Supporting Information

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Supporting Information

Sindelar et al. 10.1073/pnas.0911208107

SI Text

Supplementary Methods.Sample preparation.Preparation of the no-nucleotide K349-microtubule complex and subsequent data collection have been described (1) (only data collected in that work were used here). To generate other nucleotide states of the K349-microtubule complex,, individual drops were mixed (one drop per grid, in most cases) according to the following gen-eral protocol: an aliquot of prepared microtubules was added to an aliquot of distilled water, followed by an aliquot of the motor preparation, and subsequently aliquots of the various nucleotide-related constituents; the mixture was then incubated for 15–30 s before plunge freezing. The resulting samples were applied to homemade holey carbon grids (not exposed to glow discharge), excess solution was wicked away from the edge of the grid with filter paper, and grids were then blotted and rapidly (<0.5s later) plunge-frozen in liquid ethane.

Data processing.Films were digitized by a robotic scanning system and processed using custom SPIDER scripts that treated short microtubule segments as asymmetric single particles, as described (1). The contrast transfer function of the microscope, including astigmatism, was derived from micrographs using CTFFIND3 (2) and accounted for separately in both the alignment and the re-construction steps of the processing. A generic pdb model of the kinesin-microtubule complex, similar to our published descrip-tion of the no-nucleotide kinesin-microtubule complex (1), was used to generate a reference model for the initial round of align-ment for each nucleotide state. The FREALIGN program (3), with minor modifications to account for helical symmetry, was used to compute reconstructions and to refine the SPIDER-derived alignment parameters. Three reconstructions for each nucleotide state were generated: one using all the data, and one each using odd and even 80 Å repeats from individual micro-tubules; RMEASURE (4) was applied to the former reconstruc-tion, and Fourier Shell Correlation (FSC) to the two latter two, to estimate map resolution.

The detailed refinement scheme was as follows: a single round of refinement, using custom SPIDER scripts as described (1), was used to derive coordinates for every 80 Å repeat of the microtu-bules, between user-specified endpoints. These coordinates were used to extract a stack of images for each dataset, with one image per 80 Å repeat. The image stack, along with Euler angles and shift parameters derived from the SPIDER processing, were then input to FREALIGN for reconstruction, followed by four rounds of subsequent refinement and reconstruction. The resulting Euler angles and shift parameters were then subjected to an additional round of refinement using the SPIDER scripts, to overcome local minima in the search space. Finally, four rounds of FREALIGN refinement and reconstruction were done.

Fitting crystal structures in maps.Once preliminary fitting experi-ments had indicated isolated regions of conformational change in our maps when compared to the K349 crystal structures, crystal structure coordinates were pared to eliminate these regions. Re-gions of potential conformational change we identified included the switch loops, the neck linker, the switch II helix, andα3, all of which exhibited movements in our density maps relative to the fitted crystal models. To assess the precision of the fitting calcula-tion, each fitting step was performed three times for each nucleo-tide state: once using the full reconstruction, and once each on the independent half-dataset reconstructions used to calculate the Fourier Shell Correlation estimate of structure resolution.

Comparison of these fits suggested a positional error for pre-dicted atomic coordinates within our maps of less than 1 Å. Or-ientation errors for the fitted catalytic domain were estimated in this same way to be less than 3°.

Modeling the switch II helix extension and the phosphate tube.We modeled the switch II helix using an extended conformation of the helix found in the crystal structure of ADP-bound KIF1A (an “ADP-like” conformation; PDB ID 1I5S). Within the

“ATP-like”crystal structure model of K349 (PDB ID 1MKJ), a conserved cluster of complementary hydrophobic interactions be-tween the C-terminal end of the helix, kinesin’s catalytic core and the neck linker (5) appear to define the 3-dimensional position of this end of the helix relative to the catalytic core. We therefore fitted the Cαpositions of residues 268–275 at the C-terminal end of the switch II helix of KIF1A•ADP (PDB ID: 1I5S) to the homologous residues 261–268 of docked human conventional ki-nesin (PDB ID: 1MKJ), following fitting of 1MKJ into our ADP•Al•Fxmap of conventional kinesin (seeMethods). This procedure resulted in approximate alignment of the extended switch II helix from KIF1A•ADP with the corresponding den-sity in our ADP•Al•Fxmap.

