Eur. J. Biochem.

264, 562568 (1999) q FEBS 1999

The crystal structure of a wheat nonspecific lipid transfer protein (ns-LTP1)

complexed with two molecules of phospholipid at 2.1 A resolution
Delphine Charvolin1,2, Jean-Paul Douliez3, Didier Marion3, Claudine Cohen-Addad1 and Eva Pebay-Peyroula1,2
1

Institut de Biologie Structurale Jean-Pierre Ebel, CEA-CNRS; 2Universite Joseph Fourier, Grenoble; 3Unite de Biochimie et Technologie des
Proteines, Nantes, France

Nonspecific lipid transfer proteins (ns-LTP1) form a multigenic protein family in plants. In vitro they are able to
bind all sort of lipids but their function, in vivo, remains speculative. A ns-LTP1 isolated from wheat seed was
crystallized in the presence of lyso-myristoyl-phosphatidylcholine (LMPC). The structure was solved by
resolution to an R-factor of 16.3% and a free R-factor of 21.3%. It
molecular replacement and refined to 2.1 A
reveals for the first time that the protein binds two LMPC molecules that are inserted head to tail in a hydrophobic
cavity. A detailed study of the structure leads to the conclusion that there are two lipid-binding sites, one of which
shows a higher affinity for the LMPC than the other. Comparison with other structures of lipid-bound ns-LTP1
suggests that the presence of two binding sites is a general feature of plant ns-LTP1.
Keywords: lipid transfer protein; wheat; phospholipid; crystallography; hydrophobic cavity.

Nonspecific lipid transfer proteins (ns-LTPs) are ubiquitous

lipid binding proteins in plants. Two main groups of ns-LTPs,
ns-LTP1 and ns-LTP2, have been identified with molecular
masses of about 9 and 7 kDa, respectively. ns-LTP1 are the best
characterized group and have a basic pI and a conserved
cysteine pattern. They form a multigenic family whose genes
are spatially and temporally regulated [1]. In vivo, ns-LTP1 are
synthesized with a N-terminal signal peptide, which indicates
that they follow the secretory pathway, in agreement with their
location in extracellular layers, i.e. cell walls or cutin, and in
vacuolar structures [1,2]. Such locations are consistent with
neither a role in membrane biogenesis nor with a role in the
b-oxidation pathway of fatty acids. Sterk et al. [3] suggested
that they are involved in the formation of cutin layers by
transporting hydrophobic cutin monomers. Finally, the antimicrobial activities of ns-LTP1 observed in vitro [4] suggest a
possible role in the defence of plants against bacterial
pathogens. Although these two latter hypotheses are attractive
and consistent with most of the biological data, there is no clear
evidence for such roles in vivo (for an extensive review, see [1]
and the references cited therein).
Structural biology is a complementary approach to the search
for the biological function of proteins. Determination of threedimensional structures of ns-LTP1 has shown that these

Correspondence to E. Pebay-Peyroula, Institut de Biologie Structurale

Jean-Pierre Ebel, CEA-CNRS, 41 Avenue Jules Horowitz, 38027 Grenoble
cedex 1, France. Tel.: 33 (0) 4 76 88 95 83, Fax: 33 (0) 4 76 88 54 94,
E-mail: pebay@ibs.fr
Abbreviations: Ace-AMP1, anti-microbial protein from onion seed;
B-factor, temperature factor; CoA, coenzyme A; DMPG, di-myristoylphosphatidyl-glycerol; LMPC, lyso-myristoyl-phosphatidylcholine; LPC,
lyso-phosphatidylcholine; ns-LTP, nonspecific lipid transfer protein; PCoA,
palmitoyl coenzyme A; rms, root mean square.
Note: the novel atomic coordinates and structure factors have been
deposited with the Brookhaven Protein Data Bank (Accession numbers:
Id 1bwo and 1bwosd).
(Received 6 June 1999; revised 23 June 1999; accepted 23 June 1999)

