The Ndc80 kinetochore complex forms
oligomeric arrays along microtubules
Gregory M. Alushin1, Vincent H. Ramey1, Sebastiano Pasqualato2, David A. Ball3, Nikolaus Grigorieff4, Andrea Musacchio2,5
& Eva Nogales3,6
TheNdc80complexisakeysiteofregulated kinetochore–microtubule attachment(aprocessrequired forcelldivision),
but the molecular mechanism underlying its function remains unknown. Here we present a subnanometre-resolution
cryo-electron microscopy reconstruction of the human Ndc80 complex bound to microtubules, sufficient for precise
docking of crystal structures of the component proteins. We find that the Ndc80 complex binds the microtubule with a
tubulin monomer repeat, recognizing a- and b-tubulin at both intra- and inter-tubulin dimer interfaces in a manner
that is sensitive to tubulin conformation. Furthermore, Ndc80 complexes self-associate along protofilaments through
interactions mediated by the amino-terminal tail of the NDC80 protein, which is the site of phospho-regulation by
Aurora B kinase. The complex’s mode of interaction with the microtubule and its oligomerization suggest a mechanism
by which Aurora B could regulate the stability of load-bearing kinetochore–microtubule attachments.
The Ndc80 complex is a member of the conserved KMN kinetochore
network, which also includes the Knl-1 and Mis12 complexes1. The
Ndc80 complex is the key site for kinetochore–microtubule attach-
ment1–3and a landing pad for the spindle assembly checkpoint4–6.
Although extensively characterized genetically7,8and biochemi-
cally1–3,9,10, the mechanisms by which the Ndc80 complex effects
and coordinates these activities remain elusive.
The complex is an elongated, 57-nm heterotetramer composed of
NDC80 (also known as HEC1), NUF2, SPC24 and SPC25, each hav-
ization: SPC24 with SPC25, and NDC80 with NUF2 (refs 9–12).
Tetramerization via the dimerized coiled-coils9,10results in a dumb-
bell architecture, with the SPC24–SPC25 globular head at one end
mediating kinetochore association1,3, and the NDC80–NUF2 head at
the other mediating microtubule binding1–3,12. The NDC80–NUF2
Crystallographic structures of both globular head domains have
been obtained2,11, as well as that of a chimaeric version of the human
SPC25 and NUF2 to SPC24 (ref. 12). This 17-nm ‘bonsai’ complex,
which we refer to as Ndc80(bonsai), retained microtubule binding
and kinetochore localization. Both NDC80 and NUF2 contain a cal-
ponin homology domain (CHD), which is also present in other
microtubule binding proteins14,15. The unstructured, positively
charged 80-amino-acid N-terminal tail of the NDC80 protein is
required for high-affinity microtubule binding2,3,12,16,17, probably by
interaction with the acidic carboxy-terminal tails of tubulin (also
known as ‘E-hooks’). This region of the NDC80 protein, the site of
phospho-regulation by the Aurora B kinase3,12,18, is absent from all
and remains attached to microtubules during microtubule depoly-
merization and how this attachment is regulated during mitosis
Ndc80 binds tubulin with a novel monomeric repeat
We used cryo-electron microscopy to obtain a structure of
Ndc80(bonsai) (ref. 12), including the N-terminal tail bound to
microtubules. We used an implementation19of the iterative helical
sification to sort helical segments based on symmetry and sample
quality (Supplementary Figs 1, 2). Class averages show densities pro-
truding fromthe microtubule (Supplementary Fig. 1b,top right) with
the chevron-like orientation also reported for the Caenorhabditis ele-
gans complex1,21. Power spectra of class averages show layer lines at
1/40A˚21(and subsequent orders), corresponding to the spacing of
the tubulin monomer(Supplementary Fig. 1b,bottomright), but lack
the 1/80A˚21layer line typically observed for microtubule-binding
proteins which recognize the tubulin heterodimer22–24. This result
suggested that the Ndc80 complex binds to each tubulin monomer.
ating binding of the two CHDs to strong and weak sites present in
each tubulin dimer21. In order to determine the arrangement of the
Ndc80 complex on the microtubule lattice without imposing any
symmetry or averaging, we obtained tomographic reconstructions
of negatively stained microtubules saturated with Ndc80(bonsai)
(Supplementary Fig. 1c). Single volume slices allow us to visualize
individual tubulin monomers and bound Ndc80 complexes, which
are found with a 40A˚spacing within the thickness of a single proto-
Microtubule site recognition of the Ndc80 complex
obtained a reconstruction of the Ndc80 complex-bound microtubule
at 8.6A˚resolution (Fourier shell correlation (FSC) 0.143 criterion,
Supplementary Fig. 4a), allowing us to visualize secondary structure
1Biophysics Graduate Group, University of California, Berkeley, California 94720, USA.2Department of Experimental Oncology, European Institute of Oncology, 20139 Milan, Italy.3Life Sciences Division,
Lawrence Berkeley National Laboratory, Berkeley, California 94720, USA.4Howard Hughes Medical Institute, Rosenstiel Basic Medical Research Center, Brandeis University, Waltham, Massachusetts
02453, USA.5Research Unit of the Italian Institute of Technology at the IFOM-IEO Campus, 20139 Milan, Italy.6Howard Hughes Medical Institute, Department of Molecular and Cell Biology, University of
California, Berkeley, California 94720, USA.
1 4 O C T O B E R 2 0 1 0 | V O L 4 6 7 | N A T U R E | 8 0 5
Macmillan Publishers Limited. All rights reserved
13 and 14 protofilament microtubules were essentially identical (Sup-
plementary Fig. 5). Docking of the crystal structures of Ndc80(bonsai)
lacking the N terminus of the NDC80 protein—which we refer to as
atomic model of this interface (Fig. 1b). The excellent correspondence
against major rearrangements in the globular domains of the complex
upon microtubule binding.
