the sequence alignment and color-coded for the four repeats consistent with the color scheme for the overall structure. Invariant amino acids are shaded yellow and conserved residues are colored blue. The critical DEKA residues are colored red and shaded yellow. The gating charge residues (labeled R/K1-R6) in the S4 segment of each repeat are colored white and shaded red. The An1, An2, and occluding residues on S2 and conserved Asp on S3 in each repeat are colored blue and shaded pale cyan. The Cys residues that form intra-repeat disulfide bonds and Asn that are glycosylated are indicated by brown dots and blue triangles, respectively, below the sequences. The Uniprot IDs for the aligned sequences are: Na v PaS: D0E0C2; hna v 1.1: P35498; hna v 1.2: Q99250; hna v 1.3: Q9NY46; hna v 1.4: P35499; hna v 1.5: Q14524; hna v 1.6: Q9UQD0; hna v 1.7: Q15858; hna v 1.8: Q9Y5Y9; hna v 1.9: Q9UI33; hna v X: Q01118; Na v PaL (previously designated PaNa v 1, a better characterized Na v channel from Periplaneta Americana): D0E0C1; Na v Bg (the Na v channel from Blattella germanica): O

6 Figure S2 Expression, purification, and cryo-em analysis of Na v PaS. (A) Cell surface expression pattern of recombinant Strep-tagged Na v PaS in HEK293 cells. Immunostaining of Na v PaS with an antibody against the Strep-tag suggests that the protein can be trafficked to plasma membrane even in the absence of any β subunit. The surface distribution pattern is seen in all cells examined. (B) The last step purification of Na v PaS. Shown here is a representative chromatogram of gel filtration purification. The indicated fractions are resolved by SDS-PAGE and visualized by Coomassie blue staining. (C) A representative electron micrograph of Na v PaS with some typical particles marked by green circles. (D) Representative two-dimensional class averages of the electron micrographs. (E) Angular distribution for the final reconstruction. Each column represents one view and the size of the column is proportional to the number of particles in that view. (F) Local resolution variations of the EM reconstruction. The resolution map is estimated with RELION 2.0. (G) FSC curves of the refined model versus the overall 3.8 Å map that it was refined against (black); of the model refined in the first of the two independent maps used for the gold-standard FSC versus that same map (red); and of the model refined in the first of the two independent maps versus the second independent map (green). The small difference between the red and green curves indicates that the refinement of the atomic coordinates did not suffer from overfitting. 6

7 Figure S3 The flowchart for EM data processing. Details can be found in Materials and Methods. 7

8 Figure S4 EM maps for representative segments of Na v PaS. (A) The EM maps for the S1-S6 segments in each repeat. Bulky residues that were used to facilitate sequence assignment during model building are labeled. (B) The EM map for the intracellular gate. (C) The EM map for the III-IV linker and the CTD. The side groups of representative bulky residues are shown in the insets on the right. The maps were prepared in PyMol and contoured at 4-5 σ. 8

9 Figure S5 EM maps for the selectivity filter and pore helices of Na v PaS. (A) The EM map for the selectivity filter (SF) and the supporting P1 and P2 helices. Due to radiation damage, the densities for the carboxylate groups of some Asp/Glu residues are missing, but those for the backbone and alkyl groups are mostly discernible. A top view of the EM map for the signature residues DEKA are shown in the insets on the right. (B) The EM map for the critical residues that line up the selectivity filter vestibule. The discontinuous densities within the SF vestibule are shown as light brown meshes. It remains to be investigated whether these are noises or belong to Na + ions. (C) The density for Lys1061 observed in the map when applied with a B-factor at 300. The maps are contoured at 5 σ if not otherwise indicated. 9

10 Figure S6 Mapping of the conserved residues of Na v channels on the structure of Na v PaS. The conserved residues between Na v PaS and human Na v 1.4 are mapped to the structure of Na v PaS using ConSurf. 10

