The crystal structure of NlpI
A prokaryotic tetratricopeptide repeat protein with a globular fold
Christopher G. M. Wilson
1
, Tommi Kajander
1
and Lynne Regan
1,2
1 Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT, USA
2 Department of Chemistry, Yale University, New Haven, CT, USA
Repeat proteins in general, and tetratricopeptide
repeats (TPRs) in particular, have recently attracted
interest from the perspectives of structure, function,
folding and design [1–6]. The TPR was first identified
during sequence analysis of proteins CDC23 and
nuc2+ from yeast [7,8], and has subsequently been
found in a wide variety of polypeptides from all gen-
era. It is a degenerate 34-residue motif, which adopts a
helix-turn-helix structure. The first helix is usually
termed the ‘A’ helix, while the second is referred to as
the ‘B’ helix [6]. The most common number of tandem
TPRs within a single protein is three, but as many as
16 have been predicted on the basis of sequence analy-
sis [2]. Natural and designed TPRs whose structures
have been determined share a common tertiary organ-
ization, which is dominated by interactions between A
helices and the preceding AB pair (Fig. 1A). Local
AB, BA¢ and nonlocal AA¢ helix packing generate an
extended superhelical array with right-handed twist.
The motif is often terminated by an additional A, or
‘capping helix’, whose exposed edge is hydrophilic in

The nonglobular, extended structures that result are particularly well suited
to present a large surface area and to function as interaction domains.
Many repeat proteins have been demonstrated experimentally to fold and
function as independent domains. In tetratricopeptide (TPR) repeats, the
repeat unit is a helix-turn-helix motif. The majority of TPR motifs occur as
three to over 12 tandem repeats in different proteins. The majority of TPR
structures in the Protein Data Bank are of isolated domains. Here we pre-
sent the high-resolution structure of NlpI, the first structure of a complete
TPR-containing protein. We show that in this instance the TPR motifs do
not fold and function as an independent domain, but are fully integrated
into the three-dimensional structure of a globular protein. The NlpI struc-
ture is also the first TPR structure from a prokaryote. It is of particular
interest because it is a membrane-associated protein, and mutations in it
alter septation and virulence.
Abbreviations
HOP, Hsp organizing protein; Hsp, heat shock protein; SUPR, superhelical peptide repeat; TPR, tetratricopeptide repeat.
166 FEBS Journal 272 (2005) 166–179 ª 2004 FEBS
the full-length HOP, they act to facilitate the assembly
of multichaperone regulatory complexes. The struc-
tural independence of these TPR domains, and the
presence of independent ligand-binding sites in each,
has been assumed to be characteristic properties of
TPR domains. Methods that identify motifs from
amino acid sequence (e.g. pFAM [11]) readily predict
TPRs, with the implication that they are discrete
domains. TPR domains, or even subsets of TPRs
within a domain [12], are often studied independently.
In the course of a wider effort toward understanding
TPR structure and function, a number of related
observations intrigued us. First, the only structures of

Following translocation across the inner plasma mem-
brane, the prosequence and lipobox cysteine are
recognized, enzymatically modified and proteolytically
processed by components of the lipoprotein biosyn-
thetic pathway. This yields an N-acyl-S-sn-1,2-diacyl-
glyceryl-cysteine (residue 19) as the N-terminus of the
mature, 276 residue, membrane-anchored protein [22].
The identity of the residue at the +2 position (Ser20)
in the mature protein suggests that NlpI is not retained
at the inner membrane, but is likely to be anchored at
the outer membrane [23–25]. The precise topological
location (periplasmic or extracellular face) is not
known. NlpI has been proposed to play a role in
bacterial septation, or regulation of cell wall degrada-
tion during cell division [22]. Disruption of the chro-
mosomal copy of the nlpI gene, or plasmid-mediated
overexpression of the protein, both lead to altered cell
morphology and to osmotic sensitivity.
NlpI is of potential clinical interest, because loss of
the nlpI gene affects the synthesis of pili and flagellae,
leading to changes in extracellular adhesion properties
which are correlated with an invasive, pathogenic
phenotype [26]. A BLAST search for similar sequences
Fig. 1. Canonical TPR structure and NlpI sequences. (A) Example of
TPR extended helical structure, from the consensus design
1NA0.pdb [6]. Three repeats are the most common number seen.
The AB and AA¢Wpacking angles are responsible for curvature and
superhelicity of the motif. (B) Amino acid sequence of NlpI from
the translation of the nlpI gene [22], including the signal
prosequence (underscored) and lipobox cysteine modification site