At the N-terminal end of the helix, we observed a conserved side chain in the switch II helix extension (Ile 254) with near ideal complementarity to the switch pocket. We therefore improved the alignment manually, by performing a minor (∼3°) rigid-body rotation of the KIF1A helix about theβ-carbon of the conserved L275 side chain (conventional kinesin: L268) near the C-terminal end of the helix. This rotation approximately preserved inter-actions between hydrophobic residues found at the interface be-tween the C-terminal end of the helix and the docked conforma-tion of the neck linker as found in the 1MKJ structure, and simultaneously aligned the helical axis with the apparent axis ob-served in our density map.

Thus, docking Ile 254 into the switch pocket while simulta-neously keeping the C terminus of the switch II helix fixed in space provided two geometric constraints, uniquely defining the position and orientation of the extended switch II helix. This operation produced a minor reorientation of the switch II helix (∼3°), relative to the initial crystal structure model, which simul-taneously aligned the helical axis with the corresponding density feature in our cryoEM density maps (Fig. 2E).

To propagate this switch II helix model to the ADP-bound, no-nucleotide, and AMPPNP maps, we first aligned these other maps to the ADP•Al•Fxmap by using the Fit in Map function of UCSF Chimera, restricting the region bounds used for the fit to include only the tubulin dimer density. As described in the text, this procedure aligned the switch II helix density features from the various maps with each other. We subsequently fitted crystal structures of the K349 kinesin motor domain into these maps, as described above, to derive the relative positioning of the ex-tended switch II helix and the motor domain.

The conformation of kinesin’s phosphate tube was approxi-mated by closed switch I and switch II loops taken from crystal structures of ATP analog-bound myosin. For the switch I loop, residues 228–239 from the ADP•Al•F_xcomplex of Dictyoste-lium myosin (6) (PDB ID: 1MMD) were aligned to kinesin coordinates 1MJK as described (7), by aligning P-loop residues 179–186 (myosin) to P-loop residues 85–92 (kinesin). To approx-imate the closed conformation of the closed switch II loop (con-ventional kinesin: residues 231–236, DLAGSE), the P-loop of the ADP•Al•Fx complex of Dictyostelium myosin (6) (PDB ID:

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1MND) was superposed onto the P-loop of the 1MKJ kinesin structure to obtain aligned coordinates of this myosin structure’s homologous switch II loop segment (454–459, DISGFE). We de-leted the gamma oxygen of the myosin’s S456 residue to approx-imate the conformation of A233 in conventional kinesin, and also the phenyl group of myosin’s F458. We found that this modeled loop conformation could be subtly refined to yield better connec-tivity to the kinesin core domain, and better complementarity to the switch II helix extension, by substituting the conformation of the N-terminal residues of the loop (conventional kinesin: 231–233, DLA) by identically conserved residues 226–228 from the closed switch II loop conformation found in the crystal struc-ture of the nonmotile kinesin NOD, complexed with AMPPNP (8) (PDB ID: 3DCB). P-loop residues of NOD (87–94) were superposed with the P-loop of the 1MKJ structure to perform this latter substitution. Subsequently, we superposed the backbone atoms of myosin’s S456 onto the backbone atoms of NOD A228 to yield a model for the closed conformation of residues GSE, which in the NOD structure significantly departed from the closed conformation seen in myosin, likely due to the absence of a closed switch I loop in the NOD structure. Our final model for the switch II loop was thus a chimera derived from the myosin and NOD crystal structures; this model thus possessed backbone and side chain conformations that came exclusively from these two crystal structures (no other refinement was done).

