Crystal structure of importin-a bound to a peptide bearing
the nuclear localisation signal from chloride intracellular
channel protein 4
Andrew V. Mynott
1
, Stephen J. Harrop
1
, Louise J. Brown
2
, Samuel N. Breit
3
, Bostjan Kobe
4,5
and
Paul M. G. Curmi
1,3
1 School of Physics, University of New South Wales, Sydney, NSW, Australia
2 Department of Chemistry and Biomolecular Sciences, Macquarie University, Sydney, NSW, Australia
3 St Vincent’s Centre for Applied Medical Research, St Vincent’s Hospital and University of New South Wales, Sydney, NSW, Australia
4 School of Chemistry and Molecular Biosciences and Centre for Infectious Disease Research, University of Queensland, Brisbane, Qld,
Australia
5 Institute for Molecular Bioscience, University of Queensland, Brisbane, Qld, Australia
Introduction
The importin-a:b nuclear import pathway is one of the
best understood nuclear trafficking systems in the cell
[1]. The pathway operates via the importin-a receptor,
an armadillo (ARM) repeat protein, that recognizes and
binds directly to cargo protein in the cytoplasm. The im-
portin-a:importin-b:cargo complex travels through the
nuclear pore, with importin-b primarily responsible for
negotiating passage through the nuclear pore complex.

to the importin-a backbone. This structural evidence supports the hypothe-
sis that CLIC4 translocation to the nucleus is governed by the importin-a
nuclear import pathway, provided that CLIC4 can undergo a conforma-
tional rearrangement that exposes the NLS in an extended conformation.
Database
Structural data are available in the protein Data Bank under the accession number
3OQS.
Structured digital abstract
l
CLIC4 and importin alpha bind by x-ray crystallography (View interaction)
Abbreviations
ARM, armadillo; CLIC, chloride intracellular channel; NLS, nuclear localization signal; RSCC, real space correlation coefficient; TAg, simian
virus 40 (SV40) large T-antigen.
1662 FEBS Journal 278 (2011) 1662–1675 ª 2011 The Authors Journal compilation ª 2011 FEBS
affinity to clusters of basic residues in the NLS. Mono-
partite NLSs consist of a single cluster of basic amino
acids, approximately six residues long, which generally
interact with the major binding site in importin-a. Struc-
tural studies have shown that an NLS binds importin-a
in an extended conformation, suggesting that functional
NLSs need to be unfolded and flexible within the cargo
protein. Recent studies have demonstrated an interaction
between importin-a and the chloride intracellular chan-
nel (CLIC) protein, CLIC4 [2,3].
The structure of a soluble form of CLIC4 shows
that it adopts the canonical glutathione S-transferase
fold with an N-terminal thioredoxin-like domain and
an a-helical C-terminal domain [4]. CLIC4 can form
poorly selective anion channels that are sensitive to
redox conditions [5] and, like other CLIC family mem-

clude binding to importin-a (refer to Fig . 2D).
More recently, it has been shown that CLIC4
nuclear translocation is induced in mouse S1 keratino-
cytes by treatment with nitric oxide [3]. The nuclear
translocation is accompanied by S-nitrosylation of a
Cys residue in CLIC4, Cys234. The S-nitrosylation of
CLIC4 has been found to induce a conformational
change which destabilizes the native conformation.
Such a destabilization may facilitate the interaction
between the otherwise helical CLIC4 NLS and impor-
tin-a. It has been shown that S-nitrosylation of CLIC4
enhances the interaction with importin-a, as deter-
mined by immunoprecipitation [3].
In this article, we present the X-ray crystal structure
of mouse importin-a (70–529) bound to a peptide cor-
responding to the CLIC4 NLS. The importin-a (70–
529) construct used to obtain the importin-a:CLIC4
NLS complex lacks the first 69 residues that corre-
spond to the flexible importin-b binding domain. The
importin-b binding domain is known to have an
autoinhibitory function, whereby an internal NLS-like
sequence competes for the importin-a binding site,
reducing binding affinity for cargo proteins and help-
ing to facilitate the release of the cargo within the
nucleus [10,11]. The removal of the autoinhibitory
domain to create a truncated importin-a avoids possi-
ble competition for the binding site between this inter-
nal NLS and the CLIC4 NLS peptide.
The importin-a C-terminal domain (residues 70–529)
consists of 10 ARM structural repeats that form two

