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Structure of a modular polyketide synthase

Nature volume 510, pages 512–517 (2014)Cite this article

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Abstract

Polyketide natural products constitute a broad class of compounds with diverse structural features and biological activities. Their biosynthetic machinery, represented by type I polyketide synthases (PKSs), has an architecture in which successive modules catalyse two-carbon linear extensions and keto-group processing reactions on intermediates covalently tethered to carrier domains. Here we used electron cryo-microscopy to determine sub-nanometre-resolution three-dimensional reconstructions of a full-length PKS module from the bacterium Streptomyces venezuelae that revealed an unexpectedly different architecture compared to the homologous dimeric mammalian fatty acid synthase. A single reaction chamber provides access to all catalytic sites for the intramodule carrier domain. In contrast, the carrier from the preceding module uses a separate entrance outside the reaction chamber to deliver the upstream polyketide intermediate for subsequent extension and modification. This study reveals for the first time, to our knowledge, the structural basis for both intramodule and intermodule substrate transfer in polyketide synthases, and establishes a new model for molecular dissection of these multifunctional enzyme systems.

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Figure 1: Modular polyketide synthase for pikromycin.
Figure 2: Cryo-EM structures of holo-PikAIII.
Figure 3: Interaction of upstream ACP with the PKS module.
Figure 4: Interaction of intramodule ACP5 with KS5 in MM–PikAIII.

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Accession codes

Primary accessions

Electron Microscopy Data Bank

Data deposits

Cryo-EM maps have been deposited in the Electron Microscopy Data Bank under accession numbers EMD-5647 (holo-PikAIII conformation I), EMD-5648 (holo-PikAIII conformation II), EMD-5649 (PikAIII(ΔACP5)), EMD-5651 (pentaketide–ACP4–PikAIII(C209A/ΔACP5)), EMD-5653 (MM–PikAIII) and EMD-5662 (holo-ACP4–PikAIII(C209A/ΔACP5)).

References

  1. Newman, D. J. & Cragg, G. M. Natural products as sources of new drugs over the 30 years from 1981 to 2010. J. Nat. Prod. 75, 311–335 (2012)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Fischbach, M. A. & Walsh, C. T. Assembly-line enzymology for polyketide and nonribosomal peptide antibiotics: logic, machinery, and mechanisms. Chem. Rev. 106, 3468–3496 (2006)

    Article  CAS  PubMed  Google Scholar 

  3. Smith, S. & Tsai, S. C. The type I fatty acid and polyketide synthases: a tale of two megasynthases. Nat. Prod. Rep. 24, 1041–1072 (2007)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Broadhurst, R. W., Nietlispach, D., Wheatcroft, M. P., Leadlay, P. F. & Weissman, K. J. The structure of docking domains in modular polyketide synthases. Chem. Biol. 10, 723–731 (2003)

    Article  CAS  PubMed  Google Scholar 

  5. Buchholz, T. J. et al. Structural basis for binding specificity between subclasses of modular polyketide synthase docking domains. ACS Chem. Biol. 4, 41–52 (2009)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Tang, Y., Chen, A. Y., Kim, C. Y., Cane, D. E. & Khosla, C. Structural and mechanistic analysis of protein interactions in module 3 of the 6-deoxyerythronolide B synthase. Chem. Biol. 14, 931–943 (2007)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Tang, Y., Kim, C. Y., Mathews, I. I., Cane, D. E. & Khosla, C. The 2.7-Å crystal structure of a 194-kDa homodimeric fragment of the 6-deoxyerythronolide B synthase. Proc. Natl Acad. Sci. USA 103, 11124–11129 (2006)

    Article  CAS  ADS  PubMed  PubMed Central  Google Scholar 

  8. Zheng, J. & Keatinge-Clay, A. T. Structural and functional analysis of C2-type ketoreductases from modular polyketide synthases. J. Mol. Biol. 410, 105–117 (2011)

    Article  CAS  PubMed  Google Scholar 

  9. Zheng, J., Taylor, C. A., Piasecki, S. K. & Keatinge-Clay, A. T. Structural and functional analysis of A-type ketoreductases from the amphotericin modular polyketide synthase. Structure 18, 913–922 (2010)

