www.sciencemag.org/cgi/content/full/science.aas8935/DC1

Supplementary Materials for

Structural basis for the recognition of Sonic Hedgehog by human Patched1

Xin Gong, Hongwu Qian, Pingping Cao, Xin Zhao, Qiang Zhou, Jianlin Lei, Nieng Yan*

*Corresponding author. Email: nyan@princeton.edu

Published 28 June 2018 on Science First Release DOI: 10.1126/science.aas8935

This PDF file includes: Figs. S1 to S10

References

2

Fig. S1 Inhibition of the Hedgehog signaling for potential cancer treatment. Binding of ShhN to Ptch relieves the inhibitory effect on Smo, resulting in the activation of Hh signaling. Robotnikinin (32) and HL2-m5 macrocyclic peptide (33) are Hh signaling inhibitors targeting the Shh/Ptch interaction. Cyclopamine, Jervine, SANTs, Cur-61414, IPI-926, GDC-0449, LDE225, Vismodegib, Sonidegib and LY2940680 are Smo antagonist, while GANTs and HPIs are Hh signaling inhibitors targeting the pathway downstream of Smo (30, 31).

3

4

Fig. S2 Sequence alignment of the human Ptch1/2, mouse Ptch1/2 and Drosophila Ptc. Secondary structural elements of human Ptch1 are indicated above the sequence alignment and color-coded using the same scheme as for the overall structure in Figure 1. Invariant and highly conserved amino acids are shaded yellow and grey, respectively. The red arrow indicates the boundary of the construct used for structural determination. The E loop and H loop, which are engaged in ShhN binding, are indicated by blue arrows. The Uniprot IDs for the aligned sequences are: hPtch1: Q13635; hPtch2: Q9Y6C5; mPtch1: Q61115; mPtch2: O35595; dPtc: P18502. “h” for human, “m” for mouse, and “d” for Drosophila.

5

Fig. S3 Protein purification and cryo-EM reconstruction of Ptch1. (A) The domain organization of Ptch1. IH: Intracellular helix; TM: Transmembrane segment. (B) A representative chromatogram of the last step purification of Ptch1 through size exclusion chromatography. Shown on the right is the SDS-PAGE for the indicated fractions visualized by coomassie-blue staining. The peak fractions in Lanes 9-12 were pooled and concentrated for cryo-EM data acquisition. (C) The EM map of Ptch1 at an overall resolution of 3.9 Å.

6

Fig. S4 Cryo-EM analysis of Ptch1 alone and in complex with ShhN. (A) Cryo-EM analysis of Ptch1 alone. Left: A representative cryo-EM micrograph and representative 2D class averages. Middle: The gold-standard Fourier shell correlation (FSC) curve for the 3D reconstruction; Right: the FSC calculated between the refined structure and the half map used for refinement (red), the other half map (green), and the full map (black). (B,C) Cryo-EM analysis of the complex between Ptch1 and ShhN (B) and a Ptch1 variant designated Ptch1-3M (C) that contains triple point mutations (L282Q/T500F/P504L). The same type of images and plots is shown below the corresponding ones in Panel A.

7

Fig. S5 Flowchart for cryo-EM data processing. (A) The protocols for cryo-EM data processing and structure determination of Ptch1-WT and Ptch1-3M. (B) The procedures for structural determination of the Ptch1/ShhN complex. Please refer to Methods for details.

8

Fig. S6 Cryo-EM maps for representative segments. (A) The EM maps for representative segments in Ptch1 alone. (B) The representative EM maps for segments in the Ptch1/ShhN complex. (C) Representative densities of glycosylation sites in the ECDs of the Ptch1/ShhN complex. (D) The densities in SSD and ECDs that may belong to CHS molecules in both structures. The maps that are contoured at 5σ were prepared in PyMol, and colored green for Ptch1 alone and blue for the complex.