result of the binding of the CLIC4 NLS peptide.
A. V. Mynott et al. Crystal structure of importin-a:CLIC4 NLS peptide
FEBS Journal 278 (2011) 1662–1675 ª 2011 The Authors Journal compilation ª 2011 FEBS 1663
The importin-a:CLIC4 NLS structure presented in
this article adds to a growing body of knowledge on
the structural mechanisms that govern the classical
nuclear import model. It also clarifies that the CLIC4
NLS can indeed bind directly to importin-a on condi-
tion that it can unfold into an extended conformation.
Results
Structure of the importin-a:CLIC4 NLS peptide
complex
The structure of importin-a (70–529) bound to the
CLIC4 NLS peptide (198VKVVAKKYRN207) was
solved at 2.0 A
˚
resolution using synchrotron radiation
(Table 1). The model of importin-a in the CLIC4 NLS
peptide complex includes residues 72–496 and closely
resembles the full-length importin-a structure that
incorporates the N-terminal autoinhibitory domain
(PDB:1IAL, rmsd of 0.20 A
˚
across 425 C
a
atoms in
residues 72–496). The major binding site spanning
ARM repeats 2–4 has a similar conformation to the
equivalent region in apo importin-a (70–529), with an
rmsd of 0.16 A

˚
2
overall (3244 atoms). For the pep-
tide, B factors are slightly higher than those for
importin-a: 36.9 A
˚
2
for main-chain atoms, 40.2 A
˚
2
for
side-chain atoms and 38.7 A
˚
2
overall (62 total atoms).
Electron density analysis
Both the main-chain and side-chain atoms of the mod-
elled CLIC4 peptide show a good fit to the electron
density (Fig. 1B). The peptide residues 202–207 corre-
spond to the key binding positions P1–P6, with the
critical P2 position occupied by Lys203. This means
that the core basic motif, 203KKYR206, fills the
central binding pockets P2–P5 in which the majority
of peptide side-chain interactions take place with
importin-a. Therefore, the CLIC4 NLS satisfies the
accepted consensus sequence for an optimal NLS,
P2
K(K ⁄ R)·(K ⁄ R)
P5
[12]. The residue at P4, which has

interaction. The cation in this case is likely to be an
Na
+
ion from the crystallization buffer, with a low
occupancy (< 50%). As a result of the weak nature of
the Tyr205 cation–p bond, it seems unlikely that it will
have a significant effect on the CLIC4 NLS peptide
binding to importin-a.
We have also analysed the veracity of the CLIC4
NLS model built in the major binding site by inspect-
ing the F
clic4nls
o
À F
apo
o
data–data difference Fourier (see
Materials and methods). This Fourier analysis reduces
bias when interpreting the density of a peptide bound
to importin-a and thus provides additional support for
our structure. The results are shown in Fig. 1D. As
expected, the electron density is strong along the pep-
tide main chain with well-defined carbonyl and amide
backbone groups. The one exception to this is the
location of the amide group of Lys204 at P3, where
there is a break in the main-chain density at the 2.8r
map level. The corresponding position in the apo struc-
ture has particularly strong density at this point, which
may correspond to a water molecule. Peptide side-
chain density is also well defined in the F