and determined the protein structure by X-ray crys-
tallography. The structure reveals a fold in which the
TPR is not an independent domain, but is an integ-
ral part of a globular protein.
Results and Discussion
Cloning and expression of NlpI
The gene for NlpI was obtained by direct PCR ampli-
fication from E. coli DH10B [28]. The sequence corres-
ponding to the mature polypeptide (residues 20–294,
lacking the signal prosequence and Cys20) overexpres-
ses exceptionally well in BL21 (DE3), with yields of
 100 mgÆL
)1
(Fig. 2A). Purified mature NlpI is sol-
uble to at least 200 mgÆmL
)1
in 10 mm Tris ⁄ HCl
pH 8.0, 10 mm NaCl.
To investigate the anticipated 3-TPR domain of
NlpI, we subcloned residues 62–197. This region also
expresses well, but in contrast to the mature polypep-
tide, the majority of protein is found in inclusion bod-
ies (Fig. 2A). Material purified from the lysate soluble
fraction precipitated after elution off Ni-nitrilotriacetic
acid agarose. Alternative expression, purification, solu-
bilization and refolding regimes were investigated, but
we were unable to obtain soluble 3-TPR. This result
was surprising, as we had anticipated the 3-repeat to
be an independent domain. The insolubility of this
region was the first indication that the 3-TPR might

twofold axis of noncrystallographic symmetry running
through the dimer interface. We conclude that the
contents of the asymmetric unit represent the biolo-
gically active protein. The two chains together form
an arrow-shaped structure, wider than it is deep
(Fig. 2B,C). N-termini of both molecules share a
common point of origin, a feature compatible with
membrane localization through N-terminal lipid
anchors on both chains. Table 2 shows the secondary
structure components, interhelix packing geometries,
and the angle of rotation between the AB helix pairs
present. With the exception of an extended, but not
unstructured region of polypeptide (30–37), NlpI is
composed of a-helix (64%) and turn motifs (23%).
NlpI monomers can be described generally as a
superhelical array of helix-turn-helix motifs, in which
the C-terminus is folded (rolled-up) inside the
N-terminus (Fig. 2D). A depression on one side of
each monomer contains a bound Tris molecule. This
cavity, formed by the curvature and packing of heli-
ces, is highly suggestive of a ligand binding pocket
and we speculate that it may represent the functional
site of the protein.
Helix packing interactions
TPRs
Many features of the distribution of side chain con-
tacts within NlpI are typical of a TPR protein. The
side chain contact map (Fig. 4A) is dominated by a
Crystal structure of NlpI C. G. M. Wilson et al.
168 FEBS Journal 272 (2005) 166–179 ª 2004 FEBS

Fig. 2. Solubility of NlpI constructs and the structure of mature NlpI. (A) 10–20% gradient SDS ⁄ PAGE of NlpI expression products, showing
the insolubility of the 3-TPR construct vs. mature NlpI. BenchMark molecular mass markers (lanes 1 & 4); 3-TPR insoluble (lane 2, arrow-
head) and soluble (lane 3); mature NlpI insoluble (lane 5) and soluble (lane 6, arrowhead); mature NlpI following TEV protease cleavage, and
purification over Superdex 75 (lane 7, arrowhead, anticipated molecular mass of 31.8 kDa). (B) Side and (D) top views of the NlpI dimer.
Chains are coloured from N- (dark blue) to C-termini (orange). Axis of noncrystallographic rotational symmetry runs through the center ‘x’.
(C) Monomer of NlpI, showing the rolled-up array of helices with the C-terminus folding within the curvature of the N-terminus. Helix num-
bers are in brackets. Note that ‘A’ helices locate to the globular center, and the perpendicular arrangement of helices 10 and 11, against heli-
ces 8 and 9.
C. G. M. Wilson et al. Crystal structure of NlpI
FEBS Journal 272 (2005) 166–179 ª 2004 FEBS 169
HOP), because the majority of these residues partici-
pate in the protein core.
NonTPR helix motifs
Packing interactions are more complex for the two
remaining pairs of helices (8 and 9, 10 and 11). These
are of particular significance since they are responsible
for the compact structure of NlpI. Helix pair 10–11
(Fig. 4B) is, at 27 residues (17 of which are helix) too
short to be termed a TPR. The interhelix AB W packing
angle is the highest (+ 172°), bringing them close to
parallel, and is also of the opposite sign to that which
characterizes a TPR. Interactions with the following
pair of helices (12 and 13) is distinguished by the only
negative AA¢ angle within the protein. Critically, this
combination of nonTPR packing angles imparts left-
handed superhelical character to the region. The pitch
of the overall right-handed superhelix is therefore
reduced, which brings the C-terminus up toward the
N-terminus.
Helices 8 and 9 correspond to the region of sequence

