From the Institute of Crystallography, Russian
Academy of Sciences, Leninsky Prospekt 59, 117333 Russia,
Laboratoire de Microscopie Electronique
Structurale and
Laboratoire de Biophysique Moléculaire,
Institut de Biologie Structurale (CEA/CNRS), 41 rue Jules Horowitz,
F-38000 Grenoble Cedex 01, France, ** Institut Laue-Langevin B.P. 156, F-38042 Grenoble Cedex 9, France, and § European Molecular
Biology Laboratory (EMBL), EMBL c/o DESY, Notkestrasse 85, D-22603
Hamburg, Germany
Received for publication, April 23, 2001, and in revised form, May 1, 2001
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ABSTRACT |
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The quaternary structures of monomeric and
dimeric Drosophila non-claret disjunctional (ncd)
constructs were investigated using synchrotron x-ray and neutron
solution scattering, and their low resolution shapes were restored
ab initio from the scattering data. The experimental curves
were further compared with those computed from crystallographic models
of one monomeric and three available dimeric ncd structures in the
microtubule-independent ADP-bound state. These comparisons indicate
that accounting for the missing parts in the crystal structures for all
these constructs is indispensable to obtain reasonable fits to the
scattering patterns. A ncd construct (MC6) lacking the coiled-coil
region is monomeric in solution, but the calculated scattering from the
crystallographic monomer yields a poor fit to the data. A tentative
configuration of the missing C-terminal residues in the form of an
antiparallel The superfamily of kinesin motor proteins has about 265 members
thus far and can be subdivided into at least 10 different subfamilies. The two kinesin motors that have been
investigated in the most detail are conventional kinesin, a member of
the kinesin heavy chain subfamily, and Drosophila
ncd1 (Fig.
1), which belongs to the C-terminal
subfamily, which is the largest and most divergent subfamily
(1). Both proteins are dimers and have a three-domain structure. Their
motor domains, which are some 340 residues long, contain the ATP- and
microtubule-binding sites. Dimerization is mediated by a stalk domain
predicted to form an -sheet was found that significantly improves the fit.
The atomic model of a short dimeric ncd construct (MC5) without 2-fold
symmetry is found to fit the data better than the symmetric models.
Addition of the C-terminal residues to both head domains gives an
excellent fit to the x-ray and neutron experimental data, although the
orientation of the
-sheet differs from that of the monomer. The
solution structure of the long ncd construct (MC1) including complete
N-terminal coiled-coil and motor domains is modeled by adding a
straight coiled-coil section to the model of MC5.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-helical coiled-coil. In conventional kinesin,
the coiled-coil domain is predicted to be interrupted by hinge regions
(2), which increase the flexibility of the entire molecule. In
contrast, ncd has a continuous, uninterrupted coiled-coil. The third
domain is a small globular tail responsible for cargo binding.
Conventional kinesin has its motor domain at the N terminus of its
polypeptide chain and moves processively to the plus end of
microtubules, making several hundred steps before detaching (3-5). In
contrast, ncd has its motor domain at the C terminus of the polypeptide chain (6) and moves to the minus end of microtubules (7). There is
growing evidence that ncd is not processive (8, 9). Although
conventional kinesins and C-terminal motors move to opposite ends of
microtubules, the three-dimensional structures of their motor domains
are remarkably similar (10-13). Therefore, the opposite directionality
cannot be explained on the basis of the crystal structures of their
motor domains. Studies of the three-dimensional structure of dimeric
kinesin and ncd motor domains on microtubules in the presence of
5'-adenylylimidodiphosphate provided a valuable hint (14, 15). In both
studies, it was clearly shown that the motor-microtubule complexes have
one attached and one unattached head per tubulin dimer. The unattached
heads of kinesin and ncd have distinctly different conformations and
tilt toward the microtubule plus end for kinesin and toward the
microtubule minus end for ncd. This suggested at once that
directionality could be determined by differences in the dimer
conformations. The crystal structures of dimeric kinesin (16) and
dimeric ncd (17, 18), all in the microtubule-independent ADP form, do
indeed have different overall conformations. Only one crystal structure
is currently available for dimeric kinesin, and this displays an
asymmetric conformation, with the two heads being related to each other
by a rotation of about 120°. For ncd, three different dimeric crystal structures from two independent studies are available. The first dimeric ncd structure has a perfect 2-fold symmetry, with two identical
heads (17). The two ncd dimers in the asymmetric unit of the crystal
form of the second study are different (18). In one dimer, the two
heads are related by a 179° rotation about an axis defined by the
coiled-coil, whereas in the other one, the second head is rotated about
10° away from the 2-fold symmetry position.
