www.sciencemag.org/cgi/content/full/science.
aac9176
/DC1
Supplementary
Material
s for
Architecture of the fungal nuclear pore inner ring complex
Tobias
Stuwe
, Christopher J. Bley
, Karsten
Thierbach
, Stefan
Petrovic
, Sandra
Schilbach
,
Daniel J.
Mayo
, Thibaud
Perriches
, Emily J.
Rundlet
, Young E. Jeon
, Leslie N.
Collins
,
Ferdinand M. Huber
, Daniel H.
Lin
, Marcin
Paduch
, Akiko Koide
, Vincent
Lu
, Jessica
Fischer
, Ed
Hurt
, Shohei
Koide
, Anthony A. Kossiakoff
, André
Hoelz
*
*Corresponding author. E-
mail: hoelz@caltech.edu
Published
27 August
201
5 on
Science
Express
DOI:
10.1126/science.
aac9176
This PDF file includes:
Materials and Methods
Figs. S1 to
S38
Tables S1 to S
9
Captions for
Movies S1 to S
4
References
Other Supporting Online Material for this manuscript includes the following
:
(available at
www.sciencemag.org/cgi/content/full/science.
aac9176
/DC1
)
Movies S1 to S
4
Materials and Meth
ods
(
1- 41
)
Bacterial expression constructs
DNA fragments were amplified by PCR using
C. thermophilum
and
H. sapiens
cDNA. SUMO
(small
ubiquitin-
like modifier)
tagged proteins were cloned into a modified pET28a vector or a modified
pET
-
MCN vector
containing an N
-terminal hexahistidine tag followed by a
SUMO
tag using BamHI and NotI
restriction sites
(
42,
43
)
. Hexahistidine tagged proteins were
cloned into a modified pET28a vector
containing a N
-terminal hexahistidine tag followed by a PreScission protease cleavage site using NdeI
and NotI restriction sites
(
44
)
. GST tagged proteins were cloned into pGE
X-6P
1 using the BamH
I and
NotI restriction sites. Additionally, DNA coding for Nsp1 (residues 467
-674) and
Nup57 (
residues 77-
319)
or Nup57 (residues 146
-319)
were cloned sequentially into pETDuet1 (Novagen) for expression of
untagged protein. Nsp1 was cloned into the first multiple cloning site using NcoI and NotI restriction
sites while Nup57 was cloned into the second multiple cloning site using NdeI and XhoI
restriction sites.
The selected synthetic
antibody (sAB) fragments of sAB
-158
and sAB
-160
were cloned into the pSFV4
vector (Peter Loppnau, Structural Genomics Consortium, University of Toronto) using the restriction
sites NcoI and SalI, and subsequently digested using SalI and BsaI and re
-ligated to obtain the C
-
terminal hexahistidine tag.
Mutants were generated by
QuikChange mutagenesis and confirmed by
DNA sequencing.
Details of expression constructs are shown in Table
S5
.
Protein expression and purifi
cation
Proteins
were expressed in
E. coli
BL21
-CodonPlus(DE3)
-RIL cells (Stratagene) in Luria
-Bertani media
and
induced at an
OD
600
of 0.
8
with 0.5
mM IPTG
. For details of the
expression times and
temperatures
see
Table
S5
. Seleno
-L-
methionine
(SeMet)
labeled proteins were expressed
by a
methionine pathway inhibition protocol, as previously described
(
45
)
. Cells were harvested by
centrifugation and re-s
uspended
in a buffer containing 20
mM
TRIS (pH
8.0), 500
mM
NaCl, 5
mM 2
-
mercaptoethanol (
β
-ME), supplemented with
complete EDTA
-free prot
ease inhibitor
cocktail (Roche)
.
