Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Cryo-EM reveals the transition of Arp2/3 complex from inactive to nucleation-competent state

Abstract

Arp2/3 complex, a crucial actin filament nucleator, undergoes structural rearrangements during activation by nucleation-promoting factors (NPFs). However, the conformational pathway leading to the nucleation-competent state is unclear due to lack of high-resolution structures of the activated state. Here we report a ~3.9 Å resolution cryo-EM structure of activated Schizosaccharomyces pombe Arp2/3 complex bound to the S. pombe NPF Dip1 and attached to the end of the nucleated actin filament. The structure reveals global and local conformational changes that allow the two actin-related proteins in Arp2/3 complex to mimic a filamentous actin dimer and template nucleation. Activation occurs through a clamp-twisting mechanism, in which Dip1 forces two core subunits in Arp2/3 complex to pivot around one another, shifting half of the complex into a new activated position. By showing how Dip1 stimulates activation, the structure reveals how NPFs can activate Arp2/3 complex in diverse cellular processes.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Fig. 1: Overview of structures of inactive Arp2/3 complex and activated Arp2/3 complex bound to the actin filament pointed end and Dip1.
Fig. 2: Arp2 and Arp3 flatten and adopt the short-pitch helical conformation upon activation.
Fig. 3: Arp2 and Arp3 make filament-like long-pitch interactions with actin.
Fig. 4: Nucleotide-binding states of the Arps and actin.
Fig. 5: Dip1 bends the ARPC4 long helix to twist the clamp and rotate a block of subunits into the short-pitch conformation.
Fig. 6: Proposed mechanism of Arp2/3 complex activation by Dip1.

Data availability

EM maps and atomic models were deposited in the Electron Microscopy Data Bank and wwPDB, respectively, with accession entries EMD-21502 and PDB 6W17 (Dip1–Arp2/3–actin) and EMD-21503 and PDB 6W18 (inactive Arp2/3 complex).

References

  1. 1.

    Goley, E. D. & Welch, M. D. The ARP2/3 complex: an actin nucleator comes of age. Nat. Rev. Mol. Cell Biol. 7, 713–726 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  2. 2.

    Higgs, H. N. & Pollard, T. D. Regulation of actin polymerization by Arp2/3 complex and WASp/Scar proteins. J. Biol. Chem. 274, 32531–32534 (1999).

    CAS  Google Scholar 

  3. 3.

    Wagner, A. R., Luan, Q., Liu, S.-L. & Nolen, B. J. Dip1 defines a class of Arp2/3 complex activators that function without preformed actin filaments. Curr. Biol. 23, 1990–1998 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  4. 4.

    Goley, E. D., Rodenbusch, S. E., Martin, A. C. & Welch, M. D. Critical conformational changes in the Arp2/3 complex are induced by nucleotide and nucleation promoting factor. Mol. Cell 16, 269–279 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  5. 5.

    Rodal, A. A. et al. Conformational changes in the Arp2/3 complex leading to actin nucleation. Nat. Struct. Mol. Biol. 12, 26–31 (2005).

    CAS  PubMed  Google Scholar 

  6. 6.

    Boczkowska, M., Rebowski, G., Kast, D. J. & Dominguez, R. Structural analysis of the transitional state of Arp2/3 complex activation by two actin-bound WCAs. Nat. Commun. 5, 3308 (2014).

    PubMed  PubMed Central  Google Scholar 

  7. 7.

    Rodnick-Smith, M., Liu, S.-L., Balzer, C. J., Luan, Q. & Nolen, B. J. Identification of an ATP-controlled allosteric switch that controls actin filament nucleation by Arp2/3 complex. Nat. Commun. 7, 12226 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  8. 8.

    Rodnick-Smith, M., Luan, Q., Liu, S.-L. & Nolen, B. J. Role and structural mechanism of WASP-triggered conformational changes in branched actin filament nucleation by Arp2/3 complex. Proc. Natl Acad. Sci. USA 113, E3834–E3843 (2016).

    CAS  PubMed  Google Scholar 

  9. 9.