difference map. The presence of
Tyr205 is definitive evidence that the CLIC4 NLS
peptide binds importin-a (70–529).
CLIC4 NLS interactions with importin-a
The CLIC4 NLS forms an extensive network of inter-
actions with importin-a through both main-chain and
side-chain atoms, similar to other importin-a structures
with a bound monopartite NLS [13–15]. Hydrogen
Fig. 1. The importin-a:CLIC4 NLS peptide complex. (A) The F
o
) F
c
‘omit’ electron density map over all atoms in importin-a. Positive con-
tours are shown at 2.8r in grey. Density corresponding to the bound CLIC4 NLS peptide is clearly visible in the major binding site. (B) Ste-
reoimage of the CLIC4 NLS peptide and 2F
o
) F
c
map. The CLIC4 NLS peptide bound to the importin-a major binding site is shown as a
stick representation. Colour code for atoms: carbon, cyan; nitrogen, blue; oxygen, red. Electron density is contoured at 1.5r in grey. Binding
positions P1–P6 and the N- and C-termini are labelled. (C) Schematic representation of hydrogen bonds (broken lines, < 3.5 A
˚
) between
importin-a and the CLIC4 NLS peptide,
P1
AKKYRN
P6
. Backbone carbonyl oxygens and amide nitrogens are shown as red and blue spheres,
respectively. Nitrogen and oxygen side-chain atoms are shown as blue and red squares, respectively. (D) Stereoimage of the CLIC4 NLS
bound to importin-a, showing the F

N
f
) O
d
) at P2, the most energetically significant inter-
action involved in importin-a recognition of NLSs
[16,17].
Other basic residues in the peptide, Lys204 and
Arg206, fill negatively charged pockets at P3 and P5
Fig. 2. Analysis of the importin-a:CLIC4 NLS peptide complex. (A–C) Importin-a is coloured by the normalized B factor score, B
Àapo
z
, over a
blue–magenta colour spectrum ()3r to +3r). (A) The bound CLIC4 NLS is shown on the molecular surface of importin-a in the major binding
site. (B) The importin-a C
a
backbone is shown as a cartoon tube representation in the same orientation as in (A). Important residues are
shown as a stick representation. (C) Full-length images of importin-a coloured by the B
Àapo
z
score. Residues in grey have not been included
in the calculation of B
Àapo
z
. (D) The CLIC4 crystal structure (PDB:2AHE) is shown as a cartoon representation. The N-terminal thioredoxin
domain (blue) and C-terminal a-helical domain (green) are coloured separately. The CLIC4 NLS residues are highlighted in cyan. Inset: The
NLS is shown as a stick representation (carbons, cyan; oxygens, red; nitrogens, blue). Hydrogen bonds are represented by broken lines. (E)
A multiple sequence alignment of the CLIC4 NLS motif in human CLICs. Conserved residues are red, nonconserved residues are black and
perfect conservation is highlighted with red fill. The sequence of CLIC3 is added for comparison. Binding positions P1–P6 are shown in
an alignment corresponding to our importin-a:CLIC4 NLS peptide complex. Sequence alignment was performed using

impa
Trp142 and
impa
Trp184 favourably accepts Arg206, with
impa
Glu180
(4.9 A
˚
,N
f1
) O
e1
distance) positioned at the end of the
binding pocket.
In total, the CLIC4 NLS main chain and side chains
make 174 atom-to-atom van der Waals’ contacts with
importin-a and 13 hydrogen bonds, including one salt
bridge at P2. In addition, there are 10 hydrogen bonds
formed between the peptide and water molecules, three
of which involve the terminating carboxyl group. The
van der Waals’ contact area between the CLIC4 NLS
peptide and importin-a has been calculated for each
peptide residue by integrating over contact areas using
MolProbity [18,19]. Main-chain contributions to the
contact area were found to be approximately equal
(2–5 A
˚
2
). Side-chain contributions vary to a greater
extent, reflecting differences in the binding pockets.

