cargo (Fig. 6, B and C, and fig. S83A). However,
the NUP214NTD•DDX19 complex forms tighter
interactions when DDX19 is in its ADP-bound
state and would therefore be expected to exist
in the adenosine triphosphate (ATP)–depleted
environment of the purified nuclear envel-
ope ( 50 , 63 ). The placements of the CFNC-
hub model and NUP88NTD•NUP98APDwere
further supported by manual docking into
an anisotropic ~8-Å region of a compositesingle-particle cryo-EM map of theX. laevis
cytoplasmic outer-ring protomer, although
the map masking excluded the region that
includes the dumbbell-shaped density (fig.
S83B) ( 45 ).Bleyet al., Science 376 , eabm9129 (2022) 10 June 2022 12 of 18
Fig. 6. Docking analysis
reveals that NUP93 anchors
the CFNC to the cytoplasmic
outer ring.(A)Overviewof
the NPC cytoplasmic face
with isosurface rendering of
unexplained density cluster II
(cyan) of the ~12-Å cryo-ET
map of the intact human NPC.
The inset indicates the loca-
tion of the magnified view
in (B). (B andC)Twoviewsof
manually placed poly-alanine
models of CFNC-hub
segments CCS1 and CCS2,
as well as of the tentatively
placed NUP88NTD•NUP98APD
and NUP214NTD•DDX19
cocrystal structures shown
in cartoon representation.
(D) Cartoon representation of
a cytoplasmic face spoke.
The root mean square (r.m.s.)
end-to-end length estimate
for the NUP93R1-R2linker
definesa~40-Åradiusforthe
expected location of the distal
NUP93R1region. (E)Sche-
matic of a cytoplasmic face
spoke illustrating CFNC-hub
anchoring by the distal
NUP93R1positioned by the
distal NUP205-bound NUP93R2.
(F)SEC-MALSinteraction
analysis showing the binding
of SUMO-NUP93R1to the
heterohexameric CFNC or
CFNC hub and illustrating the
abolished binding of the
SUMO-NUP93R1LIL mutant
to the CFNC hub. Proteins
were visualized by Coomassie
brilliant blue staining.
(G)(Left)Analignmentof
C. thermophilumNic96 and
H. sapiensNUP93 sequences
shows the conservation of
residues targeted by the
LLLL and LIL mutations that
abolish binding to the CNT.
Residues are colored,
according to a multispecies
sequence alignment, from
white (less than 55% similarity), to yellow (55% similarity), to red (100% identity), using the BLOSUM62 weighting algorithm. (Right) Domain architectures of NUP214,
NUP88, NUP62, and NUP93. The location of the NUP93 LIL mutation is indicated (red dots). Single-letter abbreviations for the amino acid residues are as follows:
A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr. (H)Twoviewsofthe
NUP88NTD•NUP98APDcocrystal structure in cartoon representation. Inset boxes indicate regions of magnified view (right), of the NUP88NTDFGL-loop interaction with
NUP98APDand of the NUP98APDK/R-loop interaction with NUP88NTD. Black triangles indicate alanine substitutions in the NUP88NTDEMNY mutant.
RESEARCH | STRUCTURE OF THE NUCLEAR PORE