    Xu, X.-P. et al. Three-dimensional reconstructions of Arp2/3 complex with bound nucleation promoting factors. EMBO J. 31, 236–247 (2012).

    CAS  PubMed  Google Scholar 

  10. 10.

    Espinoza-Sanchez, S., Metskas, L. A., Chou, S. Z., Rhoades, E. & Pollard, T. D. Conformational changes in Arp2/3 complex induced by ATP, WASp-VCA and actin filaments. Proc. Natl Acad. Sci. USA 115, E8642–E8651 (2018).

    CAS  PubMed  Google Scholar 

  11. 11.

    Zimmet, A. et al. Cryo-EM structure of NPF-bound human Arp2/3 complex and activation mechanism. Sci. Adv. 6, eaaz7651 (2020).

    CAS  PubMed  PubMed Central  Google Scholar 

  12. 12.

    Rouiller, I. et al. The structural basis of actin filament branching by the Arp2/3 complex. J. Cell Biol. 180, 887–895 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  13. 13.

    Nolen, B. J., Littlefield, R. S. & Pollard, T. D. Crystal structures of actin-related protein 2/3 complex with bound ATP or ADP. Proc. Natl Acad. Sci. USA 101, 15627–15632 (2004).

    CAS  PubMed  Google Scholar 

  14. 14.

    Robinson, R. C. et al. Crystal structure of Arp2/3 complex. Science 294, 1679–1684 (2001).

    CAS  PubMed  Google Scholar 

  15. 15.

    Merino, F. et al. Structural transitions of F-actin upon ATP hydrolysis at near-atomic resolution revealed by cryo-EM. Nat. Struct. Mol. Biol. 25, 528–537 (2018).

    CAS  PubMed  Google Scholar 

  16. 16.

    Chou, S. Z. & Pollard, T. D. Mechanism of actin polymerization revealed by cryo-EM structures of actin filaments with three different bound nucleotides. Proc. Natl Acad. Sci. USA 116, 4265–4274 (2019).

    CAS  PubMed  Google Scholar 

  17. 17.

    Balzer, C. J., Wagner, A. R., Helgeson, L. A. & Nolen, B. J. Single-turnover activation of Arp2/3 complex by Dip1 may balance nucleation of linear versus branched actin filaments. Curr. Biol. 29, 3331–3338 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  18. 18.

    Tan, Y. Z. et al. Addressing preferred specimen orientation in single-particle cryo-EM through tilting. Nat. Methods 14, 793–796 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  19. 19.

    Luan, Q. & Nolen, B. J. Structural basis for regulation of Arp2/3 complex by GMF. Nat. Struct. Mol. Biol. 20, 1062–1068 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  20. 20.

    Oda, T., Iwasa, M., Aihara, T., Maéda, Y. & Narita, A. The nature of the globular- to fibrous-actin transition. Nature 457, 441–445 (2009).

    CAS  PubMed  Google Scholar 

  21. 21.

    Ingerman, E., Hsiao, J. Y. & Mullins, R. D. Arp2/3 complex ATP hydrolysis promotes lamellipodial actin network disassembly but is dispensable for assembly. J. Cell Biol. 200, 619–633 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. 22.

    Martin, A. C., Welch, M. D. & Drubin, D. G. Arp2/3 ATP hydrolysis-catalysed branch dissociation is critical for endocytic force generation. Nat. Cell Biol. 8, 826–833 (2006).

    CAS  PubMed  Google Scholar 

  23. 23.

    Vorobiev, S. et al. The structure of nonvertebrate actin: implications for the ATP hydrolytic mechanism. Proc. Natl Acad. Sci. USA 100, 5760–5765 (2003).

    CAS  PubMed  Google Scholar 

  24. 24.

    Dayel, M. J. & Mullins, R. D. Activation of Arp2/3 complex: addition of the first subunit of the new filament by a WASP protein triggers rapid ATP hydrolysis on Arp2. PLoS Biol. 2, E91 (2004).

    PubMed  PubMed Central  Google Scholar 

  25. 25.