proteins share a common fold, stabilized by four disulphide

bonds, and are characterized by a four helix bundle covered
partly by a long C-terminal arm with several turns. The loose
folding of the hydrophobic side chains in the protein core can
produce a cavity, even in the absence of lipids. Such cavities
have been seen in wheat, barley, rice and maize ns-LTP1 whose
structures were determined by either X-ray crystallography or
multidimensional NMR or both [59]. This cavity is able to
bind different types of monoacylated and diacylated lipid
molecules, including fatty acids [6,10,11], fatty acyl CoA
[12,13], lyso-phosphatidylcholine (LPC) [7,14] and phosphatidylglycerol [15]. Therefore these proteins can be unambiguously assigned to this group of lipid binding proteins. The
presence of this cavity should constitute an important feature
for their biological function. It is noteworthy that in Ace-AMP1,
an anti-microbial protein from onion seed with a ns-LTP1 fold,
no cavity is present and the protein cannot transfer or bind any
lipids. However, this protein interacts with lipidic membranes
and changes their permeability [16]. The core of Ace-AMP1
has many aromatic side chains including two tryptophan residues which are not found in other ns-LTP1. Previously, the
structure of a hydrophobic protein from soybean containing the
same disulphide bond pattern revealed a similar four helix fold
but did not show a hydrophobic cavity as in the ns-LTPs [17].
These divergences in lipid/protein interactions for proteins
sharing the same folding pattern could indicate that ns-LTP1
have different biological functions.
A study of the relationship between the structure of the
hydrophobic cavity and the lipid binding properties of the
protein should bring useful information on the functionality of
ns-LTP1. The volume of the cavity is variable for the lipid-free
protein and can increase when large lipids are bound. This
increase may or may not be accompanied by an increase in the
protein size [9,13,15]. Furthermore, a comparison of the structures of barley ns-LTP1 complexed with acyl CoA [13] and
maize ns-LTP1 complexed with palmitic acid [6] has revealed
opposite orientations of the lipid molecules. In this work, the
determination of the structure of wheat ns-LTP1 complexed
with two lyso-myristoyl-phosphatidylcholine (LMPC) molecules

 resolution (Eur. J. Biochem. 264) 563

Structure of wheat ns-LTP1 at 2.1 A

q FEBS 1999

provides new insight into understanding lipid binding and

ns-LTP1 function.

M AT E R I A L S A N D M E T H O D S
Purification of wheat ns-LTP1
Wheat bran (1 kg) was extracted with 5 L of deionized water.
After filtration through a Buchner funnel and centrifugation at
5000 g for 20 min, the extract was loaded on a column
(5  30 cm) packed with a cation-exchange resin (Streamline,
Pharmacia). The fractions were eluted by applying a gradient
from 0 to 0.7 m NaCl in 20 mm Mes pH 5.6 buffer. ns-LTP1
was detected by electrophoresis and immunoblotting as described
in [2]. ns-LTP1-enriched fractions were pooled, dialysed
overnight against deionized water, and freeze-dried. The dry
material was solubilized and loaded on a gel filtration column
(3  100 cm) packed with Sephadex G50 and eluted in 20 mm
Mes pH 5.6 buffer. Finally, ns-LTP1 was purified from the
enriched fractions by semipreparative C18 reversed-phase
bonded silica column (25  1 cm)
HPLC on a 5-mm, 300-A
using a gradient of water/acetonitrile/0.05% trifluoroacetic acid
(1% acetonitrile per min) at 508C and freeze dried. The purity
of the protein was checked by analytical HPLC and by mass
spectrometry as described in [18]. The binding of LMPC to the
purified protein was checked by fluorescence measurements [7].
Protein crystallization
Crystallization of wheat lipid transfer protein was only possible
in the presence of a phospholipid. Previous crystals obtained in
the presence of ammonium sulphate [19] were not reproducible
constantly and therefore not suitable for structure refinement.
New crystal forms were obtained using the hanging drop

method from a protein solution at 6 mgmL21 with a reservoir

containing 30% (w/v) polyethylene glycol 4000 and 0.2 m
ammonium acetate in 0.1 m citrate buffer, pH 5.6, in the
presence of LMPC (4 lipids per protein monomer) at 208C. The
lyophilized protein was solubilized in the lipid containing
solution prior to crystallization. Several lipid to protein ratios
were tested, ranging from 1 to 10 lipids per protein monomer;
the ratio of 4:1 led to the best crystals. Similarly, cocrystallization with three different lipid species was attempted: LMPC,
lyso-palmytoyl-phosphatidylcholine and di-heptanoyl-phosphatidylcholine, the choice of LMPC being dictated by crystal
quality. Two crystal forms were obtained in the same drops:
, b = 41.90 A
,
monoclinic P21 with cell parameters a = 48.30 A
, b = 113.38, two monomers per asymmetric unit;
c = 51.10 A
,
and orthorhombic P212121 with cell parameters a = 42.29 A

b = 53.55 A, c = 71.49 A, two monomers per asymmetric unit.