    Article  CAS  PubMed  Google Scholar 

  10. Keatinge-Clay, A. T. A tylosin ketoreductase reveals how chirality is determined in polyketides. Chem. Biol. 14, 898–908 (2007)

    Article  CAS  PubMed  Google Scholar 

  11. Keatinge-Clay, A. T. & Stroud, R. M. The structure of a ketoreductase determines the organization of the β-carbon processing enzymes of modular polyketide synthases. Structure 14, 737–748 (2006)

    Article  CAS  PubMed  Google Scholar 

  12. Keatinge-Clay, A. Crystal structure of the erythromycin polyketide synthase dehydratase. J. Mol. Biol. 384, 941–953 (2008)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Akey, D. L. et al. Crystal structures of dehydratase domains from the curacin polyketide biosynthetic pathway. Structure 18, 94–105 (2010)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Zheng, J., Gay, D. C., Demeler, B., White, M. A. & Keatinge-Clay, A. T. Divergence of multimodular polyketide synthases revealed by a didomain structure. Nature Chem. Biol. 8, 615–621 (2012)

    Article  CAS  Google Scholar 

  15. Gehret, J. J. et al. Terminal alkene formation by the thioesterase of curacin A biosynthesis: structure of a decarboxylating thioesterase. J. Biol. Chem. 286, 14445–14454 (2011)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Scaglione, J. B. et al. Biochemical and structural characterization of the tautomycetin thioesterase: analysis of a stereoselective polyketide hydrolase. Angew. Chem. Int. Edn Engl. 49, 5726–5730 (2010)

    Article  CAS  Google Scholar 

  17. Tsai, S. C., Lu, H., Cane, D. E., Khosla, C. & Stroud, R. M. Insights into channel architecture and substrate specificity from crystal structures of two macrocycle-forming thioesterases of modular polyketide synthases. Biochemistry 41, 12598–12606 (2002)

    Article  CAS  PubMed  Google Scholar 

  18. Tsai, S. C. et al. Crystal structure of the macrocycle-forming thioesterase domain of the erythromycin polyketide synthase: versatility from a unique substrate channel. Proc. Natl Acad. Sci. USA 98, 14808–14813 (2001)

    Article  CAS  ADS  PubMed  PubMed Central  Google Scholar 

  19. Bonnett, S. A. et al. Structural and stereochemical analysis of a modular polyketide synthase ketoreductase domain required for the generation of a cis-alkene. Chem. Biol. 20, 772–783 (2013)

    Article  CAS  PubMed  Google Scholar 

  20. Alekseyev, V. Y., Liu, C. W., Cane, D. E., Puglisi, J. D. & Khosla, C. Solution structure and proposed domain–domain recognition interface of an acyl carrier protein domain from a modular polyketide synthase. Protein Sci. 16, 2093–2107 (2007)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Whicher, J. R. et al. Cyanobacterial polyketide synthase docking domains, a new tool for engineering natural product biosynthesis. Chem. Biol. 20, 1340–1351 (2013)

    Article  CAS  PubMed  Google Scholar 

  22. Maier, T., Leibundgut, M. & Ban, N. The crystal structure of a mammalian fatty acid synthase. Science 321, 1315–1322 (2008)

    Article  CAS  ADS  PubMed  Google Scholar 

  23. Xue, Y., Zhao, L., Liu, H. W. & Sherman, D. H. A gene cluster for macrolide antibiotic biosynthesis in Streptomyces venezuelae: architecture of metabolic diversity. Proc. Natl Acad. Sci. USA 95, 12111–12116 (1998)

    Article  CAS  ADS  PubMed  PubMed Central  Google Scholar 

  24. Aldrich, C. C., Beck, B. J., Fecik, R. A. & Sherman, D. H. Biochemical investigation of pikromycin biosynthesis employing native penta- and hexaketide chain elongation intermediates. J. Am. Chem. Soc. 127, 8441–8452 (2005)

    Article  CAS  PubMed  Google Scholar 

  25. Aldrich, C. C., Venkatraman, L., Sherman, D. H. & Fecik, R. A. Chemoenzymatic synthesis of the polyketide macrolactone 10-deoxymethynolide. J. Am. Chem. Soc. 127, 8910–8911 (2005)