9

Fig. S7 Structural analysis of Ptch1. (A) The topological cartoon of Ptch1. Secondary structural elements are shown. “IH” stands for the intracellular helix. The dashed lines (residues 1-72, 608-728 and 1186-1305) depict the flexible segments that are invisible in the 3D reconstruction. (B) Structural superimposition of the two transmembrane domains (TMD1 and TMD2). (C) Structures of ECD1 and ECD2. Secondary structural elements are indicated and the glycosyl groups are shown as black sticks. (D) The core regions of ECD1/2 base domains share structural similarity with the ACT domain (PDB: 1ZPV). Despite similar fold in the core regions, the overall structures of ECD1 and ECD2 cannot be well overlaid (right).

10

Fig. S8 The Ptch1-binding surface of ShhN overlaps with that for multiple binding partners. From left to right are the structures of ShhN (light purple) in complex with Ptch1, CdoFn3, Hhip, 5E1 Fab, and heparin. The PDB codes for the four reported structures are 3D1M, 3HO5, 3MXW and 4C4N, respectively. The heparan sulfate proteoglycans (HSPGs) are a group of extracellular Shh regulators that promote Hedgehog signaling by facilitating Hh transport between cells (83). Shh was shown to bind to HSGPs through two sites, the positively charged N-terminal CW motif (residues 32KRRHPKK38), and the Hh structural core (83, 84). The first site is invisible in the structure of the Ptch1/ShhN complex, while the second site (including several positively charged residues Lys87/Arg123/Arg153/Arg155/Arg178) overlaps with the Ptch1 binding site.

11

Fig. S9 Sequence alignment of the sterol-sensing domain (SSD) and sterol-sensing like domain (SSDL) from representative proteins. (A) Sequence alignment of the SSDs from indicated human proteins. The TM segments of Ptch1 are shown above the sequence alignment. The red dots highlight the potential sterol-coordinating SSD residues in Ptch1. The Uniprot IDs for the aligned sequences are: hPtch1: Q13635; hNPC1: O15118; hDisp1: Q96F81; hNPC1L1: Q9UHC9; hSCAP: Q12770; hHMGCR: P04035. (B) The SSDLs, which are the SSD-corresponding domains in the symmetry-related repeat, share sequence similarity with SSD. The conserved and invariant residues are shaded grey and blue, respectively. Shown here is the sequence alignment of hPtch1-SSD with SSDLs from hPtch1, hNPC1, hDisp1, and hNPC1L1.

12

Fig. S10

Resolution maps for Ptch1-3M (A) and Ptch1-WT (B).

References

1. J. Briscoe, P. P. Thérond, The mechanisms of Hedgehog signalling and its roles in development and disease. Nat. Rev. Mol. Cell Biol. 14, 416–429 (2013). doi:10.1038/nrm3598 Medline

2. E. Pak, R. A. Segal, Hedgehog Signal Transduction: Key Players, Oncogenic Drivers, and Cancer Therapy. Dev. Cell 38, 333–344 (2016). doi:10.1016/j.devcel.2016.07.026 Medline

3. P. W. Ingham, Y. Nakano, C. Seger, Mechanisms and functions of Hedgehog signalling across the metazoa. Nat. Rev. Genet. 12, 393–406 (2011). doi:10.1038/nrg2984 Medline

4. L. Lum, P. A. Beachy, The Hedgehog response network: Sensors, switches, and routers. Science 304, 1755–1759 (2004). doi:10.1126/science.1098020 Medline

5. P. W. Ingham, A. P. McMahon, Hedgehog signaling in animal development: Paradigms and principles. Genes Dev. 15, 3059–3087 (2001). doi:10.1101/gad.938601 Medline

6. V. Marigo, R. A. Davey, Y. Zuo, J. M. Cunningham, C. J. Tabin, Biochemical evidence that patched is the Hedgehog receptor. Nature 384, 176–179 (1996). doi:10.1038/384176a0 Medline

7. D. M. Stone, M. Hynes, M. Armanini, T. A. Swanson, Q. Gu, R. L. Johnson, M. P. Scott, D. Pennica, A. Goddard, H. Phillips, M. Noll, J. E. Hooper, F. de Sauvage, A. Rosenthal, The tumour-suppressor gene patched encodes a candidate receptor for Sonic hedgehog. Nature 384, 129–134 (1996). doi:10.1038/384129a0 Medline