Fig. S2: Discussion.Binding of ATP analogs induced a substantial reorientation of the motor domain, relative to the switch II helix and an associated cluster of microtubule-binding elements which remain fixed on the microtubule surface. We quantified this movement using computational fitting procedures to dock crystal structure models of our kinesin construct into the maps (see SI Methods). The fitting analysis indicated that the motor domain orientation remains nearly unchanged upon ADP release (∼1.5° rotation, ADP to no-nucleotide transition) and changes by 14° upon ATP analog binding (no-nucleotide to ADP•Al•Fx transition). The 14° rotation occurs about an axis running through the motor domain approximately parallel to the microtubule axis, but skewed slightly so that the rotation axis intersects the micro-tubule surface near the contact point of kinesin’s microtubule-binding loop L8. These results agree with a cryoEM study com-paring ADP-bound and AMPPNP-bound states of the KIF1A ki-nesin at ∼10–11Å resolution (9). While we find a somewhat smaller rotation magnitude compared to the KIF1A study (∼16° compared with∼20° for KIF1A for the ADP to ATP analog transition), the rotation axis derived from our analysis is remark-ably similar to that described for KIF1A (Fig. S3).

In comparison with our other 3 maps, density for kinesin in the AMPPNP map exhibited signs of conformational averaging, in-cluding blurring and/or density attenuation in certain regions of the motor domain (Figs. S6CandEand S7), and also an ap-parent motor domain orientation that was intermediate between the ADP orientation and the ADP•Al•Fxorientation (Fig. S3). Nevertheless, the orientation of the AMPPNP-bound motor do-main as well as its detailed density features were most similar to those seen in the ADP•Al•F_xmap (Figs. S3 and S6), with re-spect to the other maps.

Consistent with previous studies (9–11), we directly observed density along the side of the kinesin motor domain in our ATP analog maps, absent in our ADP and no-nucleotide maps, corre-sponding to a docked conformation of the neck linker (Fig. S4). In addition, we observed length differences in helixα6 that

cor-binding of ATP analogs (Fig. 2 and Fig. S2). Also consistent with previous reports (9–11), we found that fitting the ATP-like K349 crystal structure into our ATP analog maps predicted an orientation of the switch II helix that approximately aligned our observed helical density, with similar agreement when fitting the ADP-like crystal structure model in our ADP-bound and no-nucleotide maps (Figs. 1 and 3). Thus, our maps strongly sup-ported the presence of ADP-like and ATP-like orientations of the switch II helix in the corresponding maps, as predicted by the switch II helix scheme.

Presence of the switch II helix extension in attached motor states. Density corresponding to several additional coils of the switch II helix, referred to in the main text as the switch II helix exten-sion, is present at the N-terminal end of the helix (Fig. 1,Left), relative to the crystallized conformations of our construct, in every nucleotide state examined. Presence of this extension in mi-crotubule-attached kinesin complexes has been corroborated in three other cryoEM reconstructions [Eg5•AMPPNP (10), KIF1A•ADP, and KIF1A•AMPPNP (13); however, the den-sity feature corresponding to the extension in the latter map ap-pears weak and ambiguous, and evidently was originally interpreted as a loop (9)].

Fig. S6: DiscussionWhen compared with crystal structure models of the corresponding nucleotide states, all four of our EM density maps showed substantial evidence of conformational change at the active site. Consistent with previous EM observations, the ADP-bound map featured an open nucleotide pocket, with the nu-cleotide site itself relatively exposed to solvent (14, 15). Also as reported previously (9), our no-nucleotide map indicates that the switch I loop intrudes into the nucleotide pocket and displaces ADP, accompanied by a conformational change of the nucleotide coordinating P-loop. The significance of these prior observations of the no-nucleotide state is strengthened by comparison with our other three maps. In contrast to the no-nucleotide map, density features in the ADP and ATP analog-bound maps account for both bound nucleotide as well as the P-loop, indicating that these ele-ments maintain the configurations seen in crystal structures (see Fig. S4). Thus, comparisons among our four maps validate our conclusion that the P-loop, switch I loop, and nucleotide elements are resolvable at this resolution and that the observed differences between nucleotide states are significant.