Trp231 (63.7 A
˚
2
). The surface area buried on the pep-
tide is 744.7 A
˚
2
, which corresponds to 59.2% of the
total peptide surface area. The real space correlation
coefficient (RSCC) for each residue has also been cal-
culated by comparing the importin-a:CLIC4 NLS
experimental electron density with density calculated
from the model. The CLIC4 NLS main chain fits the
density well, with an average RSCC of 0.96. Side
chains have greater RSCC variability, with an average
of 0.89 over all residues and 0.94 for those in P2-P5
(Table 2).
The CLIC4 NLS Tyr residue
By solving the structure of the importin-a:CLIC4 NLS
complex, we have shown that the major binding
pocket P4 is unambiguously occupied by a Tyr residue:
the first importin-a:NLS structure that has an aromatic
residue present in the core binding region. Tyr205
adopts a common rotamer with a score of 82.9%
(v
1
$ 180, v
2
$ 80), calculated by comparing the side
chain with a high-quality reference dataset using Mol-

hydrophobic interactions with surrounding importin-a
Table 1. Data collection and refinement statistics.
Data collection
Source (k) Australian Synchrotron
(0.95 A
˚
)
Detector ADSC Q210
Space group P2
1
2
1
2
1
Unit cell dimensions (A
˚
): a, b, c 78.6, 89.6, 100.1
Resolution (A
˚
)
a
2.0 (2.11–2.00)
Observations 318 168 (27 080)
Unique reflections 46 758 (5520)
Completeness (%)
a
96.7 (80.3)
Mean I ⁄ r
a
12.2 (1.8)

Favoured region 98.6
Allowed region 1.4
Disallowed 0
a
Outer shell statistics are shown in parentheses.
b
R
merge
= R
hkl-
R
i
|I
i
) <I>| ⁄ R
hkl
R
i
I.
c
R factor = R
hkl
||F
obs
| ) |F
calc
|| ⁄ R
hkl
|F
obs

bers of contacts are comparable with those of the basic
residues at P2 (Lys203: 31 contacts), P3 (Lys204: 20
contacts) and P5 (Arg206: 45 contacts).
Normalized B factor analysis
In order to analyse changes in the conformational
dynamics of importin-a binding site residues, as a
result of the presence of a bound CLIC4 NLS peptide,
the relative B factor score, B
Àapo
z
, was calculated. This
score represents a change in flexibility of each residue
in apo importin-a and the corresponding residue in the
importin-a:CLIC4 NLS complex. The B
Àapo
z
score was
determined by comparing normalized B factors from
the importin-a:CLIC4 NLS structure with those from
apo importin-a (see Materials and methods). A nega-
tive score represents a decrease in flexibility, a positive
score represents an increase in flexibility and a score of
zero represents no change. Residues near the importin-
a C-terminus (430–496) were considered to be outliers
(Z > 4) and are thus excluded from the analysis. The
B
Àapo
z
scores have a zero mean and standard deviation
of unity (Fig. S1). The B

sc
);
Trp184 ()3.9
sc
). In addition, Ser149 ()3.3
sc
) is notably
constrained by the CLIC4 NLS in a single conforma-
tion compared with dual rotamer conformations in the
apo structure.
The presence of the Lys203 side chain at P2 has
marginal effects on the hydrogen-bonding partners
Thr155 ()0.6
res
) and Asp192 ()0.8
res
), but a significant
effect on the third hydrogen-bonding partner, the car-
bonyl group of Gly150 ()3.1
res
). This can be explained
by the presence of a water molecule in the apo struc-
ture which is predicted to make hydrogen bonds with
Thr155 and Asp192, but not Gly150. We also note
that the Lys N
f
–Gly150 bond has the added effect of
reducing flexibility in the connecting loop between
ARM repeats 1 and 2 (Ser149–Ser152).
At the P4 binding site, we see that Tyr205 has a sta-

impa
Gln181
which has a corresponding decrease in its B
Àapo
z
score of
)1.9
sc
. This B factor analysis does not reflect significant
changes in residue flexibility as a result of long-range
electrostatic interactions between CLIC4 NLS side
chains at P3 (Lys204) or P5 (Arg206) and acidic resi-
dues in importin-a (Glu180, Glu266, Asp270).
Table 2. Characteristics of the importin-a:CLIC4 NLS peptide interaction.
NLS residue
Interactions
a
Van der Waals’ contact area (A
˚
2
)
b
Buried surface
area (A
˚
2
) RSCC
c
Main chain Side chain Main chain Side chain Total
Val201 0 ⁄ 1 ⁄ 00⁄ 0 ⁄ 0 0.4 0 0.4 26.7 0.89 ⁄ 0.74