Only the latter crystal form was used for solving and refining
the structure.
Structure determination and refinement at 2.1 A resolution
A first model was obtained by molecular replacement from the

orthorhombic crystal form and X-ray diffraction data to 2.6 A

resolution using data
resolution. It was then refined to 2.1 A
collected with a better crystal. Data were collected at room
temperature on a FAST detector mounted on a Elliot GX21
X-ray generator using a copper rotating anode (Enraf-Nonius).
Data were reduced using the madnes package [20], the procor
program [21] and the CCP4 package [22]. Data collection
statistics are summarized in Table 1. The structure was solved
by molecular replacement using a model of maize ns-LTP1
solved by X-ray diffraction at the laboratory (unpublished data)
and the amore package [23]. The self-rotation function
indicated the presence of two monomers in the asymmetric

unit. They were located using the data between 10 and 3.5 A

Table 1. Data processing and refinement statistics.

Native 2.6 A

Native 2.1 A

Resolution
)
limits (A

Number of
measurements

Rsym (%)

, I/s .

Redundancy

Completeness
(%)

30.22.6
24.32.1

12279
23492

6.0
4.8

8.8
12.6

2.6
2.6

87.0
90.5

Data collection temperature

)
Resolution range (A
F/s amplitude cutoff
Number of unique reflections
Completeness (%)
R-factor (%)a
Free R-factor (%)a
)
Rms deviation bond lengths (A
Rms deviation bond angles (8)
Rms deviation dihedral angles (8)
Rms deviation improper angles (8)
2)
Average temperature factor (A
for all atoms
for protein atoms
for lipid atoms
Number of lipids/protein
Total number of water molecules
a

20 8C
102.1
2
8525
85.0
16.3
21.3
0.005
1.05
23.50
0.59
22.5
19.0
52.9
2
145

Rsym and R-factors are defined as follows: Rsym Sh Si jIhi , Ih . j/Sh Si Ihi , where I(h) is the reflection h, Sh is the sum over all reflections and Si is
the sum over the i measurements of reflection h, R-factor = S|Fobs Fcalc|/SFobs, where Fobs and Fcalc are the observed and calculated structure factor
amplitudes, respectively. The reflection set for the free R-factor contains 5% of the total set of unique reflections.

564 D. Charvolin et al. (Eur. J. Biochem. 264)

q FEBS 1999

(program o [25]), with energy minimization and simulated

annealing (up to 3000 K) using the parameters as in Engh and
Huber [26]. Noncrystallographic symmetry restraints were
maintained between the main chain atoms of the two molecules
model was then refined using
of the asymmetric unit. The 2.6 A
data set with a bulk solvent correction (free R-factor
the 2.1 A
data set and extended to
set of reflections same as in the 2.6 A
the high resolution). All restraints were relaxed between the
two monomers. Water molecules were gradually added.
Individual B-factors (temperature factors) were refined. The
phases were improved using the ARP program [27] and lipid
molecules appeared clearly in the Fo-Fc density. Two lipids per
monomer were gradually built and refined from the coordinates
and geometric constraints of the crystallographic structure of

lyso-phosphatidyl-ethanolamine [28]. The final model at 2.1 A

resolution contains 1626 nonhydrogen atoms, including 1356
protein atoms, 124 lipid atoms and 145 water atoms. Refinement statistics are summarized in Table 1 (R-factor = 16.3%,
free R-factor = 21.3%). The Ramachandran plot calculated
with the program procheck [29] for both monomers shows no
residues in the forbidden areas and 94.5% of the residues in the
most favoured areas. The mean B-factor calculated for all atoms
2), is comparable to the value
in the asymmetric unit (22.5 A
2). Figure 1 was drawn
obtained from the Wilson plot (20.5 A
with the program molscript [30] and Figs 2, 4, and 6 were
drawn with the program grasp [31].
Fig. 1. Ribbon representation of wheat ns-LTP1 complexed with LMPC.
H1 is pictured yellow, H2, red, H3, green, H4 and loop LCter, blue. All other
loops are grey. Disulphide bonds are drawn using stick representation.