    Article  CAS  PubMed  Google Scholar 

  26. Lyon, A. M., Dutta, S., Boguth, C. A., Skiniotis, G. & Tesmer, J. J. Full-length Gαq–phospholipase C-β3 structure reveals interfaces of the C-terminal coiled-coil domain. Nature Struct. Mol. Biol. 20, 355–362 (2013)

    Article  CAS  Google Scholar 

  27. Strunk, B. S. et al. Ribosome assembly factors prevent premature translation initiation by 40S assembly intermediates. Science 333, 1449–1453 (2011)

    Article  CAS  ADS  PubMed  PubMed Central  Google Scholar 

  28. Staunton, J. et al. Evidence for a double-helical structure for modular polyketide synthases. Nature Struct. Biol. 3, 188–192 (1996)

    Article  CAS  PubMed  Google Scholar 

  29. Kao, C. M., Pieper, R., Cane, D. E. & Khosla, C. Evidence for two catalytically independent clusters of active sites in a functional modular polyketide synthase. Biochemistry 35, 12363–12368 (1996)

    Article  CAS  PubMed  Google Scholar 

  30. Gokhale, R. S., Lau, J., Cane, D. E. & Khosla, C. Functional orientation of the acyltransferase domain in a module of the erythromycin polyketide synthase. Biochemistry 37, 2524–2528 (1998)

    Article  CAS  PubMed  Google Scholar 

  31. Whicher, J. R. et al. Structural rearrangements of a polyketide synthase module during its catalytic cycle. Nature http://dx.doi.org/10.1038/nature13409 (this issue)

  32. Yin, Y., Lu, H., Khosla, C. & Cane, D. E. Expression and kinetic analysis of the substrate specificity of modules 5 and 6 of the picromycin/methymycin polyketide synthase. J. Am. Chem. Soc. 125, 5671–5676 (2003)

    Article  CAS  PubMed  Google Scholar 

  33. Yuzawa, S., Kapur, S., Cane, D. E. & Khosla, C. Role of a conserved arginine residue in linkers between the ketosynthase and acyltransferase domains of multimodular polyketide synthases. Biochemistry 51, 3708–3710 (2012)

    Article  CAS  PubMed  Google Scholar 

  34. Kittendorf, J. D., Beck, B. J., Buchholz, T. J., Seufert, W. & Sherman, D. H. Interrogating the molecular basis for multiple macrolactone ring formation by the pikromycin polyketide synthase. Chem. Biol. 14, 944–954 (2007)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Kapur, S., Chen, A. Y., Cane, D. E. & Khosla, C. Molecular recognition between ketosynthase and acyl carrier protein domains of the 6-deoxyerythronolide B synthase. Proc. Natl Acad. Sci. USA 107, 22066–22071 (2010)

    Article  CAS  ADS  PubMed  PubMed Central  Google Scholar 

  36. Kapur, S. et al. Reprogramming a module of the 6-deoxyerythronolide B synthase for iterative chain elongation. Proc. Natl Acad. Sci. USA 109, 4110–4115 (2012)

    Article  CAS  ADS  PubMed  PubMed Central  Google Scholar 

  37. Bonnett, S. A. et al. Acyl-CoA subunit selectivity in the pikromycin polyketide synthase PikAIV: steady-state kinetics and active-site occupancy analysis by FTICR-MS. Chem. Biol. 18, 1075–1081 (2011)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Huang, W. et al. Crystal structure of β-ketoacyl-acyl carrier protein synthase II from E. coli reveals the molecular architecture of condensing enzymes. EMBO J. 17, 1183–1191 (1998)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Chemler, J. A. et al. Biochemical and structural characterization of germicidin synthase: analysis of a type III polyketide synthase that employs acyl-ACP as a starter unit donor. J. Am. Chem. Soc. 134, 7359–7366 (2012)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Keatinge-Clay, A. T., Maltby, D. A., Medzihradszky, K. F., Khosla, C. & Stroud, R. M. An antibiotic factory caught in action. Nature Struct. Biol. 11, 888–893 (2004)

