8 Å structure of the outer rings of the Xenopus laevis nuclear pore complex obtained by cryo-EM and AI

Abstract

The nuclear pore complex (NPC), one of the largest protein complexes in eukaryotes, serves as a physical gate to regulate nucleocytoplasmic transport. Here, we determined the 8 Å resolution cryo-electron microscopic (cryo-EM) structure of the outer rings containing nuclear ring (NR) and cytoplasmic ring (CR) from the Xenopus laevis NPC, with local resolutions reaching 4.9 Å. With the aid of AlphaFold2, we managed to build a pseudoatomic model of the outer rings, including the Y complexes and flanking components. In this most comprehensive and accurate model of outer rings to date, the almost complete Y complex structure exhibits much tighter interaction in the hub region. In addition to two copies of Y complexes, each asymmetric subunit in CR contains five copies of Nup358, two copies of the Nup214 complex, two copies of Nup205 and one copy of newly identified Nup93, while that in NR contains one copy of Nup205, one copy of ELYS and one copy of Nup93. These in-depth structural features represent a great advance in understanding the assembly of NPCs.

Key words： nuclear pore complex; cryo-EM; Xenopus laevis; AlphaFold2; nuclear ring; cytoplasmic ring

References

1 AfoninePV, Grosse-Kunstleve RW, EcholsN, HeaddJJ, Moriarty NW, MustyakimovM, TerwilligerTC, Urzhumtsev A, ZwartPH, AdamsPD (2012) Towards automated crystallographic structure refinement with phenix.refine. Acta Crystallogr D Biol Crystallogr 68:352–367

2 AkeyCW, Radermacher M (1993) Architecture of the Xenopus nuclear pore complex revealed by three-dimensional cryo-electron microscopy. J Cell Biol 122:1–19

3 AllegrettiM, Zimmerli CE, RantosV, WilflingF, RonchiP, FungHKH, Lee C-W, HagenW, TuroňováB, KariusK et al (2020) In-cell architecture of the nuclear pore and snapshots of its turnover. Nature 586:796–800

4 AndersenKR, Onischenko E, TangJH, KumarP, ChenJZ, UlrichA, Liphardt JT, WeisK, SchwartzTU (2013) Scaffold nucleoporins Nup188 and Nup192 share structural and functional properties with nuclear transport receptors. Elife 2:e00745

5 BeckM, Forster F, EckeM, PlitzkoJM, Melchior F, GerischG, BaumeisterW, Medalia O (2004) Nuclear pore complex structure and dynamics revealed by cryoelectron tomography. Science 306:1387–1390

6 BeckM, LucicV, ForsterF, Baumeister W, MedaliaO (2007) Snapshots of nuclear pore complexes in action captured by cryoelectron tomography. Nature 449:611–615

7 BernadR, van der Velde H, FornerodM, PickersgillH (2004) Nup358/RanBP2 attaches to the nuclear pore complex via association with Nup88 and Nup214/CAN and plays a supporting role in CRM1-mediated nuclear protein export. Mol Cell Biol 24:2373–2384

8 BilokapicS, Schwartz TU (2012) Molecular basis for Nup37 and ELY5/ELYS recruitment to the nuclear pore complex. Proc Natl Acad Sci USA 109:15241–15246

9 BilokapicS, Schwartz TU (2013) Structural and functional studies of the 252 kDa nucleoporin ELYS reveal distinct roles for its three tethered domains. Structure 21:572–580

10 BoehmerT, JeudyS, BerkeIC, Schwartz TU (2008) Structural and functional studies of Nup107/Nup133 interaction and its implications for the architecture of the nuclear pore complex. Mol Cell 30:721–731

11 BrohawnSG, LeksaNC, SpearED, Rajashankar KR, SchwartzTU (2008) Structural evidence for common ancestry of the nuclear pore complex and vesicle coats. Science 322:1369–1373