8. N. Fuse, T. Maiti, B. Wang, J. A. Porter, T. M. T. Hall, D. J. Leahy, P. A. Beachy, Sonic hedgehog protein signals not as a hydrolytic enzyme but as an apparent ligand for patched. Proc. Natl. Acad. Sci. U.S.A. 96, 10992–10999 (1999). doi:10.1073/pnas.96.20.10992 Medline

9. J. Taipale, M. K. Cooper, T. Maiti, P. A. Beachy, Patched acts catalytically to suppress the activity of Smoothened. Nature 418, 892–897 (2002). doi:10.1038/nature00989 Medline

10. R. Rohatgi, L. Milenkovic, M. P. Scott, Patched1 regulates hedgehog signaling at the primary cilium. Science 317, 372–376 (2007). doi:10.1126/science.1139740 Medline

11. K. C. Corbit, P. Aanstad, V. Singla, A. R. Norman, D. Y. R. Stainier, J. F. Reiter, Vertebrate Smoothened functions at the primary cilium. Nature 437, 1018–1021 (2005). doi:10.1038/nature04117 Medline

12. P. Aza-Blanc, F. A. Ramírez-Weber, M. P. Laget, C. Schwartz, T. B. Kornberg, Proteolysis that is inhibited by hedgehog targets Cubitus interruptus protein to the nucleus and converts it to a repressor. Cell 89, 1043–1053 (1997). doi:10.1016/S0092-8674(00)80292-5 Medline

13. C. H. Chen, D. P. von Kessler, W. Park, B. Wang, Y. Ma, P. A. Beachy, Nuclear trafficking of Cubitus interruptus in the transcriptional regulation of Hedgehog target gene expression. Cell 98, 305–316 (1999). doi:10.1016/S0092-8674(00)81960-1 Medline

14. N. Méthot, K. Basler, Hedgehog controls limb development by regulating the activities of distinct transcriptional activator and repressor forms of Cubitus interruptus. Cell 96, 819–831 (1999). doi:10.1016/S0092-8674(00)80592-9 Medline

15. J. Kim, M. Kato, P. A. Beachy, Gli2 trafficking links Hedgehog-dependent activation of Smoothened in the primary cilium to transcriptional activation in the nucleus. Proc. Natl. Acad. Sci. U.S.A. 106, 21666–21671 (2009). doi:10.1073/pnas.0912180106 Medline

16. J. W. Yoon, Y. Kita, D. J. Frank, R. R. Majewski, B. A. Konicek, M. A. Nobrega, H. Jacob, D. Walterhouse, P. Iannaccone, Gene expression profiling leads to identification of GLI1-binding elements in target genes and a role for multiple downstream pathways in GLI1-induced cell transformation. J. Biol. Chem. 277, 5548–5555 (2002). doi:10.1074/jbc.M105708200 Medline

17. X. Wen, C. K. Lai, M. Evangelista, J.-A. Hongo, F. J. de Sauvage, S. J. Scales, Kinetics of hedgehog-dependent full-length Gli3 accumulation in primary cilia and subsequent degradation. Mol. Cell. Biol. 30, 1910–1922 (2010). doi:10.1128/MCB.01089-09 Medline

18. C. C. Hui, S. Angers, Gli proteins in development and disease. Annu. Rev. Cell Dev. Biol. 27, 513–537 (2011). doi:10.1146/annurev-cellbio-092910-154048 Medline

19. J. Taipale, P. A. Beachy, The Hedgehog and Wnt signalling pathways in cancer. Nature 411, 349–354 (2001). doi:10.1038/35077219 Medline

20. P. A. Beachy, S. S. Karhadkar, D. M. Berman, Tissue repair and stem cell renewal in carcinogenesis. Nature 432, 324–331 (2004). doi:10.1038/nature03100 Medline

21. L. L. Rubin, F. J. de Sauvage, Targeting the Hedgehog pathway in cancer. Nat. Rev. Drug Discov. 5, 1026–1033 (2006). doi:10.1038/nrd2086 Medline