View larger version (31K):
[in a new window]
Fig. 1.
a, bar diagram of the full-length
microtubule motor ncd from Drosophila melanogaster. The
different domains and the start positions of three ncd fragments used
in this study are indicated. b, a ribbon model of the ncd
dimer in the ADP state.
Comparison between experimental solution scattering curves and those
evaluated from crystallographic structures is an established method to
verify the structural similarity between macromolecules in crystals and
in solution (19-21). The present x-ray and neutron scattering study
aims to check the three available dimeric ncd crystal structures
against the solution scattering data. The structures of several
monomeric and dimeric ncd constructs are analyzed, and an attempt is
made to model the conformation of the C-terminal region, which has not
been resolved in any of the known ncd crystal structures thus far.
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MATERIALS AND METHODS |
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Purification of Recombinant Proteins--
The plasmids pET/MC1,
pET/MC5, and pET/MC6 were a kind gift of Sharon Endow (Duke Medical
Center, Durham, NC). All protein purification steps were
performed at 4 °C. The purification of monomeric MC6 (corresponding
to residues Met333-Lys700 of ncd and 14 additional amino acids), dimeric MC5 (corresponding to residues
Ala295-Lys700 and 14 additional amino acids),
and dimeric MC1 (corresponding to residues
Leu209-Lys700 of ncd and 13 additional amino
acids) was performed as described previously (6), with some
modifications. Cells were grown overnight at 37 °C in 2 liters of
Luria-Bertani medium supplemented with 150 mg/ml ampicillin and induced
with 0.05 mM/ml
isopropyl--D-thiogalactopyranoside. The temperature was
lowered to 20 °C, and the cells were further induced for 12 h.
The cells were centrifuged and passed twice through a French press and
centrifuged at 15,000 × g for 1 h. Further
purification steps were performed as described previously (6).
As an additional step, the proteins were concentrated using a CENTRICON
YM-30 (Amicon) and loaded onto a self-packed gel filtration column
(Sephacryl S-300; Amersham Pharmacia Biotech) previously equilibrated
in buffer A (20 mM PIPES, pH 7.4, 200 mM NaCl,
1 mM MgCl2, 1 mM dithiothreitol,
and 1 mM EGTA). The eluted peak fractions were
concentrated, and fractions of 150 µl/tube in the concentration range
of 1-20 mg/ml were frozen in liquid nitrogen and stored at 80 °C
until use.
Albumin (67 kDa), ovalbumin (43 kDa), chymotrypsinogen (25 kDa), and ribonuclease A (13.7 kDa) were used as standards.
For data collection, the freshly thawed proteins were either measured directly in 100-µl quartz cuvettes, centrifuged at 15,000 × g for 30 min at 4 °C, or subjected to an additional gel filtration to obtain the protein solutions under the best possible monodisperse conditions. Equilibrium binding studies performed at 24.7 °C showed that in the presence of different nucleotides, MC1 has a tendency to aggregate (22). Additional experiments indicated that the protein is more stable at lower temperatures. Therefore, the x-ray scattering measurements were performed at 12 °C, and the neutron scattering experiments were performed at 6 °C.
Scattering Experiments and Data Processing--
The synchrotron
radiation x-ray scattering data were collected on the X33 camera
(23-25) of the European Molecular Biology Laboratory on the storage
ring DORIS III of the Deutsches Elektronen Synchrotron (DESY) with
multiwire proportional chambers with delay line readout (26). The
scattering patterns were recorded at a sample-detector distance of
2.0 m to cover the range of momentum transfer 0.23 nm1 < s < 3.5 nm
1 (s = 4
sin
/
, 2
is the scattering angle, and
= 0.15 nm is the wavelength). The
data were normalized to the intensity of the incident beam and
corrected for the detector response, the scattering of the buffer was
subtracted, and the difference curves were scaled for protein
concentration. All procedures involved statistical error propagation
using the SAPOKO program.2
The data obtained on samples with protein concentrations between 1 and
20 mg/ml were extrapolated to zero concentration following standard
procedures (27). For MC5 and MC1, the data were also collected at a
sample-detector distance of 3.5 m covering the range 0.13 nm
1 < s < 2.0 nm
1 and merged with the higher angle data to
yield the final composite scattering curves. The molar masses of the
solutes were calculated by comparison with the forward scattering from
reference solutions of bovine serum albumin (molar mass = 66 kDa).