    Le Clainche, C., Pantaloni, D. & Carlier, M.-F. ATP hydrolysis on actin-related protein 2/3 complex causes debranching of dendritic actin arrays. Proc. Natl Acad. Sci. USA 100, 6337–6342 (2003).

    PubMed  Google Scholar 

  26. 26.

    Dalhaimer, P. & Pollard, T. D. Molecular dynamics simulations of Arp2/3 complex activation. Biophys. J. 99, 2568–2576 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  27. 27.

    Luan, Q., Liu, S.-L., Helgeson, L. A. & Nolen, B. J. Structure of the nucleation-promoting factor SPIN90 bound to the actin filament nucleator Arp2/3 complex. EMBO J. 37, 100005 (2018).

  28. 28.

    Ti, S.-C., Jurgenson, C. T., Nolen, B. J. & Pollard, T. D. Structural and biochemical characterization of two binding sites for nucleation-promoting factor WASp-VCA on Arp2/3 complex. Proc. Natl Acad. Sci. USA 108, E463–E471 (2011).

    CAS  PubMed  Google Scholar 

  29. 29.

    Luan, Q., Zelter, A., MacCoss, M. J., Davis, T. N. & Nolen, B. J. Identification of Wiskott–Aldrich syndrome protein (WASP) binding sites on the branched actin filament nucleator Arp2/3 complex. Proc. Natl Acad. Sci. USA 115, E1409–E1418 (2018).

    CAS  PubMed  Google Scholar 

  30. 30.

    Padrick, S. B., Doolittle, L. K., Brautigam, C. A., King, D. S. & Rosen, M. K. Arp2/3 complex is bound and activated by two WASP proteins. Proc. Natl Acad. Sci. USA 108, E472–E479 (2011).

    CAS  Google Scholar 

  31. 31.

    Hetrick, B., Han, M. S., Helgeson, L. A. & Nolen, B. J. Small molecules CK-666 and CK-869 inhibit actin-related protein 2/3 complex by blocking an activating conformational change. Chem. Biol. 20, 701–712 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  32. 32.

    Alekhina, O., Burstein, E. & Billadeau, D. D. Cellular functions of WASP family proteins at a glance. J. Cell Sci. 130, 2235–2241 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  33. 33.

    Basu, R. & Chang, F. Characterization of Dip1p reveals a switch in Arp2/3-dependent actin assembly for fission yeast endocytosis. Curr. Biol. 21, 905–916 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  34. 34.

    Balzer, C. J., Wagner, A. R., Helgeson, L. A. & Nolen, B. J. Dip1 co-opts features of branching nucleation to create linear actin filaments that activate WASP-bound Arp2/3 complex. Curr. Biol. 28, 3886–3891 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  35. 35.

    Jurgenson, C. T. & Pollard, T. D. Crystals of the Arp2/3 complex in two new space groups with structural information about actin-related protein 2 and potential WASP binding sites. Acta Crystallogr. F Struct. Biol. Commun. 71, 1161–1168 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  36. 36.

    Marchand, J. B., Kaiser, D. A., Pollard, T. D. & Higgs, H. N. Interaction of WASP/Scar proteins with actin and vertebrate Arp2/3 complex. Nat. Cell Biol. 3, 76–82 (2001).

    CAS  PubMed  PubMed Central  Google Scholar 

  37. 37.

    Smith, B. A. et al. Three-color single molecule imaging shows WASP detachment from Arp2/3 complex triggers actin filament branch formation. Elife 2, e01008 (2013).

    PubMed  PubMed Central  Google Scholar 

  38. 38.

    Scheres, S. H. W. RELION: implementation of a Bayesian approach to cryo-EM structure determination. J. Struct. Biol. 180, 519–530 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  39. 39.

    Zhang, K. Gctf: real-time CTF determination and correction. J. Struct. Biol. 193, 1–12 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  40. 40.

    Punjani, A., Rubinstein, J. L., Fleet, D. J. & Brubaker, M. A. cryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination. Nat. Methods 14, 290–296 (2017).

    CAS  Google Scholar 

  41. 41.