22. J. Xie, M. Murone, S.-M. Luoh, A. Ryan, Q. Gu, C. Zhang, J. M. Bonifas, C.-W. Lam, M. Hynes, A. Goddard, A. Rosenthal, E. H. Epstein Jr., F. J. de Sauvage, Activating Smoothened mutations in sporadic basal-cell carcinoma. Nature 391, 90–92 (1998). doi:10.1038/34201 Medline

23. R. K. Mann, P. A. Beachy, Novel lipid modifications of secreted protein signals. Annu. Rev. Biochem. 73, 891–923 (2004). doi:10.1146/annurev.biochem.73.011303.073933 Medline

24. J. A. Porter, K. E. Young, P. A. Beachy, Cholesterol modification of hedgehog signaling proteins in animal development. Science 274, 255–259 (1996). doi:10.1126/science.274.5285.255 Medline

25. J. A. Porter, S. C. Ekker, W.-J. Park, D. P. von Kessler, K. E. Young, C.-H. Chen, Y. Ma, A. S. Woods, R. J. Cotter, E. V. Koonin, P. A. Beachy, Hedgehog patterning activity: Role of a lipophilic modification mediated by the carboxy-terminal autoprocessing domain. Cell 86, 21–34 (1996). doi:10.1016/S0092-8674(00)80074-4 Medline

26. R. B. Pepinsky, C. Zeng, D. Wen, P. Rayhorn, D. P. Baker, K. P. Williams, S. A. Bixler, C. M. Ambrose, E. A. Garber, K. Miatkowski, F. R. Taylor, E. A. Wang, A. Galdes, Identification of a palmitic acid-modified form of human Sonic hedgehog. J. Biol. Chem. 273, 14037–14045 (1998). doi:10.1074/jbc.273.22.14037 Medline

27. P. M. Lewis, M. P. Dunn, J. A. McMahon, M. Logan, J. F. Martin, B. St-Jacques, A. P. McMahon, Cholesterol modification of sonic hedgehog is required for long-range signaling activity and effective modulation of signaling by Ptc1. Cell 105, 599–612 (2001). doi:10.1016/S0092-8674(01)00369-5 Medline

28. Z. Chamoun, R. K. Mann, D. Nellen, D. P. von Kessler, M. Bellotto, P. A. Beachy, K. Basler, Skinny hedgehog, an acyltransferase required for palmitoylation and activity of the hedgehog signal. Science 293, 2080–2084 (2001). doi:10.1126/science.1064437 Medline

29. K. P. Williams, P. Rayhorn, G. Chi-Rosso, E. A. Garber, K. L. Strauch, G. S. Horan, J. O. Reilly, D. P. Baker, F. R. Taylor, V. Koteliansky, R. B. Pepinsky, Functional antagonists of sonic hedgehog reveal the importance of the N terminus for activity. J. Cell Sci. 112, 4405–4414 (1999). Medline

30. B. Z. Stanton, L. F. Peng, Small-molecule modulators of the Sonic Hedgehog signaling pathway. Mol. Biosyst. 6, 44–54 (2010). doi:10.1039/B910196A Medline

31. S. Peukert, K. Miller-Moslin, Small-molecule inhibitors of the hedgehog signaling pathway as cancer therapeutics. ChemMedChem 5, 500–512 (2010). doi:10.1002/cmdc.201000011 Medline

32. B. Z. Stanton, L. F. Peng, N. Maloof, K. Nakai, X. Wang, J. L. Duffner, K. M. Taveras, J. M. Hyman, S. W. Lee, A. N. Koehler, J. K. Chen, J. L. Fox, A. Mandinova, S. L. Schreiber, A small molecule that binds Hedgehog and blocks its signaling in human cells. Nat. Chem. Biol. 5, 154–156 (2009). doi:10.1038/nchembio.142 Medline

33. A. E. Owens, I. de Paola, W. A. Hansen, Y.-W. Liu, S. D. Khare, R. Fasan, Design and Evolution of a Macrocyclic Peptide Inhibitor of the Sonic Hedgehog/Patched Interaction. J. Am. Chem. Soc. 139, 12559–12568 (2017). doi:10.1021/jacs.7b06087 Medline