The surface of the Ndc80 complex binding the microtubule lattice
is minimal, and includes the C-terminal end of helix G and the short
helices B and F in the NDC80 protein. This ‘toe’ recognizes a site
between two tubulin monomers, a ‘toe-print’ present at both intra-
and inter-dimer interfaces. The toe-print is composed of the short
helix H119 in one tubulin subunit and the loop connecting helix H8
with b-strand S7 in the other (Fig. 2a, purple and orange, respec-
tively). Superposition of the crystal structures of a- and b-tubulin
and multiple-sequence alignments show the toe-print to be highly
conserved between the two tubulin monomers (Fig. 2a, bottom),
consistent with the ability of the Ndc80 complex to recognize them
both. The end of the tubulin crystal structure marking the beginning
of the disordered E-hook of tubulin (Fig. 2a, red), required for high-
affinity Ndc80 complex binding1,12, is adjacent to the toe-print, from
where we propose it extends and acts as a second, distinct binding site.
The interface we observe is largely in agreement with mutagenesis
analysis of NDC80 residues important for binding (for example,
K123, K166, H176)12. Interestingly, the NUF2 CHD is not in contact
with the surface of the microtubule, yet mutations in this region also
disrupt binding12. The positively charged surface of this domain is
approximately 15 A˚from the E-hook of a laterally adjacent tubulin
monomer and thus could be engaged in an electrostatic interaction.
The Ndc80 toe is a tubulin conformation sensor
During microtubule disassembly, protofilaments bend outwards by
kinking of tubulin at intra- and inter-dimer interfaces. In spite of its
We therefore hypothesized that the NDC80 toe acts as a sensor of the
conformational state of tubulin, and that the complex would bind
preferentially to straight protofilaments.
Using co-sedimentation assays, we investigated the ability of
Ndc80(bonsai) to bind vinblastine-induced tubulin spirals (Sup-
plementary Fig. 6), a polymer analogous to the peels observed at
microtubule ends27. Because spirals retain the E-hooks, which make
a major contribution to binding affinity, we expected the interaction
to be reduced, not eliminated. Indeed, we observed a modest but
for this form of tubulin versus the straight microtubule conformation
(Fig. 2b top, Fig. 2c). We next sought to delineate the relative con-
tributions of the toe versus the N terminus of NDC80 to tubulin
binding. Ndc80(bonsai(7D)), a phosphomimetic construct of the
seven Aurora B phosphorylation sites confirmed in vitro12(Online
Methods, Supplementary Fig. 7), showed significantly reduced affin-
ity towards the straight microtubule conformation and negligible
affinity towards the vinblastine-induced, bent conformation; this
reduction reflects the increased relative contribution of the toe-print
interaction to affinity when the N terminus–E-hook interaction is
impaired (Fig. 3b, c). This result is consistent with a bipartite binding
mechanism, with the NDC80 N terminus providing affinity without
to affinity that is exquisitely sensitive to tubulin conformation.
We next investigated the effect of the complex’s small bias in affin-
ity towards straight tubulin. We polymerized tubulin into dynamic
microtubules, then initiated depolymerization by cooling in the pres-
ence or absence of Ndc80(bonsai). Cold-stable microtubules and
straight tubulin sheets were observed only in the presence of the
Ndc80 complex(Fig. 3d,e).Together, theseresults areconsistent with
the Ndc80 complex favouring a straight tubulin conformation, and
with this specificity being mediated by the toe. Our studies also indi-
cate that the complex has a stabilizing effect on microtubules.
Ndc80 self-assembles on microtubules
In our cryo-electron microscopy reconstruction, connections appear
between Ndc80 complexes along protofilaments (Fig. 1a, b), support-
microtubules12,17. We collected tomograms of negatively stained
microtubules with non-saturating amounts of Ndc80 complex to test
for the presence of Ndc80–Ndc80 complex interactions under more
physiological conditions. We found that the complex forms clusters
along protofilaments that retain tubulin monomer spacing (Fig. 3a, e;
Supplementary Fig. 8). Decoration was heterogeneous, with some
microtubules approaching saturation and others almost undecorated
(Fig. 3a, Supplementary Fig. 9a); this is a direct manifestation of
cooperativity. Wedonotobserve orderedself-associationofthecom-
Figure 1 | Structure of the Ndc80 complex–microtubule interface. a, End on
(from the plus-end) and side views of the microtubule–Ndc80 complex cryo-
electron microscopy reconstruction (tubulin, green; NDC80–NUF2 head, blue;
disordered SPC24–SPC25 head, red). b, Orthogonal views of docked crystal
structures (NDC80, blue; NUF2, gold; tubulin, green). In ball and stick
representation are residues adjacent to the absent N terminus of NDC80 in
Ndc80(bonsai(DN)) (magenta), and ordered residues in tubulin preceding the
E-hooks (red). The region ofthemap occupied by NUF2(right panel) is further
from the Ndc80 complex–microtubule interface and thus is of lower resolution.
c, Orthogonal views of the positive difference density (magenta) between the
cryo-electron microscopy reconstruction and the docked crystal structures,
8 0 6 | N A T U R E | V O L 4 6 7 | 1 4 O C T O B E R 2 0 1 0
Macmillan Publishers Limited. All rights reserved
of the pooled distributions of cluster number and size indicates that it
is most probable for a complex to be found in a cluster of four mole-
cules or in large clusters of more than ten molecules (Fig. 3i,
Supplementary Fig. 9b). Given the reported number (6–8) of Ndc80
complexes per microtubule at the kinetochore, the larger clusters are
probably not physiologically relevant28,29. Our data are therefore con-
sistent with the formation of two or three clusters per kinetochore
Analysis of our docking results shows densities not accounted for
by the crystal structures. We attempted to visualize these regions by
calculating a difference map between the experimental density map
and the docked crystal structures (Fig. 1c; Supplementary Fig. 11).