    Zivanov, J., Nakane, T. & Scheres, S. H. W. A Bayesian approach to beam-induced motion correction in cryo-EM single-particle analysis. IUCrJ. 6, 5–17 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  42. 42.

    Bai, X., Rajendra, E., Yang, G., Shi, Y. & Scheres, S. H. W. Sampling the conformational space of the catalytic subunit of human γ-secretase. Elife 4, e11182 (2015).

    PubMed  PubMed Central  Google Scholar 

  43. 43.

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

    CAS  Google Scholar 

  44. 44.

    Källberg, M., Margaryan, G., Wang, S., Ma, J. & Xu, J. RaptorX server: a resource for template-based protein structure modeling. Methods Mol. Biol. 1137, 17–27 (2014).

    PubMed  Google Scholar 

  45. 45.

    Roy, A., Kucukural, A. & Zhang, Y. I-TASSER: a unified platform for automated protein structure and function prediction. Nat. Protoc. 5, 725–738 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  46. 46.

    Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D Biol. Crystallogr. 66, 486–501 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  47. 47.

    Afonine, P. V. et al. Towards automated crystallographic structure refinement with phenix.refine. Acta Crystallogr. D Biol. Crystallogr. 68, 352–367 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  48. 48.

    Zhou, N., Wang, H. & Wang, J. EMBuilder: a template matching-based automatic model-building program for high-resolution cryo-electron microscopy maps. Sci. Rep. 7, 2664 (2017).

    PubMed  PubMed Central  Google Scholar 

  49. 49.

    Chen, V. B. et al. MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr. D Biol. Crystallogr. 66, 12–21 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  50. 50.

    Berman, H., Henrick, K., Nakamura, H. & Markley, J. L. The worldwide Protein Data Bank (wwPDB): ensuring a single, uniform archive of PDB data. Nucleic Acids Res. 35, D301–D303 (2007).

    CAS  PubMed  Google Scholar 

  51. 51.

    Nolen, B. J. & Pollard, T. D. Insights into the influence of nucleotides on actin family proteins from seven structures of Arp2/3 complex. Mol. Cell 26, 449–457 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  52. 52.

    Nolen, B. J. & Pollard, T. D. Structure and biochemical properties of fission yeast Arp2/3 complex lacking the Arp2 subunit. J. Biol. Chem. 283, 26490–26498 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  53. 53.

    Nolen, B. J. et al. Characterization of two classes of small molecule inhibitors of Arp2/3 complex. Nature 460, 1031–1034 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  54. 54.

    Otterbein, L. R., Graceffa, P. & Dominguez, R. The crystal structure of uncomplexed actin in the ADP state. Science 293, 708–711 (2001).

    CAS  PubMed  Google Scholar 

  55. 55.

    Graceffa, P. & Dominguez, R. Crystal structure of monomeric actin in the ATP state. Structural basis of nucleotide-dependent actin dynamics. J. Biol. Chem. 278, 34172–34180 (2003).

    CAS  PubMed  Google Scholar 

  56. 56.

    Krissinel, E. & Henrick, K. Inference of macromolecular assemblies from crystalline state. J. Mol. Biol. 372, 774–797 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

Cryo-EM data were collected at the Stony Brook University (SBU) Cryo-EM center, and we thank G. Hu for providing support at this center. Computational infrastructures were provided by the cryo-EM and High-Performance Computing facilities at SBU. We thank N. Samaroo and B. Ding for help with experiments, and acknowledge K. Prehoda and members of the Nolen and Chowdhury laboratory for comments on the manuscript. We also thank G. C. Lander for providing access to the Scripps Research cryo-EM facility for determining the feasibility of the project. We thank D. Kovar for the capping protein expression vector. This work was supported by SBU start-up funds to S.C., and by NIH grant nos. R01GM092917 and R35GM136319 to B.J.N. The SBU cryo-EM center is supported by NIH grant no. S10 OD012272. M.S. was supported by the Fulbright Association.