12 BuiKH, von Appen A, DiGuilioAL, OriA, SparksL, MackmullM-T, Bock T, HagenW, Andrés-PonsA, Glavy JS et al (2013) Integrated structural analysis of the human nuclear pore complex scaffold. Cell 155:1233–1243

13 CronshawJM, Krutchinsky AN, ZhangW, ChaitBT, Matunis MJ (2002) Proteomic analysis of the mammalian nuclear pore complex. J Cell Biol 158:915–927

14 DeblerEW, MaY, SeoH-S, Hsia K-C, NoriegaTR, BlobelG, HoelzA (2008) A fence-like coat for the nuclear pore membrane. Mol Cell 32:815–826

15 DelavoieF, SoldanV, RinaldiD, Dauxois JY, GleizesPE (2019) The path of pre-ribosomes through the nuclear pore complex revealed by electron tomography. Nat Commun 10:497

16 EibauerM, Pellanda M, TurgayY, DubrovskyA, WildA, MedaliaO (2015) Structure and gating of the nuclear pore complex. Nat Commun 6:7532

17 EmsleyP, Lohkamp B, ScottWG, CowtanK (2010) Features and development of Coot. Acta Crystallogr D Biol Crystallogr 66:486–501

18 Fernandez-MartinezJ, Kim SJ, ShiY, UplaP, Pellarin R, GagnonM, ChemmamaIE, WangJ, NudelmanI, Zhang W et al (2016) Structure and function of the nuclear pore complex cytoplasmic mRNA export platform. Cell 167:1215–1228

19 GaikM, Flemming D, von AppenA, KastritisP, Mücke N, FischerJ, StelterP, OriA, BuiKH, Baßler J et al (2015) Structural basis for assembly and function of the Nup82 complex in the nuclear pore scaffold. J Cell Biol 208:283–297

20 GoddardTD, Goddard TD, HuangCC, MengEC, Pettersen EF, CouchGS, MorrisJH, FerrinTE (2018) UCSF ChimeraX: Meeting modern challenges in visualization and analysis. Protein Sci 27:14–25

21 HampoelzB, Andres-Pons A, KastritisP, BeckM (2019) Structure and assembly of the nuclear pore complex. Annu Rev Biophys 48:515–536

22 HinshawJE, Carragher BO, MilliganRA (1992) Architecture and design of the nuclear pore complex. Cell 69:1133–1141

23 HoelzA, DeblerEW, BlobelG (2011) The structure of the nuclear pore complex. Annu Rev Biochem 80:613–643

24 HsiaKC, Stavropoulos P, BlobelG, HoelzA (2007) Architecture of a coat for the nuclear pore membrane. Cell 131:1313–1326

25 HuangJ, Rauscher S, NawrockiG, RanT, FeigM, de GrootBL, Grubmüller H, MacKerell JrAD (2017) CHARMM36m: an improved force field for folded and intrinsically disordered proteins. Nat Methods 14:71–73

26 HuangG, ZhangY, ZhuX, ZengC, WangQ, Zhou Q, TaoQ, LiM, LeiM, YanC et al (2020) Structure of the cytoplasmic ring of the Xenopus laevis nuclear pore complex by cryo-electron microscopy single particle analysis. Cell Res 30:520–531

27 HuttenS, Kehlenbach RH (2006) Nup214 is required for CRM1-dependent nuclear protein export in vivo. Mol Cell Biol 26:6772–6785

28 JumperJ, EvansR, PritzelA, Green T, FigurnovM, RonnebergerO, Tunyasuvunakool K, BatesR, ŽídekA, Potapenko A et al (2021) Highly accurate protein structure prediction with AlphaFold. Nature 596:583–589

29 KampmannM, BlobelG (2009) Three-dimensional structure and flexibility of a membrane-coating module of the nuclear pore complex. Nat Struct Mol Biol 16:782–788

30 KassubeSA, StuweT, LinDH, Antonuk CD, NapetschnigJ, BlobelG, HoelzA (2012) Crystal structure of the N-terminal domain of Nup358/RanBP2. J Mol Biol 423:752–765