The map shows positive density peaks adjacent to the C-terminal end
of the tubulin crystal structure, corresponding to the extension of the
H12 helices in each monomer. Stabilization of these residues upon
Ndc80 complex binding is consistent with this region of tubulin con-
tributing to affinity1,12. Small extra density is also observed by the toe-
NUF2 and the aH–aI helical hairpin of NDC80, with aseries of small
peaks running along the groove between two longitudinally adjacent
complexes, connecting this region with the N-terminal end of the
Ndc80(bonsai(DN)) crystal structure.
corresponds to the N terminus of NDC80. We propose that the N
terminus mediates weak contacts between the globular heads of
NDC80 and NUF2 before making a strong contact, corresponding
(necessary for high affinity), probably in an adjacent protofilament.
these densities, which probably correspond to ordered points of con-
tact formed by this mostly unstructured polypeptide.
In agreement with this proposal, we found that Ndc80(bonsai(DN))
was deficient in cluster formation (Fig. 3b, f, i). The presence of some
clusters of two complexes suggests that the N terminus is the major
but not sole molecular determinant of clustering. We found that
Ndc80(bonsai(7D)) showed a clustering phenotype similar to
Ndc80(bonsai(DN)), with a cluster size of one molecule being the
most probable, despite a higher average surface density, and a few
clusters of moderate size (Fig. 3c, g,i). We therefore conclude that the
N terminus of NDC80 mediates Ndc80–Ndc80 complex interactions,
aspreviouslyspeculated12,andthatphosphorylation atAurora Bsites
is capable of modulating this binding.
Next, we dissected the differential contributions of the N terminus
to affinity and cooperativity. Subtilisin cleavage of the E-hooks from
tubulin significantly reduces the affinity of the Ndc80 complex for
microtubules1,12,17. As expected, after this treatment we found a sig-
nificantly lower average surface density of Ndc80 complexes com-
pared to uncleaved microtubules (Fig. 3d, h, i). If the N terminus of
NDC80 were only contributing indirectly to cluster formation by
increasing microtubule-binding affinity, the cluster size distribution
for this condition should be similar to the N terminus mutants.
Instead, we observe a most probable cluster size of three molecules,
(Fig. 3i). This observation strongly supports our hypothesis that the
NDC80 N terminus forms specific intermolecular interactions between
occurring after microtubule binding, as the average cluster size on sub-
tilisin-cleaved microtubules is smaller than on uncleaved microtubules.
Role of Ndc80 clusters in mitosis
It was recently demonstrated that Aurora B’s activity is governed by
spatial localization rather than directly by tension30, confirming pre-
vious proposals31,32. On the basis of this finding, we suggest a scheme
that starts with phosphorylated Ndc80 complexes in an unattached
kinetochore (Fig. 4a)33. When the kinetochore encounters a micro-
tubule, individual NDC80-NUF2 heads would initially bind the
microtubule through a low-affinity interaction. The bound heads
would then escape the Aurora B phosphorylation zone, provided that
intra-kinetochore stretching increased as microtubule binding by
additional heads allowed the site to come under tension34. This could
P[ FY] PR
[ RK] [ RK] AF[ VL] HW Y
S P S P S P S P
Wild type Ndc80(bonsai(7D))
Wild type Ndc80(bonsai(7D))
S P S P S P
Figure 2 | The NDC80 toe-print is a tubulin conformation sensor.
a, Superposition of the tubulin intra-dimer and inter-dimer interfaces, viewed
from theoutsideofthemicrotubule (a-tubulin,green;b-tubulin,blue; ordered
orange). Consensus sequences are indicated, with deviations between the
monomers in parenthesis. b, SDS–PAGE of co-sedimentation assays with
straight (taxol) and curved (vinblastine) tubulin polymers with the indicated
Ndc80(bonsai) constructs. S, supernatant; P, pellet. [Tubulin
monomer]56mM, [Ndc80(bonsai)]50.5mM. c, Quantification of b. Error
depolymerization of dynamic microtubules in the presence and absence of
Ndc80(bonsai). e, Negative-stain electron microscopy of Ndc80 complex-
induced cold-stable microtubules and straight tubulin sheets. Scale bar, 50nm.
1 4 O C T O B E R 2 0 1 0 | V O L 4 6 7 | N A T U R E | 8 0 7
Macmillan Publishers Limited. All rights reserved
occur via straightening of the flexible Ndc80 complex itself13(Fig. 4a)
or by the stretching of a compliant link in the inner kinetochore35. As
the phosphorylation zone is cleared, phosphatase activity (possibly
KMN-network-localized Protein phosphatase 1; ref. 36) would
dephosphorylate the N terminus of NDC80, resulting in the forma-
tion of high-affinity Ndc80 complex clusters.
Once assembled, the cluster arrangement is consistent with the
complex maintaining load-bearing attachments via biased diffusion37
(Fig. 4b), as recently proposed on the basis of functional studies38. An
alternative model, based on shuffling, is also consistent with our data
port a biased-diffusion model with significant differences from pre-
vious studies that presumed either a continuous sleeve37or sites
longitudinal subunit spacing of the microtubule. This arrangement
would arise only after the kinetochore comes under tension.
Consistent with this hypothesis, outer kinetochore rearrangement
on microtubule interaction has been observed in vivo39.
A cluster could diffuse on the microtubule lattice, but its diffusion
would become biased at a microtubule end. Thus, a shrinking micro-
tubule would pull the attached chromatid polewards. Cluster dif-
fusion should be facilitated by the 40A˚rather than 80A˚spacing of
the Ndc80 complexes, with a shorter distance to the transition state
between binding sites during diffusion. Thus, monomer binding may
rapid kinetics (40A˚is the smallest step a microtubule-binding ele-
tubule). Our finding that the NDC80 toe serves as a tubulin
conformation sensor suggests that microtubule subunit loss is not
required to bias diffusion: the curving of protofilaments at a depoly-
merizing end would suffice40. Depolymerizing ends have been
observed at metaphase kinetochores in vivo, apparently stabilized
by attached filaments of unknown identity (Fig. 4b)41.
