1
Supplementary Figure Legends:
Supplementary Fig. 1. The Ncd neck position is not influenced by adjacent motors.
a, Cryo-EM map of the 281-700 a.a. Ncd construct with a SH3 N-terminal tag in the
presence of apyrase. An elongate neck density pointing towards the microtubule plusend is observed. For this construct, the distal part of the neck/SH3 density (labeled N)
appears to overlap with the unbound head density of another motor on the adjacent
filament (HA), implying a potential interaction with the SH3 domain which could
contribute to the stability of the neck. b, Cryo-EM map of the 281-700 a.a. Ncd construct
without SH3 tag (N-termianl His6 tag only) shown in the same orientation as in (a). For
this construct, the distal part of the neck density (N) does not overlap with the head
density of an adjacent motor (HA). Difference density maps also confirm that the position
of the neck relative to the motor domains is identical in (A) and (B) (not shown). This
rules out the possibility that an artifactual SH3-head interaction contributed to the
position and/or stability of the neck. c, Cryo-EM map of the Ncd-SH3 construct in the
presence of ADP- AlF4-, shows no density overlap between the neck density (N) and
adjacent motors (HA).
Supplementary Fig. 2: Comparisons of WT maps reveal similar neck conformation
for AMPPNP and ADP-AlF4- states. a, Overlay of WT 3D map AMPPNP (blue) with
the WT ADP-AlF4- 3D map (red), shows that the density for the neck and unbound heads
are in the same orientation in the two structures. b,c Statistical difference maps represent
statistically significant differences (red, P<0.0005) between 3D maps of Ncd-microtubule
2
complexes compared using a students t test. (b) Statistical difference maps comparing
WT nucleotide-free and the WT AMPPNP 3D maps (red) superimposed on the WT
AMPPNP map (transparent blue). The two peaks labeled H0 and HATP are associated with
the rotation of the unbound head between the nucleotide-free and AMPPNP states. The
two peaks labeled and N0 and NATP, reflect the position of the neck in the nucleotide-free
and AMPPNP states respectively. (c) Statistical difference maps comparing WT
nucleotide-free and WT ADP-AlF4- 3D maps (red) superimposed on the ADP-AlF4- map
(transparent blue). Peaks are labeled as in (b). The same difference peaks observed in the
comparison between the nucleotide-free and AMPPNP maps (b) can be seen here,
implying that the head and neck occupy similar positions in both nucleotide states.
Supplementary Figure 3. Binding interface between Ncd and microtubule
corresponding to the docking experiments shown in Figs. 1d-f. The microtubule is
oriented so the plus-end is on the left. a, Wildtype Ncd-ADP structure5 (dark blue)
docked to nucleotide-free 3D maps as shown previously (Fig. 1d.). Tubulin6 (orange)
was docked separately to the remaining density. Docking shows a good fit for both
tubulin and Ncd density. b, Close-up view of the region boxed in (A) shows more
detailed view of the interface between the bound motor domain and tubulin. Predicted
microtubule binding elements are highlighted, L8 (yellow), L11-4-L12 (light blue) and
L2 (red) (4,5).
Supplementary Figure 4. Comparisons of Ncd N340K EM maps consistant with
model for bidirectional motility (see text). a, 3D maps of N340K in the presence of 5
3
mM AMPPNP, show a clear definition of the microtubule-bound head, but no neck
density and a less clearly defined detached head. b, Overlay between N340K ADP-AlF4(blue) and AMPPNP (red) show that the orientation of the heads in the two structures are
identical. c,d Statistical difference maps representing statistically significant differences
(red, P<0.0005) between 3D maps of N340K-microtubule complexes compared using a
students t test. c, Statistically significant differences between N340K nucleotide-free and
N340K AMPPNP 3D maps (red) superimposed on the AMPPNP map (transparent blue).