R E S U LT S
Overall protein structure

resolution, with a correlation of 41.0% and an R-factor of

with the program
45.2%. The structure was refined up to 2.6 A
xplor 3.8.1 [24] and the model was progressively improved by
alternating manual corrections from 2Fo-Fc and Fo-Fc maps

The asymmetric unit is composed of two monomers of 90

residues. Two molecules of LMPC are bound per monomer. The
wheat ns-LTP1 consists of four helices, H1 (residues 317), H2
(residues 2537), H3 (residues 4155), and H4 (residues 6373)

Fig. 2. Section view of the wheat ns-LTP1

structure (brown worm) and its molecular
surface, showing the hydrophobicity of the
internal tunnel. Residues bordering the
tunnel are drawn as white sticks.

q FEBS 1999

resolution (Eur. J. Biochem. 264) 565

Structure of wheat ns-LTP1 at 2.1 A

Fig. 3. View of the 2Fo-Fc electron density map contoured at 1 s

around the four lipid molecules of the asymmetric unit. (a) Lipids in
monomer A; (b) lipids in monomer B.

and a long C-terminal loop LCter (residues 7490) (Fig. 1).

The helices were defined using the program DSSP [32]. They
are linked by three loops, L1-2 (residues 1824), L2-3
(residues 3840) and L3-4 (residues 5662). Helix H1 is
significantly bent around Pro13 and helix H4, at Ile69, before
the doublet Pro70-Pro71. LCter is a long winding loop made of
two successive turns (residues 7480 and residues 8190)
maintained close to the core of the structure by the disulphide
bridge Cys48-Cys87. The structure contains two pairs of
disulphide bridges located by pairs at opposite sides of the
protein (Fig. 1). Electron density maps reveal clear unique
conformations for the disulphide bridges Cys13-Cys27, Cys28Cys73 and Cys48-Cys87, while two alternate conformations are
observed for the Cys3-Cys50 disulphide bond. In all loops, the
electron density is well defined and atomic B-factors for the
2. The protein is best
main chain atoms are below 30 A
described as a helix bundle with H1/H2 and H3/H4 (including
LCter) forming two antiparallel helix sets, surrounding a
strikingly large tunnel which runs through the protein (Fig. 2)
covered predominantly with hydrophobic residues: Val10 and
Cys13 from helix H1, Cys27, Val31 and Leu34 from helix H2,
Ala38 from loop L23, Ala47, Cys48, Leu51, Lys52 and Ala55
from helix H3, Leu61 from loop L34, Ala66, Arg67, Ile69 and
Pro70 from helix H4 and Val75, Leu77, Tyr79, Ile81, Ser82,
Leu83 and Ile85 from loop LCter. The two extremities of the
tunnel are exposed to the solvent and include both hydrophobic
or hydrophilic residues: Asp7, Arg11, Leu14, Ile54 and Ile58
on one side, and His35, Arg44, Pro78 and Val90 on the other
side. The two monomers of the asymmetric unit, which were
refined independently, have similar conformations with root
for the main chain
mean square (rms) deviations of 0.32 A
for all atoms.
atoms and 0.9 A

Fig. 4. Representation of the two molecules of LMPC inserted in the

hydrophobic cavity of wheat ns-LTP1. Superposition of the two
monomers of the asymmetric unit and the corresponding lipid molecules:
Monomer A and lipids A1 and A2 are coloured blue, monomer B and lipids
B1 and B2 are red.

The polar head groups are exposed to solvent areas, between

LCter and the beginning of H3 for lipid 1 and between L34
and the end of H1, for lipid 2. We notice that the choline group
is only visible in lipid B1 (Fig. 3). This is consistent with the
observed B-factors. In the aliphatic chains, the atomic B-factors
are similar to the mean B-factor of the protein (Fig. 5). In the
polar head groups, they are much higher, especially in lipids A2
and B2, increasing from the carbonyl to the choline, with
2. Choline groups are likely to be
maximum values of 80100 A
multiconformational. The comparison between the two monomers A and B of the asymmetric unit shows that lipids in site 1

Binding of LMPC
In both monomers (A and B), the lipid molecules correspond to
well defined electron densities which show unambiguously the
presence of two alkyl chains complexed to each monomer
(Fig. 3). Lipid 1 (A1 and B1 for monomers A and B,
respectively) and lipid 2 (A2 and B2) are positioned head to
tail, crossing the protein through the hydrophobic tunnel
(Fig. 4). The aliphatic chains are buried inside the tunnel.