Whereas the KMN network and the Ndc80 complex are conserved
from yeast to humans, the mechanism of microtubule attachment
may not be. We propose that the fungal Ndc80 complex has diverged
to act as a coupler to the Dam1 complex, and our structural results,
coupled with conservation analysis, suggest apossiblebinding site for
Wild type Ndc80(bonsai(ΔN)) Ndc80(bonsai(7D))
Percentage of observed molecules
Ndc80(bonsai(7D)), N = 5, n = 210
Ndc80(bonsai(ΔN)), N = 4, n = 62
SMTs, N = 4, n = 102
WT, N = 8, n = 862
Average no. Ndc80s per MT
Figure 3 | ClusterformationrequirestheNterminusoftheNDC80protein.
a–d, Central slices of tomograms of microtubule-bound Ndc80(bonsai)
constructs under subsaturating conditions. Ndc80(bonsai):tubulin monomer
ratio was1:2 forwild-typeand subtilisin-cleaved microtubules (SMTs) and2:1
for NDC80 N terminus mutants. Scale bar, 25nm; black dots, gold fiducials.
e, Serial slices of the wild-type reconstruction 4.5nm apart show cooperative
binding and cluster formation only along (not between) protofilaments. Scale
bar, 10nm. f–h, Selected views of clusters in Ndc80(bonsai(DN)),
Ndc80(bonsai(7D)), and SMT reconstructions, respectively. Black lines
indicate position and orientation of Ndc80 complexes. i, Quantification of
cluster size populations. N, number of reconstructed microtubule segments; n,
total number of Ndc80 complexes observed; MT, microtubule; WT, wild type.
Asterisks, the most probable cluster size for each of the populations (wild-type
hastwopeaks).See SupplementaryTable1 for pair-wise statisticalcomparison
of these distributions.
8 0 8 | N A T U R E | V O L 4 6 7 | 1 4 O C T O B E R 2 0 1 0
Macmillan Publishers Limited. All rights reserved
the Dam1 complex on microtubule-bound Ndc80 (Supplementary
Discussion, Supplementary Fig. 13).
We have used cryo-electron microscopy and molecular docking to
attachment. Our studies also demonstrate the microtubule-mediated
oligomerization of the Ndc80 complex, which directly involves the
Aurora B-regulated N terminus of the NDC80 protein.
Cryo-electron microscopy and helical reconstruction. Taxol-stabilized micro-
tubules were decorated with Ndc80(bonsai) after application to glow-discharged
C-flat grids (Protochips), then plunge-frozen in ethane slush. Images were col-
lectedon KodakSO-163film with a Tecnai F20 electronmicroscopeoperating at
200kVat anominalmagnificationof50,0003withadoseof15electronsper A˚2.
Micrographs were digitized with a Nikon Super CoolScan 8000 scanner with a
step size of 6.35mm. Image processing and projection-matching alignment were
carried out using programs from the EMAN, IMAGIC and SPIDER packages,
and final refinement and CTF correction was performed with a version of
Visualization and molecular docking was performed with UCSF Chimera.
Amplitude-weighted difference maps were calculated using the program
DIFFMAP (http://emlab.rose2.brandeis.edu/software). References for all image
analysis and visualization software can be found in the Online Methods section.
Tubulin co-sedimentation assays. Tubulin polymerized with or without con-
formation-stabilizing drugs was mixed with Ndc80(bonsai), then layered on to a
50% glycerol cushion supplemented with additives and pelleted by ultracentri-
and pellet fractions were analysed by SDS–polyacrylamide gel electrophoresis
Electron tomography. Samples were prepared on C-flat grids augmented with a
continuous carbon layer, and stained with uranyl formate. Tilt series were col-
lected on 2k32k CCD cameras using a JEOL 3100 microscope operating at
300kV with the SerialEM package, or a Phillips CM200 microscope operating
at 200kV with Digital Micrograph (Gatan). Processing was carried out with
programs from the EMAN, SPIDER and IMOD software packages. References
for all image analysis software can be found in the Online Methods section.
Full Methods and any associated references are available in the online version of
the paper at www.nature.com/nature.
Received 10 February; accepted 13 August 2010.
1.Cheeseman, I. M., Chappie, J. S., Wilson-Kubalek, E. M. & Desai, A. The conserved
KMN network constitutes the core microtubule-binding site of the kinetochore.
Cell 127, 983–997 (2006).
Wei, R. R., Al-Bassam, J. & Harrison, S. C. The Ndc80/HEC1 complex is a contact
point for kinetochore-microtubule attachment. Nature Struct. Mol. Biol. 14, 54–59
regulated by Hec1. Cell 127, 969–982 (2006).
Martin-Lluesma, S., Stucke, V. M. & Nigg, E. A. Role of Hec1 in spindle checkpoint
signaling and kinetochore recruitment of Mad1/Mad2. Science 297, 2267–2270
DeLuca, J. G. et al. Nuf2 and Hec1 are required for retention of the checkpoint
proteins Mad1 and Mad2 to kinetochores. Curr. Biol. 13, 2103–2109 (2003).
Kemmler, S. et al. Mimicking Ndc80 phosphorylation triggers spindle assembly
checkpoint signalling. EMBO J. 28, 1099–1110 (2009).
Chen, Y., Riley, D. J., Chen, P. L. & Lee, W. H. HEC, a novel nuclear protein rich in
leucine heptad repeats specifically involved in mitosis. Mol. Cell. Biol. 17,
Wigge, P. A. et al. Analysis of the Saccharomyces spindle pole by matrix-assisted
Wei, R. R., Sorger, P. K. & Harrison, S. C. Molecular organization of the Ndc80
complex, an essential kinetochore component. Proc. Natl Acad. Sci. USA 102,
10. Ciferri, C. et al. Architecture of the human ndc80-hec1 complex, a critical
constituent of the outer kinetochore. J. Biol. Chem. 280, 29088–29095 (2005).
11. Wei, R. R. et al. Structure of a central component of the yeast kinetochore: the
Spc24p/Spc25p globular domain. Structure 14, 1003–1009 (2006).
12. Ciferri, C. et al. Implications for kinetochore-microtubule attachment from the
structure of an engineered Ndc80 complex. Cell 133, 427–439 (2008).