Fig. S7: Discussion.One of the novel aspects of our maps was the exceptional clarity of density features observed for the switch I loop; this clarity substantially contributed to our identification of a “phosphate tube” conformation for this loop, in the ATP analog states. However, the switch I loop density was somewhat attenuated in our AMPPNP map (Fig. S6), rendering the phos-phate tube identification more ambiguous in this nucleotide state. Evidence presented in this figure and in Figs. S3 and S4 sug-gests an explanation for this attenuation of the phosphate tube density. As shown in Fig. S3, crystal structures fit into the AMPPNP map exhibit an orientation intermediate between the no-nucleotide orientation and the ADP•Al•Fxorientation. Furthermore, less density was apparent for the docked conforma-tion of the neck linker in the AMPPNP map compared with the ADP•Al•Fx map (compare panels in Fig. S4; also see Mo-vies S3 and S4). All of these observations for the AMPPNP state could be accounted for by conformational averaging of multiple motor orientations, some similar to the ADP/no-nucleotide

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(17). Possibly related discrepancies between AMPPNP and ADP•Al•F_x states have also been seen in studies of the ncd kinesin variant (18). We therefore suggest that ADP•Al•Fx may be a more suitable analog for structural studies of kinesin’s ATP states.

While it is unclear which stage of hydrolysis ADP•Al•Fx ap-proximates, the mechanism presented here predicts that the over-all active site structure, including that of the phosphate tube, remains intact throughout hydrolysis and may persist throughout one or more rounds of resynthesis. Further experiments will be required to test such aspects of the kinesin mechanism.

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0 0.5 1 0 0.02 0.04 0.06 0.08 0.1 0.12 0.14 0.16 0 0.5 1 0 0.02 0.04 0.06 0.08 0.1 0.12 0.14 0.16 0 0.5 1 0 0.02 0.04 0.06 0.08 0.1 0.12 0.14 0.16 ADP No-Nucleotide AMPPNP 0 0.5 1 0 0.02 0.04 0.06 0.08 0.1 0.12 0.14 0.16 10Å 9Å 8Å 10Å 9Å 8Å 10Å 9Å 8Å 10Å 9Å 8Å FSC FSC FSC FSC Spatial Frequency (1/Å)

Fig. S1. Fourier Shell Correlation curves for the four kinesin-microtubule complex maps. Compared with other comparable published cryoEM reconstructions of kinesin-microtubule complexes (reported resolutions 9–11 Å), a substantially higher signal-to-noise ratio is found in our reconstructions at resolutions higher than∼12Å. This improvement is likely due in part to the large amount of data collected (135,000–240,000 kinesin molecules per dataset, Table S1). Earlier cryoEM work on kinesin has been based on much smaller datasets, by at least a factor of 2–4. The no-nucleotide map, which was produced using data we presented previously, exhibits a higher signal-to-noise ratio relative to our own prior analysis. We attribute this difference to improved data processing tech-niques. As indicated in the text, we find that reconstructions produced by the methods used here compare favorably with synthetic maps derived from atomic models (see Movie S7), indicating near optimal reconstruction quality at the current resolution of 8–9 Å.

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Fig. S2. Evidence of ATP analog-induced tilting and accompanying neck linker docking. (A), (B) View down the microtubule axis, looking towards the micro-tubule plus end, showing tilting of the catalytic core domain in response to ATP analog binding. The arrows indicate tilting of the catalytic cores, which occurs about an axis approximately parallel to the microtubule axis (see Fig. S3). The position of residue N255, where the crystallized switch II helix terminates, is indicated. (C), (D) Lengthening of helixα6, found in a comparison of no-nucleotide and ADP•Al•F_xmaps, respectively. The position of residue N255, where the crystallized switch II helix terminates, is indicated. The corresponding density features of theα6 in ADP-bound and AMPPNP-bound maps, respectively, were similar but less distinct, possibly due to noise and/or conformational averaging.

Fig. S3. Movements of the catalytic core, found in fitted K349 crystal structures for our four cryoEM maps. Axes of rotation defined by these movements are indicated with black rods. All transitions from (no-nucleotide or ADP) to (AMPPNP or ADP•Al•F_x) are represented. The axis of rotation found in a prior comparison of ADP-bound and AMPPNP-bound nucleotide states of KIF1A (see text) is indicated by the gray rod.

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Fig. S4. Oblique view with the microtubule plus end pointing towards the right and into the paper, showing evidence of docking by the neck linker. Predicted position of the docked neck linker (Magenta Ribbon) is indicated, as found by fitting crystal structure 1MKJ into our density maps.