For purification, the cell suspension
s of all proteins and protein complexes were
supplemented with
1 mg DNase I (Roche) and lysed using a cell disruptor (Avestin). Cell lysate
s were
cleared by
centrifugation at 30,000
x
g
for 1
hour
. The supernatants were filtered through a 0.45
-
μ
m filter
(Millipore) and
purified using standard chromatography methods
; for details see
Table
S6
. Proteins
were concentrat
ed
to
~10-
20
mg/
ml for bioc
hemical interaction experiments, complex
reconstitution
,
and crystallization.
Purification of Nup192
•
Nic96
R2-
SOL
.
Nup192
and
GST
tagged
Nic96
R2-
SOL
were
expressed
individually. After harvesting cells
, were
re-s uspended in 50
mM TRIS (pH
8.0), 500
mM NaCl,
5 mM
β
-
ME, 10
%
(v/v)
glycerol supplemented with
complete
EDTA
-free protease inhibitor cocktail (Roche)
,
2
μ
M
bovine lung aprotinin (Sigma)
and 1
mM
phenylmethanesulfonyl
fluoride (
PMSF; Sigma)
. Prior to
lysis,
cell
suspension
s of GST-
Nic96
R2-
SOL
and Nup192
were mixed in a 1:2
ratio
. Clarified lysate was
loaded onto a Ni
-NTA column equilibrated in
a buffer containing 25
mM TRIS (pH 8.0), 500
mM NaCl,
5 mM
β
-ME, 20
mM imidazole
, and
5 %
(v/v)
glycerol and eluted with an
imidazole gradient. Peak
fractions were pooled and
loa
ded onto a Glutathione S
epharose
4 Fast F
low
(GE Healthcare)
column
and eluted using a glutathione gradient. Peak
fraction
s were pooled and dialyzed over
night against
a
buffer containing 20
mM TRIS (pH
8.0), 1
50
mM NaCl,
5 mM
DTT,
and
5 %
(v/v)
glycerol
in
the
presence of PreScission
protease. The protein was concentrated and further purified over a
HiLoad
Superdex
200 16/60 PG
column
equilibrated in a
buffer containing 20
mM TRIS (pH
8.0),
150
mM
NaCl
, and
5 mM DTT.
Peak
fractions were pooled and concentrated to ~20
mg/
ml for bioc
hemical
interaction experiments
.
Purification of
CNT
•
Nic96.
Cells
containing CNT and Nic96 were
grown individually
and re-
suspended in 50
mM TRIS (pH
8.0), 500
mM NaCl,
4 mM
β
-ME, 10
%
(v/v) glycerol supplemented with
complete
EDTA
-free
protease inhibitor cocktail (Roche)
, 2
μ
M
bovine lung aprotinin (Sigma)
and 1
mM
phenylmethanesulfonyl
fluoride (
PMSF; Sigma)
. Prior to lysis,
cell
suspension
containing CNT and
Nic96 were mixed in a 3:1 ratio. Clarified lysate was loaded onto a Ni
-NTA column equilibrated in
a
buffer containing 2
5 mM TRIS (pH
8.0), 500
mM NaCl,
5 mM
β
-ME, 20
mM imidazole
, and 5
%
(v/v)
glycerol
and
eluted with an imidazole gradient. Peak fractions were pooled and desalted using a
HiPrep
2
26/20 Desalting
(GE Healthcare)
column equilibrated in a buffer containing 20
mM TRIS (pH
8.0),
150
mM NaCl, 5
mM DTT, and 5
%
(v/v) glycerol in the presence of ULP
1 protease. After SUMO
cleavage, the protein was loaded onto a MonoQ 5/50 GL (GE Healthcare) column equilibrated in
20
mM TRIS (pH
8.0), 150
mM NaCl, 5
mM DTT, and 5
%
(v/v) glycerol and eluted with a sodium
chloride gradient.
Protein
-containing fractions were pooled and concentrated to ~
5 mg/
ml for
biochemical interaction experiments.
Purification of sABs.