˚
) Peak (0.9785) Inflection (0.9795) Remote (0.9500)
Resolution (A
˚
) 30–2.05 30–2.0 30–2.0
Total number of reflections 408241 506497 282547
Number of unique reflections
a
85218 91502 91856
Completeness (%)
a
96.1 (84.0) 96.0 (83.6) 94.9 (81.2)
I ⁄ Sigma
b
33.8 (4.7) 40.4 (4.8) 29.3 (2.7)
R
merge
b
(%) 5.4 (30.7) 4.9 (35.5) 5.3 (46.6)
Redundancy
a
4.8 5.5 3.1
FOM after
SOLVE 0.58
FOM after
RESOLVE 0.72
FOM after
DM 0.64
R ⁄ R
free

by an unusual association with the next pair of helices,
10 and 11. These pack against 8 and 9 at an angle of
96° (visible in Fig. 2D), which is the highest inter-
repeat rotation angle within the structure. A unique,
nonTPR interaction takes place where the indole ring
of Trp200 (helix 10) inserts between helices 8 and 9,
against Phe190 and the amide backbone of Phe165.
This locks the pairs together (Fig. 4D). The abrupt
increase in helical array curvature is the second factor
responsible for bringing distal regions of sequence back
toward proximal ones.
Long-range interactions & globularity
The presence of long-range contacts within NlpI is
revealed by clusters in the contact map far from the
diagonal (Fig. 4A). These interactions take place only
between A helices, which dominate the inside of the
NlpI helical roll (Fig. 2C). The clusters can be consid-
ered as four overlapping groups (Fig. 4E). Cluster 1
involves helices 12 and 14, packing against the
N-terminal region of NlpI. These constitute the most
distant interactions between elements of primary
structure, and include a hydrogen bond between the
backbone carbonyl of Asn263 and the backbone amide
of Leu34. The positive AA¢Wangle between helix 12
and helix 10 is in part responsible for this. Cluster 2
consists of loop against loop interactions between
TPR1 and TPR2, with helix 14 (including a Ca back-
bone contact between Gly76 and His266). From func-
tional perspectives this is perhaps significant, as this
first TPR is more open than any other, forming an

AB pair W°
c
AB W°
c
AA¢
Rotation
d
(AB)(A¢B¢)°
Helix pair sequence
(signature TPR residues underscored)
0 27–29 – – – – – consensus motif
1 38–51 6.4 – – – – W LG Y A F A P
2 58–74 7.6 1 )160.5 +31.3 57.6 DDERAQ
LLYERGVLYDSLGLRALARNDFSQALAIRPDM
3 78–91 5.9 (TPR1) (pair 1–2)
4 96–108 5.9 2 )165.5 +16.7 59.0 PEV
FNYLGIYLTQAGNFDAAYEAFDSVLELDPTY
5 112–125 5.1 (TPR2) (pair 2–3)
6 131–142 8.9 3 )153.0 +26.5 16.2
YAHLNRGIALYYGGRDKLAQDDLLAFYQDDPND
7 146–159 12.2 (TPR3) (pair 3–4)
8 164–177 14.6 4 +163.3 +26.9 96.2 PFRSLWLYLAEQKLDEKQAKEVLKQHFEKSDKEQW
9 179–192 3.9 (pair 4–5)
10 199–206 12.9 5 +172.6 )26.0 40.4 GWNIVEFYLGNISEQTLMERLKADATD
11 212–222 7.4 (pair 5–6)
12 226–246 19.4 6 )158.3 +38.2 261
NTSLAEHLSETNFYLGKYYLSLGDLDSATALFKLAVANNVHNF
13 250–261 8.6 (TPR4) (pair 1–6)
14 269–283 7.9 – – – –
a