Fig. S5. (A), (B) Predicted interactions of the switch loops, in ADP/AMPPNP states, giving similar geometries as found for the no-nucleotide∕ADP•Al•F_xstates depicted in Fig. 2AandC, respectively. (C) Result of superposing the crystal structure of KAR3•ADP (19) on our fitted K349 crystal structure for the ADP map. Ribbon diagram and space-filled models in this panel are exclusively from the KAR3 structure. The switch II helix from KAR3 is aligned nearly perfectly with the modeled switch II helix in panel (A) (propagated from our ADP•Al•F_xmap by aligning tubulin, see Methods). Also, interactions of R598 and A628 with I649 in the KAR3 structure are nearly identical to interactions of the R203/A233/I649 residue triad we derived in our K349•ADP∕no-nucleotide models.

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Fig. S7. Blurring/attenuation of features in the AMPPNP map compared to the other maps, suggesting conformational averaging. Black arrowheads in (A) and (C) indicate well-defined features corresponding to helicesα1 andα2. Bracket in (B) indicates apparent horizontal blurring of theα2 feature. The hollow arrowhead in (B) indicates a region of low density at the predicted location of helixα1. Lines in (B) indicate helix positions from (A) and (C). Both helices α1 andα2 have highly conserved, ordered conformations in crystal structures, so that disorder in this specific region of the protein chain is unlikely.

Fig. S8. Prediction of a novel ATP-bound conformation of myosin, with closed actin-binding cleft. (A) Crystal structure of the ADP•Al•F_xcomplex of Dic-tyostelium myosin II (20), with open actin-binding cleft. Homologous elements to kinesin are colored with the same scheme as in previous figures. The re-mainder of the myosin molecule is rendered in gray, except for the lower 50 kD domain (pink). (B) Depiction of a hypothetical myosin conformation in which the switch II helix of the crystal structure in (A) is aligned to match the same orientation, relative to the nucleotide pocket, as seen in our ATP•kinesin•microtubule model (Fig. 3C). This operation leads to a seesaw-like movement between the lower 50 kD domain (associated with the switch II helix) and the upper 50 kD domain, closing the actin-binding cleft.

Movie S1. 360° view of ADP map of microtubule-complexed kinesin, rendered as a semitransparent isosurface. The crystal structure of the human kinesin K349 construct (PDB ID 1BG2) is shown in itsﬁtted orientation in our map (seeMethods). Rendering details and color scheme are identical to those in Figs. 1 and 2. Movie S1 (MOV)

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Movie S2 360° view of no-nucleotide map of microtubule-complexed kinesin, rendered as in Movie S1. The crystal structure of the human kinesin K349 construct (PDB ID 1BG2) is shown in itsﬁtted orientation.

Movie S2 (MOV)

Movie S3 360° view of AMPPNP map of microtubule-complexed kinesin, rendered as in Movie S1. The crystal structure of the human kinesin K349 construct (PDB ID 1MKJ) is shown in itsﬁtted orientation.

Movie S3 (MOV)

Movie S4 360° view of ADP•Al•F_xmap of microtubule-complexed kinesin, rendered as in Movie S1. The crystal structure of the human kinesin K349 con-struct (PDB ID 1MKJ) is shown in itsﬁtted orientation.

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Movie S5 Three-dimensional rendering of Fig. 1E. Movie S5 (MOV)

Movie S6 360° view of our ADP•Al•F_xmap, shown with the fitted coordinates of our completed model for kinesin’s ATP-bound, microtubule-attached conformation.

Movie S6 (MOV)

Movie S7 360° view of a synthetic density map rendered from our completed model for kinesin’s ATP-bound, microtubule-attached conformation. Map is filtered to 8 Å resolution.

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Table S1. Reconstruction statistics Reconstruction RMEASURE FSC Number of particles Kinesin•ADP 8.8 Å 9.0 Å 156, 507 Kinesin no-nucleotide 7.9 Å 8.5 Å 137, 059 Kinesin•AMPPNP 8.6 Å 8.6 Å 171, 184 Kinesin•ADP•Al•F_x 8.8 Å 8.9 Å 238,498

Resolution of reconstructions was determined by two independent meth-ods: RMEASURE was applied to the final reconstructed volume, and the Four-ier Shell Correlation (FSC) was also calculated (seeMethods). Values for the resolution estimates corresponding to the 0.5 criterion. The number of kinesin molecules per reconstruction is also indicated.