31 KelleyK, Knockenhauer KE, KabachinskiG, SchwartzTU (2015) Atomic structure of the Y complex of the nuclear pore. Nat Struct Mol Biol 22:425–431

32 KimSJ, Fernandez-Martinez J, NudelmanI, ShiY, ZhangW, RavehB, Herricks T, SlaughterBD, HoganJA, UplaP et al (2018) Integrative structure and functional anatomy of a nuclear pore complex. Nature 555:475–482

33 KosinskiJ, Mosalaganti S, von AppenA, TeimerR, DiGuilio AL, WanW, BuiKH, HagenWJH, BriggsJAG, Glavy JS et al (2016) Molecular architecture of the inner ring scaffold of the human nuclear pore complex. Science 352:363–365

34 LiebschnerD, Afonine PV, BakerML, BunkócziG, Chen VB, CrollTI, HintzeB, HungLW, JainS, McCoy AJ et al (2019) Macromolecular structure determination using X-rays, neutrons and electrons: recent developments in Phenix. Acta Crystallogr D Struct Biol 75:861–877

35 LinDH, HoelzA (2019) The structure of the nuclear pore complex (an update). Annu Rev Biochem 88:725–783

36 LinDH, Zimmermann S, StuweT, StuweE, HoelzA (2013) Structural and functional analysis of the C-terminal domain of Nup358/RanBP2. J Mol Biol 425:1318–1329

37 LinDH, StuweT, SchilbachS, Rundlet EJ, PerrichesT, MobbsG, FanY, ThierbachK, Huber FM, CollinsLN et al (2016) Architecture of the symmetric core of the nuclear pore. Science 352:1015

38 MaimonT, EladN, DahanI, Medalia O (2012) The human nuclear pore complex as revealed by cryo-electron tomography. Structure 20:998–1006

39 MastronardeDN (2005) Automated electron microscope tomography using robust prediction of specimen movements. J Struct Biol 152:36–51

40 MosalagantiS, Kosinski J, AlbertS, SchafferM, Strenkert D, SaloméPA, MerchantSS, Plitzko JM, BaumeisterW, EngelBD et al (2018) In situ architecture of the algal nuclear pore complex. Nat Commun 9:2361

41 NapetschnigJ, BlobelG, HoelzA (2007) Crystal structure of the N-terminal domain of the human protooncogene Nup214/CAN. Proc Natl Acad Sci USA 104:1783–1788

42 NapetschnigJ, Kassube SA, DeblerEW, WongRW, BlobelG, HoelzA (2009) Structural and functional analysis of the interaction between the nucleoporin Nup214 and the DEAD-box helicase Ddx19. Proc Natl Acad Sci USA 106:3089–3094

43 OriA, Banterle N, IskarM, Andrés-PonsA, Escher C, BuiHK, SparksL, Solis-Mezarino V, RinnerO, BorkP et al (2013) Cell type-specific nuclear pores: a case in point for context-dependent stoichiometry of molecular machines. Mol Syst Biol 9:648

44 PettersenEF, Goddard TD, HuangCC, CouchGS, Greenblatt DM, MengEC, FerrinTE (2004) UCSF Chimera–a visualization system for exploratory research and analysis. J Comput Chem 25:1605–1612

45 PhillipsJC, BraunR, WangW, Gumbart J, TajkhorshidE, VillaE, ChipotC, SkeelRD, Kalé L, SchultenK (2005) Scalable molecular dynamics with NAMD. J Comput Chem 26:1781–1802

46 PortSA, Monecke T, DickmannsA, SpillnerC, HofeleR, UrlaubH, Ficner R, KehlenbachRH (2015) Structural and functional characterization of CRM1-Nup214 interactions reveals multiple FG-binding sites involved in nuclear export. Cell Rep 13:690–702

47 PunjaniA, Rubinstein JL, FleetDJ, BrubakerMA (2017) cryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination. Nat Methods 14:290–296