The maximum dimensions of the particles in solution
(Dmax) were estimated using the orthogonal expansion program ORTOGNOM (28). The forward scattering I(0) and the
radii of gyration (Rg) were evaluated using the
Guinier approximation (27) and the indirect transform package GNOM (29, 30), the latter of which also provided the distance distribution function p(r) of the particles.
Neutron scattering experiments on MC5 were performed on the D22
small-angle scattering instrument at the Institut Laue-Langevin in
Grenoble, France (31). Samples with protein concentrations between 1 and 10 mg/ml were contained in quartz cells (Helma) of 1.0- and 2.0-mm
optical path length for H2O and D2O solutions, respectively. The data were collected using neutrons with a wavelength of = 0.6 nm and a spectral width
(
/
) of 8% with a sample detector
distance of 4.0 m, to cover the range of momentum transfer 0.1 nm
1 < s < 2.3 nm
1. Data were corrected for buffer
scattering and normalized to the scattering of 1.0 mm of
H2O, in the standard manner. The molar mass of the solute
was computed from the normalized data extrapolated to infinite dilution
and to zero scattering angle (32, 33).
Data Analysis--
The atomic model of the monomeric ncd was
taken from the kinesin home page, and the models of dimeric ncd were
taken from the Protein Data Bank (34), entries 2NCD and 1CZ7,
respectively. The scattering curves from the atomic models were
calculated using the CRYDAM
program,3 an enhanced version
of the CRYSOL (35) and CRYSON programs (36) that takes the scattering
from the solvation shell into account. The macromolecule is surrounded
by a 0.3-nm-thick hydration layer with an adjustable density
(b) that may differ from that of the bulk solvent
(
s). The scattering from the particle in
solution is
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(Eq. 1) |
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(Eq. 2) |
The low resolution shapes of the ncd constructs were determined
ab initio from the x-ray scattering curves. To remove the scattering contribution due to the internal structure of the particles, appropriate constants were subtracted from the experimental data to
ensure that the intensity decays as s4,
following Porod's law (37) for homogeneous particles. This procedure
yields an approximation of the shape scattering curve (i.e.
scattering due to the excluded volume of the particle filled by
constant density), and these data were used in the ab initio analysis (38). A sphere of diameter Dmax is
filled by densely packed small spheres (dummy atoms) of radius
r0
Dmax. The
structure of this dummy atoms model is defined by configuration vector
X, assigning an index to each atom corresponding to solvent (0) or solute particle (1). The scattering intensity from the dummy atoms
model is computed as
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(Eq. 3) |
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(Eq. 4) |
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RESULTS |
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Construct MC6--
This construct includes residues
Met333-Lys700 of ncd (6) and corresponds to the
complete motor domain and part of 7 forming the core-linker
interface. This well-characterized ncd motor fragment is known to be
monomeric in solution at low concentrations. At concentrations of up to
10 µM, there is no indication that MC6 forms aggregates
(22). A very similar construct (Arg335-Lys700)
has been crystallized, and its structure has been solved in the
ADP form to 2.5 Å resolution (11). Compared with MC6, the crystallographic model includes 321 amino acids, lacking the first 14 N-terminal and the last 33 C-terminal residues. The coordinates of a
head domain from the dimeric ncd structure (18) that includes the
N-terminal
7 allowed us to complement the model at the N terminus of
the protein starting from residue Met333. Additionally,
loop L11, which is present in the monomeric structure (11) but absent
in the two dimeric crystal structures (17, 18), was added. The
completeness of the crystallographic model was thus increased from 87%
to 91%, and the modified atomic model of monomeric MC6 is presented in
Fig. 2, top row.
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The processed scattering curve from MC6 in Fig.