34. P. A. Beachy, S. G. Hymowitz, R. A. Lazarus, D. J. Leahy, C. Siebold, Interactions between Hedgehog proteins and their binding partners come into view. Genes Dev. 24, 2001–2012 (2010). doi:10.1101/gad.1951710 Medline

35. J. E. Hooper, M. P. Scott, The Drosophila patched gene encodes a putative membrane protein required for segmental patterning. Cell 59, 751–765 (1989). doi:10.1016/0092-8674(89)90021-4 Medline

36. Y. Nakano, I. Guerrero, A. Hidalgo, A. Taylor, J. R. S. Whittle, P. W. Ingham, A protein with several possible membrane-spanning domains encoded by the Drosophila segment polarity gene patched. Nature 341, 508–513 (1989). doi:10.1038/341508a0 Medline

37. R. L. Johnson, A. L. Rothman, J. Xie, L. V. Goodrich, J. W. Bare, J. M. Bonifas, A. G. Quinn, R. M. Myers, D. R. Cox, E. H. Epstein Jr., M. P. Scott, Human homolog of patched, a candidate gene for the basal cell nevus syndrome. Science 272, 1668–1671 (1996). doi:10.1126/science.272.5268.1668 Medline

38. H. Hahn, C. Wicking, P. G. Zaphiropoulous, M. R. Gailani, S. Shanley, A. Chidambaram, I. Vorechovsky, E. Holmberg, A. B. Unden, S. Gillies, K. Negus, I. Smyth, C. Pressman, D. J. Leffell, B. Gerrard, A. M. Goldstein, M. Dean, R. Toftgard, G. Chenevix-Trench, B. Wainwright, A. E. Bale, Mutations of the human homolog of Drosophila patched in the nevoid basal cell carcinoma syndrome. Cell 85, 841–851 (1996). doi:10.1016/S0092-8674(00)81268-4 Medline

39. D. Carpenter, D. M. Stone, J. Brush, A. Ryan, M. Armanini, G. Frantz, A. Rosenthal, F. J. de Sauvage, Characterization of two patched receptors for the vertebrate hedgehog protein family. Proc. Natl. Acad. Sci. U.S.A. 95, 13630–13634 (1998). doi:10.1073/pnas.95.23.13630 Medline

40. P. G. Zaphiropoulos, A. B. Undén, F. Rahnama, R. E. Hollingsworth, R. Toftgård, PTCH2, a novel human patched gene, undergoing alternative splicing and up-regulated in basal cell carcinomas. Cancer Res. 59, 787–792 (1999). Medline

41. T. T. Tseng, K. S. Gratwick, J. Kollman, D. Park, D. H. Nies, A. Goffeau, M. H. Saier Jr., The RND permease superfamily: An ancient, ubiquitous and diverse family that includes human disease and development proteins. J. Mol. Microbiol. Biotechnol. 1, 107–125 (1999). Medline

42. A. Yamaguchi, R. Nakashima, K. Sakurai, Structural basis of RND-type multidrug exporters. Front. Microbiol. 6, 327 (2015). doi:10.3389/fmicb.2015.00327 Medline

43. P. E. Kuwabara, M. Labouesse, The sterol-sensing domain: Multiple families, a unique role? Trends Genet. 18, 193–201 (2002). doi:10.1016/S0168-9525(02)02640-9 Medline

44. X. Gong, H. Qian, X. Zhou, J. Wu, T. Wan, P. Cao, W. Huang, X. Zhao, X. Wang, P. Wang, Y. Shi, G. F. Gao, Q. Zhou, N. Yan, Structural Insights into the Niemann-Pick C1 (NPC1)-Mediated Cholesterol Transfer and Ebola Infection. Cell 165, 1467–1478 (2016). doi:10.1016/j.cell.2016.05.022 Medline

45. X. Li, J. Wang, E. Coutavas, H. Shi, Q. Hao, G. Blobel, Structure of human Niemann-Pick C1 protein. Proc. Natl. Acad. Sci. U.S.A. 113, 8212–8217 (2016). doi:10.1073/pnas.1607795113 Medline