axis of symmetry. The dimer interface consists of the
extended N-terminal region, helix 1 and TPR helices
2, 3, 11, 12, 13 and 14 (Table 3, Figs 5 and 6B). The
values obtained for interface surface area, interaction
type (two-thirds hydrophobic, but also hydrogen
bonds and salt-bridges), gap volume index and planar-
ity (which relate to the complementarity of the inter-
face surfaces) fall within the ranges associated with
known homodimeric states [29]. Three aspects are
especially noteworthy. First, rotational symmetry
places the N-termini of both monomers spatially close
to each other. A lipid-modified dimer will therefore be
anchored to a plasma membrane in a specific orienta-
tion (N-termini ‘face down’ toward the membrane).
This is significant, because the potential ligand binding
AB
C
D
EF
Fig. 4. Contact map of mature NlpI and packing interactions. (A) Backbone (upper left from diagonal) and side chain (lower right from diago-
nal) contacts within 5 A
˚
. Long-range contact clusters are boxed. (B) Packing interactions between nonTPR helices 10 (red) and 11 (blue), and
(C) helices 8 (red) and 9 (blue). Space-filling atoms shown are large and small hydrophobic residues (F, Y, W, I, L, V, A and G). Bulky groups
of helix 8 point toward the protein core. (D) View of NlpI helices 8 and 9 (with helix 7 removed), showing diminished association between
the pair, and the insertion of Trp200 from helix 10. Right-handed superhelical curvature imparted by the first three TPRs appears to cease,
allowing the subsequent structure to roll-up. (C) Location of long-range packing clusters from (A), which define the core of NlpI. (F) Aromatic
and bulky side chains surrounding Trp169 (orange).
Table 3. NlpI dimer interface statistics. Values were obtained with
SURFNET [29,55]. SA ¼ surface area.

noted that the first TPR (helices 2 and 3) participates
in long-range interactions within a monomer through
loop residues (Asp73, Ser74, Leu75, Arg78), while the
majority of the inner front-face assumes an open ‘lip’
conformation (Fig. 4D). In contrast, seven residues of
the outer back-face participate in the dimer interface,
packing against C-terminal portions of polypeptide
from the partner molecule. Consideration of mono-
meric NlpI alone gives the impression that these heli-
ces make few molecular contacts when in fact they
make many, albeit with a separate polypeptide chain.
The insolubility of NlpI 3-TPR (fragment 62–197) is
therefore understandable, in terms of the failure of an
isolated motif to form critical intra- and intermole-
cular contacts. These observations demonstrate the
capacity, and on occasion the necessity, of TPRs to
participate at all levels of structure organization, and
suggest that the fold is more versatile than was previ-
ously thought.
We now know the structure of NlpI, and observe
that the TPRs in this protein do not form an inde-
pendent domain. One could therefore ask if there
are any features of the TPR sequences that hint at
differences between these TPRs, and those that fold in-
dependently. Unfortunately, with the limited sequence–
structure data available at this stage, there are no
correlations strong enough to allow us to predict, or
subclassify, which TPR sequences will form an exten-
ded array and which will adopt globular structures.
Implications for function: a putative binding cleft

ions. The structure of Tris, also a compo-
nent of the crystallization mother liquor, was found to
fit the density envelope, making hydrogen bonds with
carboxylate groups of Glu235 and Glu270, and with
the back bone amide of Val269. Phe165 and Phe268
face each other, flanking the two carboxylates and Tris
(Fig. 6D).
NlpI is thought to play a role in the regulation of the
cell wall and extracellular surface, but its exact function
is not known, and no ligand interactions have yet been
described. There has been some suggestion that the
C-terminus may associate with the periplasmic protease
Tsp, and it has been proposed that removal of residues
beyond Gly282 serves to activate the protein [27]. How-
ever, the C-terminus of NlpI does not contain a motif
that resembles the canonical ‘WVAAA’ associated with
Fig. 5. NlpI dimer interface. Chain B has been translated and rota-
ted to expose the surface in contact (yellow). The interface is com-
posed of remote regions of sequence from the N- and C-termini.
Val32 is indicated to illustrate the rotational symmetry of the inter-
face. Contacts were obtained with
SURFNET [55] and CONTACT from
the CCP4 [42,43] suite of programs.
C. G. M. Wilson et al. Crystal structure of NlpI
FEBS Journal 272 (2005) 166–179 ª 2004 FEBS 173
Tsp recognition [30]. Our structure suggests the func-
tionality of NlpI in fact lies within the cleft associated
with globular body of the fold.
Structural homologs
A DALI search for homologous structures finds two