Fig. 5. Atomic B-factor of the four lipid molecules of the asymmetric

unit. The lipid atoms are labelled according to the Protein Data Bank
coordinates file. The arrows indicate the value of the mean B-factor
calculated from all atoms of monomer A and monomer B.

566 D. Charvolin et al. (Eur. J. Biochem. 264)

q FEBS 1999

(A1 and B1) superimpose all along the aliphatic chain up to the
phosphate group (Fig. 4). Lipids in site 2 (A2 and B2) super translation.
impose only along the aliphatic chains with a 1-A
There are more lipid/protein interactions in site 1 than in site 2.
This is highlighted by several hydrophobic interactions in site 1
with His35, Cys48, Leu51, Ala55, Ala66, Asn76, Leu77,
Pro78, Ile81, Ser82, Leu83, Val90, and by a hydrogen bond
between the carbonyl of the ester bond in LMPC and the
hydroxy group of Tyr79. In site 2, only a few hydrophobic
interactions are observed with Val10, Leu14, Val31, Ile54, Ile58
and His59.

DISCUSSION
Lipid binding in wheat ns-LTP1
Our crystallographic study reveals for the first time that wheat
ns-LTP1 binds two molecules of LMPC. In the present
structure, the cavity described in the lipid-free protein [5]
becomes a tunnel formed by the displacement of helix H1 and
loop LCter. Both lipids are located head to tail inside the
hydrophobic core with their aliphatic chains in the highly
hydrophobic part of the tunnel and their polar head groups
directed towards the solvent areas, at each end of the tunnel. In
site 1, LMPC strongly interacts with the protein through
hydrophobic interactions and through a hydrogen bond with
Tyr79. In site 2, LMPC is involved only in few hydrophobic
interactions. The absence of hydrogen bond linking lipid 2 to
the protein, as well as the lower number of hydrophobic
interactions suggest that lipid 2 has a lower affinity for the
protein than lipid 1. These findings are in agreement with
fluorescence experiments which show that wheat ns-LTP1 can
bind more than one lipid molecule per protein [33]. Fluorescence experiments under resonance energy transfer conditions suggest that the maize LTP is also able to bind two lipids
in the cavity [34].

Comparison with wheat ns-LTP1 bound to di-myristoylphosphatidyl-glycerol (DMPG). The solution structure of
wheat ns-LTP1 complexed with DMPG has been determined
by NMR [15]. Comparison with the present structure shows that
the orientation of DMPG corresponds to site 1 of LMPC with
the polar head group lying in a similar position (Fig. 6a). In the
NMR structure, the Tyr79 side chain is directed towards the
solvent, preventing the formation of a hydrogen bond between
this residue and the lipid. This is in contradiction with our
structure, as well as with the maize ns-LTP1 structure complexed with palmitate [6], in which a hydrogen bond links the
hydroxyl group of Tyr81 to the carboxyl group of the fatty acid.
As in the NMR structures, aliphatic chains of DMPG were
modelled without any experimental constraints [15]; this may
explain the discrepancies in the localization of DMPG and
LMPC aliphatic chains.
Comparison with maize ns-LTP1 bound to palmitate. In the
crystal structure of maize-ns-LTP1 complexed with palmitate
[6], the palmitate has its polar head group in site 1, close to the
ester group of LMPC, and its aliphatic chain in site 2 (Fig. 6b).
Similarly to our structure, the polar head group forms a
hydrogen bond with Tyr81. This indicates that this tyrosine,
which is highly conserved in ns-LTP1 sequences could play a
role in lipid binding to site 1.
Comparison with barley ns-LTP1 bound with palmitoyl
coenzyme A (PCoA). In the structure of PCoA-bound-barley
ns-LTP1 determined by NMR [13], PCoA is oriented according
to site 2 and its palmitoyl aliphatic chain is strongly curved
inside the hydrophobic pocket, extending out of site 2 (Fig. 6c).
The polar head of PCoA is directed towards site 2, is highly
disordered and shows multiple conformations. Both PCoA in
barley ns-LTP1, and LMPC (site 2) in wheat ns-LTP1, only
interact through hydrophobic contacts with the protein.