13. Wang, H. W. et al. Architecture and flexibility of the yeast Ndc80 kinetochore
complex. J. Mol. Biol. 383, 894–903 (2008).
14. Hayashi, I., Wilde, A., Mal, T. K. & Ikura, M. Structural basis for the activation of
15. Slep, K. C. & Vale, R. D. Structural basis of microtubule plus end tracking by
XMAP215, CLIP-170, and EB1. Mol. Cell 27, 976–991 (2007).
Aurora B zone: phosphorylated Ndc80
Attachment, maturation and affinity
Spindle forces pull
bound heads out of
or dimers bind
with low affinity
40 Å diffusion steps,
minimizing kinetic barrier
Site detaches upon tubulin
Filaments observed to contact
Figure 4 | Proposedmodelsofattachmentmaturationandbiaseddiffusion.
a, Cartoon illustrating the phospho-regulated formation of Ndc80 complex
clusters in vivo concomitant with stable kinetochore–microtubule attachment
machinery is regulated by the spatial localization of Aurora B and a
counterbalancing phosphatase rather than directly by tension. b, Diagrams of
the proposed biased diffusion process for coupling chromosome movement to
microtubule depolymerization via the Ndc80 complex. Colours as in Fig. 1b,
except that the NDC80–NUF2 head is shown in gold and unidentified
filaments are shown in grey.
1 4 O C T O B E R 2 0 1 0 | V O L 4 6 7 | N A T U R E | 8 0 9
Macmillan Publishers Limited. All rights reserved
16. Guimaraes, G. J., Dong, Y., McEwen, B. F. & Deluca, J. G. Kinetochore-microtubule
interaction between unstructured tails on microtubules and Ndc80(Hec1). Curr.
Biol. 18, 1785–1791 (2008).
18. Cheeseman, I. M. et al. Phospho-regulation of kinetochore-microtubule
attachments by the Aurora kinase Ipl1p. Cell 111, 163–172 (2002).
19. Ramey, V.H.,Wang, H. W.& Nogales, E.Ab initioreconstructionofhelicalsamples
with heterogeneity, disorder and coexisting symmetries. J. Struct. Biol. 167,
20. Egelman, E. H. The iterative helical real space reconstruction method:
surmounting the problems posed by real polymers. J. Struct. Biol. 157, 83–94
21. Wilson-Kubalek, E. M., Cheeseman, I. M., Yoshioka, C., Desai, A. & Milligan, R. A.
Orientation andstructureofthe Ndc80complex onthemicrotubule lattice. J.Cell
Biol. 182, 1055–1061 (2008).
of cytoplasmic dynein bound to microtubules. Proc. Natl Acad. Sci. USA 104,
23. Hoenger, A. & Gross, H. Structural investigations into microtubule-MAP
complexes. Methods Cell Biol. 84, 425–444 (2008).
24. des Georges, A. et al. Mal3, the Schizosaccharomyces pombe homolog of EB1,
changes the microtubule lattice. Nature Struct. Mol. Biol. 15, 1102–1108 (2008).
25. Lo ¨we,J.,Li,H.,Downing,K.H.&Nogales,E.Refinedstructureofalphabeta-tubulin
at 3.5 A resolution. J. Mol. Biol. 313, 1045–1057 (2001).
26. Wang, H. W. & Nogales, E. Nucleotide-dependent bending flexibility of tubulin
regulates microtubule assembly. Nature 435, 911–915 (2005).
27. Wilson, L., Jordan, M. A., Morse, A. & Margolis, R. L. Interaction of vinblastine with
steady-state microtubules in vitro. J. Mol. Biol. 159, 125–149 (1982).
28. Joglekar, A. P., Bouck, D. C., Molk, J. N., Bloom, K. S. & Salmon, E. D. Molecular
architecture of a kinetochore-microtubule attachment site. Nature Cell Biol. 8,
29. Joglekar, A. P. et al. Molecular architecture of the kinetochore-microtubule
attachmentsite isconserved between point and regionalcentromeres.J.CellBiol.
181, 587–594 (2008).
30. Liu, D., Vader, G., Vromans, M. J., Lampson, M. A. & Lens, S. M. Sensing
chromosome bi-orientation by spatial separation of aurora B kinase from
kinetochore substrates. Science 323, 1350–1353 (2009).
31. Tanaka, T. U. et al. Evidence that the Ipl1-Sli15 (Aurora kinase-INCENP) complex
promotes chromosome bi-orientation by altering kinetochore-spindle pole
connections. Cell 108, 317–329 (2002).
33. Santaguida, S. & Musacchio, A. The life and miracles of kinetochores. EMBO J. 28,
34. Maresca, T. J. & Salmon, E. D. Intrakinetochore stretch is associated with changes
in kinetochore phosphorylation and spindle assembly checkpoint activity. J. Cell
Biol. 184, 373–381 (2009).
35. Wan, X. et al. Protein architecture of the human kinetochore microtubule
attachment site. Cell 137, 672–684 (2009).
by KNL1 opposes Aurora B kinase. J. Cell Biol. 188, 809–820 (2010).
37. Hill, T. L. Theoretical problems related to the attachment of microtubules to
kinetochores. Proc. Natl Acad. Sci. USA 82, 4404–4408 (1985).
38. Powers, A. F. et al. The Ndc80 kinetochore complex forms load-bearing
in vertebrate kinetochores is a flexible network with multiple microtubule
interactions. Nature Cell Biol. 9, 516–522 (2007).
40. Lombillo, V. A., Stewart, R. J. & McIntosh, J. R. Minus-end-directed motion of
kinesin-coated microspheres driven by microtubule depolymerization. Nature
373, 161–164 (1995).