Author information

Affiliations

Authors

Contributions

B.J.N. and S.C. conceived the project. Biochemical conditions for preparation of samples were determined by B.J.N., S.C. and M.S. Cryo-EM data collection and data processing were performed by M.S. and S.C. Atomic models were built by M.S. B.J.N performed structural analysis. All authors participated in manuscript preparation.

Corresponding authors

Correspondence to Saikat Chowdhury or Brad J. Nolen.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Peer review information Peer review reports are available. Inês Chen was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 Cryo-Electron Microscopy of Arp2/3 complex in active and inactive states.

a, A representative micrograph containing actin filaments polymerized from Dip1-activated Arp2/3 complex. Circles mark the knob of density corresponding to Dip1-bound S. pombe Arp2/3 complex at the pointed ends of filaments. Dip1 and Arp2/3 complex bind weakly to the ends of spontaneously nucleated actin filaments but strongly to Dip1–Arp2/3–nucleated filaments34, so we concluded that most or all filaments with density for Arp2/3 complex were nucleated by Dip1-activated Arp2/3 complex. b, Reference-free 2D class averages obtained from particles corresponding to the knob shaped densities highlighted by circles in a. These class averages show Dip1–Arp2/3–actin-filament complex adopts different orientations in vitrified ice. c, Two different views of the reconstructed 3D map of active Arp2/3 complex bound to Dip1 and pointed end of nucleated actin filament, colored based on local resolution values of each voxel. d, Euler angle distribution plot for particles contributing to the reconstructed map in c (map colored in gray at the center). e, A micrograph of S. pombe Arp2/3 complex sample collected by tilting the stage to 40˚ showing uniform distribution of particles in ice. f, Representative reference free 2D class averages of Arp2/3 complex showing different orientations of the complex obtained from particles extracted from tilted and non-tilted micrographs. g, Two different rotated views of the 3D reconstruction of Arp2/3 complex in an inactive state colored based on per-voxel local resolution values. h, Plot showing the Euler angle distribution of particles that went into the final reconstructed inactive Arp2/3 complex map (colored in gray). Scale bars (white line) for the micrographs in a and e represent distance of 25 nm and for the 2D class averages b and f represent 12.5 nm distance.

Extended Data Fig. 2 Single-particle cryo-EM data processing workflow for 3D reconstructions of activated and inactive Arp2/3 complex.

a, Data processing schematic for Dip1–Arp2/3–actin-filament complex. Particle stack was initially binned by factor of four and subjected to multiple rounds of 2D and 3D classification to discard particles that did not correspond to the complex. The cleaned particle stack was re-extracted without binning and further subjected to downstream 3D processing. All 3D classifications were performed without image alignment (referred to as clustering). Final composite map was generated by combining focused maps corresponding to the Dip1-Arp2/3 cap (Region-1, colored purple) and the Arp-actin filament (Region-2, colored blue). b, Schematic for processing of Arp2/3 complex (inactive state) cryo-EM data. Particles extracted from all micrographs (tilted and non-tilted) were cleaned by multiple rounds of 2D classification and then subjected to multiple ab initio 3D reconstructions. Particles corresponding to intact Arp2/3 complex were further subjected to downstream data processing that lead to a 4.2 Å 3D reconstruction of Arp2/3 complex in inactive state.

Extended Data Fig. 3 Reconstructed maps and atomic models of subunits.

Fourier shell correlation (FSC) plots between masked (yellow) and unmasked (orange) half maps, and final map and model (blue) for global resolution estimates for a, Dip1–Arp2/3–actin-filament complex, and b, inactive Arp2/3 complex. EM density for each subunit is shown as gray mesh. c, Arp3, d, Arp2, e, ARPC1, f, ARPC2, g, ARPC3, h, ARPC4, i, ARPC5, j, Dip1, and k, actin maps with built models. l, Maps and models corresponding to the long helices from the ‘clamp’ subunits, ARPC2 (cyan) and ARPC4 (blue) in the inactive state (left) and active state (right).