The expression of sABs
was
induced at an
OD
600
of 0.9 with 0.25
mM
IPTG and grown at
37
°C for
3 hours. The lysate was incubated at 65
°C for 30
minutes and then
cooled on ice for 15
minutes before centrifugation
at
30,000
x
g
for 1
hour
. The supernatant
was
filtered
through a 0.45
-
μ
m filter (Millipore) and loaded onto a
Ni-NTA
column equilibrated in 20
mM TRIS
(pH
8.0), 500
mM NaCl,
and 20
mM imidazole. Protein was eluted with a linear gradient of 20
mM TRIS
(pH
8.0), 500
mM NaCl,
and 500
mM imidazole. Protein
-containing fractions from the Ni
-NTA affinity
purification were pooled and loaded onto a 5
ml HiTrap MabSelect SuRe
(GE Healthcare)
column
equilibrated in a buffer containing 20
mM TRIS (pH
8.0) and 500
mM NaCl
and
eluted using a linear
gradient of an elution buffer containing 0.1
M acetic acid. The eluted fractions were
dialyzed
against a
buffer containing 20
mM TRIS (pH
8.0) and 100
mM NaCl
and concentrated to ~50
mg/
ml for
biochemical interaction experiments, complex reconstitution, and crystallization.
Purification
of
CNT•Nic96
R1
.
Purified CNT was mixed with 2
-fold molar
excess of
purified
SUMO
-Nic96
R1
and incubated on ice for 30
minutes.
The
mixture
was then loaded onto a
HiLoad
Superdex 200 16/60
PG
column
equilibrated in buffer containing 20
mM TRIS (pH
8.0), 100
mM NaCl
,
and
5 mM DTT. To obtain untagged CNT
•
SUMO
-Nic96
R1
, the proteins were mixed in the presence of
ULP1 protease
. Fractions containing either CNT•SUMO
-Nic96
R1
or untagged CNT•Nic96
R1
were pooled
and concentrated to ~
30
mg/
ml for biochemical
studies.
Purification
of
CNT•Nic96
R1
•
sA
B- 158.
Purified CNT was mixed with 2
-fold molar excess of
purified SUMO
-Nic96
R1
, 2
-fold molar excess of purified sAB
-158, and
ULP1 protease
, followed by
incubation
on ice for 30
minutes.
The
mixture
was then loaded onto a
Superdex 200 10/300 GL column
equilibrated in buffer containing 20
mM TRIS (pH
8.0), 100
mM
NaCl, and 5
mM DTT. Fractions
containing
CNT•Nic96
R1
•sA
B- 158
were pooled and concentrated to ~
50
mg/
ml for biochemical studies
and crystallization.
Purification of
Nup82
NTD
•
Nup159
T
•
Nup145N
APD
.
Purified Nup82
NTD
•
Nup159
T
was mixed
with a
1.2
-fold molar
excess of Nup145N
APD
and
incubated on ice for 30
minutes.
The
mixture
was then
loaded onto a
Superdex 200 16/60
PG
column
equilibrated in buffer containing 20
mM TRIS (pH
8.0),
100
mM NaCl, and 5
mM DTT. Peak fractions
containing a stoichiometric
Nup82
NTD
•
Nup159
T
•
Nup145N
APD
hetero-
trimer were pooled and concentrated to ~
30
mg/
ml for
biochemical studies
and crystallization.
Reconstitution of hsNup49
CCS2+3
*•
hsNup57
CCS3
* complexes.
Purified
hs
Nup49
CCS2+
3
* was
mixed
with an equimolar amount of
hs
Nup57
CCS3
* and incubated on ice for 30 minutes.
The
mixture
was then
loaded onto a
HiLoad
Superdex 200 16/60 PG
column
equilibrated in buffer containing 20
mM TRIS
(pH
8.0), 100
mM NaCl, and 5
mM DTT. Peak fractions containing a
stoichiometric
hs
Nup49
CCS2+
3
*•
hs
Nup57
CCS3
* complex
were pooled and concentrated to ~20
mg/
ml for crystallization.
Reconstitution of CNT
•
Nic96
•Nup192•Nup53.
