Tai L et al. (2022), 8 Å structure of the outer rings of the Xenopus...

XB-ART-58805

Protein Cell 2022 Oct 01;1310:760-777. doi: 10.1007/s13238-021-00895-y.

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

Tai L , Zhu Y , Ren H , Huang X , Zhang C , Sun F .

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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.

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Species referenced: Xenopus tropicalis Xenopus laevis
Genes referenced: ahctf1 elavl2 nup107 nup133 nup160 nup188 nup205 nup214 nup37 nup43 nup62 nup85 nup88 nup93 nup98 psmd6 ranbp2 sec13
GO keywords: nuclear pore outer ring [+]

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References [+] :

Afonine, Towards automated crystallographic structure refinement with phenix.refine. 2012, Pubmed

	Figure 1. The pseudoatomic model of the CR and NR from the X. laevis oocyte NPC. (A) Overall view of the X. laevis NPC CR and NR structure, displaying the inner and outer Y complexes in each asymmetric unit, as well as the densities other than the Y complexes. (B) Model-map overlay of the NPC CR subunit. The map density is displayed in gray with transparency. Models of the inner Y complex, outer Y complex and extra densities are in the same colors as in (A). (C) Model-map overlay of the NPC NR subunit. The map density is displayed in gray with transparency. Models of the inner Y complex, outer Y complex and extra densities are in the same colors as in (A). (D) Model display of two Y complexes in the NR subunit. All Nups are colored differently and labeled. The major differences compared with the previous 2015-model are highlighted in the transparent gray surface. Nup107 region and Y complex hub regions are shown in zoomed-in views. The 4th zoomed-in view is rotated for better display. Important regions are labeled with the sequence and secondary structure numbers.
	Figure 2. The structures and interaction details of five copies of Nup358 in each CR subunit. (A) Domain assignment of modeled part of Nup358. (B) Location of 40 copies of Nup358 in CR, while the 5 copies of Nup358 in each subunit are colored differently. (C) Location of 5 copies of Nup358 in the CR subunit, colored differently. (D) Interactions of 5 copies of Nup358 with surrounding Nups. Some zoomed-in views are rotated for better display.
	Figure 3. The structures and interaction details of two copies of Nup214 complexes in each CR subunit. (A) Domain assignment of modeled parts of Nup214, Nup88 and Nup62. (B) Location of 16 copies of the Nup214 complex in CR. The Nup214 complex in each subunit is colored differently. (C) Location of 2 copies of the Nup214 complex in the CR subunit. The left Nup214 complex is colored in chartreuse, and the right Nup214 complex is colored in light pink. (D) Model of the left and right Nup214 complex attached to two CR subunits. (E) Domain display of left and right Nup214 complexes. (F) Interaction sites of left and right Nup214 complexes.
	Figure 4. ELYS connects Nup160, Nup96 and Nup85 within the same NR subunit and Nup133 in an adjacent subunit. (A) Domain assignment of X. laevis ELYS. (B) Location of ELYS in the NR. (C) Location of ELYS in the NR asymmetric unit. (D) Interaction of ELYS with the convex inner Nup160 and inner Y complex hub in the same NR subunit and the inner Nup133 in the adjacent subunit. All discussed Nups are colored differently. Interaction sites are labeled in detail. Some zoomed-in views are rotated for better display.
	Figure 5. The locations of Nup205 in CR and NR. (A) Domain assign- ment of Nup205 and Nup188. (B) Location of 8 copies of Nup205 in NR. (C) Location of Nup205 in NR subunit. (D) Location of 16 copies of Nup205 in CR, showing the relative positions of inner and outer Nup205. Nup205 within each subunit is colored differently. (E) Location of 2 copies of Nup205 in the CR subunit.
	Figure 6. The interaction details of Nup205 in CR and NR. (A) Interaction of Nup205 with surrounding Nups in the same NR subunit and in an adjacent (ad.) subunit. (B) Interaction sites of inner and outer Nup205 to surrounding Nups. All discussed Nups are colored differently. Interaction sites are labeled in detail. All zoomed-in views are rotated for better display.
	Figure 7. Nup93 acts as a bridge to connect the stems of the inner and outer Y complexes in NR and CR. (A) Domain assignment of the modeled part of Nup93. (B) Location of 8 copies Nup93 in NR. (C) Location of Nup93 in the NR subunit. (D) Loca- tion of 8 copies Nup93 in CR. (E) Location of Nup93 in the CR subunit.
	Figure 8. The interaction details of Nup93 in NR and CR. (A) Interaction sites of Nup93 with surrounding Nups in NR. Some zoomed-in views are rotated for better display. (B) Interaction sites of Nup93 with surrounding Nups in CR. (C) Model comparison between Nup93 in CR and NR, showing spatial conflicts between outer right Nup358 and the NTD of Nup93 from NR. All discussed Nups are colored differently. Interaction sites are labeled in detail.
	Figure S1. Data processing workflow of the CR from the X. laevis oocyte NPC.
	Figure S2. Data processing workflow of the NR from the X. laevis oocyte NPC.
	Figure S3. Assessments of cryo-EM maps of the CR subunit. (A, B, C) Map display, local resolution estimation, FSC, 3D-FSC and directional FSC of the CR subunit region, CR core region and CR Nup358 region. (D) Map model overlay of the CR subunit, and map versus model FSC estimation.
	Figure S4. Assessments of cryo-EM maps of the NR subunit. (A, B, C) Map display, local resolution estimation, FSC, 3D-FSC and directional FSC of the NR subunit region, NR core region and NR Nup133 region. (D) Map model overlay of the NR asymmetric unit, and map versus model FSC estimation.
	Figure S5. Model building quality and secondary structure assignment of the CR subunit. (A-J) Model building quality of Nup85, Seh1, Nup43, Nup160, Nup37, Sec13, Nup96, Nup107, Nup133, Nup205, Nup93, Nup358 and Nup214 complexes.
	Figure S5. Model building quality and secondary structure assignment of the CR subunit. (A-J) Model building quality of Nup85, Seh1, Nup43, Nup160, Nup37, Sec13, Nup96, Nup107, Nup133, Nup205, Nup93, Nup358 and Nup214 complexes.
	Figure S5. Model building quality and secondary structure assignment of the CR subunit. (A-J) Model building quality of Nup85, Seh1, Nup43, Nup160, Nup37, Sec13, Nup96, Nup107, Nup133, Nup205, Nup93, Nup358 and Nup214 complexes.
	Figure S5. Model building quality and secondary structure assignment of the CR subunit. (A-J) Model building quality of Nup85, Seh1, Nup43, Nup160, Nup37, Sec13, Nup96, Nup107, Nup133, Nup205, Nup93, Nup358 and Nup214 complexes.
	Figure S5. Model building quality and secondary structure assignment of the CR subunit. (A-J) Model building quality of Nup85, Seh1, Nup43, Nup160, Nup37, Sec13, Nup96, Nup107, Nup133, Nup205, Nup93, Nup358 and Nup214 complexes.
	Figure S6. Model building quality and secondary structure assignment of the NR asymmetric unit. (A-G) Model building quality of Nup85, Seh1, Nup43, ELYS, Nup160, Sec13, Nup96, Nup133, Nup107, Nup205 and Nup93.
	Figure S6. Model building quality and secondary structure assignment of the NR asymmetric unit. (A-G) Model building quality of Nup85, Seh1, Nup43, ELYS, Nup160, Sec13, Nup96, Nup133, Nup107, Nup205 and Nup93.
	Figure S6. Model building quality and secondary structure assignment of the NR asymmetric unit. (A-G) Model building quality of Nup85, Seh1, Nup43, ELYS, Nup160, Sec13, Nup96, Nup133, Nup107, Nup205 and Nup93.
	Figure S7. Comparisons of the refined structure and predicted structure by AlphaFold2 (AF) of CR Nups from the X. laevis NPC.
	Figure S8. Comparisons of the refined structure and predicted structure by AlphaFold2 (AF) of NR Nups from the X. laevis NPC.

