RESUMEN
Molecular motors employ chemical energy to generate unidirectional mechanical output against a track while navigating a chaotic cellular environment, potential disorder on the track, and against Brownian motion. Nevertheless, decades of nanometer-precise optical studies suggest that myosin-5a, one of the prototypical molecular motors, takes uniform steps spanning 13 subunits (36 nm) along its F-actin track. Here, we use high-resolution interferometric scattering microscopy to reveal that myosin takes strides spanning 22 to 34 actin subunits, despite walking straight along the helical actin filament. We show that cumulative angular disorder in F-actin accounts for the observed proportion of each stride length, akin to crossing a river on variably spaced stepping stones. Electron microscopy revealed the structure of the stepping molecule. Our results indicate that both motor and track are soft materials that can adapt to function in complex cellular conditions.
Asunto(s)
Actinas , Miosina Tipo V , Actinas/química , Miosinas/química , Citoesqueleto de Actina/química , Movimiento (Física) , Miosina Tipo V/químicaRESUMEN
Fuelled by ATP hydrolysis, dyneins generate force and movement on microtubules in a wealth of biological processes, including ciliary beating, cell division and intracellular transport. The large mass and complexity of dynein motors have made elucidating their mechanisms a sizable task. Yet, through a combination of approaches, including X-ray crystallography, cryo-electron microscopy, single-molecule assays and biochemical experiments, important progress has been made towards understanding how these giant motor proteins work. From these studies, a model for the mechanochemical cycle of dynein is emerging, in which nucleotide-driven flexing motions within the AAA+ ring of dynein alter the affinity of its microtubule-binding stalk and reshape its mechanical element to generate movement.
Asunto(s)
Dineínas/metabolismo , Animales , Dineínas/química , Humanos , Modelos BiológicosRESUMEN
Dynein ATPases power diverse microtubule-based motilities. Each dynein motor domain comprises a ring-like head containing six AAA+ modules and N- and C-terminal regions, together with a stalk that binds microtubules. How these subdomains are arranged and generate force remains poorly understood. Here, using electron microscopy and image processing of tagged and truncated Dictyostelium cytoplasmic dynein constructs, we show that the heart of the motor is a hexameric ring of AAA+ modules, with the stalk emerging opposite the primary ATPase site (AAA1). The C-terminal region is not an integral part of the ring but spans between AAA6 and near the stalk base. The N-terminal region includes a lever-like linker whose N terminus swings by approximately 17 nm during the ATPase cycle between AAA2 and the stalk base. Together with evidence of stalk tilting, which may communicate changes in microtubule binding affinity, these findings suggest a model for dynein's structure and mechanism.
Asunto(s)
Dictyostelium/ultraestructura , Dineínas/metabolismo , Proteínas Protozoarias/metabolismo , Animales , Dictyostelium/metabolismo , Dineínas/ultraestructura , Proteínas Fluorescentes Verdes/metabolismo , Microscopía Electrónica , Proteínas Protozoarias/ultraestructuraRESUMEN
Using electron microscopy and image processing, we have observed myosin 5a modified with lever arms of different lengths (four, six, and eight calmodulin-binding IQ domains) and orientations walking along actin filaments. Step lengths were dependent on lever length: 8IQ > 6IQ > 4IQ, which is consistent with myosin 5a having evolved to walk straight along actin. Lead heads were mostly in the prepowerstroke state, tethered there by the trail head. However, improved image processing showed that in 5-10% of molecules the lead motor was in the postpowerstroke state. This is a unique attached state of myosin, where the motor domain has completed its powerstroke at the expense of severe lever distortion, but with little cargo movement. Postpowerstroke lead heads were seen in both wild-type and modified lever molecules, mostly where there was least strain. These data allow the strain dependence of the equilibrium between pre- and postpowerstroke conformations to be measured. Slow rates of ADP dissociation observed from lead heads of these molecules can be explained by the unfavorable equilibrium between the pre- and postpowerstroke conformations preceding ADP loss.