The hetero
-nonameric IRC protomer including the
CFC proteins Nup82
NTD
and Nup159
T
was reconstituted as follows. A Nup192•Nup53 complex was
formed by mixing purified Nup192 and Nup53 with a 1.2 fold molar excess of Nup53, incubated on ice
for 30 min
utes
and purified over a
HiLoad
Superdex 200 16/60
PG
column equilibrated in
a buffer
con
taining 20
mM TRIS (pH
8.0), 100
mM NaCl and 5
mM DTT. The
Nup192•Nup53 dimer was
subsequently mixed with
a 1.2
-
fold molar excess of CNT•Nic96, incubated for 30
minutes on ice and
further purified over a
HiLoad
Superdex 200 16/60 PG gel filtration column
equilibrated in 20
mM TRIS
(pH
8.0), 100
mM NaCl and 5
mM DTT
. Complex
-containing fractions were pooled and concentrated to
~8 mg/
ml for biochemical studies.
Reconstitution of CNT
•
Nic96
•Nup192•Nup53•Nup145N•Nup82
NTD
•Nup159
T
.
To reconstitute
CNT•Nic96•Nup192•Nup53•Nup145N•Nup82•Nup159, 1
mg of
purified
CNT•Nic96•Nup192•Nup53 was
mixed
with 0.8
mg of
purified
Nup82
NTD
•Nup159
T
•Nup145N
,
incubated
for 30
minutes on ice
, and
3
purified over
a Superose
6 10/300
GL column
equilibrated in
20
mM TRIS (pH
8.0), 100
mM NaCl and
5 mM DTT
.
Generation of synthetic antibodies and monobodies
The selection of synthetic binders was performed using avi
-tagged CNT,
CNT•Nic96
R1
, and
CNT•Nic96
139-
201
, containing
Nup49 with an N
-terminal avi
-tag.
Biotinylation was carried out
using
40
μ
M
protein
in
a buffer containing
50
mM BICINE (pH
8.3), 100
mM biotin, 10
mM ATP, 10
mM
magnesium acetate, and
30
μ
g biotin ligase (BirA) at 30
°C for 2
hours
in a total volume of 2
ml. After
labeling, protein was buffer exchanged using a 5
ml HiTrap Desalting (GE Healthcare)
column
equilibrated with a buffer containing 20
mM TRIS (pH
8.0), 100
mM NaCl, and 5
mM DTT and purified
again using a HiLoad Superdex 200 16/60 PG
column equilibrated in the same
buffer. The extent of
biotinyl
ation and efficiency of capture were tested by
incubating 10
μ
g protein with 50
μ
l of
Streptavidin
MagneSphere
Paramagnetic Particles (
Promega),
followed by a single wash
ing
step
with 50
μ
l buffer
,
containing 20
mM TRIS (pH
8.0), 100
mM NaCl, and 5
mM DTT, and resolving the bound proteins
by
SDS
-PAGE and visualizing by Coomassie Brilliant Blue staining.
For sAB identification, f
our rounds of competitive selection were performed using 100
nM
(round
1), 50
nM (round
2), 10
nM (round
3), and 10
nM (round
4) biotinylated
CNT,
CNT•Nic96
R1
, or
CNT•Nic96
139-
201
a nd a phage display library according to previously published protocols
(
46,
47
)
. After
successful selection, the specificity of candidate sABs was tested against the biotinylated
CNT,
CNT•Nic96
R1
, and CNT•Nic96
139-
201
using a single point competitive ELISA assay
(
46
)
. Only sequence
-
unique sABs with the desired binding properties were
chosen
for further biochemical characterization.
Monobody libraries were screened with a similar protocol and
affinities determined
by yeast display
, as
previously described
(
48,
49
).