X-ray crystal structure of the CLIC4 NLS peptide
(198VKVVAKKYRN207) bound to importin-a (70–
529). The monopartite NLS peptide binds in the major
binding groove of importin-a in an extended confor-
mation consistent with previously solved structures [1].
In the case of CLIC4, this extended NLS conformation
differs greatly from its helical conformation as seen in
the soluble CLIC4 structure (Fig. 2D). This important
feature means that the putative CLIC4 NLS needs to
undergo a structural transition if it is to be a biologi-
cally active NLS. It also suggests that there is tighter
control over CLIC4 translocation to the nucleus in
comparison with other nuclear-destined proteins that
do not require their NLS to undertake a structural
rearrangement.
The F
clic4nls
o
ÀF
apo
o
data–data difference Fourier was
inspected to ensure that electron density in the impor-
tin-a major binding site is not confused with residual
density from the apo importin-a structure. The results
of this difference Fourier show that all peptide side
chains in P1–P6 positions have well-defined
F
clic4nls
o

by strong, unambiguous density in the F
clic4nls
o
ÀF
apo
o
difference map. We note that a structure of the phos-
pholipid scramblase 1 NLS bound to importin-a has
been reported, in which a Trp residue is located three
residues downstream from P4 [20].
The Tyr residue is found to interact with importin-a
through both hydrophobic and hydrogen-bonding
interactions. Although it is an unusual NLS residue
containing a bulky phenol side group, it neatly fits at
its location with the hydroxyl group reaching the im-
portin-a main chain near the C-terminus of the ARM1
H3 helix. Most of the hydrophobic contacts with im-
portin-a residues lining the P4 pocket are equivalent to
those made by the aliphatic portion of an Arg residue
in the TAg NLS, whereas the hydrogen bonds shared
between the Tyr hydroxyl group and carbonyl groups
from
impa
Leu104 and
impa
Arg106 are reproduced in the
importin-a:TAg NLS P4 binding position involving the
N
f
atom of

The CLIC4 NLS contains an Arg residue (Arg206)
in binding position P5 which adopts a common and
extended rotamer conformation. The P5 position is
defined by importin-a residues Trp142 and Trp184,
which both adopt a rare rotamer conformation, facili-
tating the formation of this hydrophobic pocket. The
site is normally occupied by a NLS Lys residue in
A. V. Mynott et al. Crystal structure of importin-a:CLIC4 NLS peptide
FEBS Journal 278 (2011) 1662–1675 ª 2011 The Authors Journal compilation ª 2011 FEBS 1669
importin-a:NLS structures, where the aliphatic portion
of the side chain fits into the hydrophobic alcove and
allows the charged head group access to the solvent on
the other side. The extended side chain of Arg206
allows for the same favourable interactions as a Lys
residue, including a hydrogen bond to
impa
Gln181 and
the formation of a network of solvent interactions.
The positively charged guanidinium group of Arg206
is also compensated by the nearby acidic residue
impa
Glu180, with an interaction distance (N
f1
) O
e1
)of
4.9 A
˚
.
Conservation of the CLIC NLS