Asunto(s)
Citoesqueleto de Actina/ultraestructura , Microscopía Electrónica/métodos , Miosina Tipo V/metabolismo , Miosina Tipo V/ultraestructura , Citoesqueleto de Actina/metabolismo , Adenosina Difosfato/metabolismo , Adenosina Trifosfato/metabolismo , Secuencia de Aminoácidos , Animales , Línea Celular , Etilmaleimida/farmacología , Cinética , Ratones , Modelos Moleculares , Mutación , Miosina Tipo V/genética , Unión Proteica/efectos de los fármacos , SpodopteraRESUMEN
Molecular motors employ chemical energy to generate unidirectional mechanical output against a track. By contrast to the majority of macroscopic machines, they need to navigate a chaotic cellular environment, potential disorder in the track and Brownian motion. Nevertheless, decades of nanometer-precise optical studies suggest that myosin-5a, one of the prototypical molecular motors, takes uniform steps spanning 13 subunits (36 nm) along its F-actin track. Here, we use high-resolution interferometric scattering (iSCAT) microscopy to reveal that myosin takes strides spanning 22 to 34 actin subunits, despite walking straight along the helical actin filament. We show that cumulative angular disorder in F-actin accounts for the observed proportion of each stride length, akin to crossing a river on variably-spaced stepping stones. Electron microscopy revealed the structure of the stepping molecule. Our results indicate that both motor and track are soft materials that can adapt to function in complex cellular conditions.
RESUMEN
The myosin 2 family of molecular motors includes isoforms regulated in different ways. Vertebrate smooth-muscle myosin is activated by phosphorylation of the regulatory light chain, whereas scallop striated adductor-muscle myosin is activated by direct calcium binding to its essential light chain. The paired heads of inhibited molecules from myosins regulated by phosphorylation have an asymmetric arrangement with motor-motor interactions. It was unknown whether such interactions were a common motif for inactivation used in other forms of myosin-linked regulation. Using electron microscopy and single-particle image processing, we show that indistinguishable structures are indeed found in myosins and heavy meromyosins isolated from scallop striated adductor muscle and turkey gizzard smooth muscle. The similarities extend beyond the shapes of the heads and interactions between them: In both myosins, the tail folds into three segments, apparently at identical sites; all three segments are in close association outside the head region; and two segments are associated in the same way with one head in the asymmetric arrangement. Thus, these organisms, which have different regulatory mechanisms and diverged from a common ancestor >600 Myr ago, have the same quaternary structure. Conservation across such a large evolutionary distance suggests that this conformation is of fundamental functional importance.
Asunto(s)
Evolución Molecular , Miosina Tipo II/química , Pecten , Estructura Cuaternaria de Proteína , Pavos , Animales , Microscopía Electrónica , Músculo Liso/ultraestructura , Músculo Estriado/ultraestructura , Miosina Tipo II/antagonistas & inhibidores , Pliegue de ProteínaRESUMEN
We have used electron microscopy and single-particle image processing to study head conformation in myosin V molecules. We find that in the presence of ATP, many heads have a sharply angled conformation that is rare in its absence. The sharply angled conformation is similar to a myosin II atomic structure proposed to mimic the prepower stroke state. The leading head in molecules attached to actin by both heads has a similar conformation, but is also sharply angled in a second plane by tethering through the trail head. The lead head lever joins the motor domain approximately 5 nm axially from where it joins the trail motor. These positions locate the converter subdomain and show the lead motor is in the prepower stroke conformation. Tethering by the trail head places the lead head motor domain at the correct axial position along the actin for binding, but at the wrong orientation. Attachment is achieved either by bending the lead head lever throughout its length or at the pliant point. The microscopy shows that most of the walking stride is produced by changes in lever angle brought about by converter movement, but is augmented by distortion produced by thermal energy.