To evaluate the binding affinity and specificity of the selected sABs
and MBs, 1.5
-fold molar
excess of sAB or MB was incubated with
CNT, CNT•Nic96
R1
, or CNT•Nic96
139-
201
a nd loaded onto a
MonoQ 5/50 GL
column
equilibrated in a buffer containing 20
mM TRIS (pH
8.0), 100
mM NaCl, and
5 mM DTT and eluted using a NaCl gradient. Interacting sABs
or MBs eluted with the CNT,
CNT•Nic96
R1
, or CNT•Nic96
139-
201
, whereas non
-interacting sABs or MBs eluted prior to the gradient
step.
The
screened
sABs
or MBs formed stable complexes with
CNT, CNT•Nic96
R1
, and CNT•Nic96
139-
201
, but failed to differentiate between them.
Multi-
angle
light
scattering coupled
to
analytical size
-exclusion chromatography
Purified proteins and complex formation were characterized by inline multi
-angle light scatt
ering
following separation on
Superdex 200 10/300 GL or Superose 6 10/300 GL
columns (
50
)
. All proteins
and complexes were analyzed in a
buffer containing
20
mM TRIS
( pH
8.0
), 100
mM
NaCl
and
5 mM
DTT, except
those involving Nup192
TAIL
, which were carried out in a buffer
containing
20
mM TRIS
(pH
8.0), 200
mM
NaCl
and
5 mM DTT. The chromatography system was connected in series with an
18-
angle light scattering detector (DAWN HELEOS II, Wyatt Technology), a dynamic light scattering
detector (DynaPro Nanostar, Wyatt Technology), and a refractive index detector (Optilab t
-rEX, Wyatt
Technology). Data
were
collected every 1
second at a flow rate of 0.5
ml/min
at 25
º C and analyzed
using ASTRA
6 software
, yielding molar mass and mass distribution (polydispersity) of the samples.
For interaction studies, proteins were mixed and incubated on ice for 30
minutes prior to being applied
to the
gel filtration column. Protein containing frac
tions were analyzed by SDS
-PAGE followed by
Coomassie brilliant blue staining.
Thermal stability assay
Purified
CNT
( 10
μ
g) was
incubated
i
n a total volume of
50
μ
l for 30
minutes
at
40-
61 ºC,
increased in
3.5
ºC
increments
. Soluble
and pellet fractions were separated by centrifugation
at 30,000
x
g
for
35
minutes
at 4
ºC
, resolved
by
SDS
-PAGE,
and visualized by Coomassie brilliant blue
staining.
GST
pull
-down
protein-
protein interaction analysis
Bait protein (5
μ
g,
purified GST
-Nic96
R1
or GST) was incubated
for 30
minutes
with
an equimolar
amount
of
target proteins in
a buffer containing 20
mM TRIS
(pH
8.0)
, 100
mM NaCl, 4
mM DTT. The
4
mixtures
were then incubated for 30
minutes
with 20
μ
l pre
-washed
Glutathione Sepharose 4B
resin
(GE Healthcare). The beads were collected by
centrifugation at 500
x
g
for 2
min
utes
and washed
five
times with 200
μ
l binding
buffer.
Bound
proteins
were
released from the resin by addition of
0.5
μ
g
PreScission protease and incubation
for 20
minutes
at room temperature.
Samples from the load,
PreScission
eluted supernatant
, and post
-elution beads
were analyzed by SDS
-PAGE and visualized
by
Coomassie brilliant blue
staining.
Because Nup49 and Nup57 were poorly behaved when expressed
individually, they
were not tested in isolation.
Yeast strains, media and miscellaneous genetic methods
Preparation of all media (yeast peptone dextrose, YPD; synthetic dextrose complete,
SDC), LiAc
-
mediated yeast transformation and genetic manipulation was carried out
according to standard
protocols.
