RESEARCH ARTICLE
◥NUCLEAR PORE COMPLEX
Structure of the cytoplasmic ring of theXenopus
laevisnuclear pore complex
Xuechen Zhu1,2,3†, Gaoxingyu Huang1,2,3†, Chao Zeng4,5†, Xiechao Zhan1,2,3†, Ke Liang1,2,3†,
Qikui Xu1,2,3, Yanyu Zhao1,2,3, Pan Wang4,5, Qifan Wang1,2,3, Qiang Zhou1,2,3, Qinghua Tao^4 ,
Minhao Liu^4 , Jianlin Lei^4 , Chuangye Yan4,5, Yigong Shi1,2,3,4,5
The nuclear pore complex (NPC) mediates nucleocytoplasmic cargo transport. Here, we present a single-
particle cryo–electron microscopy reconstruction of the cytoplasmic ring (CR) subunit from theXenopus laevis
NPC at 3.7- to 4.7-angstrom resolution. The structure of an amino-terminal domain of Nup358 has been
resolved at 3.0 angstroms, facilitating the identification of five Nup358 molecules in each CR subunit. Our final
model of the CR subunit included five Nup358, two Nup205, and two Nup93 molecules in addition to the two
previously characterized Y complexes. The carboxyl-terminal fragment of Nup160 served as an organizing
center at the vertex of each Y complex. Structural analysis revealed how Nup93, Nup205, and Nup358 facilitate
and strengthen the assembly of the CR scaffold that is primarily formed by two layers of Y complexes.
T
he nuclear pore complex (NPC) resides
on the nuclear envelope (NE) and medi-
ates nucleocytoplasmic cargo transport
( 1 – 3 ). As one of the largest cellular ma-
chineries ( 4 – 6 ), a vertebrate NPC has a
molecular mass of >100 MDa and consists of
multiple cytoplasmic filaments (CF), a cytoplasmic
ring (CR), an inner ring (IR), a nuclear ring (NR),
a nuclear basket (NB), and a luminal ring (LR)
( 5 – 9 ) (Fig. 1A). Each NPC has eight repeating
subunits known as the spokes ( 7 ). Our present
study concerns the structure of the CR constitu-
ents in each spoke, referred to as the CR subunits.
For structural elucidation of the NPC, cryo–
electron tomography used to be the main-
stream approach. The CR subunit of the human
NPC was reconstructed through subtomogram
averaging to a highest resolution of ~15 Å ( 10 ).
Each CR subunit is featured with two Y-shaped
multicomponent complexes known as the inner
and the outer Y complexes. Sixteen Y complexes,
eight inner and eight outer, in the CR assemble
in a head-to-tail fashion to build the scaffold in
the form of the proximal and distal concentric
ring, respectively (Fig. 1, A and B) ( 11 , 12 ). The
tripartite Y complex consists of a short arm
(Nup85, Nup43, and Seh1), a long arm (Nup160
and Nup37), and a stem (Nup96, Sec13, Nup107,
and Nup133) ( 11 , 13 )(Fig.1,AandD).
To achieve higher resolution of the CR as-
sembly, we used single-particle cryo–electron
microscopy (cryo-EM) reconstruction to exam-
ine the CR subunit from theXenopus laevis
NPC. During data processing, two relatively
independent and rigid structural entities stood
out, designated as the core region and the
Nup358 region ( 14 ). The core region contains
the long arm from the inner Y complex and
the short arms from both Y complexes, and the
Nup358 region covers the stems of the two Y
complexes (Fig. 1B). An overall resolution of
5 to 8 Å was achieved after masked refinement
for these two regions, enabling assignment of
some components, such as two Nup205 and two
Nup358 molecules ( 14 ). However, reliable dock-
ing and modeling requires higher resolution.
In this study, we achieved an average res-
olution of 3.7 Å for the core region and 4.7 Å
for the Nup358 region. We also solved a cryo-
EM structure of the N-terminala-helical do-
main of Nup358 at 3.0-Å resolution. Relying
ontheimprovedresolutionsandfacilitatedby
AlphaFold prediction, we were able to gener-
ateastructuralmodelfortheentireCRofthe
X. laevisNPC. In addition to the previously
characterized Y complexes, five Nup358, two
Nup205, and two Nup93 molecules were iden-
tified to be constituents of the CR subunit. Our
current structure expands the molecular mass
by 80% compared with the previously reported
composite models of the human CR ( 10 , 12 ).Overall structure of a CR subunit
To reduce conformational heterogeneity, we
spread the opened NE onto the grids with
minimal force and used the chemical cross-
linker glutaraldehyde to stabilize the NPC.