Lipid binding in other ns-LTP1

CONCLUSION

Several lipid-free or lipid-bound structures of ns-LTP1 have

previously been determined by NMR spectroscopy or X-ray
diffraction. The rms differences between these structures and
the present one are listed in Table 2 and show high similarities
between the structures, although a systematic difference is
observed in the loop LCter from residue 76 to the end. This
allows the protein structure to adjust to the presence of different
lipids by modifying the position of the loop LCter. The size of
the cavity increases in order to accommodate the binding of one
or two lipid molecules.

Our present study shows for the first time, that a ns-LTP1 is
able to bind two monoacylated lipids inserted head to tail in the
hydrophobic cavity. This structural analysis highlights the
existence of two lipid binding sites of different affinity for
lipids, with more lipidprotein interactions in site 1 than in
site 2. Orientations of the lipid molecules which are found to be
opposite in barley and maize lipid-bound structures are shown
here to be both possible. This explains why only one LPC
molecule was observed in previous NMR studies of maize
ns-LTP1 [7]. A large excess of lipid would have been necessary
to saturate site 2. However, in barley ns-LTP1 complexed with
PCoA, steric constraints and additional interactions between
PCoA and the protein are more favourable to the binding
of PCoA in site 2.
ns-LTP1 function has not been clearly determined yet. In this
regard, it is interesting to note that some structural and binding
properties are shared between plant ns-LTP1 and serum
albumin, a protein involved in the transport of fatty acids in
the blood of mammals. Serum albumin is a protein with a
higher molecular mass than ns-LTP1 but each helical domain
encloses a tunnel which is the binding site for fatty acid.
Furthermore as in ns-LTP1, one of these domains can bind two
fatty acid molecules and the lipid carboxylate is stabilized by
both hydrogen and electrostatic interactions with arginine,
lysine and tyrosine side chains [35]. Therefore, these similarities and the data obtained in this work sustain the role of

Table 2. Comparison with other ns-LTP1 structures. The numbers

) calculated with program lsqkab [22]
indicate the rms displacement (A
on main chain atoms, and in parentheses on all atoms. References are
shown in square brackets.
X-ray diffraction

NMR spectroscopy

Wheat

Free
Bound with DMPG

2.39 (2.90) [5]

2.47 (3.12) [15]

Maize

Free
Bound with palmitate

1.40 (1.86) [6]

1.33 (1.73) [6]

2.18 (2.57) [7]

Barley

Free
Bound with PCoA

2.50 (2.90) [13]

2.55 (3.16) [13]

q FEBS 1999

resolution (Eur. J. Biochem. 264) 567

Structure of wheat ns-LTP1 at 2.1 A

Fig. 6. Superposition of ligand molecules

in the wheat ns-LTP1 structure and other
known ns-LTP1 structures. LMPC
molecules are coloured green, superimposed:
(a) with DMPG (blue) from the NMR-solved
wheat ns-LTP1 structures [15]; (b) with
palmitate (violet) from the X-ray-solved
maize ns-LTP1 structure [6]; (c) with PCoA
(orange) from the NMR-solved barley
ns-LTP1 structures [13]. In each case, Tyr79
and its equivalent in the other structures are
represented. Phosphorus atoms in lipids are
yellow, oxygen atoms red, and nitrogen atoms
blue.

plant ns-LTP as a transporter of fatty acids or fatty acid

derivatives (e.g. hydroxy-fatty acids, acyl-CoA) necessary
during the formation of cutin layers or during lipid mobilization
in germinating seedlings, the current hypothesis for the
biological function of plant ns-LTP [1].

ACKNOWLEDGEMENTS
We are grateful to Mogens Lehmann for having initiated this work and for
useful discussions during this study, to Richard Kahn and Emile Duee for
helpful advice. We thank Christine Saint Pierre and Eric Forest for mass
spectrometry analysis. We also thank Denize Sy, Patrick Sodano, Francoise
Vovelle and Marius Ptak for providing unpublished coordinates of the
solution structures of lipid-free and lipid-bound wheat ns-LTP1 and for
stimulating comments.

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