46. M. C. Harvey, A. Fleet, N. Okolowsky, P. A. Hamel, Distinct effects of the mesenchymal dysplasia gene variant of murine Patched-1 protein on canonical and non-canonical Hedgehog signaling pathways. J. Biol. Chem. 289, 10939–10949 (2014). doi:10.1074/jbc.M113.514844 Medline

47. A. Fleet, J. P. Lee, A. Tamachi, I. Javeed, P. A. Hamel, Activities of the Cytoplasmic Domains of Patched-1 Modulate but Are Not Essential for the Regulation of Canonical Hedgehog Signaling. J. Biol. Chem. 291, 17557–17568 (2016). doi:10.1074/jbc.M116.731745 Medline

48. X. Lu, S. Liu, T. B. Kornberg, The C-terminal tail of the Hedgehog receptor Patched regulates both localization and turnover. Genes Dev. 20, 2539–2551 (2006). doi:10.1101/gad.1461306 Medline

49. S. Murakami, R. Nakashima, E. Yamashita, A. Yamaguchi, Crystal structure of bacterial multidrug efflux transporter AcrB. Nature 419, 587–593 (2002). doi:10.1038/nature01050 Medline

50. J. S. McLellan, X. Zheng, G. Hauk, R. Ghirlando, P. A. Beachy, D. J. Leahy, The mode of Hedgehog binding to Ihog homologues is not conserved across different phyla. Nature 455, 979–983 (2008). doi:10.1038/nature07358 Medline

51. B. Bishop, A. R. Aricescu, K. Harlos, C. A. O’Callaghan, E. Y. Jones, C. Siebold, Structural insights into hedgehog ligand sequestration by the human hedgehog-interacting protein HHIP. Nat. Struct. Mol. Biol. 16, 698–703 (2009). doi:10.1038/nsmb.1607 Medline

52. I. Bosanac, H. R. Maun, S. J. Scales, X. Wen, A. Lingel, J. F. Bazan, F. J. de Sauvage, S. G. Hymowitz, R. A. Lazarus, The structure of SHH in complex with HHIP reveals a recognition role for the Shh pseudo active site in signaling. Nat. Struct. Mol. Biol. 16, 691–697 (2009). doi:10.1038/nsmb.1632 Medline

53. H. R. Maun, X. Wen, A. Lingel, F. J. de Sauvage, R. A. Lazarus, S. J. Scales, S. G. Hymowitz, Hedgehog pathway antagonist 5E1 binds hedgehog at the pseudo-active site. J. Biol. Chem. 285, 26570–26580 (2010). doi:10.1074/jbc.M110.112284 Medline

54. F. Lu, Q. Liang, L. Abi-Mosleh, A. Das, J. K. De Brabander, J. L. Goldstein, M. S. Brown, Identification of NPC1 as the target of U18666A, an inhibitor of lysosomal cholesterol export and Ebola infection. eLife 4, e12177 (2015). doi:10.7554/eLife.12177 Medline

55. N. Boutet, Y.-J. Bignon, V. Drouin-Garraud, P. Sarda, M. Longy, D. Lacombe, P. Gorry, Spectrum of PTCH1 mutations in French patients with Gorlin syndrome. J. Invest. Dermatol. 121, 478–481 (2003). doi:10.1046/j.1523-1747.2003.12423.x Medline

56. K. Fujii, Y. Kohno, K. Sugita, M. Nakamura, Y. Moroi, K. Urabe, M. Furue, M. Yamada, T. Miyashita, Mutations in the human homologue of Drosophila patched in Japanese nevoid basal cell carcinoma syndrome patients. Hum. Mutat. 21, 451–452 (2003). doi:10.1002/humu.9132 Medline

57. G. R. Hime, H. Lada, M. J. Fietz, S. Gillies, A. Passmore, C. Wicking, B. J. Wainwright, Functional analysis in Drosophila indicates that the NBCCS/PTCH1 mutation G509V results in activation of smoothened through a dominant-negative mechanism. Dev. Dyn. 229, 780–790 (2004). doi:10.1002/dvdy.10499 Medline