For
details of
the
yeast expression constructs and
haploid
S. cerevisiae
strains
see
Tables S7
and S8
. To generate the listed shuffle strains the respective essential gene
s were
replaced
with
either the
HIS3
or
the
natNT2
casse
tte
by homologous recombination
, as previously
described
(
51,
52
)
. Due to the lethality of the deletions, the indicated parental
strain was complemented with a
pRS416 construct carrying the respective full
-length
S. cerevisiae
gene with an N
-terminal mCherry tag
under the control of the P
NOP1
promoter prior to chromosomal integration. Subsequently, pRS415
-eGFP
or pRS415-
mCherry constructs carrying the various
Nup
variants were introduced. The transformants
were selected twice on SDC
-leucine (Leu) plates followed by plating twice on SDC+5
-fluoroorotic acid
(5-FOA) to ensure the loss of the pRS416
-mCher
ry construct prior to analysis.
T he
NUP192
shuffle
constructs and
the
nup49-
313
mutant
(EVPIP)
were described previously
(
8, 37
).
Yeast cell growth
analyses and fluorescence microscopic assays
For viability and growth analysis,
S. cerevisiae
strains carrying eGFP
-tagged variants of the respective
scN
UP
were grown at 30
°C.
Ten
-fold dilution series were generated, of which 20
μ
l were
spotted on
SDC
-Leu and SDC+5
-FOA plates and grown at 23
°C for 5
days. For growth analysis of the shuffled
strains, 7.5
μ
l of the dilution series were spotted on YPD plates and grown at 23
°C, 30
°C, and
37 °C
for 2
-4 d
ays.
For
sc
Nup57 co-
localization analysis, cells with
a genomic
, C-
terminal GFP-
tagged
scN
UP57
and pRS415-
mCherry constructs carrying the various
NUP
variants in the respective
knockout background were grown at 30
°C to mid
log phase and after
wards shifted for 6
h ours
to
37
°C.
The large ribosomal subunit
sc
R
pl25 reporter assay was performed as described previously
(
53
).
The pRS411 (for
scN
UP192
and
scN
IC96
mutant
analysis) or the pRS413 (for
scN
UP49
mutant
analysis) vectors carrying m
Cherry
-tagged
scRPL25
were co-
transformed with the respective pRS415
-
eGFP
variant
plasmids (see above) into the indicated shuffle strain. The transformants were selected
twice on SDC
-LEU
-MET or SDC
-LEU
-HIS plates followed by plating twice on SDC
-MET+5-
FOA or
SDC
-HIS+5
-FOA to ensure the loss of the pRS416
-mCherry construct. For analysis, cells were grown
in selective SDC
-MET or SDC
-HIS medium at 30
°C to mid
-log phase.
For fluorescence microscopy,
live cells were washed once with water, re
-suspended and analyzed using a Carl Zeiss Observer Z.1
equipped with a Hamamatsu camera C10600 Orca
-R2.
The statistical analysis was carried out using
three
sets of independent images with at least 500
cells each for every analyzed sample
. Error bars
represent standard deviation
.
FISH mRNA Export Assay
Liquid cultures of deletion yeast strains were grown at 30
°C (23
°C for the
nic96
Δ
/Nup57-
GFP
strain)
in YPD media to an OD
600
of 0.4 and subsequently shifted to 37
°C for 4
h ours
prior to
fixation in
formaldehyde. These cells were then analyzed by FISH using an Alexa647
-labeled
50-
mer oligo(dT)
probe
as previously described (
54,
55
)
. The statistical analysis was carried out using
three
sets of
independent images with at least 500
cells each for every analyzed sample
. Error bars represent
standard de
viation
.
Crystallization and structure determination
Crystals of Nup192
TAIL
, N
up188
TAIL
,
hsNup49
CCS3
*,
hsNup57
CCS3
*, Nup57
CCS3
*,
hsNup49
CCS2+3
*
•hs
Nup57
CCS3
* with
2:4
and 2:2 stoichiometry,
Nup82
NTD
•Nup159T•Nup145N
APD
, and
5
CNT•Nic96
R1
•sAB
-158
were grown at
21 °
C in hanging drops containing 1
μ
l of protein and 1
μ
l of a
reservoir solution.
SeMet
-labeled crystals were grown under
similar
conditions.