    Article  CAS  Google Scholar 

  41. Ferrer, J. L., Jez, J. M., Bowman, M. E., Dixon, R. A. & Noel, J. P. Structure of chalcone synthase and the molecular basis of plant polyketide biosynthesis. Nature Struct. Biol. 6, 775–784 (1999)

    Article  CAS  PubMed  Google Scholar 

  42. Leibundgut, M., Jenni, S., Frick, C. & Ban, N. Structural basis for substrate delivery by acyl carrier protein in the yeast fatty acid synthase. Science 316, 288–290 (2007)

    Article  CAS  ADS  PubMed  Google Scholar 

  43. Beck, B. J., Yoon, Y. J., Reynolds, K. A. & Sherman, D. H. The hidden steps of domain skipping: macrolactone ring size determination in the pikromycin modular polyketide synthase. Chem. Biol. 9, 575–583 (2002)

    Article  CAS  PubMed  Google Scholar 

  44. Rowe, C. J. et al. Engineering a polyketide with a longer chain by insertion of an extra module into the erythromycin-producing polyketide synthase. Chem. Biol. 8, 475–485 (2001)

    Article  CAS  PubMed  Google Scholar 

  45. Zheng, J., Fage, C. D., Demeler, B., Hoffman, D. W. & Keatinge-Clay, A. T. The missing linker: a dimerization motif located within polyketide synthase modules. ACS Chem. Biol. 8, 1263–1270 (2013)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Bunkoczi, G. et al. Mechanism and substrate recognition of human holo ACP synthase. Chem. Biol. 14, 1243–1253 (2007)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Masoudi, A., Raetz, C. R., Zhou, P. & Pemble, C. W. IV Chasing acyl carrier protein through a catalytic cycle of lipid A production. Nature 505, 422–426 (2014)

    Article  CAS  ADS  PubMed  Google Scholar 

  48. Nguyen, C. et al. Trapping the dynamic acyl carrier protein in fatty acid biosynthesis. Nature 505, 427–431 (2014)

    Article  CAS  ADS  PubMed  Google Scholar 

  49. Wu, N., Tsuji, S. Y., Cane, D. E. & Khosla, C. Assessing the balance between protein–protein interactions and enzyme–substrate interactions in the channeling of intermediates between polyketide synthase modules. J. Am. Chem. Soc. 123, 6465–6474 (2001)

    Article  CAS  PubMed  Google Scholar 

  50. Beck, B. J., Aldrich, C. C., Fecik, R. A., Reynolds, K. A. & Sherman, D. H. Iterative chain elongation by a pikromycin monomodular polyketide synthase. J. Am. Chem. Soc. 125, 4682–4683 (2003)

    Article  CAS  PubMed  Google Scholar 

  51. Pfeifer, B. A., Admiraal, S. J., Gramajo, H., Cane, D. E. & Khosla, C. Biosynthesis of complex polyketides in a metabolically engineered strain of E. coli. Science 291, 1790–1792 (2001)

    Article  CAS  ADS  PubMed  Google Scholar 

  52. Sánchez, C., Du, L., Edwards, D. J., Toney, M. D. & Shen, B. Cloning and characterization of a phosphopantetheinyl transferase from Streptomyces verticillus ATCC15003, the producer of the hybrid peptide–polyketide antitumor drug bleomycin. Chem. Biol. 8, 725–738 (2001)

    Article  PubMed  Google Scholar 

  53. Ohi, M., Li, Y., Cheng, Y. & Walz, T. Negative staining and image classification— powerful tools in modern electron microscopy. Biol. Proced. Online 6, 23–34 (2004)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Ludtke, S. J., Baldwin, P. R. & Chiu, W. EMAN: semiautomated software for high-resolution single-particle reconstructions. J. Struct. Biol. 128, 82–97 (1999)

    Article  CAS  PubMed  Google Scholar 

  55. Tang, G. et al. EMAN2: an extensible image processing suite for electron microscopy. J. Struct. Biol. 157, 38–46 (2007)

    Article  CAS  PubMed  Google Scholar 

  56. Baker, M. L., Zhang, J., Ludtke, S. J. & Chiu, W. Cryo-EM of macromolecular assemblies at near-atomic resolution. Nature Protocols 5, 1697–1708 (2010)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. Rosenthal, P. B. & Henderson, R. Optimal determination of particle orientation, absolute hand, and contrast loss in single-particle electron cryomicroscopy. J. Mol. Biol. 333, 721–745 (2003)