3 yields a molecular mass of 41 ± 3 kDa, in good agreement with the value estimated from the primary
sequence of the monomeric protein (44 kDa). In contrast, the scattering
curve computed by the CRYDAM program from the model in Fig. 2
(top row) gives a poor fit to the experimental data (Fig. 3,
;
= 2.1). The maximum dimension and the radius of gyration
calculated from the crystallographic model (7.5 and 2.29 nm,
respectively) are significantly smaller than their experimental
counterparts (9.0 ± 0.5 and 2.58 ± 0.04 nm, respectively).
An attempt to improve the fit by assuming partial dimerization of MC6
in solution yielded an only marginally lower discrepancy (
= 1.9 at 4% of dimers). It can thus be concluded that MC6 is monomeric
in solution, even at relatively high (up to 20 mg/ml) concentrations,
but it has a significantly more extended conformation than the modified
crystallographic model in Fig. 2 (top row).
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To obtain independent information about the overall structure of MC6 in
solution, its shape was restored ab initio from the experimental data as described under "Materials and Methods." Several independent restorations starting from different random approximations yielded reproducible results, and a typical solution is
presented in Fig. 2 (middle row) in the orientation that
best matches the crystallographic model. The calculated scattering curve from the ab initio model (Fig. 2, ) deviates
significantly from the raw experimental data at higher angles, as it
should, but neatly fits the shape scattering curve with
= 0.73. The ab initio restoration thus confirms that the
solution structure of MC6 is more anisometric than the crystallographic model.
This discrepancy is not surprising given that about 30 C-terminal
residues are still missing in the model in Fig. 2 (top row). These residues are disordered in all ncd crystal structures solved thus
far (11, 17, 18). The missing region represents about 9% of the molar
mass of the protein and should make a significant contribution to the
solution scattering pattern. To find a plausible configuration of this
region in solution, the C-terminal residues were added to the atomic
model of MC6 in several possible conformations (a two- or
three-stranded antiparallel -sheet or a helix-turn-helix motif) and
in different orientations with respect to the atomic model. A dozen
models were generated, and for each model, the agreement with the
experimental data was computed using the CRYDAM program. The solution
scattering patterns were rather sensitive to the configuration and
orientation of this region, and the discrepancy computed for these
models ranged from
= 0.96 to
= 2.7. The models in
which the last C-terminal residues form an antiparallel two-stranded
-sheet yielded systematically better fits than those forming a
three-stranded antiparallel
-sheet or a helix-turn-helix motif. The
best model in Fig. 2 (shown in the bottom row) provides an
excellent fit (Fig. 3,
) to the experimental data with
= 0.96 and represents a probable configuration of the missing C-terminal region in solution.
MC5 Construct, X-ray Scattering--
The ncd construct MC5 (6)
contains residues Ala295 -Lys700 of ncd and 14 additional residues at the N terminus of the protein. The protein
contains part of the N-terminal coiled-coil region, linker, and the
complete motor domain. MC5 has previously been shown to be a dimer in
solution (6). The molecular mass computed from the composite x-ray
scattering curve of MC5 in Fig. 4 was 75 ± 8 kDa, confirming that the protein forms dimers in solution (the value estimated from the primary sequence of the dimer is 80 kDa).
The Dmax and Rg values
are 13 ± 1 and 4.05 ± 0.04 nm, respectively. Three
available crystallographic models were tested against the scattering
data. The first published crystal structure (17) has been solved using
a similar ncd construct encoding residues
Glu281-Lys700. The final model contains
residues Leu303-Met672, thus missing the
N-terminal residues 281-302, the loop L11 region (residues
Lys588-Thr596), and the C-terminal residues
Thr673-Lys700. The crystal structure contains
one head domain per asymmetric unit. This dimeric ncd structure (dimer
1) has a perfect 2-fold symmetry about the coiled-coil axis. The second
study (18) is based on a different crystal form containing two ncd
dimers per asymmetric unit. The overall conformation of the two heads
in one of the dimers (dimer 2) is similar to that of dimer 1. In the
second dimer (dimer 3), the head domains are not related by crystallographic symmetry. The second head domain is rotated about 10° away from the 2-fold symmetry position. The scattering curves computed from dimers 1 and 2 yielded the fits to the experimental data
with = 1.53 (the models and the fits are not shown). Dimer 3 (Fig. 5, top row) provided the
fit to the experimental data (Fig. 4,
) with
= 1.28 at
V = 107 nm3 and
b = 46 electrons/nm3. Because this model, like that of MC6
in the previous section, lacked the L11 loop and C-terminal region, an
attempt was made to improve the fit by adding these regions to each of
the monomers. Using the same orientation of the C-terminal loop as in
Fig. 3, however, worsened the fit (
= 1.41; curve not shown).