11 Figure S7 The asymmetric pore domain of Na v PaS has only one small fenestration in the current conformation. (A) The structure of the pore domain of Na v PaS is shown in four perpendicular side views. The disulfide bonds and glycosyl moieties are shown as spheres and sticks, respectively. (B) The polar and charged residues within the central cavity of the pore domain. The Asn or Lys at the corresponding S6 locus of the four repeats is highly conserved in human Na v channels (fig. S1). (C) The S5 and S6 segments have different conformations in the four repeats. The pore forming segments are superimposed relative to the SF and pore helices P1 and P2 (P1-SF-P2). The deviation points on S5 and S6 are indicated by the black arrows. (D) Closed pore fenestrations. Estimation of the size of the central cavity along the two diagonal directions by HOLE suggests closure of fenestrations on three side surfaces. The diameter of the only fenestration enclosed by S6 III and S6 IV is approximately 2.3 Å, too narrow for local anesthetics entrance. Shown here are one top and two perpendicular side views of the central cavity. 11

12 Figure S8 Sequence Alignment of Na v PaS with the rabbit Ca v 1.1. The sequences of Na v PaS, the rabbit Ca v 1.1, and human Na v 1.4 are aligned using ClustalW. The sequence of Na v 1.4 is then omitted for clarity. Invariant and conserved residues are shaded yellow and pale cyan, respectively. The secondary structural elements of Na v PaS are shown above the sequences. The Uniprot ID for the rabbit Ca v 1.1 is P

13 Figure S9 Conformational changes between Na v PaS and representative bacterial Na v channels. (A) Structural comparison of Na v PaS with Na v Rh and the Na v Ab-1.7 VSD4 chimera. The three structures are aligned relative to the P1-SF-P2 funnel. The same structural elements are used as reference for all superimpositions presented in this figure. The orange arrows indicate the positional rotation of VSDs from Na v PaS to Na v Rh. (B) Conformational changes of the S4-S5 constriction ring and the preceding S4 segments between Na v PaS and the Na v Ab-1.7 VSD4 chimera. The side view suggests that the conformational changes of the S4-S5 segments mainly involve horizontal, but not vertical shifts. The grey band indicates the cytoplasmic boundary of the membrane. (C) The S4-S5 constriction ring and the S5 segments appear to be tightened in Na v PaS compared to those in the bacterial Na v channels. The corresponding segments in the four repeats undergo right-handed rotation around the central pore axis from bacterial Na v channels to Na v PaS. (D) Structural comparison of the S6 tetrahelical bundle between Na v PaS and bacterial Na v channels. The S6 bundle in the present structure of Na v PaS exhibits a righthanded iris-like twisting compared to that of the closed Na v Ab and the open Na v MS. An intracellular view is shown on the right. 13

14 Figure S10 Structural mapping of disease-associated mutations identified in the human Na v channels. The disease-related mutations identified in the human Na v channels are mapped to the structure of Na v PaS, color coded for different subtypes of Na v channels. 14

15 Table S1. Data collection and model statistics Data collection EM equipment FEI Titan Krios Voltage (kv) 300 Detector Gatan K2 Summit Pixel size before magnification distortion correction (Å) Pixel size after magnification distortion correction Electron dose (e - /Å 2 ) 50 Defocus range (µm) -1.7 ~ -2.6 Number of collected micrographs 13,757 Number of selected micrographs 11,843 Reconstruction Software RELION 2.0 Number of used Particles 1,373,581 Symmetry C1 Resolution (before post-processing, Å) 4.2 Resolution (after post-processing, Å) 3.8 Map sharpening B-factor (Å 2 ) 270 Refinement Software Phenix-dev-2405 Cell dimensions a=b=c (Å) α=β=γ ( ) Model composition Number of protein residues 1,323 Side chains assigned 1,177 Number of sugar moieties 20 R.m.s deviations Bonds length (Å) 0.01 Bonds Angle ( ) 1.17 Ramachandran plot statistics (%) Preferred 84.0 Allowed 15.3 Outlier