For CNT
•
Nic96
R1
•sAB
-
158, native crystals were derivatized with
p-
chloromercuribenzoic acid (PCMB),
ethyl mercury
thiosalicylate (EMTS), potassium hexachloroosmate (K
2
OsCl
6
),
and
potassium osmate (K
2
OsO
4
).
Crystals were cryoprotected by supplementing the drop with cryo protectant
and flash-
frozen in liquid
nitrogen.
For details of the crystallization and
cryo protection see Table
S9
. X-
ray diffraction data were
collected at 100
K at beaml
ine 8.2.2 at the Advanced Light Source (ALS), Lawrence Berkley National
Laboratory (LBNL), beamline BL12-
2 at the Stanford Synchrotron Radiation Source (SSRL), and
beamline GM/CA
-CAT 23ID
-D at the Advanced Photon Source (APS). The X
-ray diffraction data were
pro
cessed using the HKL2000 and XDS
(
56,
57
).
Initial phases for all structures
were calculated in PHASER using single anomalous dispersion
(SAD)
X- ray diffraction data obtained from
SeMet
derivatives
(
58
)
.
Solvent flattening and NCS
averaging
were performed in
RESOLVE,
yielding improve
d phases and
experimental maps of excellent
quality
. In
the case
of CNT
•
Nic96
R1
•
sAB
-158,
the best
experimental map was obtained
using
SAD
X-
ray diffraction data obtained from the K
2
OsCl
6
derivative
and
inclusion of the data in the highest
resolution shell was beneficial for
its quality.
Iterative rounds of model building and refinement were
performed
with COOT and PHENIX
(
59,
60
)
. In the cases of Nup188
TAIL
and
hs
Nup49
CCS2+3
*,
the
refinements did
not converge in higher symmetry space groups and were therefore carried out in P1.
The unambiguous assignment of the CNT•Nic96
R1
•sAB
-158 sequence register was aided by
anomalous difference Fourier map calculations
using the derivatives.
Subsequently, the assigned
sequence register was
verified
by
methionine mutants yielding
22 additional selenium sites (Nup49:
I263M, H276M, L31M, A307M, L332M, E438M; Nup57
: I82M, H109M, L113M, E127M, I138
M, L198M,
I233M, L253M, V304M; Nsp1
: L527M, L548M, A628M; and Nic96
R1
: L140M, L158M, L161M, L178M).
In total
78 heavy
atom sites were identified in anomalous difference Fourier maps
( fig.
S29
).
For all structures, the final models
possess
excellent R
work
and R
free
values and the assessment
of their stereochemical qualities with
MolProbity revealed no Ramachandran outliers
(
61
)
. For details
of
the data collection and refinement statistics see
Table
s S2
to S4
.
Illustrations and figures
Gel filtration profiles and MALS graphs were generated in IGOR (WaveMetrics) and assembled in
Adobe Illustrator. Sequence alignments were generated using ClustalX
(
62
)
and colored with
ALSCRIPT
(
63
)
. Electrostatic potentials were
calculated with APBS (Adaptive Poisson-
Blotzmann
Solver) software (
64
)
. Structure figures were generated with PyMOL (www.pymol.org).
Coordinates and Structure Factors
The coordinates
and structure factors have been deposited with the Protein Data Bank with accession
codes 5CWV
(Nup192
TAIL
),
5CWU
(Nup188
TAIL
), 4JQ5 (
hs
Nup49
CCS2+3
*), 4JNV and 4JNU
(
hs
Nup57
CCS3
*),
5CWT
(Nup57
CCS3
*), 4JO7 (
hs
Nup49
CCS2+3
*•
hs
Nup57
CCS3
*, 2:2 stoichiometry),
4JO9
(
hs
Nup49
CCS2+3
*•
hs
Nup57
CCS3
*, 2:4 stoichiometry),
5CWW
(Nup82
NTD
•Nup159
T
•Nup145N
APD
) and
5CWS
(CNT•Nic96
R1
•sAB
-158).
6
7
8
Fig. S1.
Reconstitution and dissection of the IRC.