Asunto(s)
Miosina Tipo V/química , Actinas/química , Adenosina Trifosfato/metabolismo , Animales , Línea Celular , Cristalografía por Rayos X , Calor , Hidrólisis , Insectos , Ratones , Microscopía Electrónica , Modelos Moleculares , Miosina Tipo II/química , Unión Proteica , Conformación Proteica , Estructura Terciaria de ProteínaRESUMEN
Remodelling the contractile apparatus within smooth muscle cells allows effective contractile activity over a wide range of cell lengths. Thick filaments may be redistributed via depolymerisation into inactive myosin monomers that have been detected in vitro, in which the long tail has a folded conformation. Using negative stain electron microscopy of individual folded myosin molecules from turkey gizzard smooth muscle, we show that they are more compact than previously described, with heads and the three segments of the folded tail closely packed. Heavy meromyosin (HMM), which lacks two-thirds of the tail, closely resembles the equivalent parts of whole myosin. Image processing reveals a characteristic head region morphology for both HMM and myosin, with features identifiable by comparison with less compact molecules. The two heads associate asymmetrically: the tip of one motor domain touches the base of the other, resembling the blocked and free heads of this HMM when it forms 2D crystals on lipid monolayers. The tail of HMM lies between the heads, contacting the blocked motor domain, unlike in the 2D crystal. The tail of whole myosin is bent sharply and consistently close to residues 1175 and 1535. The first bend position correlates with a skip in the coiled coil sequence, the second does not. Tail segments 2 and 3 associate only with the blocked head, such that the second bend is near the C-lobe of the blocked head regulatory light chain. Quantitative analysis of tail flexibility shows that the single coiled coil of HMM has an apparent Young's modulus of about 0.5 GPa. The folded tail of the whole myosin is less flexible, indicating interactions between the segments. The folded tail does not modify the compact head arrangement but stabilises it, indicating a structural mechanism for the very low ATPase activity of the folded molecule.
Asunto(s)
Subfragmentos de Miosina , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Miosinas del Músculo Liso , Actinas/metabolismo , Animales , Simulación por Computador , Microscopía Electrónica , Modelos Moleculares , Subfragmentos de Miosina/química , Subfragmentos de Miosina/ultraestructura , Pliegue de Proteína , Miosinas del Músculo Liso/química , Miosinas del Músculo Liso/metabolismo , Miosinas del Músculo Liso/ultraestructura , PavosRESUMEN
Dyneins are the largest and most complex of the three classes of linear motor proteins in eukaryotic cells. The mass of the dynein motor domain is about ten times that of the other microtubule motor, kinesin. Dynein's homology with the AAA+ superfamily of mechanoenzymes distinguishes it from both kinesin and myosin, which share a common fold and ancestry as members of the G-protein superfamily. In contrast to the other motor proteins, little is known about the mechanism of dynein; its three-dimensional structure is unknown even at low resolution. Recent two-dimensional images from electron microscopy have revealed new details of its structure and how this changes to produce movement. These and the recently solved crystal structure of another AAA+ protein, ClpB, offer tantalising hints about dynein's mechanism, suggesting it may act like a molecular winch.
Asunto(s)
Dineínas/química , Proteínas de Escherichia coli/química , Proteínas de Choque Térmico/química , Cinesinas/química , Proteínas Motoras Moleculares/química , Animales , Microscopía por Crioelectrón , Endopeptidasa Clp , Unión Proteica , Conformación ProteicaRESUMEN
Here, we report on the structure and in situ location of arthrin (monoubiquitinated actin). Labelling of insect muscle thin filaments with a ubiquitin antibody reveals that every seventh subunit along the filament long-pitch helices is ubiquitinated. A three-dimensional reconstruction of frozen-hydrated arthrin filaments was produced. This was based on a novel algorithm that divides filament images into short segments that are used for single-particle image processing. Difference maps with an actin filament reconstruction locate ubiquitin at the side of actin sub-domain 1 opposite where myosin binds. Consistent with the reconstructions, peptide mapping places the ubiquitin linkage on lysine 118 in actin. Molecular modelling was used to generate arthrin monomers from ubiquitin and actin crystal structures. Filament models constructed from these monomers were compared with the arthrin reconstruction. The reconstruction suggests ubiquitin attached to Lys118 adopts one or a few conformers, stabilized by a small interface with actin. The function of actin ubiquitination is not known, but may involve regulation of muscle contractile activity.
Asunto(s)
Actinas/química , Proteínas de Drosophila/química , Proteínas de Microfilamentos/química , Proteínas Musculares/química , Ubiquitina/metabolismo , Actinas/metabolismo , Algoritmos , Secuencia de Aminoácidos , Animales , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citología , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Proteínas de Insectos , Proteínas de Microfilamentos/metabolismo , Modelos Moleculares , Fibras Musculares Esqueléticas/química , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/ultraestructura , Proteínas Musculares/metabolismo , Estructura Cuaternaria de ProteínaRESUMEN
Cytoplasmic dynein is a dimeric AAA(+) motor protein that performs critical roles in eukaryotic cells by moving along microtubules using ATP. Here using cryo-electron microscopy we directly observe the structure of Dictyostelium discoideum dynein dimers on microtubules at near-physiological ATP concentrations. They display remarkable flexibility at a hinge close to the microtubule binding domain (the stalkhead) producing a wide range of head positions. About half the molecules have the two heads separated from one another, with both leading and trailing motors attached to the microtubule. The other half have the two heads and stalks closely superposed in a front-to-back arrangement of the AAA(+) rings, suggesting specific contact between the heads. All stalks point towards the microtubule minus end. Mean stalk angles depend on the separation between their stalkheads, which allows estimation of inter-head tension. These findings provide a structural framework for understanding dynein's directionality and unusual stepping behaviour.