The importin-a:CLIC4 NLS complex presented here
supports the hypothesis that CLIC4 can enter the
nucleus via an importin-a-mediated nuclear import
pathway. However, the recognition of full-length
CLIC4 by importin-a is strictly dependent on its abil-
ity to undergo a change in conformation that exposes
a linear NLS ready for binding.
Experiments showing that CLIC4 translocates to the
nucleus are not clear on the exact mechanism by which
translocation occurs. Although mutagenesis of the
putative CLIC4 NLS implies an interaction with im-
portin-a, immunoprecipitation experiments have shown
that CLIC4 also associates with nuclear transport fac-
tor-2 and Ran [2]. The nuclear transport factor-2 path-
way is normally used for the nuclear import of
RanGDP [28], but nuclear transport factor-2 can also
interact with cargo complexes [29]. The nuclear trans-
port of CLIC4 may therefore reveal new general para-
digms for nucleocytoplasmic transport processes.
Triggers for structural change
Typically, NLSs are located in solvent-exposed regions
of unstructured domains, such as flexible ends or loop
regions. The CLIC4 NLS motif, as seen in the soluble
CLIC4 crystal structure, is unusual for an NLS as it is
neither unfolded nor situated at domain termini
(Fig. 2D). As we have shown that the CLIC4 NLS
binds to importin-a in an extended conformation, this
confirms a previous hypothesis [4] that structural
changes in CLIC4 are required to expose the NLS
region. The NLS in CLIC4 forms a structurally stable

rotamer assisting nitric oxide transfer. In this model, a
strong steric clash is expected between the S-nitrosylat-
ed Cys234 and His196 in helix 6, which can be allevi-
ated by a shift of at least 1 A
˚
in the interdomain
interface. Accordingly, experiments have shown that
the S-nitrosylation of CLIC4 induces a structural
change [3]. We note that an S-nitrosylation trigger
leading to conformational change has been observed
previously in blackfin tuna myoglobin, where the
S-nitrosylation of a Cys residue causes a structural
domain shift [33]. Whether the S-nitrosylation of
Cys234 results in a similar structural rearrangement of
CLIC4, and provides a trigger for NLS conforma-
tional change, requires further investigation.
The possibility of trigger events to expose NLSs
offers an interesting criterion for cargo selection by
nuclear import receptors. Typically, NLSs are identifi-
able as a result of their high content of basic residues,
which allows them to bind tightly to an acidic binding
site in importin-a. However, it is clear that the prerequi-
site for NLS binding, based on sequence alone, is very
limited because a core region of just four residues is nec-
essary for importin-a peptide recognition. Furthermore,
there is a certain degree of tolerance to nonbasic resi-
dues within the importin-a binding region, particularly
at P4, where we find the Tyr residue in CLIC4 NLS. In
order to select specific cargo proteins destined for the
nucleus, it is likely that importin-a screens additional

lated with 10 mL of the overnight culture. The cells were
grown at 37 °C until an absorbance at 600 nm of 1.0 cm
)1
was reached, at which point the culture was induced with
1mm isopropyl thio-b-d-galactoside. The temperature was
then lowered to 30 °C and expression continued for approxi-
mately 5 h. Cells were harvested by centrifugation at
11 000 g for 8 min and resuspended in 35 mL NaCl ⁄ P
i
with
2mm dithiothreitol. At this stage, protease inhibitors were
also added (Complete Protease Inhibitor Cocktail; Roche
Applied Science, Penzberg, Germany). The cells were lysed
using a French pressure cell and the resulting cell debris was
centrifuged at 39 000 g for 45 min at 4 °C.
The supernatant was added to a 3-mL solution of either
nickel nitrilotriacetic acid agarose resin (Qiagen, Valencia,
CA, USA) or Profinity immobilized metal affinity chro-
matography (IMAC) resin (Bio-Rad, Hercules, CA, USA),
and was mixed under gentle rotation for 1 h. The resin
was then loaded into an Econopak gravity flow column
(Bio-Rad) and unbound protein was eluted and discarded.
The resin was washed with 200 mL of 20 mm Hepes, pH 7.0,
500 mm NaCl, 1 mm MgCl
2
, 0.1 mm dithiothreitol and up
to 20 mm imidazole. Bound protein was then step eluted
with 10 mL of a buffer containing 20 mm Hepes,
pH 7.0, 500 mm NaCl, 0.1 mm dithiothreitol and 150 mm
imidazole.

