RESEARCH ARTICLE SUMMARY
◥NUCLEAR PORE COMPLEX
Structure of the cytoplasmic ring of theXenopus
laevisnuclear pore complex
Xuechen Zhu†, Gaoxingyu Huang†, Chao Zeng†, Xiechao Zhan†, Ke Liang†, Qikui Xu, Yanyu Zhao,
Pan Wang, Qifan Wang, Qiang Zhou, Qinghua Tao, Minhao Liu, Jianlin Lei,
Chuangye Yan, Yigong Shi
INTRODUCTION:The nuclear pore complex (NPC)
resides on the nuclear envelope (NE) and me-
diates nucleocytoplasmic cargo transport. As
one of the largest cellular machineries, a ver-
tebrate NPC consists of cytoplasmic filaments,
a cytoplasmic ring (CR), an inner ring, a nuclear
ring, a nuclear basket, and a luminal ring. Each
NPC has eight repeating subunits. Structure de-
termination of NPC is a prerequisite for under-
standing its functional mechanism. In the past
two decades, integrative modeling, which com-
bines x-ray structures ofindividual nucleoporins
and subcomplexes with cryo–electron tomogra-
phy reconstructions, has played a crucial role in
advancing our knowledge about the NPC.
The CR has been a major focus of structural
investigation. The CR subunit of human NPC
was reconstructed by cryo–electron tomography
through subtomogram averaging to an overall
resolution of ~20 Å, with local resolution up to
~15 Å. Each CR subunit comprises two Y-shaped
multicomponent complexes known as the inner
and outer Y complexes. Eight inner and eight
outer Y complexes assemble in a head-to-tail
fashion to form the proximal and distal rings,
respectively, constituting the CR scaffold. To
achieve higher resolution of the CR, we used
single-particle cryo–electron microscopy (cryo-
EM) to image the intact NPC from the NE of
Xenopus laevisoocytes. Reconstructions of the
core region and the Nup358 region of the
X. laevisCR subunit had been achieved at
average resolutions of 5 to 8 Å, allowing iden-
tification of secondary structural elements.RATIONALE:Packing interactions among the
components of the CR subunit were poorly
defined by all previous EM maps. Additional
components of the CR subunit are strongly
suggested by the EM maps of 5- to 8-Å res-
olution but remain to be identified. Address-
ing these issues requires improved resolution
of the cryo-EM reconstruction. Therefore, we
mayneedtoenhancesamplepreparation,
optimize image acquisition, and develop an
effective data-processing strategy.RESULTS:To reduce conformational heteroge-
neityofthesample,wespreadtheopenedNE
onto the grids with minimal force and used
the chemical cross-linker glutaraldehyde to sta-
bilize the NPC. To alleviate orientation bias ofthe NPC, we tilted sample grids and imaged the
sample with higher electron dose at higher
angles. We improved the image-processing
protocol. With these efforts, the average res-
olutions for the core and the Nup358 regions
have been improved to 3.7 and 4.7 Å, respec-
tively. The highest local resolution of the core
region reaches 3.3 Å. In addition, a cryo-EM
structure of the N-terminala-helical domain of
Nup358 has been resolved at 3.0-Å resolution.
These EM maps allow the identification of five
copies of Nup358, two copies of Nup93, two
copies of Nup205, and two copies of Y com-
plexes in each CR subunit. Relying on the EM
maps and facilitated by AlphaFold prediction,
we have generated a final model for the CR of
the X. laevisNPC. Our model of the CR subunit
includes 19,037 amino acids in 30 nucleoporins.
A previously unknown C-terminal fragment
of Nup160 was found to constitute a key part of
the vertex, in which the short arm, long arm,
and stem of the Y complex meet. The Nup160
C-terminal fragment directly binds theb-propeller
proteins Seh1 and Sec13. Two Nup205 mole-
cules, which do not contact each other, bind
the inner and outer Y complexes through dis-
tinct interfaces. Conformational elasticity of
the two Nup205 molecules may underlie their
versatility in binding to different nucleoporins
in the proximal and distal CR rings. Two Nup93
molecules, each comprising an N-terminal ex-
tended helix and an ACE1 domain, bridge the Y
complexes and Nup205. Nup93 and Nup205
together play a critical role in mediating the
contacts between neighboring CR subunits.
Five Nup358 molecules, each in the shape of a
shrimp tail and named“the clamp,”hold the
stems of both Y complexes. The innate con-
formational elasticity allows each Nup358 clamp
to adapt to a distinct local environment for
optimal interactions with neighboring nucle-
oporins. In each CR subunit, thea-helical
nucleoporins appear to provide the confor-
mational elasticity; the 12b-propellers may
strengthen the scaffold.CONCLUSION:Our EM map–based model of
theX. laevisCR subunit substantially ex-
pands the molecular mass over the reported
composite models of vertebrate CR subunit. In
addition to the Y complexes, five Nup358, two
Nup205, and two Nup93 molecules constitute
thekeycomponentsoftheCR.Theimproved
EM maps reveal insights into the interfaces
among the nucleoporins of the CR.
▪STRUCTURE OF THE NUCLEAR POREZhuet al., Science 376 , 1177 (2022) 10 June 2022 1of1
The list of author affiliations is available in the full article online.
*Corresponding author. Email: huanggaoxingyu@westlake.
edu.cn (G.H.); [email protected] (Y.S.)
†These authors contributed equally to this work.
Cite this article as X. Zhuet al., Science 376 , eabl8280
(2022). DOI: 10.1126/science.abl8280READ THE FULL ARTICLE AT
https://doi.org/10.1126/science.abl8280Cryo-EM structure of the double-layered CR of theX. laevisNPC.TheX. laevisCR, containing eight
repeating subunits, is modeled on the basis of cryo-EM reconstructions (top left panel). One CR subunit is
shown in two different views to highlight nucleoporins of key interest (bottom left and right panels). The inner
and outer Y complexes are colored dark and light gray, respectively. Two Nup205, two Nup93, and five
Nup358 molecules are colored blue, red, and purple, respectively.