Asunto(s)
Adenosina Trifosfato/metabolismo , Dineínas Citoplasmáticas/metabolismo , Microtúbulos/metabolismo , Tubulina (Proteína)/metabolismo , Animales , Microscopía por Crioelectrón , Dineínas Citoplasmáticas/ultraestructura , Dictyostelium , Microscopía Electrónica , Microscopía Fluorescente , Microtúbulos/ultraestructura , Modelos Moleculares , Proteínas Motoras Moleculares , Porcinos , Tubulina (Proteína)/ultraestructuraRESUMEN
We show that negative-stain electron microscopy and image processing of nucleotide-free (apo) striated muscle myosin-2 subfragment-1 (S1), possessing one light chain or both light chains, is capable of resolving significant amounts of structural detail. The overall appearance of the motor and the lever is similar in rabbit, scallop and chicken S1. Projection matching of class averages of the different S1 types to projection views of two different crystal structures of apo S1 shows that all types most commonly closely resemble the appearance of the scallop S1 structure rather than the methylated chicken S1 structure. Methylation of chicken S1 has no effect on the structure of the molecule at this resolution: it too resembles the scallop S1 crystal structure. The lever is found to vary in its angle of attachment to the motor domain, with a hinge point located in the so-called pliant region between the converter and the essential light chain. The chicken S1 crystal structure lies near one end of the range of flexion observed. The Gaussian spread of angles of flexion suggests that flexibility is driven thermally, from which a torsional spring constant of ~23 pN·nm/rad² is estimated on average for all S1 types, similar to myosin-5. This translates to apparent cantilever-type stiffness at the tip of the lever of 0.37 pN/nm. Because this stiffness is lower than recent estimates from myosin-2 heads attached to actin, we suggest that binding to actin leads to an allosteric stiffening of the motor-lever junction.
Asunto(s)
Cadenas Ligeras de Miosina/química , Subfragmentos de Miosina/química , Animales , Pollos , Procesamiento de Imagen Asistido por Computador , Metilación , Microscopía Electrónica , Cadenas Ligeras de Miosina/metabolismo , Subfragmentos de Miosina/metabolismo , Pecten/química , Conformación Proteica , ConejosRESUMEN
Flagellar dyneins are essential microtubule motors in eukaryotes, as they drive the beating motions of cilia and flagella. Unlike myosin and kinesin motors, the track binding mechanism of dyneins and the regulation between the strong and weak binding states remain obscure. Here we report the solution structure of the microtubule-binding domain of flagellar dynein-c/DHC9 (dynein-c MTBD). The structure reveals a similar overall helix-rich fold to that of the MTBD of cytoplasmic dynein (cytoplasmic MTBD), but dynein-c MTBD has an additional flap, consisting of an antiparallel b sheet. The flap is positively charged and highly flexible. Despite the structural similarity to cytoplasmic MTBD, dynein-c MTBD shows only a small change in the microtubule- binding affinity depending on the registry change of coiled coil-sliding, whereby lacks the apparent strong binding state. The surface charge distribution of dynein-c MTBD also differs from that of cytoplasmic MTBD, which suggests a difference in the microtubule-binding mechanism.