	Figure S10. CR and NR models of X. laevis NPC fitted in human NPC map (EMD- 3103).
	Figure S11. Nup205, instead of Nup188, is located in NR and CR. (A) Model comparison of Nup205 and Nup188 onto local densities in NR subunit. Major differences are enlarged and displayed in detail. (B) Model comparison of Nup205 and Nup188 onto local densities in CR subunit. The cryo-EM density maps are shown as a black mesh.
	Figure S12. Comparisons of bridge-like densities in the NPC NR for different species.
	Figure S13. Sequence alignments of Nups in CR and NR from X. laevis (or X. tropicalis) and human NPCs.
	Figure S13. Sequence alignments of Nups in CR and NR from X. laevis (or X. tropicalis) and human NPCs.
	Figure S13. Sequence alignments of Nups in CR and NR from X. laevis (or X. tropicalis) and human NPCs.
	Figure S13. Sequence alignments of Nups in CR and NR from X. laevis (or X. tropicalis) and human NPCs.
	Figure S13. Sequence alignments of Nups in CR and NR from X. laevis (or X. tropicalis) and human NPCs.
	Figure S13. Sequence alignments of Nups in CR and NR from X. laevis (or X. tropicalis) and human NPCs.
	Figure S13. Sequence alignments of Nups in CR and NR from X. laevis (or X. tropicalis) and human NPCs.
	Figure S13. Sequence alignments of Nups in CR and NR from X. laevis (or X. tropicalis) and human NPCs.
	Figure S13. Sequence alignments of Nups in CR and NR from X. laevis (or X. tropicalis) and human NPCs.
	Figure S13. Sequence alignments of Nups in CR and NR from X. laevis (or X. tropicalis) and human NPCs.