48 RoloffS, Spillner C, KehlenbachRH (2013) Several phenylalanineglycine motives in the nucleoporin Nup214 are essential for binding of the nuclear export receptor CRM1. J Biol Chem 288:3952–3963

49 RoutMP, Aitchison JD, SupraptoA, HjertaasK, ZhaoY, ChaitBT (2000) The yeast nuclear pore complex: composition, architecture, and transport mechanism. J Cell Biol 148:635–651

50 SampathkumarP, KimSJ, UplaP, Rice WJ, PhillipsJ, TimneyBL, PieperU, BonannoJB, Fernandez-Martinez J, HakhverdyanZ et al (2013) Structure, dynamics, evolution, and function of a major scaffold component in the nuclear pore complex. Structure 21:560–571

51 SeoHS, MaY, DeblerEW, Wacker D, KutikS, BlobelG, HoelzA (2009) Structural and functional analysis of Nup120 suggests ring formation of the Nup84 complex. Proc Natl Acad Sci USA 106:14281–14286

52 StuweT, LinDH, CollinsLN, Hurt E, HoelzA (2014) Evidence for an evolutionary relationship between the large adaptor nucleoporin Nup192 and karyopherins. Proc Natl Acad Sci USA 111:2530–2535

53 StuweT, BleyCJ, ThierbachK, Petrovic S, SchilbachS, MayoDJ, Perriches T, RundletEJ, JeonYE, Collins LN et al (2015) Architecture of the fungal nuclear pore inner ring complex. Science 350:56–64

54 SuM (2019) goCTF: geometrically optimized CTF determination for single-particle cryo-EM. J Struct Biol 205:22–29

55 TanYZ, Baldwin PR, DavisJH, WilliamsonJR, PotterCS, CarragherB, Lyumkis D (2017) Addressing preferred specimen orientation in single-particle cryo-EM through tilting. Nat Methods 14:793–796

56 TegunovD, CramerP (2019) Real-time cryo-electron microscopy data preprocessing with Warp. Nat Methods 16:1146–1152

57 von AppenA, Kosinski J, SparksL, OriA, DiGuilio AL, VollmerB, MackmullM-T, Banterle N, ParcaL, KastritisP et al (2015) In situ structural analysis of the human nuclear pore complex. Nature 526:140–143

58 WuJ, Matunis MJ, KraemerD, BlobelG, Coutavas E (1995) Nup358, a cytoplasmically exposed nucleoporin with peptide repeats, Ran-GTP binding sites, zinc fingers, a cyclophilin A homologous domain, and a leucine-rich region. J Biol Chem 270:14209–14213

59 WuC, HuangX, ChengJ, Zhu D, ZhangX (2019) High-quality, high-throughput cryo-electron microscopy data collection via beam tilt and astigmatism-free beam-image shift. J Struct Biol 208:107396

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

61 ZhangY, LiS, ZengC, Huang G, ZhuX, WangQ, WangK, ZhouQ, Yan C, ZhangW et al (2020) Molecular architecture of the luminal ring of the Xenopus laevis nuclear pore complex. Cell Res 30:532–540

62 ZhengSQ, Palovcak E, ArmacheJ-P, VerbaKA, ChengY, AgardDA (2017) MotionCor2: anisotropic correction of beam-induced motion for improved cryo-electron microscopy. Nat Methods 14:331–332

63 ZhuD, WangX, FangQ, Van Etten JL, RossmannMG, RaoZ, ZhangX (2018) Pushing the resolution limit by correcting the Ewald sphere effect in single-particle Cryo-EM reconstructions. Nat Commun 9:1552

64 ZimmerliC, Allegretti M, RantosV, GoetzSK, Obarska-Kosinska A, ZagoriyI, HalavatyiA, Mahamid J, KosinskiJ, BeckM (2020) Nuclear pores constrict upon energy depletion.

65 ZivanovJ, NakaneT, ForsbergBo, Kimanius D, HagenWJH, LindahlE, Scheres SHW (2018) New tools for automated high-resolution cryo-EM structure determination in RELION-3. Elife 7: e42166