Asunto(s)
Chlamydomonas reinhardtii/metabolismo , Dineínas/química , Microtúbulos/metabolismo , Proteínas de Plantas/química , Sitios de Unión , Dineínas/metabolismo , Simulación de Dinámica Molecular , Proteínas de Plantas/metabolismo , Estructura Secundaria de ProteínaRESUMEN
Dynein ATPases are the largest known cytoskeletal motors and perform critical functions in cells: carrying cargo along microtubules in the cytoplasm and powering flagellar beating. Dyneins are members of the AAA+ superfamily of ring-shaped enzymes, but how they harness this architecture to produce movement is poorly understood. Here, we have used cryo-EM to determine 3D maps of native flagellar dynein-c and a cytoplasmic dynein motor domain in different nucleotide states. The structures show key sites of conformational change within the AAA+ ring and a large rearrangement of the "linker" domain, involving a hinge near its middle. Analysis of a mutant in which the linker "undocks" from the ring indicates that linker remodeling requires energy that is supplied by interactions with the AAA+ modules. Fitting the dynein-c structures into flagellar tomograms suggests how this mechanism could drive sliding between microtubules, and also has implications for cytoplasmic cargo transport.
Asunto(s)
Adenosina Trifosfato/química , Dineínas Axonemales/química , Chlamydomonas reinhardtii/enzimología , Dictyostelium/enzimología , Adenosina Difosfato/química , Dineínas Axonemales/ultraestructura , Axonema/ultraestructura , Microscopía por Crioelectrón , Microscopía por Video , Microtúbulos/química , Microtúbulos/ultraestructura , Modelos Moleculares , Proteínas de Plantas/química , Proteínas de Plantas/ultraestructura , Unión Proteica , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Proteínas Protozoarias/química , Proteínas Protozoarias/ultraestructura , Homología Estructural de ProteínaRESUMEN
Despite more than 40 years of investigation since the discovery of dynein [Gibbons, I. R. and Rowe, A. J. (1965). Science149, 424-426] our understanding of how this microtubule-based motor generates force and movement remains frustratingly incomplete at the atomic level. Electron microscopy (EM) has played a major role in establishing dynein's complex architecture and its nucleotide-dependent conformational changes. In this chapter we review recent structural studies and describe in detail negative stain EM and computational single-particle image processing techniques that have been used to investigate dynein. We describe studies of both Chlamydomonas flagellar inner arm dynein-c and recombinant cytoplasmic dynein from Dictyostelium. We also detail methods for locating green fluorescent protein (GFP) and blue fluorescent protein (BFP) tags inserted at specific locations within the dynein motor, which can be used to map subdomains and conformational changes.
Asunto(s)
Dineínas/ultraestructura , Procesamiento de Imagen Asistido por Computador/métodos , Microscopía Electrónica/métodos , Microscopía por Crioelectrón/instrumentación , Microscopía por Crioelectrón/métodos , Dineínas/genética , Microscopía Electrónica/instrumentación , Modelos Moleculares , Conformación Proteica , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/ultraestructuraRESUMEN
Coupling between ATPase and track binding sites is essential for molecular motors to move along cytoskeletal tracks. In dynein, these sites are separated by a long coiled coil stalk that must mediate communication between them, but the underlying mechanism remains unclear. Here we show that changes in registration between the two helices of the coiled coil can perform this function. We locked the coiled coil at three specific registrations using oxidation to disulfides of paired cysteine residues introduced into the two helices. These trapped ATPase activity either in a microtubule-independent high or low state, and microtubule binding activity either in an ATP-insensitive strong or weak state, depending on the registry of the coiled coil. Our results provide direct evidence that dynein uses sliding between the two helices of the stalk to couple ATPase and microtubule binding activities during its mechanochemical cycle.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Dineínas/química , Dineínas/metabolismo , Microtúbulos/metabolismo , Proteínas Protozoarias/química , Proteínas Protozoarias/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Dictyostelium , Locomoción , Modelos Moleculares , Datos de Secuencia Molecular , Unión Proteica , Estructura Secundaria de ProteínaRESUMEN
Inner-arm dynein-f of Chlamydomonas flagella is a heterodimeric dynein. We performed conventional in vitro motility assays showing that dynein-f translocates microtubules at the comparatively low velocity of approximately 1.2 microm/s. From the dependence of velocity upon the surface density of dynein-f, we estimate its duty ratio to be 0.6-0.7. The relation between microtubule landing rate and surface density of dynein-f are well fitted by the first-power dependence, as expected for a processive motor. At low dynein densities, progressing microtubules rotate erratically about a fixed point on the surface, at which a single dynein-f molecule is presumably located. We conclude that dynein-f has high processivity. In an axoneme, however, slow and processive dynein-f could impede microtubule sliding driven by other fast dyneins (e.g., dynein-c). To obtain insight into the in vivo roles of dynein-f, we measured the sliding velocity of microtubules driven by a mixture of dyneins -c and -f at various mixing ratios. The velocity is modulated as a function of the ratio of dynein-f in the mixture. This modulation suggests that dynein-f acts as a load in the axoneme, but force pushing dynein-f molecules forward seems to accelerate their dissociation from microtubules.