    Article  CAS  PubMed  Google Scholar 

  58. Fernández, J. J., Luque, D., Caston, J. R. & Carrascosa, J. L. Sharpening high resolution information in single particle electron cryomicroscopy. J. Struct. Biol. 164, 170–175 (2008)

    Article  PubMed  Google Scholar 

  59. Scheres, S. H. & Chen, S. Prevention of overfitting in cryo-EM structure determination. Nature Methods 9, 853–854 (2012)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  60. Li, X. et al. Electron counting and beam-induced motion correction enable near-atomic-resolution single-particle cryo-EM. Nature Methods 10, 584–590 (2013)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  61. Chen, S. et al. High-resolution noise substitution to measure overfitting and validate resolution in 3D structure determination by single particle electron cryomicroscopy. Ultramicroscopy 135, 24–35 (2013)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  62. Henderson, R. et al. Tilt-pair analysis of images from a range of different specimens in single-particle electron cryomicroscopy. J. Mol. Biol. 413, 1028–1046 (2011)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  63. Murray, S. C., Flanagan, J., Popova, O. B., Chiu, W., Ludtke, S. J. & Serysheva, I. I. Validation of cryo-EM structure of IP3R1 channel. Structure 21, 900–909 (2013)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  64. Pettersen, E. F. et al. UCSF Chimera—a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004)

    CAS  PubMed  Google Scholar 

  65. Hansen, D. A. et al. Biocatalytic synthesis of pikromycin, methymycin, neomethymycin, novamethymycin, and ketomethymycin. J. Am. Chem. Soc. 135, 11232–11238 (2013

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

We thank R. Henderson for sharing the phase-randomization program and for valuable advice on the implementation of the tilt-pair validation, and Y. Cheng and X. Li for sharing the image sub-frame alignment program. This work was supported by the Pew Scholar Program in Biomedical Sciences (G.S.), the University of Michigan Biological Sciences Scholars Program (G.S.), Rackham Merit and American Foundation for Pharmaceutical Education predoctoral fellowships (D.A.H.), a National Research Service Award postdoctoral fellowship (J.A.C.), the Life Sciences Research Foundation (A.R.H.N.), National Institutes of Health grants 1R21CA138331-01A1 (K.H.), GM076477 (D.H.S. and J.L.S.), DK042303 (J.L.S.) and DK090165 (G.S.), and the Hans W. Vahlteich Professorship (to D.H.S.).

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Author notes

  1. Somnath Dutta and Jonathan R. Whicher: These authors contributed equally to this work.

Authors and Affiliations

  1. Life Sciences Institute, University of Michigan, Ann Arbor, 48109, Michigan, USA

    Somnath Dutta, Jonathan R. Whicher, Douglas A. Hansen, Joseph A. Chemler, Grady R. Congdon, Alison R. H. Narayan, David H. Sherman, Janet L. Smith & Georgios Skiniotis

  2. Chemical Biology Graduate Program, University of Michigan, Ann Arbor, 48109, Michigan, USA

    Jonathan R. Whicher

  3. Department of Medicinal Chemistry, University of Michigan, Ann Arbor, 48109, Michigan, USA

    Douglas A. Hansen & David H. Sherman

  4. Department of Chemistry, University of Michigan, Ann Arbor, 48109, Michigan, USA

    Wendi A. Hale, Kristina Håkansson & David H. Sherman

  5. Department of Microbiology & Immunology, University of Michigan, Ann Arbor, 48109, Michigan, USA

    David H. Sherman

  6. Department of Biological Chemistry, University of Michigan, Ann Arbor, 48109, Michigan, USA

    Janet L. Smith & Georgios Skiniotis

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  1. Somnath Dutta
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Contributions

S.D. carried out cryo-EM data collection and processing. J.R.W. produced PikAIII variants and conducted enzyme assays. G.R.C. assisted with cryo-EM image processing. W.A.H., A.R.H.N. and K.H. carried out mass-spectrometry analysis. D.A.H. synthesized the thiophenol-activated pentaketide and pentaketide–CoA substrates. J.A.C. produced initial PikAIII samples and prepared Fig. 1. S.D., J.R.W., J.L.S. and G.S. analysed the data and interpreted results. K.H., D.H.S., J.L.S. and G.S. designed research. S.D., J.R.W., D.H.S., J.L.S. and G.S. wrote the manuscript.