41. McIntosh, J. R. et al. Fibrils connect microtubule tips with kinetochores: a
mechanism to couple tubulin dynamics to chromosome motion. Cell 135,
Supplementary Information is linked to the online version of the paper at
Acknowledgements We are grateful to K. H. Downing for supporting the work carried
out by D.A.B.,to C. Ciferri for his knowledge and advice about the Ndc80 complex and
critical reading of the manuscript, and to P. Grob and S. Lipscomb for
electron-microscopy and computer support, respectively. We also acknowledge
D. Typke and B. Glaeser for advice on data collection, and C. Sindelar for discussion of
ofGeneralMedicalSciences (E.N.). E.N.andN.G.are Howard Hughes MedicalInstitute
Author Contributions G.M.A. performed research. G.M.A. and V.H.R. developed data
processing tools. G.M.A. and S.P. generated and purified Ndc80(bonsai) mutants.
D.A.B. generated the tomograms displayed in Supplementary Fig. 1. N.G. adapted
Frealign software for helical samples and generated the final refined reconstruction.
G.M.A., A.M. and E.N. wrote the Article.
docking model have been deposited at the EMDB and PDB under accession numbers
5223 and 3IZ0, respectively. Reprints and permissions information is available at
www.nature.com/reprints. The authors declare no competing financial interests.
Readers are welcome to comment on the online version of this article at
www.nature.com/nature. Correspondence and requests for materials should be
addressed to E.N. (email@example.com).
8 1 0 | N A T U R E | V O L 4 6 7 | 1 4 O C T O B E R 2 0 1 0
Macmillan Publishers Limited. All rights reserved
Mutagenesis. Ndc80(bonsai) point mutations were generated using standard
procedures for site-directed mutagenesis. The Ndc80(bonsai(7D)) mutant was
generated as follows: Synonymous mutations were performed that destroyed the
StuI restriction site at base pair (bp) 81–86 of the NDC80–SPC25 protein coding
sequence and subsequently reintroduced it at bp 250–255. An NdeI restriction
site was also created 59 to the beginning of the coding sequence. This construct
seven in vitro verified12Aurora B phosphorylation sites mutated to aspartic acid
(S4D, S8D, S15D, S44D, S55D, S62D, S69D) was synthesized (GENEART) and
cloned into the swappable N terminus construct using standard protocols.
Biochemical sample preparation. Ndc80(bonsai) complex was purified as
described12. 10mgml21bovine brain tubulin (Cytoskeleton) was polymerized
in CB1 buffer (80mM PIPES pH 6.8, 1mM EGTA, 1mM MgCl2, 1mM GTP,
60min of further incubation. We have found this procedure primarily produces
tabletop microcentrifuge for 20min, then resuspended in room temperature EM
buffer (80mM PIPES pH 6.8, 1mM EGTA, 1mM MgCl2, 1mM DTT, 0.05%
Nonidet P-40) supplemented with 160mM taxol. Tubulin concentration was
assayed by A280after depolymerization on ice with 50mM CaCl2.
Ndc80(bonsai) at 2mgml21was rapidly thawed before storage on ice, diluted
1:1 with EM buffer, then desalted into EM buffer using a Zeba spin desalting
column (Pierce). The removal of salt resulted in a rapid precipitation of the
sample, which was clarified by ultracentrifugation in a Beckman TLA100 rotor
for 20min at 61,734g at 4uC. The sample was then warmed to 25uC for approxi-
mately 15min before electron microscopy grid preparation, which resulted in a
by negative-stain electron microscopy did not reveal any ordered assemblies
(Supplementary Fig. 10). The sample was once again clarified for 3min at
17,000g in a tabletop microcentrifuge. Protein concentration was estimated with
the Coomassie Plus protein assay reagent (Pierce) using bovine serum albumin
on the state of the NDC80 N terminus: the Ndc80(bonsai(DN)) and
Cryo-sample preparation. C-flatgrids (Protochips) were glow-discharged using
an Edwards Carbon Evaporator. Taxol-stabilized microtubules were diluted to
0.25mgml21in EM buffer supplemented with 20mM taxol, and 2ml was applied
ratio of ,2:1 Ndc80 complex:tubulin monomer, and incubated for 1min. The
grid was then briefly blotted before a second 4ml addition of Ndc80 complex and
2s and plunged into ethane slush. This protocol is essentially similar to that used
for visualization of the C. elegans complex21, with minor modification.
Cryo-electron microscopy. 100 micrographs were collected on Kodak SO-163
film with a Tecnai F20 electron microscope operating at 200kV at a nominal
2.2mm underfocus. Micrographs were digitized with a Nikon Super CoolScan
8000 scanner with a step size of 6.35mm. After digitization, the power spectra of
carbon presentin each imagewas examined, and imageslacking Thonrings to at
least 8A˚were excluded.
Image processing and IHRSR. We carried out helical processing essentially as
described19, with some modifications. Unless otherwise indicated, all processing
samples of tobacco mosaic virus (TMV) imaged under identical conditions. CTF
parameters were estimated for each image using the program CTFFIND343.
Images were divided into three approximate defocus groups (1.2mm, 1.7mm,
2.2mm). Segments 768 pixels long were extracted from the micrographs with
the helix option in BOXER44, using 90% overlap20. Pixel intensities were normal-
ized, and large-scale gradients in intensity due to variations in ice thickness were
subtracted. A two-dimensional Wiener filter was then applied to each segment,
with the intention of aiding the detection of highly ordered segments, which
should diffract to high resolution. Subsequent experience has demonstrated that
in fact be detrimental in cases with a low signal-to-noise ratio in the low spatial
frequency regime, as amplification of high-resolution spatial frequency compo-
nents in raw images drives alignment based on noise rather than signal. The
segments were then masked such that each image contained approximately 2
turns of the 1-start helix, corresponding to 78 asymmetric units, and decimated
threefold to a pixel size of 3.72A˚.
We then subjected the data to reference-free two-dimensional classification as
described19. This method successfully sorts segments on the basis of protofila-
ment number and helical quality, as is apparent in Supplementary Fig. 2. As
microtubules can incorporate varying numbers of protofilaments in vitro45, and
segments vary in their quality, this method is used as an alternative to assessing
each individual microtubule by manual inspection of Moire ´ patterns and power
spectra. Out of an initial set of 10,253 segments, only 1,475 were members of
classes that corresponded to well-ordered 13 protofilament microtubules and
were selected for further processing.