Extended Data Fig. 4 The structure of inactive S. pombe Arp2/3 complex is nearly identical to other inactive Arp2/3 complex structures.

a, Structural superposition of inactive S. pombe Arp2/3 complex (colored subunits) from the cryo-EM reconstruction presented here with inactive Bos taurus Arp2/3 complex (semi-transparent gray subunits, PDB 4JD218). Structures were superposed using Arp3 subdomains 1 and 2, Arp2, ARPC1, ARPC2 and ARPC4. b, Structural superposition showing that Arp3 from the S. pombe inactive Arp2/3 complex structure shows a more open nucleotide binding cleft than other ATP-bound Arp3 structures. Subdomains 1 and 2 of inactive S. pombe Arp3 (orange) were superposed on inactive BtArp3 (transparent gray, PDB 4JD2). Cyan arrow shows the direction of rotation of subdomains 3 and 4 toward subdomains 1 and 2 during cleft closure. Red arrow shows the axis of rotation of cleft closure. A rotation of 10.3° about this axis is required to close the S. pombe nucleotide cleft to the same extent as the BtArp2/3 structure. The open cleft in the inactive S. pombe Arp2/3 complex is the major structural difference between the two structures.

Extended Data Fig. 5 Comparison of Arp2 and Arp3 in the activated complex to actin in the nucleated filament.

a, Superposition of Arp3 Cα atoms from the active structure to actin subunit n in the nucleated filament. The RMSD between 256 pruned atom pairs is 1.31 Å (across all 354 pairs: 3.45). b, Superposition of Arp2 Cα atoms from the active structure to actin subunit n in the nucleated filament. The RMSD between 314 pruned atom pairs is 0.92 Å (across all 352 pairs: 2.74).

Extended Data Fig. 6 Partial flattening allows Arp3 to make short pitch interactions that mimic those of a short pitch actin dimer.

a, Left, Arp3 from the active (orange) and inactive (semi-transparent gray) Arp2/3 complex structures, with definition of measurements in plot. Top, the twist angle (𝜑) is the dihedral angle between the centers of mass (blue spheres) of each of the four subdomains (see Methods). Bottom, distance x1 between Y92 Cα and K235 Cα in S. pombe Arp3. Right, plot of the twist (𝜑) versus distance x1 in inactive or active structures of actin, Arp2 or Arp3. Data points highlighted in yellow are from the activated Arp2/3 complex structure. A subset of structures are labeled: em (from cryo-EM reconstructions here); PDB 6DEC (Spin90 bound to Bos taurus (Bt)Arp2/3 complex, chain ID in parentheses21); PDB 4JD2 (BtArp2/3 complex bound to GMF18); PDB 1NWK and PDB 1J6Z (representative actin monomer structures bound to ATP or ADP, respectively53,54). b, Superposition of inactive (twisted) Arp3 from the inactive S. pombe Arp2/3 complex cryo-EM structure and Arp3 from the activated structure overlaid using subdomains 3 and 4. Arp2 in the short pitch conformation from the activated structure is shown in pink. Partial flattening of Arp3 upon activation allows subdomains 1 and 2 to make closer contacts with Arp2 in the short pitch conformation. c, Surface area buried at the short pitch interface between Arp2 and Arp3 in the active structure (model 1) or theoretical models in which the Arps are modeled in the short pitch conformation and either Arp2 (model 2), Arp3 (model 3), or both (model 4) adopt the twisted conformation. Models 2, 3 and 4 were constructed by individually superposing either Arp2, Arp3, or both subunits from the inactive structure onto their corresponding subunit in the activated structure using Cα atoms from subdomains 3 and 4. d, Comparison of the short pitch interface between Arp3 and Arp2 (left panel) or actin (n) and actin (n + 2) right panel. The 9° rotation of Arp2 brings the helix harboring E231 from Arp2 closer to subdomain 1 of Arp3, allowing E231 to interact with R418.