Asunto(s)
Chlamydomonas reinhardtii/enzimología , Dineínas/química , Flagelos/química , Animales , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , ATPasa de Ca(2+) y Mg(2+)/metabolismo , Movimiento Celular , Chlamydomonas reinhardtii/química , Chlamydomonas reinhardtii/fisiología , Dineínas/ultraestructura , Flagelos/fisiología , Microscopía Electrónica , Modelos Moleculares , Fragmentos de Péptidos/químicaRESUMEN
The p7 protein of hepatitis C virus functions as an ion channel both in vitro and in cell-based assays and is inhibited by amantadine, long alkyl chain imino-sugar derivatives, and amiloride compounds. Future drug design will be greatly aided by information on the stoichiometry and high resolution structure of p7 ion channel complexes. Here, we have refined a bacterial expression system for p7 based on a glutathione S-transferase fusion methodology that circumvents the inherent problems of hydrophobic protein purification and the limitations of chemical synthesis. Rotational averaging and harmonic analysis of transmission electron micrographs of glutathione S-transferase-FLAG-p7 fusion proteins in liposomes revealed a heptameric stoichiometry. The oligomerization of p7 protein was then confirmed by SDS-PAGE and mass spectrometry analysis of pure, concentrated FLAG-p7. The same protein was also confirmed to function as an ion channel in suspended lipid bilayers and was inhibited by amantadine. These data validate this system as a means of generating high resolution structural information on the p7 ion channel complex.
Asunto(s)
Canales Iónicos/química , Proteínas Virales/fisiología , Amantadina/química , Animales , Cromatografía Líquida de Alta Presión , Diseño de Fármacos , Electroforesis en Gel de Poliacrilamida , Escherichia coli/metabolismo , Glutatión Transferasa/metabolismo , Membrana Dobles de Lípidos/química , Membrana Dobles de Lípidos/metabolismo , Microscopía Electrónica de Transmisión , Plásmidos/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Porcinos , Proteínas Virales/químicaRESUMEN
Flexible macromolecules pose special difficulties for structure determination by crystallography or NMR. Progress can be made by electron microscopy, but electron cryo-microscopy of unstained, hydrated specimens is limited to larger macromolecules because of the inherently low signal-to-noise ratio. For three-dimensional structure determination, the single particles must be invariant in structure. Here, we describe how we have used negative staining and single-particle image processing techniques to explore the structure and flexibility of single molecules of two motor proteins: myosin and dynein. Critical for the success of negative staining is a hydrophilic, thin carbon film, because it produces a low noise background around each molecule, and stabilises the molecule against damage by the stain. The strategy adopted for single-particle image processing exploits the flexibility available within the SPIDER software suite. We illustrate the benefits of successive rounds of image alignment and classification, and the use of whole molecule averages and movies to analyse and display both structure and flexibility within the dynein motor.
Asunto(s)
Microscopía por Crioelectrón/métodos , Dineínas/química , Miosinas/química , Animales , Colorantes , Dineínas/ultraestructura , Moluscos , Miosinas/ultraestructuraRESUMEN
Dynein ATPases are microtubule motors that are critical to diverse processes such as vesicle transport and the beating of sperm tails; however, their mechanism of force generation is unknown. Each dynein comprises a head, from which a stalk and a stem emerge. Here we use electron microscopy and image processing to reveal new structural details of dynein c, an isoform from Chlamydomonas reinhardtii flagella, at the start and end of its power stroke. Both stem and stalk are flexible, and the stem connects to the head by means of a linker approximately 10 nm long that we propose lies across the head. With both ADP and vanadate bound, the stem and stalk emerge from the head 10 nm apart. However, without nucleotide they emerge much closer together owing to a change in linker orientation, and the coiled-coil stalk becomes stiffer. The net result is a shortening of the molecule coupled to an approximately 15-nm displacement of the tip of the stalk. These changes indicate a mechanism for the dynein power stroke.