Segments from defocus groups were combined before IHRSR with SPIDER.
Using a naked microtubule as a reference, the reconstruction converged after 10
rounds of refinement. Particles with cross-correlation scores less than one stand-
ard deviation below the mean were excluded, corresponding to approximately
taxol-stabilized microtubules46, but could result from an error in our pixel size
microtubule was applied after refinement was complete by real-space averaging.
The resolution of the reconstruction at this stage was limited to approximately
20A˚(‘bronze’ reconstruction, Supplementary Fig. 3), as filtering the volume at
higher resolution did not reveal any additional features.
This limit in resolution was found to be at least partially due to limited pixel
sampling. With no further alignment of the particles, simply reducing the
decimation factor of the original CTF-corrected data from threefold to twofold
(2.48A˚per pixel) resulted in a discrete jump in resolution, to approximately 12–
particle was removed at this stage, increasing the number of asymmetric units
incorporated into the reconstruction, as not all segments incorporated into the
reconstruction were from adjacent positions in microtubule filaments.
Approximately 50,000 asymmetric units were incorporated at this stage, and
secondary structure elements began to be visualized when a B-factor of
2450A˚2was applied with the program bfactor (http://emlab.rose2.brandei-
s.edu/software) using the cosine edge mask option with a radius of 8.5A˚. This
model was used as the input for final refinement in Frealign.
Final refinement with Frealign. We used the computer program Frealign47to
refine our reconstruction. Frealign was originally designed to work with single
particle images. To work with helical particles, we implemented a helical sym-
algorithm described previously48. Masking of the helical sections is done using a
Using the helical symmetry operator, a set of Euler angles and shifts producing
equivalent views for each section can be calculated. Using this set of alignment
parameters, each section is inserted multiple times into the reconstruction. The
numberofinsertionsis matchedto thedegree ofoverlapbetweenhelical sections
such that it corresponds to the number of symmetry-related subunits contained
in the non-overlapping part of each section. In the case of the Ndc80 complex-
decorated microtubules, the unique, non-overlapping area of the filament was
chosen to contain approximately one helical turn of 13 subunits. In addition to
this, the 3-start helical symmetry of the 13 protofilament microtubule increased
the number of unique subunits per segment threefold, to 39. Therefore, in the
present case, each helical segment is inserted into the three-dimensional recon-
the particle. The final reconstruction is masked using a cylindrical mask with
user-defined radius, and the helical symmetry is imposed in real space (option
BEAUTIFY in Frealign) to remove the small density gradient along the helical
axis due to the reconstruction algorithm48.
For the refinement of Euler angles and shifts for each helical section, the
reference projections from the helical reference are masked with a soft-edged
mask to reset the image density to background level in a 10% margin near the
edges of the image. This is necessary to avoid truncation of reference projections
at the edges when applying the small shifts typically observed during alignment.
Furthermore, the refinement is carried out using the weighted correlation coef-
ficient49, but without the use of the absolute value.For helical structures, the low-
resolution correlation terms are essentially insensitive to shifts along the helical
axis and, therefore, if the absolute value is used, the high-resolution layer lines
ing in-plane and out-of-plane alignments of each section are restrained as prev-
iously described50, but using separate statistics for each segment. No restraints
profile as theinput reconstruction, and is shownin gold in Supplementary Fig. 3.
Difference map calculation. The experimental map was segmented, and an
appropriate number of Ndc80(bonsai(DN)) and tubulin crystal structures were
docked into the map to approximately account for the total mass with UCSF
Macmillan Publishers Limited. All rights reserved
Chimera51. These crystal structures were converted into SPIDER volume format Download full-text
procedure retained the orientation of the crystal structures relative to each other,
this volume had to be re-docked into the experimental map, which was accomp-
lished using a cross-correlation search with SPIDER. The amplitude-weighted
difference map was calculated using the program DIFFMAP (http://emlab.ro-
se2.brandeis.edu/diffmap). Amplitude weighting was critical for a meaningful
comparison, as the amplitudes of the volume derived from the crystal structures
were down-weighted several hundred fold in all resolution shells.
Tubulin co-sedimentation assays. For biochemical assays Ndc80(bonsai) pro-
teins were desalted into binding buffer (80mM PIPES pH 6.8, 1mM EGTA,
1mM MgCl2, 1mM DTT, 5% sucrose). We found that this buffer increased the
solubility of the protein in the absence of salt while allowing for accurate deter-
mination of protein concentration by A280. The phenyl group of Nonidet P-40
results in significant absorbance of EM buffer at 280nm, which is refractory to
reproducible protein concentration measurement by this method. The presence
of sucrose significantly reduces contrast in the electron microscope, and thus
imaging experiments, we have found no differences in Ndc80 complex–micro-
tubule interactions or tubulin polymer behaviour between the two buffers (data
To generate vinblastine spirals, we diluted tubulin stored in CB1 buffer to
3mgml21in binding buffer supplemented with 1mM vinblastine sulphate
(Sigma). After two hours incubation at 25uC, robust formation of spiral aggre-
gates was observed (Supplementary Fig. 6).
Taxol microtubules or vinblastine spirals (6mM tubulin monomer) and
Ndc80(bonsai) proteins (0.5mM) were mixed in a 50ml reaction volume and
The binding reactions were layered on to a 100ml 50% glycerol cushion contain-
ing buffer components and the appropriate drug and polymers were pelleted by
ultracentrifugation at 312,530g. in a Beckman TLA 100 rotor for 10min at 25uC,
essentially as described12. Supernatant and pellet fractions were collected and
precipitated in 90% ethanol at 220uC for 16h before analysis by SDS–PAGE.
a Typhoon Trio (GE Healthcare). Apparent slight degradation of the Ndc80
complex was observed in the presence of 1mM vinblastine sulphate (Fig. 2b),
but this did not cause spuriouspelleting ofthe complexin the absenceoftubulin.