Corresponding author

Correspondence to Georgios Skiniotis.

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The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 PikAIII sample preparation and raw EM images.

a, SDS–polyacrylamide gel electrophoresis (SDS–PAGE) gel of each purified form of PikAIII examined by cryo-EM. The numbers on the left indicate molecular weight in kDa. b, Raw EM image of holo-PikAIII particles embedded in negative stain. c, Raw cryo-EM image of holo-PikAIII particles. d, Boxed-out particle projections of holo-PikAIII.

Extended Data Figure 2 PikAIII initial cryo-EM three-dimensional reconstructions.

a, Generation of initial MM–PikAIII reconstructions using 3,600 particle projections and employing three different starting models. Top, generation of a MM–PikAIII reconstruction using a randomized Gaussian sphere from makeinitialmodel.py in EMAN2 (ref. 56). Middle, refinement of a starting model obtained by the EMAN2 initial-model-generation program e2initialmodel.py55. Bottom, refinement starting from a low-pass filtered model of the excised KS–AT di-domain crystal structure7. These three different starting models converged to similar structures that are also in agreement with reference-free class averages. b, Comparison of re-projections of a low-resolution cryo-EM three-dimensional map (iteration 28 of top panel in this figure) with reference-free two-dimensional class averages and raw particle projections from both cryo-EM and negative-stain MM–PikAIII data. c, Tilt-pair parameter plot of PikAIII(ΔACP5) with a tilt angle differential of 30° (+15°/−15°). Black dots represent each particle pair’s tilt axis and tilt angle based on the cryo-EM map of PikAIII(ΔACP5). Most particle projection pairs cluster in a region (red circle) centred at 29.5° with a root mean squared deviation (r.m.s.d.) tilt angle of 8.3° and tilt axis of 90.23°.

Extended Data Figure 3 Cryo-EM analysis of MM–PikAIII.

a, Refinement of MM–PikAIII reconstruction: a three-dimensional model obtained ab initio (see Extended Data Fig. 2) was used as a starting reference for initial projection matching of ∼29,000 cryo-EM particle views to provide a map at 8.1 Å resolution (middle), which was subsequently used for the refinement of the entire MM–PikAIII data set (56,292 projections) to obtain a final map at 7.3 Å resolution. The panels on the bottom show vertical and horizontal cross-sections of the final MM–PikAIII cryo-EM map. b, Cross-correlation values between the overall pseudo-atomic-resolution model and the three-dimensional maps of various states of PikAIII.

Extended Data Figure 4 Estimation of EM map resolution and assessment of over-refinement.

Conventional Fourier shell correlation (FSC) curves (blue), gold-standard FSC (red) and high-resolution phase randomization tests (green) for PikAIII reconstructions. For conventional FSC calculations (full data-set refinement) we have used the conservative FSC = 0.5 criterion as a resolution indicator, whereas for gold-standard FSC calculations (half data-set independent refinements) we have used the FSC = 0.143 criterion, as previously applied59. Consistently, the indicated resolution at FSC = 0.5 of the conventional FSC is in close agreement with FSC = 0.143 of the gold-standard FSC. The FSC calculations with phase randomized data show a sharp drop-off at the expected resolution level (10 Å, or 12 Å for holo-PikAIII) and a lack of noise refinement. Additionally, we measured the FSC between the average map of the two gold-standard half-maps and the conventional map (full data set) as implemented previously60 (see Extended Data Fig. 10). The agreement between the conventional and the average gold-standard map by this method is also fully consistent with conventional and gold-standard FSC calculations. The table inset summarizes the number of projections used for each reconstruction, the conventional and gold-standard FSC resolution indications, and the agreement by FSC between the average map of the two gold-standard half-maps and the conventional map.