58. G. Tate, M. Li, T. Suzuki, T. Mitsuya, A new germline mutation of the PTCH gene in a Japanese patient with nevoid basal cell carcinoma syndrome associated with meningioma. Jpn. J. Clin. Oncol. 33, 47–50 (2003). doi:10.1093/jjco/hyg005 Medline

59. J. L. Goldstein, M. S. Brown, A century of cholesterol and coronaries: From plaques to genes to statins. Cell 161, 161–172 (2015). doi:10.1016/j.cell.2015.01.036 Medline

60. K. L. Luskey, B. Stevens, Human 3-hydroxy-3-methylglutaryl coenzyme A reductase. Conserved domains responsible for catalytic activity and sterol-regulated degradation. J. Biol. Chem. 260, 10271–10277 (1985). Medline

61. M. T. Vanier, Complex lipid trafficking in Niemann-Pick disease type C. J. Inherit. Metab. Dis. 38, 187–199 (2015). doi:10.1007/s10545-014-9794-4 Medline

62. B. R. Myers, L. Neahring, Y. Zhang, K. J. Roberts, P. A. Beachy, Rapid, direct activity assays for Smoothened reveal Hedgehog pathway regulation by membrane cholesterol and extracellular sodium. Proc. Natl. Acad. Sci. U.S.A. 114, E11141–E11150 (2017). doi:10.1073/pnas.1717891115 Medline

63. L. Guan, T. Nakae, Identification of essential charged residues in transmembrane segments of the multidrug transporter MexB of Pseudomonas aeruginosa. J. Bacteriol. 183, 1734–1739 (2001). doi:10.1128/JB.183.5.1734-1739.2001 Medline

64. S. C. Goetz, K. V. Anderson, The primary cilium: A signalling centre during vertebrate development. Nat. Rev. Genet. 11, 331–344 (2010). doi:10.1038/nrg2774 Medline

65. F. R. Taylor, D. Wen, E. A. Garber, A. N. Carmillo, D. P. Baker, R. M. Arduini, K. P. Williams, P. H. Weinreb, P. Rayhorn, X. Hronowski, A. Whitty, E. S. Day, A. Boriack-Sjodin, R. I. Shapiro, A. Galdes, R. B. Pepinsky, Enhanced potency of human Sonic hedgehog by hydrophobic modification. Biochemistry 40, 4359–4371 (2001). doi:10.1021/bi002487u Medline

66. H. Tukachinsky, K. Petrov, M. Watanabe, A. Salic, Mechanism of inhibition of the tumor suppressor Patched by Sonic Hedgehog. Proc. Natl. Acad. Sci. U.S.A. 113, E5866–E5875 (2016). doi:10.1073/pnas.1606719113 Medline

67. J. Lei, J. Frank, Automated acquisition of cryo-electron micrographs for single particle reconstruction on an FEI Tecnai electron microscope. J. Struct. Biol. 150, 69–80 (2005). doi:10.1016/j.jsb.2005.01.002 Medline

68. X. Fan, L. Zhao, C. Liu, J.-C. Zhang, K. Fan, X. Yan, H.-L. Peng, J. Lei, H.-W. Wang, Near-Atomic Resolution Structure Determination in Over-Focus with Volta Phase Plate by Cs-Corrected Cryo-EM. Structure 25, 1623–1630.e3 (2017). doi:10.1016/j.str.2017.08.008

69. X. Li, P. Mooney, S. Zheng, C. R. Booth, M. B. Braunfeld, S. Gubbens, D. A. Agard, Y. Cheng, Electron counting and beam-induced motion correction enable near-atomic-resolution single-particle cryo-EM. Nat. Methods 10, 584–590 (2013). doi:10.1038/nmeth.2472 Medline

70. S. Q. Zheng, E. Palovcak, J.-P. Armache, K. A. Verba, Y. Cheng, D. A. Agard, MotionCor2: Anisotropic correction of beam-induced motion for improved cryo-electron microscopy. Nat. Methods 14, 331–332 (2017). doi:10.1038/nmeth.4193 Medline