Quantification was performed with ImageJ52.
To test for cold stabilization, 10mgml21tubulin in CB1 buffer was polymer-
buffer was set up on ice, and then heated to 37uC for 1min. Dynamic micro-
and 3.3mM NDC80 and incubated at 37uC for 10min. The reaction was then
shifted to ice for 30min, and subsequently analysed by pelleting assay as above,
except ultracentrifugation was performed at 4uC.
To analyse the outcomes of the described experiments by negative stain elec-
tron microscopy, we repeated them substituting EM buffer for binding buffer.
Samples were prepared on continuous carbon grids, stained with 2% uranyl
scope operating at 120kV between approximately 1mm and 2mm underfocus.
Negative stain sample preparation for tomography. Samples were prepared as
described for cryo-electron microscopy, except that the grids were augmented
International) before sample application. To achieve sub-saturating binding,
wild-type Ndc80(bonsai) was diluted to 0.15mgml21and only a single applica-
tion was performed, corresponding to a 1:2 Ndc80 complex:tubulin monomer
subtilisin A (Calbiochem) in EM buffer for 30min at 37uC. The reaction was
stopped with 2mM PMSF, and the SMTs were pelleted and resuspended in EM
buffer before sample preparation.
of Ndc80(bonsai(DN)) and Ndc80(bonsai(7D)), double-application of 0.6–
0.7mgml21Ndc80 complex was performed, corresponding to a 2:1 Ndc80 com-
plex:tubulin monomer ratio. Samples were stained with 2% uranyl formate.
Electron tomography and cluster quantification. The tomograms displayed in
Supplementary Fig. 1 were derived from tilt series collected on a 2k32k CCD
camera from 260u to 60u using a JEOL 3100 microscope operating at 300kV at
approximately 2mm underfocus with a nominal magnification of 40,0003. The
acquisition was performed semi-automatically using a version of SerialEM
graphic image stacks were aligned either manually with the eTomo suite of
programs, or automatically using the software RAPTOR53. Tomographic recon-
structions were constructed using the eTomo suite of programs and visualized
using the IMOD software package54.
The tomograms displayed in Fig. 3 were derived from tilt series collected from
265u to 65u on a 2k32k CCD camera using a Phillips CM200 microscope
tionof39,0003. Imageswerefilteredto 25–30A˚,before thefirstphase-inversion
Cluster quantificationwas performed by manual inspection of the reconstruc-
comparisons between the distributions towards spurious difference. Pair-wise
comparisons were performed using Welch’s t-test55, which is appropriate for
samples featuring both different numbers of observations and possibly unequal
variances (which the unbiased estimator of the variance of the distributions
suggested in this case), and P-values are shown in Supplementary Table 1. In
all but one of the cases P,0.0015, suggesting that the data derived from each of
the conditions shown in Fig. 3 and Supplementary Fig. 9 do indeed sample
Ndc80(bonsai(DN)), where we find a probability of 0.61 of sampling the same
distribution.This supports our assertion that these two mutants phenocopy each
Conservation analysis and structure alignments. We performed multiple-
sequence alignments using CLUSTALW256, and mapped this analysis on to the
Ndc80(bonsai(DN)) crystal structure using the CONSURF server57. The align-
ments included 48 fungi sequences and 39 metazoan sequences in the case of
NDC80and 52 fungi sequences and 22 metazoan sequences in the case of NUF2,
and are available on request. Sequences of a- and b-tubulin from six represent-
ative organisms (Saccharomyces cerevisiae, Caenorhabditis elegans, Drosophila
melanogaster, Xenopus laevis, Bos taurus and Homo sapiens) were compared.
Since the Ndc80(bonsai(DN)) crystal structure is of the human complex, fungal
sequences had to be threaded on to the structure by alignment with the human
sequence. As we observe a similar pattern of conservation at the NDC80 toe, we
performed with the MatchMaker function in UCSF Chimera51.
42. Frank, J. et al. SPIDER and WEB: processing and visualization of images in 3D
electron microscopy and related fields. J. Struct. Biol. 116, 190–199 (1996).
tilt in electron microscopy. J. Struct. Biol. 142, 334–347 (2003).
44. Ludtke, S. J., Baldwin, P. R. & Chiu, W. EMAN: semiautomated software for high-
resolution single-particle reconstructions. J. Struct. Biol. 128, 82–97 (1999).
45. Wade, R. H., Chretien, D. & Job, D. Characterization of microtubule protofilament
numbers. How does the surface lattice accommodate? J. Mol. Biol. 212, 775–786
Struct. Biol. 157, 117–125 (2007).
48. Sachse, C.et al.High-resolution electron microscopyofhelicalspecimens: a fresh
look at tobacco mosaic virus. J. Mol. Biol. 371, 812–835 (2007).
49. Stewart, A. & Grigorieff, N. Noise bias in the refinement ofstructures derived from
single particles. Ultramicroscopy 102, 67–84 (2004).
50. Chen, J. Z. et al. Molecular interactions in rotavirus assembly and uncoating seen
by high-resolution cryo-EM. Proc. Natl Acad. Sci. USA 106, 10644–10648 (2009).
interactive visualization of large molecular assemblies. Structure 13, 473–482
52. Abramoff, M. D., Magelhaes, P. J. & Ram, S. J. Image Processing with ImageJ.
Biophotonics International 11, 36–42 (2004).
53. Amat,F.etal.Markovrandomfield based automaticimagealignment forelectron
tomography. J. Struct. Biol. 161, 260–275 (2008).
dimensional image data using IMOD. J. Struct. Biol. 116, 71–76 (1996).
55. Welch, B. L. The generalisation of student’s problems when several different
population variances are involved. Biometrika 34, 28–35 (1947).
56. Larkin, M. A. et al. Clustal W and Clustal X version 2.0. Bioinformatics 23,
of residues on protein structures. Nucleic Acids Res. 33, W299–302 (2005).
Macmillan Publishers Limited. All rights reserved