As in the comparisons of the WT maps (Supplementary Fig. 2 b,c), there are two peaks
(H0 and HATP) attributable the position of the unbound head in the absence of nucleotide
(H0) and the presence of AMPPNP (HATP) implying some net rotation of the unbound
head between nucleotide states. However, in contrast to the WT maps, only a single peak
(N0), associated with the neck density in its pre-powerstroke position, is seen with the
NcdN340K mutant. As described in the text, the details in the raw maps of the
NcdN340K protein suggest that the neck occupies both pre- and post-powerstroke
positions in the nucleotide-free state. The presence of a pre-powerstroke peak (N0) is
consistent with the notion that the pre-powerstroke state is significantly populated in the
nucleotide-free state but not significantly populated in the AMPPNP state. The absence
of a peak corresponding to a post-powerstroke position (NATP in Supplementary Fig.
S2b,c) indicates that this conformation is equally populated in the nucleotide-free and
AMPPNP states. d, Statistical difference maps comparing N340K nucleotide-free and
N340K ADP-AlF4- 3D maps (red) superimposed on the ADP-AlF4- map (transparent
blue), are nearly identical to the N430K nucleotide-free/ AMPPPNP difference maps (c),
4
implying no significant conformational changes between the AMPPNP and ADP-AlF4states, as was the case for the wild-type motor (Supplementary Fig. 2).
Supplementary Figure 5. Heterodimer purification and motility experiments. a,
Separate gel filtration runs of purified biotin-tagged Ncd homodimer (N281, black),
untagged Ncd homodimer (N280notag, orange), biotin-tagged Ncd neck homodimer
(N280_Neck, purple), biotin-tagged Ncd monomer (N325, blue), and the nickel column
elution from the heterodimer prep (red) are shown here aligned and scaled (see Methods).
Purified homodimers elute in the expected order based on their sizes, whereas the Ncd
monomer N325 elutes much later. The nickel column elution from the heterodimer prep,
which should contain a mixture of single-headed heterodimer (N280_Het) and tagged
neck homodimer (N281_Neck), shows two peaks. As expected, one of the peaks elutes
at the same position as N281_Neck. The second peak, indicated with an arrow, is unique
to this preparation and elutes in a position intermediate between N281_Neck and
N280notag, as would be predicted for N280_Het. Gel electrophoresis of these fractions
confirmed that both the motor domain and the tagged neck domain elute in the N280_Het
fraction (not shown). The fraction indicated by this peak was used in all N280_Het
experiments. b, Diagram of typical gliding assay shown (see also methods). Glass
surface represented with light blue, grey circles represents combination of Streptavidin
and biotinylated BSA bound to the glass, and a rhodamine-labeled microtubule is shown.
The gel filtration purified heterodimer (N280_Het) and two possible contaminants
(N280notag and N281_Neck) are shown here to demonstrate how they would be
predicted to interact with the glass and or microtubule in the gliding assay. The figure
demonstrates that only N280_Het would be predicted to support microtubule gliding
5
under these conditions, since it is the only dimer in this mixture that has both a biotin tag
to couple it to the glass slide and a motor domain to bind microtubules. Table showing
results of gliding assays with the constructs purified constructs shown in (a), which
confirm that only N280_Het,
N281, and N325 (the tagged Ncd homodimer and
monomer respectively) can support gliding of microtubules as predicted from design of
gliding assay. The absence of microtubule binding in the presence of both N280notag
and N281_neck suggest that recombination of the polypeptide does not occur and that the
aggregation of the two homodimers is not responsible for the motility observed in the
single-headed heterodimer containing fractions. Comparison of N280_Het gliding
velocity to that of the Ncd dimer (N281) and monomer (N325) show that the singleheaded heterodimer behaves more like the dimer, implying that coiled-coil interactions
play a larger role then the unbound head in determining the velocity of the motor. The
slow movement observed in the monomer (N325) is not likely to be random since
experiments with polarity-marked microtubules indicate these motors move towards the
minus-end.
6
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