To further understand this discrepancy, a low resolution model of the
particle shape was restored ab initio, assuming P2 symmetry
(this assumption is justified at low resolution). The ab
initio model is presented in Fig. 5 (middle row), and
the fit is presented in Fig. 4 (
). This model suggests that the
orientation of the C-terminal loop makes the particle more anisometric
in the plane of the 2-fold axis. Assuming that the orientation of
the loop may change upon dimerization, several models differing with
regard to orientation of the
-sheet were constructed and validated
against the scattering data. The best model presented in Fig. 5,
bottom row, yielded a fit to the experimental data with
= 0.98 at V = 111 nm3 and
b = 44 electrons/nm3 (Fig. 4,
).
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MC5 Construct, Neutron Scattering--
The molecular mass of the
protein computed from the neutron scattering of MC5 in H2O
was 78 ± 5 kDa, in good agreement with the x-ray data and with
the estimated molecular mass of the dimeric protein. The maximum
dimension of the particle was found to be 12 ± 1 nm in both
H2O and D2O. The radii of gyration were
3.96 ± 0.12 and 3.88 ± 0.05 nm in H2O and
D2O, respectively. The difference between the x-ray and
neutron scattering curves should be attributed to the influence of the
hydration shell. As demonstrated previously (34), bound water around
the protein surface is denser than bulk water, and this makes the
particles appear larger for x-rays, smaller for neutrons in
D2O, and nearly unchanged for neutrons in H2O
(the scattering length density of the solvent is close to zero in the
last case). A direct comparison of the three scattering patterns in
Fig. 6 displays the same trend as
observed earlier for other proteins and clearly illustrates the
influence of the hydration shell (36). The neutron scattering patterns
in H2O and D2O computed from the atomic model
of MC5 in Fig. 5, top row, fitted the experimental data with
= 1.40 and 2.47, respectively (Fig.
7). Addition of the missing C-terminal
residues in the conformation determined from the x-ray data (Fig. 5,
bottom row) improves the agreement for both neutron data
sets, yielding
= 1.06 and
= 1.78 for the curves
recorded in H2O and D2O, respectively. This improvement of the fit to the neutron data lends further credit to the
model in Fig. 5, bottom row, deduced solely from x-ray measurements.
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|
MC1 Construct--
MC1 contains residues
Leu209-Lys700 (including the complete predicted
coiled-coil region and motor domain) of full-length ncd and 13 additional residues at the N terminus of the protein (6). This
construct has been shown to be dimeric in solution. The composite x-ray
scattering curve from MC1 in Fig. 8
yields a molecular mass of 123 ± 12 kDa, which agrees well with
the expected value of 114 kDa. The maximum dimension of the construct
(25 ± 3 nm) and its radius of gyration (6.7 ± 0.3 nm)
suggest that the particle is extremely elongated. Compared with MC5
(Dmax = 13 ± 1 nm), MC1 has 86 additional
residues in the N-terminal region predicted to form an -helical
coiled-coil. Because the pitch of the helix is 0.54 nm, the expected
length of the additional 86 residues would be 12.9 nm, yielding
Dmax = 25.9 nm for the complete MC1 protein, in
good agreement with the experimental value. An ab initio low
resolution model of MC1 in Fig. 9
(middle row) also suggests a linearly extended form of the
additional coil-coil region. A tentative model of the MC1 was therefore
built by extending the coiled-coil portion of the MC5 construct in Fig.