Please refer to the materials and methods,
figs. S1 to S4, and table S1 for details of cryo-
EM sample preparation, image acquisition,data processing, and structural modeling and
refinement.Wepreviouslydefinedthecore
and the Nup358 regions in each CR unit based
on their spatial arrangement but were unable
to reliably assign nucleoporins (Fig. 1B) ( 14 ).
The average resolutions for these two regions
now reach 3.7 and 4.7 Å, with the highest local
resolution of the core region up to 3.3 Å
(Fig. 1C, Movie 1, and fig. S5).
Structural modeling for the best-resolved
components within the CR subunit, including
the C-terminal fragment of Nup160 (Nup160-
CTF), Nup93a5, Nup205, and other previously
recognized structural components of the CR,
was aided by x-ray structures ( 12 )orAlphaFold
predictions ( 15 ). Assignment of the two Nup93
a5 helices was facilitated by several bulky side
chains that are readily discernible in the EM
map. In addition, the model of this long helix
predicted by AlphaFold matches the EM map
exceptionally well (fig. S5). Assignment of the
residues in eachX. laevisnucleoporin was as-
sisted by sequence alignment with its functional
orthologs. The EM density in the Nup358 region
and the periphery of the core region, includ-
ing all five Nup358 clamps and two Ancestral
Coatomer Element 1 (ACE1) domains that be-
long to Nup93 ( 16 ), is less well resolved and
lacks side chain features. Modeling in these
areas was aided by the separately determined
atomic structure of Nup358-NTD2 (residues 222
to 738) (figs. S6 to S9), previous analyses of ver-
tebrate NPC ( 14 ), and AlphaFold predictions ( 15 ).
Altogether, our final model of the CR sub-
unit consists of 19,037 amino acids in 30
nucleoporins (Fig. 1, A and D, Movie 1, and
tables S2 and S3). Except for Nup358, Nup88,
Nup155, and Nup98, all protein components
in the current model have two distinct copies
in each CR subunit ( 14 ). For description clarity,
the copy closer to and away from the central
channel will be denoted with -I (inner) and -O
(outer), respectively. In the following sections,
we will focus on previously uncharacterized
structural features of the CR for illustration.Nup160-CTF at the vertex of the Y complex
The Y complexes have been the most extensively
characterized, with most of their constituents
known. On the basis of the improved EM map
of the core region, the previously unknown
Nup160-CTF, comprising helicesa38 toa47,
was found to sit at the center of the vertex,
wheretheshortarm,longarm,andstemof
the Y complex meet ( 11 , 17 ) (Fig. 2, A and B).
This observation helps to define the organi-
zation of the vertex in vertebrates.
Seh1-I from the short arm and Sec13-I from
the stem are connected to the CTF of Nup160-I
(Fig.2,AandC).FortheinnerYcomplex,the
4CD loop (the loop between strands C and D of
blade 4) of Seh1 contacts helixa40 of Nup160-
CTF, whereas theb-3D strand (the D strand of
blade 3) of Seh1 interacts with helixa42 ofSTRUCTURE OF THE NUCLEAR POREZhuet al., Science 376 , eabl8280 (2022) 10 June 2022 1of10
(^1) Westlake Laboratory of Life Sciences and Biomedicine,
Westlake University, 310024 Hangzhou, China.^2 Key
Laboratory of Structural Biology of Zhejiang Province, School
of Life Sciences, Westlake University, 310024 Hangzhou,
China.^3 Institute of Biology, Westlake Institute for Advanced
Study, 310024 Hangzhou, China.^4 Beijing Advanced
Innovation Center for Structural Biology and Frontier
Research Center for Biological Structure, Tsinghua
University, 100084 Beijing, China.^5 Tsinghua University-
Peking University Joint Center for Life Sciences, School of
Life Sciences, Tsinghua University, 100084 Beijing, China.
*Corresponding author. Email: [email protected].
cn (G.H.); [email protected] (Y.S.)
†These authors contributed equally to this work.