Extended Data Fig. 7 Uncurling of the W-loop facilitates long-pitch interactions with actin.

a, Close up of long pitch interactions between the Arp3 barbed end groove (BEG) and the actin D-loop. Activated Arp3 (orange) is overlaid on an inactive (transparent) structure using subdomains 3 and 4. A structure of Bos taurus Arp2/3 complex (PDB 4JD2) was used as the inactive structure in this and all analyses in this figure as some sidechains at the barbed end of Arp3 are missing in the inactive S. pombe Arp2/3 complex structure presented here. b, Long pitch interactions between the Arp2 BEG in the activated (pink) or inactive (transparent) structure (PDB 4JD2). Arp2 from each structure was superposed using subdomains 3 and 4. c, Structure of Arp3 from activated Arp2/3 complex showing the definitions of the measurements made in panel d. d, Plot of the twist angle (𝜑) versus distance x2—which measures uncurling of the W loop—for inactive or active structures of actin, Arp2 or Arp3. em: structures from the cryo-EM reconstructions presented here; PDB 6DEC: structure of Spin90 bound to Bos taurus (Bt)Arp2/3 complex26; PDB 4JD2: BtArp2/3 complex bound to GMF18; PDB 1NWK and PDB 1J6Z: representative actin monomer structures bound to ATP or ADP, respectively53,54.

Extended Data Fig. 8 Flattening causes changes in the barbed end groove that facilitate long pitch interactions with actin subunits.

a, Molecular surface representation of active Arp3 showing perspective of close up views of the barbed end grooves (BEGs) depicted in b. b, BEG of inactive (PDB 4JD2) or active Arp2 or Arp3 with engaged D-loop from actin subunit n + 1 or n. Residue numbers are for the S. pombe Arp2/3 complex. Green spheres and dashed line show distance x3, which measures the distance between the C-terminus and the front of the BEG. c, Plot of the twist angle versus distance x3 for inactive or active structures of actin, Arp2 or Arp3. Individually labeled data points are as described in Extended Data Fig. 7d.

Extended Data Fig. 9 Close up of nucleotide clefts of Arp2 and Arp3.

a, Stereo image of the nucleotide binding cleft of Arp3 showing the density of the modeled ATP phosphates and conserved catalytic residue Q160. PG: gamma phosphate. b, Stereo image of the nucleotide binding cleft of Arp2 showing the density of the modeled ATP phosphates and conserved catalytic residue Q137. c, Stereo image of Arp2 from the active structure (pink) superposed with actin (cyan) from the cryo-EM structure of AMP-PNP bound actin filaments PDB 6DJM15 showing the nucleotide and the nucleotide binding cleft.

Extended Data Fig. 10 The clamp subunits twist during activation of Arp2/3 complex.

a, Model-based surface representation of ARPC2 and ARPC4 in active or inactive structure (PDB 4JD2) with residues contacting Arp2 or Arp3 (within 3.7 Å) colored magenta or orange, respectively. The Bos taurus inactive structure (PDB 4JD2) was used for the inactive structure in this analysis because the inactive S. pombe Arp2/3 complex structure presented here is missing some sidechains at the interface with the clamp. b, Overlay of ARPC2 and ARPC4 from the active structure on ARPC2 and ARPC4 from the inactive S. pombe Arp2/3 complex structure, respectively. Residues 1-271 of ARPC2 and 4-140 of ARPC4 were used to overlay the structures.

Supplementary information

Supplementary Information

Supplementary Figs. 1 and 2.

Reporting Summary

Peer Review Information

Supplementary Video 1

Model of Dip1-activated Arp2/3 complex on the end of the nucleated actin filament. Arp2 and Arp3 mimic actin subunits to make filament-like contacts with the first two actin subunits in the nucleated actin filament.

Supplementary Video 2

Morph showing movement of Arp2/3 complex from the inactive to active conformation. Twisting of the ‘clamp subunits’ in the Arp2/3 complex during activation shifts half of the subunits to a new activated position.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Shaaban, M., Chowdhury, S. & Nolen, B.J. Cryo-EM reveals the transition of Arp2/3 complex from inactive to nucleation-competent state. Nat Struct Mol Biol 27, 1009–1016 (2020). https://doi.org/10.1038/s41594-020-0481-x

Download citation

Further reading

Search

Quick links