hydrophobic). (C) The surface of an NlpI monomer, showing the putative ligand binding cleft and bound Tris molecule. (D) Tris molecule,
conserved acidic and aromatic side chains within the cleft. Orange dashes indicate hydrogen bonds between Tris and the amide
backbone of Val269, and conserved side chain carboxylates of Glu235 and Glu270.
Crystal structure of NlpI C. G. M. Wilson et al.
174 FEBS Journal 272 (2005) 166–179 ª 2004 FEBS
rmsd of 4.3 A
˚
over 212 residues), consists of eight
superhelical peptide repeat (SUPR) motifs that assume
a superhelical fold [31]. SUPRs resemble TPRs, but
their helices are slightly longer (16–18 residues) and
the sequence consensus is more degenerate. The
N-terminal region of MalT is responsible for func-
tional dimerization, while the C-terminus is thought to
contain a maltotriose binding site, formed by the con-
cave surface of four SUPR repeats. Close structural
similarity can be seen between the first three AB
repeats of NlpI and MalT (Fig. 7B). However, by the
sixth repeat the reduction in NlpI superhelical pitch
has folded the protein back onto itself, while MalT
continues in a more regular superhelix (and in conse-
quence lacks hydrophobic core interactions).
In terms of their biological roles, p67
phox
and MalT
both mediate intermolecular interactions, and are
responsible for the assembly of multiprotein com-
plexes. It is therefore interesting to speculate, on the
basis of structural identity and the presence of a con-
served surface cleft, whether NlpI participates in ana-

aaagtgaagtcc-3¢ and 5¢-attattggatccctattgctggtccgattctgccag-3¢.
3-TPR NlpI primers (residues 62–197) were 5¢-aataatccatgg
gggcacagcttttatatgagcgcggag-3¢ and 5¢-aataatggatcctcactgttc
cttatccgatttttcgaagtgc-3¢. PCR products were doubly diges-
ted with NcoI and BamHI (New England Biolabs, Beverly,
MA, USA), and purified by agarose gel electrophoresis
onto dialysis membrane, prior to ligation into doubly diges-
ted, dephosphorylated expression vector pET11a-HT. This
vector was assembled in-house from vectors pProEX-HTa
(Invitrogen) and pET11a [32] (Novagen, San Diego, CA,
USA), and places cloned sequences under T7 promoter con-
trol. Expression in an E. coli DE3 bacterial host produces
an N-terminal hexahistidine-tagged protein, cleavable with
TEV protease. Ligation products were transformed into
electrocompetent E. coli DH10B (Invitrogen), and trans-
formants sequenced by the W. M. Keck Facility.
Expression and purification
Plasmids, verified by DNA sequencing, were transformed
into E. coli BL21 (DE3) Gold (Stratagene), and grown in
Luria–Bertani medium supplemented with 100 lgÆmL
)1
car-
benicillin at 37 °C until cell culture absorbance at 600 nm
was 0.5. The temperature was reduced to 25 ° C before
induction with 100 lm isopropyl thio-b-d-galactoside. Cells
were harvested by centrifugation (6000 g, 20 min) after 4 h
further growth, and stored at )80 °C. Selenomethione
(SeMet)-labelled NlpI was expressed in E. coli methionine
auxotroph B834 (DE3), grown in M9 medium [33] supple-
mented with 50 mgÆL