(
A
to
H
)
SEC
-MA
LS and SDS
-PAGE analysis
corresponding to
Figure
1 . SEC
-MALS profiles of nucleoporins or nup complexes are shown
individually (red and blue) and after their preincubation (green). Measured molecular masses are
indicated
for the peak fractions. Gray bars in
dicate fractions that were resolved on SDS
-PAGE gels and
visualized by Coomassie staining.
9
10
Fig. S2.
Reconstitution and dissection of the IRC (continued).
(
A
to
F
)
SEC
-MALS and SDS
-PAGE analysis
corresponding to
Figure
1 . SEC
-MALS profiles of nucleoporins
or nup complexes are shown
individually (red and blue) and after their preincubation (green). Measured molecular masses are
indicated
for the peak fractions. Gray bars indicate fractions that were resolved on SDS
-PAGE gels and
visualized by Coomassie stai
ning.
11
12
Fig.
S 3.
Structure
of the minimal CFC in complex with Nup145N
APD
.
(
A
) Structure of the
Nup82
NTD
•Nup159
T
•Nup145N
APD
hetero-
trimer
shown in cartoon representation. Nup145N
APD
(green),
Nup159
T
(red), and Nup82
NTD
(blue), the Nup82
NTD
helical 4CD (grey) and 6CD (orange) insertions and
FGL
loop (yellow), and the conserved Nup145N
APD
K loop (purple) are illustrated. (
B
) Structure of the
previously determined
sc
Nup82
NTD
•
sc
Nup159
T
•
mm
Nup145N
APD
hetero-
trimer (PDB
ID:
3TKN
) shown
in
cartoon representation
(
29
)
. (
C
) Supe
rposition
of the two hetero
-trimers
in cartoon representation
,
revealing
the evolutionary conservation of their architecture
s, and differing mainly by an angular
displacement of Nup145
APD
. (
D
) The
Inset
marks t
he location of the two close
-up views, depicting the
molecular details of the Nup145N
APD
-Nup82
NTD
interaction (top) and the Nup159
T
-Nup82
NTD
interaction
(bottom). The D pocket that binds the K loop is indicated.
(
E
) Sequence
alignments of the conserved
S. cerevisiae
and
C. thermophilum
Nup159
T
, the Nup82
NTD
FGL loop, and the Nup145N K/R loop
.
Sequence
similarity is shaded from white (less than 60
% similarity), to yellow (
60
% similarity), to red
(100
% identity)
using the BLOSUM62 matrix. Numbering below alignment is according
to
the
C. thermophilum
proteins.
S econdary structure is shown above the alignment.
13
14
Fig. S4.
The CNT does not interact with Nic96
R2
or Nup192
TAIL
, nor does Nic96
R1
bind Nup192
TAIL
.
(
A
to
C
)
SEC
-MALS
profiles of nucleoporins or nup complexes are shown individually (red and blue) and after
their preincubation (green). Measured molecular masses are indicated
for the peak fractions. Gray bars
indicate fractions that were resolved on SDS
-PAGE gels and visualized by Coomassie staining.
Aste
risks indicate degradation products or contamination.
15
16
Fig.
S 5.
Nup
188
TAIL
forms an alternative complex with the CNT
.
(
A
)
Reconstitution of SUMO
-
Nup188
TAIL
•
SUMO
-Nic96
R1-
R2
•
CNT
. (
B
) Reconstitution of SUMO
-Nup188
TAIL
•
SUMO
-Nic96
R2
. (
C, D
)
SEC analysis of Nup
192
TAIL
•
SUMO
-Nic96
R2
and
SUMO
-Nup188
TAIL
•
SUMO
-Nic96
R2
carried out in a 1
M
NaCl buffer. SEC profiles of
nucleoporins or nup complexes are shown individually (red and blue) and
after their preincubation (green).
Gray bars indicate fractions that were resolv
ed on SDS
-PAGE gels
and visualized by Coomassie staining.
Asterisks indicate degradation products or contamination.
17
18