Extended Data Figure 5 Partial mass spectra of active-site PikAIII and ACP4–PikAIII(C209A/ΔACP5) peptides from LC/FT-ICR MS of trypsin-digested proteins.

a–d, ACP5 active-site peptides in their apo (a, b) and holo (with phosphopantetheine (Ppant) (c, d) states at 2+ and 3+ charge states. On the basis of integrated peak abundances from multiple LC/MS runs, greater than 97% of the ACP5 Ser 1438-containing peptides were modified with Ppant. e, f, Confirmation of the C209A mutation of the KS5 active site. The mutated active-site peptide was detected in the 4+ (e) and 3+ (f) charge states. g–i, Example mass spectra of Ser 3605-containing active-site ACP4-derived peptides following enzymatic loading of the pentaketide from pentaketide–CoA. Both apo- (g), holo- (with Ppant) (h) and pentaketide–ACP4 (i) were detected. j–l, Example mass spectra of active-site ACP4-derived peptides from a control experiment in which pentaketide–CoA was absent. The majority of the ACP4 active-site peptides were detected in the apo and holo states, while a very small percentage (<1%) contained the pentaketide intermediate. m–p, ACP5 active-site peptides following incubation with MM–CoA. The MM building block was detected in high abundance on ACP5 Ser 1438 (o, p) with some unloaded holo-protein as well (m, n). q–s, AT5 active-site peptides following incubation with MM–CoA. The MM building block was detected on AT Ser 655.

Extended Data Figure 6 PikAIII domain organization and connectivity.

a, Crystal structure of excised DEBS module 5 KS–AT di-domain7. KS (blue, yellow active site) and AT (green with red active site) domains interact differently than in the full module (Fig. 2), and the post-AT linker (red) lies on the surface of the KS domain. b, Localization of post-ACP5 dimerization helices. Top, stereo view of holo-PikAIII conformer I with the density ascribed to the post-ACP5 dimerization helices (rendered in cyan) observed between the ACP5 domains (orange). Bottom, overview of localization and enlarged cut-out densities of post-ACP5 dimerization helices (cyan) in holo-PikAIII conformer I. c, Stereo view of holo-PikAIII conformer II with the density ascribed to the post-ACP5 dimerization helices (rendered in cyan) observed between the ACP5 domains (orange). d, Proposed connectivity of domains in PikAIII determined by distances between domain termini and linker lengths. The catalytic domains are coloured (green or blue) according to the assigned polypeptide chain. The AT interacts with the KS of the opposite monomer whereas the AT–KR interaction is within the monomer. Active-site locations are indicated in yellow.

Extended Data Figure 7 Domain interfaces in PikAIII.

a, Stereo view of the docked crystal structures of KS (blue) and AT (green) in the holo-PikAIII cryo-EM map reveals an extensive interface. The red star marks the side entrance to the KS active site where the catalytic Cys 209 (yellow spheres) resides. b, The interface of KS (blue) and AT (green) is less than 20 Å from the KS active-site Cys 209 (spheres; blue C and yellow S). This is the only region of steric clashes between the KS and AT crystal structures rigidly docked in the three-dimensional maps (KS5 amino acids 350–357 clash with AT5 488–498 and 526–531). Asp 352 (spheres; blue C and red O) of the KS and Lys 490 and Arg 525 (spheres; green C and blue N) of the AT were substituted with Ala in PikAIII–TE. D352A and K490A, which are located in the clash zone, resulted in 0% and 50% activity, respectively, relative to wild-type PikAIII–TE. The R525A substitution abolished product formation even though this residue is outside the clash zone. The sensitivity of Arg 525 and insensitivity of Lys 490 to Ala substitution is consistent with a structural rearrangement at the KS–AT interface. c, The docked crystal structures of AT (green) and KR (purple) in the holo-PikAIII cryo-EM map. The interface is formed primarily by a loop of KR (residues 928–936) and an α-helix of AT (residues 760–775). d, The KR domain of PikAIII(ΔACP5) (right) is rotated by 165° compared to holo-PikAIII (left). e, View of the unobstructed path and proximity of Ser 1438 (red) and Cys 209 (yellow) in the docked structures of KS and ACP in the MM–PikAIII cryo-EM map.