ity purification was performed with slow rocking at 4 °C
overnight, with one-fifth volume of Ni-charged nitrilotri-
acetic acid agarose slurry (Qiagen). Washing steps were
performed at room temperature in a disposable standing
column (Bio-Rad, Hercules, CA, USA), with 5 bed vol-
umes buffer A and 5 bed volumes buffer B (50 mm
Tris ⁄ HCl pH 8.0, 300 mm NaCl, 5 mm imidazole). Bound
protein was eluted in buffer C (50 mm Tris ⁄ HCl pH 8.0,
150 mm NaCl, 300 mm imidazole). Hexahistidine tags were
removed by treatment with 10 units of AcTEV protease
(Invitrogen) overnight at room temperature, followed by
dialysis against buffer D (50 mm Tris ⁄ HCl pH 8.0, 150 mm
NaCl). TEV protease and uncleaved fusion were removed
by gravity flow through a 1 mL bed of fresh Ni-nitrilotri-
acetic acid agarose. Protein was loaded onto a Superdex
S200 16 ⁄ 60 prep grade column (Amersham Biosciences),
equilibrated in buffer D. Fractions containing NlpI were
pooled, dialysed against buffer E (10 mm Tris ⁄ HCl pH 8.0,
10 mm NaCl) and concentrated with a Centriprep YM10
spin concentrator (Millipore, Billerica, MA, USA). Mature
NlpI mass was verified by MALDI mass spectrometry.
Protein concentration was estimated by SDS ⁄ PAGE
against BenchMark Protein Ladder (Invitrogen), and by
absorbance at 280 nm assuming a calculated e
mature NlpI
¼
43 240 m
)1
Æcm
)1

2
, 27% (v ⁄ v) PEG 400, 0.8% (v ⁄ v)
n-butanol, at 22 ° C. SeMet-labelled NlpI crystallized iso-
morphously, under identical conditions. Rectangular crys-
tals grew over 3 days at 22 °C to 0.3 · 0.5 · 1.0 mm.
Data collection & phasing
Crystals were flash-frozen from mother liquor in a nitrogen
gas cryo-stream. In-house data collection used a Mar345
image plate detector (MAR Research), coupled to a Rigaku
CuKa rotating anode source. NlpI crystallized in the space-
group P2
1
2
1
2
1
, with unit-cell dimensions a ¼ 64.35 A
˚
,b¼
81.65 A
˚
,c¼ 136.66 A
˚
, with two monomers in the asym-
metric unit and a Matthews coefficient of 2.82 A
˚
3
ÆDa
)1
(solvent content ¼ 56.36%) [35,36]. A three-wavelength

well as expected. Solvent flattening, NCS averaging with an
operator (obtained from the resolve dimer model, and
refined with imp [40]), and phase extension, were repeated
with dm [41] in 100 steps, using the CCP4 graphical user
interface [42,43] and the phases from solve. This resulted
in significant improvement in map quality.
Model refinement
Several schemes were tested for refinement. The best results
were obtained using cns [44] for rigid body refinement at
2.0 A
˚
, and conjugate gradient minimization at 2.5 A
˚
with
maximum likelihood target, resulting in R-factors of
R ⁄ R
free
¼ 30.0 ⁄ 30.9%. The model was then manually
improved and completed in o [45]. Thereafter the structure
was refined with refmac 5.0 [46] through the ccp4 graphi-
cal user interface [42,43], with overall anisotropic B-factor
Crystal structure of NlpI C. G. M. Wilson et al.
176 FEBS Journal 272 (2005) 166–179 ª 2004 FEBS
refinement of data and bulk solvent to 2.0 A
˚
resolution.
The first round of refinement converged to R-factors of
R ⁄ R
free
¼ 21.1 ⁄ 25.2%. The model was once more inspected

sourceforge.net).
Model coordinates
Coordinates and structure factors have been submitted to
the RCSB Protein Databank, under accession code
1XNF.pdb.
Acknowledgements
This work was supported in part by NIH grants
GM62413 and GM57265 (L.R.). C.G.M.W. is a James
Hudson Brown–Alexander Brown Coxe Postdoctoral
Fellow (Yale University School of Medicine). T.K. is
supported by a postdoctoral fellowship from Helsingin
Sanomat Centennial Foundation (Finland). Data for
this study were measured at beamline X12C of the
National Synchrotron Light Source. We are grateful
for the assistance of Dr Anand Saxena in the use of
this beamline. The National Synchrotron Light Source,
Brookhaven National Laboratory, is supported by the
US Department of Energy, Division of Materials
Sciences and Division of Chemical Sciences, under
Contract No. DE-AC02–98CH10886. X12C is suppor-
ted principally by the Offices of Biological and Envi-
ronmental Research and of Basic Energy Sciences of
the US Department of Energy, and from the National
Center for Research Resources of the National Insti-
tutes of Health.
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