71. T. Grant, N. Grigorieff, Measuring the optimal exposure for single particle cryo-EM using a 2.6 Å reconstruction of rotavirus VP6. eLife 4, e06980 (2015). doi:10.7554/eLife.06980 Medline

72. K. Zhang, Gctf: Real-time CTF determination and correction. J. Struct. Biol. 193, 1–12 (2016). doi:10.1016/j.jsb.2015.11.003 Medline

73. D. Kimanius, B. O. Forsberg, S. H. Scheres, E. Lindahl, Accelerated cryo-EM structure determination with parallelisation using GPUs in RELION-2. eLife 5, e18722 (2016). doi:10.7554/eLife.18722 Medline

74. G. Tang, L. Peng, P. R. Baldwin, D. S. Mann, W. Jiang, I. Rees, S. J. Ludtke, EMAN2: An extensible image processing suite for electron microscopy. J. Struct. Biol. 157, 38–46 (2007). doi:10.1016/j.jsb.2006.05.009 Medline

75. P. B. Rosenthal, R. Henderson, Optimal determination of particle orientation, absolute hand, and contrast loss in single-particle electron cryomicroscopy. J. Mol. Biol. 333, 721–745 (2003). doi:10.1016/j.jmb.2003.07.013 Medline

76. S. Chen, G. McMullan, A. R. Faruqi, G. N. Murshudov, J. M. Short, S. H. W. Scheres, R. Henderson, High-resolution noise substitution to measure overfitting and validate resolution in 3D structure determination by single particle electron cryomicroscopy. Ultramicroscopy 135, 24–35 (2013). doi:10.1016/j.ultramic.2013.06.004 Medline

77. P. Emsley, B. Lohkamp, W. G. Scott, K. Cowtan, Features and development of Coot. Acta Crystallogr. D 66, 486–501 (2010). doi:10.1107/S0907444910007493 Medline

78. P. D. Adams, P. V. Afonine, G. Bunkóczi, V. B. Chen, I. W. Davis, N. Echols, J. J. Headd, L.-W. Hung, G. J. Kapral, R. W. Grosse-Kunstleve, A. J. McCoy, N. W. Moriarty, R. Oeffner, R. J. Read, D. C. Richardson, J. S. Richardson, T. C. Terwilliger, P. H. Zwart,

PHENIX: A comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D 66, 213–221 (2010). doi:10.1107/S0907444909052925 Medline

79. A. Amunts, A. Brown, X. C. Bai, J. L. Llácer, T. Hussain, P. Emsley, F. Long, G. Murshudov, S. H. W. Scheres, V. Ramakrishnan, Structure of the yeast mitochondrial large ribosomal subunit. Science 343, 1485–1489 (2014). doi:10.1126/science.1249410 Medline

80. E. F. Pettersen, T. D. Goddard, C. C. Huang, G. S. Couch, D. M. Greenblatt, E. C. Meng, T. E. Ferrin, UCSF Chimera—a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004). doi:10.1002/jcc.20084 Medline

81. W. L. DeLano, The PyMOL Molecular Graphics System; www.pymol.org. (2002).

82. O. S. Smart, J. G. Neduvelil, X. Wang, B. A. Wallace, M. S. Sansom, HOLE: A program for the analysis of the pore dimensions of ion channel structural models. J. Mol. Graph. 14, 354–360 (1996). doi:10.1016/S0263-7855(97)00009-X Medline

83. D. M. Whalen, T. Malinauskas, R. J. Gilbert, C. Siebold, Structural insights into proteoglycan-shaped Hedgehog signaling. Proc. Natl. Acad. Sci. U.S.A. 110, 16420–16425 (2013). doi:10.1073/pnas.1310097110 Medline

84. P. Farshi, S. Ohlig, U. Pickhinke, S. Höing, K. Jochmann, R. Lawrence, R. Dreier, T. Dierker, K. Grobe, Dual roles of the Cardin-Weintraub motif in multimeric Sonic hedgehog. J. Biol. Chem. 286, 23608–23619 (2011). doi:10.1074/jbc.M110.206474 Medline