Extended Data Figure 8 PikAIII functional assays.

a, Example high-performance liquid chromatography (HPLC) traces of the PikAIII–TE assay. The levels of 10-dml produced by wild-type (WT) PikAIII–TE (red trace), D352A PikAIII–TE (green), K490A PikAIII–TE (blue), R525A PikAIII–TE (orange), and a no enzyme control (yellow) are shown. b, Activity of PikAIII–TE mutants. ND, not detectable. All reactions were completed in triplicate and the data are presented as average ± standard deviation. c, Example HPLC traces of the PikAIII/PikAIV assay. The levels of 10-dml and nbl produced by wild-type PikAIII/PikAIV (red trace), wild-type PikAIII with PikAIV(R147E) (green), wild-type PikAIII with PikAIV(R320E) (blue), and a no enzyme control (yellow) are shown. d, Activity of PikAIV mutants. All reactions were completed in triplicate and the data are presented as average ± standard deviation.

Extended Data Figure 9 Analysis of ACP-less PikAIII.

a, Overlay of gel filtration chromatography elution profiles of PikAIII(ΔACP5) (blue) and PikAIII(Δ1403–1562) (red). PikAIII(ΔACP5) includes the post-ACP dimerization helices and elutes as a dimer whereas PikAIII(Δ1403–1562) lacks the dimerization helices and elutes as a monomer. The first peak in the red trace is apparently aggregated protein in the void volume of the S300 column. b, Solid rendering (left) and transparent representation with modelled structures (right) of the cryo-EM map of PikAIII(ΔACP5) at a resolution of 7.8 Å. c, Example HPLC traces of chromophore–CoA loading experiments. The blue trace (280 nm) indicates the level of protein and the red trace (550 nm) indicates the chromophore from CoA 547 (New England Biolabs). Incubation of apo-ACP4–PikAIII(C209A/ΔACP5) with SVP and CoA 547 indicates that 100% of the ACP4 was in the apo form, based on molar extinction coefficients for protein and chromophore. d, Incubation of pentaketide–ACP4–PikAIII(C209A/ΔACP5) with SVP and CoA 547 indicates that 80% of the ACP4 was loaded with pentaketide. e, Conventional FSC curve for the three-dimensional reconstruction of holo-ACP4–PikAIII(C209A/ΔACP5) (no pentaketide added). f, Orthogonal views of solid rendering (top) and transparent representations with modelled structures (bottom) of the cryo-EM three-dimensional reconstruction of holo-ACP4–PikAIII(C209A/ΔACP5) (no pentaketide added). No density for the upstream ACP4 was observed in the cryo-EM map even though densities corresponding to the N-terminal docking domains are clearly visible (compare with Fig. 3b). Fit into the three-dimensional maps shown in b and f are the structures of DEBS module 5 KS (blue; PDB accession 2HG4), DEBS module 5 AT (green; PDB accession 2HG4) and DEBS module 1 KR (purple; PDB accession 2FR0).

Extended Data Figure 10 Cryo-EM map refinement and resolution validation scheme.

The flow chart shows the overall three-dimensional reconstruction scheme and resolution calculation by conventional and gold-standard FSC procedures using MM–PikAIII as an example. The procedure was applied for every high-resolution three-dimensional reconstruction in this study. Besides the conventional full data set refinement (left), each data set was split into two separate half data sets, which were employed for two independent reconstructions using the 50 Å filtered EM map as an initial reference (right; gold-standard procedure). The final two gold half reconstructions were compared by FSC, and the indicated resolution by gold-standard FSC with the 0.143 criterion59 showed excellent agreement with the value indicated at the 0.5 level of the conventional FSC (Extended Data Fig. 4). In addition, the two gold-standard half-maps were averaged, and the resulting average gold-standard map was compared by FSC to the corresponding conventional map, again showing very good agreement at the same resolution range (Extended Data Fig. 4). These tests, along with the phase randomization tests (Extended Data Fig. 4), reveal the lack of over-refinement and accurate resolution values reported in this study.

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Dutta, S., Whicher, J., Hansen, D. et al. Structure of a modular polyketide synthase. Nature 510, 512–517 (2014). https://doi.org/10.1038/nature13423

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