(CH
3
)
3
charged groups of the Q-SepharoseÔ HP resin
in Buffer A (50 mm NaCl, 20 mm Tris, pH 8.0, 2 mm dith-
iothreitol), before being eluted over a salt gradient from
50 mm to 1 m at a flow rate of 4 mLÆmin
)1
(total volume
A. V. Mynott et al. Crystal structure of importin-a:CLIC4 NLS peptide
FEBS Journal 278 (2011) 1662–1675 ª 2011 The Authors Journal compilation ª 2011 FEBS 1671
of 400 mL). Eluted fractions with the highest concentration
of protein were pooled and concentrated using an Amicon
YM-10 Centriprep centrifugal concentrator (Amicon,
Billerica, MA, USA) in a Sorvall SH-3000 swinging bucket
rotor (Sorvall, Waltham, MA, USA) at 1800 g. The con-
centrated protein from ion exchange chromatography was
loaded on a Superdex 200 10 ⁄ 300 GL (GE Healthcare)
analytical size exclusion column and eluted with 20 mm
Tris, pH 8.0, 100 mm NaCl and 2 mm dithiothreitol.
Fractions corresponding to importin-a (70–529) were pooled
and concentrated to approximately 18 mgÆmL
)1
, flash
frozen in liquid nitrogen and stored at )80 °C until use.
NLS peptide synthesis
The CLIC4 NLS peptide was synthesized (Sigma-Genosys,
Sydney, Australia) and used in the crystallization of impor-
tin-a:CLIC4 peptide complexes. Although the proposed

tals were gradually transferred into a cryoprotectant solu-
tion consisting of the reservoir solution supplemented with
20% glycerol. The importin-a:CLIC4 NLS crystal was flash
cooled in liquid nitrogen and stored in a cryogenic dry ship-
ping dewar. It was then mounted at 100 K in a nitrogen
cryostream on beamline PX-1 (3BM-1B) at the Australian
Synchrotron [34], and data collection was performed using
an ADSC Quantum 210 (Q210) detector.
A number of initial images were screened at orthogonal
u angles to gauge crystal quality before autoindexing and
determining the optimum rotation range using strategy
from within mosflm [35]. Crystals were found to have the
symmetry of the orthorhombic space group P2
1
2
1
2
1
with
unit cell dimensions of approximately a =79A
˚
, b =90A
˚
and c = 100 A
˚
(see Table 1). The final diffraction dataset
was obtained over 180 images with a 1° oscillation angle
(u) and 5-s exposure time per image. The crystal to detector
distance was set at 220 mm and the beam width was set to
200 lm · 200 lm.

Electron density analysis
Fourier analysis was used to compare the importin-
a:CLIC4 NLS peptide complex (observed structure factors
F
pep
o
) with an apo structure of importin-a (70–529)
(observed structure factors F
apo
o
). This structure is described
in ref. [42]. The structure factors from each dataset were
scaled using the ccp4 program scaleit [36]. This calculates
a scaling R factor of 16.3%, supporting isomorphism
between the two crystals. We refer to the Fourier difference
of these structure factors as a data–data difference Fourier,
F
pep
o
ÀF
apo
o
, which is calculated as:
F ¼ F
pep
o





,
with similar lattice dimensions (CLIC4 NLS crystal:
a = 78.6 A
˚
, b = 89.6 A
˚
, c = 100.1 A
˚
; apo crystal:
a = 79.0 A
˚
, b = 89.8 A
˚
, c = 100.3 A
˚
), and the scaling R
factor is relatively low (16.3%). We have calculated a nor-
malized B factor score using a method described previously
[43]. The B factors of atoms in structure j are first normal-
ized according to the following equation:
B
z
¼
B
i
À l
j
r
j
where B

References
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Supporting Information
The following supplementary material is available:
Fig. S1. Analysis of the importin-a:CLIC4 NLS com-
plex.
This supplementary material can be found in the
online version of this article.
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