Figure 4. Model for MT depolymerization by kinesin 13. (A) The NM construct can assume an extended (top) or compact (bottom) conformation. These correspond to the crystal structures (Protein Data Bank reference codes 2HEH and 1v8k) shown in Fig. 3 B. Both conformations can deco- rate adjacent binding sites on straight protofilaments (left), but the extended conformation decorates alternate binding sites on a curved protofilament (top right). (B) The dimeric FL construct is designed to bind two adjacent protofilaments instead of binding adjacent heterodimers on a single proto- filament. Because of its size and design, this motor is postulated to bind alternate binding sites on both straight and curved protofilaments, although our data only provide evidence for the latter. alternate site binding with processivity to separate terminal tubulin from a curling protofilament via steric strain. However, our obser- vations that both NM and FL engage in alternate site binding and that FL forms longer stacks than NM seem to argue against this possibility. A further potential concern with the proposed model is that staggered FL dimers could stabilize (as opposed to destabilize) the MT end via intraprotofilament cross-bridging. We believe this to be an unlikely scenario for the following reasons: (a) the alter- nate site-binding requirement guarantees that the terminal two heterodimers of every pair of protofilaments are free to peel away from its neighbors even with cross-linking of multiple kinesin 13s, (b) high and low affinity kinesin 13–binding cycles in the presence of ATP reduce the probability of saturating the MT end with stag- gered kinesin 13s, and (c) intraprotofilament cross-linking would not inhibit kinesin 13 activity, so this scenario is more likely to re- sult in multiprotofilament depolymerization products than to pre- vent depolymerization altogether. The model presented in this study represents a divergence from the processivity along a single protofilament observed for conventional kinesin. As such, it is vital to extend and validate these observations in the context of MTs; such experiments are ongoing in our laboratory. Materials and methods Protein expression and purification Constructs were prepared as described previously: M is from Plasmodium falciparum (Moores et al., 2002) and represents the kinesin 13 catalytic motor core. NM includes residues 182–583 of kinesin 13 from Cricetulus 56 JCB • VOLUME 185 • NUMBER 1 • 2009 griseus (Ovechkina et al., 2002). FL (also known as Q710) contains all but the last eight residues of kinesin 13 from C. griseus (Moore and Wordeman, 2004). ∆N (residues 182–718) and ∆C (residues 1–583) are N- and C-terminal deletions of C. griseus MCAK, prepared similar to FL. M, NM, and ∆C exist as monomers in solution, whereas FL and ∆N exist as dimers (Hertzer et al., 2006; Ems-McClung et al., 2007). The use of M from P. falciparum was necessitated by the fact that M from C. griseus was incurably prone to aggregation, which prevented us from performing ex- periments with it (our unpublished observations). However, using a human MCAK M construct (88% sequence identity with C. griseus MCAK) with MT depolymerization activity, we observed adjacent site binding to, and de- gree of stack formation of, dolastatin rings in a manner indistinguishable from the P. falciparum M construct (Fig. S3). Thus, the phenomena that we describe are unlikely to be a result of species-specific properties of our con- structs but instead relate to a generalized mechanism for kinesin 13 MT de- polymerization. The human M construct (residues 257–593) was provided by Cytokinetics. Kinesin 13 and ring complex formation Rings were formed by incubating 0.04 mM dolastatin-10 dissolved in DMSO with 2 mg/ml GTP-tubulin (Cytoskeleton, Inc.) in 40 mM Pipes, pH 6.8, 1.50 mM MgCl2, and 12% (vol/vol) DMSO for 20 min at room tem- perature. Kinesin 13 constructs were dialyzed into BrB80-KCl buffer (80 mM Pipes, pH 6.8, 2 mM MgCl2, 1 mM EGTA, and 100 mM KCl) at 4°C stir- ring for 3–4 h. Constructs were mixed with 5 mM AMPPNP and 0.5 µM rings in BrB80-KCl at 3 µM for 2–3 min at room temperature. Sedimenta- tion assay reaction mixtures were ultracentrifuged in a rotor (TLA-100; Beckman Coulter) at 100,000 rpm at room temperature for 20 min. The super- natant and pellet were separated, subjected to SDS-PAGE analysis, and the amount of protein in each lane was estimated using ImageJ (National Insti- tutes of Health; Abramoff et al., 2004) and a BSA standard curve. EM sample preparation and data collection EM samples were prepared on 400-mesh copper continuous carbon grids that were cleaned using a plasma cleaner (5 s; 25% O2 and 75% Ar; So- larus) immediately before use. A 5-µl drop of sample was applied to the grid, washed with BrB80-KCl, and negatively stained with 1% uranyl acetate. EM was performed at room temperature using a transmission electron micro- scope (Tecnai F20; FEI) at 120 kiloelectronvolts (keV) with a 4,000 × 4,000 charge-coupled device camera (Gatan), and data were collected in low dose at 2 µm defocus at 50,000× magnification using Leginon (Suloway et al., 2005) in manual mode. Image processing Image processing was done in SPIDER (Frank et al., 1996) with functions avail- able from Appion (Lander et al., 2009). In Appion, micrograph quality was assessed, particles were picked and boxed, and initial reference-free align- ment and classification was performed on 2,000 rings for each dataset. Classes with well-stained particles underwent reference-based alignment in SPIDER to sort out 13-, 14-, and 15-mer rings. Only 14-mer rings (80% of all particles) were processed further. Particles underwent reference-based align- ment, correspondence analysis, and hierarchical ascendant classification in SPIDER to sort out alternate binding rings (Fig. S2; Frank et al., 1996). Particles were considered alternate binders for cross-correlation coefficients ≥1,800, resulting in 20%, 80%, and 80% alternate binders for M, NM, and FL, respectively. Particles that did not reach stable alignment or failed to pass visual inspection were thrown out, resulting in 4.4, 69.4, and 53.9% alter- nate binders for M, NM, and FL datasets. Individual motor–tubulin com- plexes were picked manually in the EMAN Boxer program (Ludtke et al., 1999) from alternate binding class averages. These particles underwent two rounds of reference-based alignment and classification in SPIDER (Frank et al., 1996). Difference maps and Student’s t tests were calculated in SPIDER (Frank et al., 1996) and Phoelix (Whittaker et al., 1995). Fitting kinesin 13 crystal structure into tubulin rings Chimera 1.2470 (Pettersen et al., 2004) was used for all fitting experiments. An EM density map of Ncd (nonclaret disjunctional kinase-like protein)-MT (Endres et al., 2006) was the scaffold for fitting - and -tubulin (Nogales et al., 1998) and Ncd (Sablin et al., 1998) crystal structures using the “fit model into map” feature. The two crystal structures of truncated NM, KIF2C (Ogawa et al., 2004), were fitted using the “matchmaker” feature until all atoms agreed within 0.345 Å. To model the interaction with a curved proto- filament, the fitted model of KIF2C and straight -tubulin were used as a scaffold for fitting a crystal structure of stathmin-bound tubulin dimers (Ravelli et al., 2004) until all atoms of -tubulin agreed within 0.345 Å. As our ex- perimental data did not yield 3D density information, we did not use our 2D