accommodate the passage of small cytosolic
domains of inner nuclear membrane inte-
gral membrane proteins (INM-IMPs), sug-
gesting the mechanism by which inner ring
constriction upon energy depletion might
interfere with the path of diffusion of these
proteins between outer and inner nuclear
envelope membranes ( 78 – 80 ). It remains to
be established whether the extent of inner ring
dilation observed by current cryo-ET recon-
structions of the human andS. cerevisiae
NPCs capture the maximally achievable lateral
channel dimensions. However, the previous
observations that karyopherin-mediated ac-
tive INM-IMP transport requires unstructured
tethers spanning the distance between the
nuclear envelope and the central transport
channel is consistent with the observed lateral
channel dimensions (fig. S83C) ( 81 – 85 ). Tak-
ing advantage of the composite NPC struc-
tures, future studies are expected to elucidate
the nucleocytoplasmic translocation pathways
and the impact of inner ring dilation for in-
dividual INM-IMPs. Similarly, the transloca-
tion of perinuclear domains on the opposite
side of the nuclear envelope is expected to be
limited by the luminal ring that encircles the
NPC midplane formed by Pom152/POM210
Ig-like domains ( 37 , 47 , 86 ).
Finally, the capacity of the NPC to exist in
dilated and constricted states dependent on
tension imparted by the surrounding nuclear
envelope portrays the NPC as the cell’slargest
mechanosensitive channel, with implications
in cellular energy–state sensing and transport
of transcriptional regulators in response to
mechanical stress on the cell ( 46 , 87 ). The con-
striction and dilation of the inner ring may not
only affect the distribution and local concen-
tration of FG repeats in the central transport
channel, a determinant of karyopherin-mediated
transport efficiency ( 88 ) but also sterically mod-
ulate the flux of INM-IMPs and large cargos
such as preribosome or messenger ribonucleo-
protein particles (mRNPs). Future studies will
have to establish the causal links between the
transmission of tension from the nuclear envel-
ope to the NPC, the dilation of its inner ring, and
mechanisms by which dilation and constriction
may modulate nucleocytoplasmic transport.
Our results illuminate the elusive linker-
scaffold molecular interactions that main-
tain the integrity of the NPC, providing a
comprehensive characterization of a final
major aspect of the NPC symmetric core. Build-
ing on this roadmap, future studies can address
NPC assembly and disassembly mechanisms,
the emergence of NPC polarity and attachment
of the asymmetric cytoplasmic filaments and
nuclear basket to the symmetric core, mecha-
nisms of NPC-associated diseases, and mecha-
nisms of NPC dilation and constriction along
with their implications in nucleocytoplasmic
transport.
Methods summary
Comprehensive materials and methods are
presented in the supplementary materials.
Briefly, the source of materials and reagents is
listed in table S1. Summaries of bacterial ex-
pression constructs and conditions (table S2),
protein purification procedures (table S3), an-
alytical SEC-MALS protein interaction analyses
(table S4), and ITC binding affinity measure-
ments (table S5) are provided. Experimental
details of x-ray crystallography and single-
particle cryo-EM structure determination pro-
cedures are described, including summaries of
crystallization and cryo-protection conditions
(table S6), as well as data collection, process-
ing, and refinement statistics (tables S7 to S12).
S. cerevisiaeconstructs (table S13) and strains
(table S14), as well as the experimental details
of the viability and growth assay, subcellular
nup localization analysis, 60S preribosome
export assay, and mRNA export fluorescence
in situ hybridization (FISH) assay, which es-
tablish the physiological relevance of the bio-
chemical and structural findings, are provided.
Details of the incremental quantitative dock-
ing procedures for nup and nup complex
crystal and single-particle cryo-EM structures
into ~12- and ~23-Å cryo-ET maps of the intact
human NPC (constricted state) ( 38 , 47 ), as well
as into an ~37-Å in situ cryo-ET map of the
human NPC ( 45 )andan~25-Åinsitumapof
theS. cerevisiaeNPC ( 36 ) (dilated states), are
provided. Inventories of nup and nup complex
experimental structures used to generate
the near-atomic composite structures of the
S. cerevisiae(table S15) and human (table
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RESEARCH | STRUCTURE OF THE NUCLEAR PORE