5 by 86 residues, as illustrated in Fig. 9, top row. The
scattering pattern computed from this model yields a reasonable fit to
the experimental data (Fig. 8,
) with
= 1.47 at
V = 125 nm3 and
b = 42 electrons/nm3. To establish the configuration of the
missing C-terminal region, several plausible models were tested as
described above for the MC5 construct. Addition of the C-terminal
region in the conformation found for the monomeric MC6 (Fig. 3)
worsened the fit to the experimental data, but the configuration
identical to that for MC5 (Fig. 5, right panel) yielded the
best fit (Fig. 8,
) with
= 0.95 at V = 135 nm3 and
b = 36 electrons/nm3. The final model of MC1 shown in Fig.
9, bottom row, suggests that the relative orientation of the
two heads is independent of the length of the coiled-coil.
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DISCUSSION |
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The present analysis of monomeric and dimeric constructs of the
microtubule-dependent molecular motor ncd indicates that
parts missing in the crystal structures must be taken into account for successful fitting of the solution scattering data. The modeling of the
missing C-terminal region up to Lys700, which has not been
resolved in any of the ncd crystal structures thus far, was crucial in
this respect. It was found that the most probable configuration of this
region is an antiparallel -sheet. Of course, the orientations of
this region presented in Figs. 2, 5, and 9 should be considered as
tentative models because they were based on fitting solution scattering
data and not on crystallographic information. With this caveat in mind,
it is interesting to note that the probable orientation of the
C-terminal fragment changes upon dimerization (i.e. from MC6
to MC5) but remains stable in the dimeric protein independent of the
coiled-coil lengths in the ncd constructions (MC5 to MC1).
One of the most important findings of the present study is that the published crystallographic structures of the dimeric ncd in the ADP-bound form (17, 18) are compatible with the solution scattering data from the MC5 construct. This agrees with the results of Stone et al. (42) obtained on three other dimeric Drosophila ncd constructs. Although the differences between the three available crystal structures are rather small, the structure lacking the 2-fold symmetry axis along the coiled-coil domain yields a somewhat better fit. It is interesting that a nonsymmetric crystallographic model of dimeric rat kinesin has recently been found to provide better agreement with the solution scattering data of Drosophila kinesin (43).
The longest dimeric ncd construct analyzed in the present study, MC1, covers the entire N-terminal coiled-coil domain and catalytic motor core. The model obtained by addition of a straight coiled-coil region to the above-mentioned model of MC5 yields a remarkably good fit to the solution scattering data. This suggests that MC1 has a straight coiled-coil domain, whereas the relative orientation of the two heads is independent of the length of the coiled-coil domain. This creates some confidence that working with truncated motor protein fragments instead of full-length proteins does not alter the overall shape of these molecules.
It has been demonstrated in the present study that the overall
conformation of the dimeric ncd in solution agrees with the crystallographic models (17, 18). A tentative configuration of the
C-terminal loop in monomeric and dimeric ncd is proposed, based on the
scattering data. Future x-ray and neutron scattering experiments will
include the investigation of nucleotide-dependent conformational changes of dimeric ncd in solution. In addition, different members of the highly divergent C-terminal subfamily should
be compared to determine whether they have the same overall conformation.
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ACKNOWLEDGEMENT |
---|
We thank Sharyn Endow for plasmids pET/MC1, pET/MC5, and pET/MC6.
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FOOTNOTES |
---|
* This work was supported by grants from the Association pour le Recherche sur le Cancer and CNRS and by Grant 00-243 from the International Association for the Promotion of Cooperation with Scientists from New Independent States of the Former Soviet Union.
¶ To whom correspondence should be addressed: European Molecular Biology Laboratory (EMBL), EMBL c/o DESY, Notkestrasse 85, D-22603 Hamburg, Germany. Tel.: 49-40-89902-125; Fax: 49-40-89902-149; E-mail: Svergun@EMBL-Hamburg.DE.
§§ Experimental work of this author in Hamburg was supported under the Training and Mobility of Researchers/Large Scale Facilities program to the European Molecular Biology Laboratory Hamburg Outstation (reference number ERBFMGECT980134).
Published, JBC Papers in Press, May 2, 2001, DOI 10.1074/jbc.M103618200
2 D. I. Svergun and M. Koch, unpublished observations.
3 M. Malfois and D. I. Svergun, manuscript in preparation.
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ABBREVIATIONS |
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The abbreviations used are: ncd, non-claret disjunctional; PIPES, piperazine-N,N'-bis(2-ethanesulfonic acid), e/nm3, electrons/nm3.
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