Journal of

Structural

Journal of Structural Biology 146 (2004) 205–216

Biology

www.elsevier.com/locate/yjsbi

The structure of dynein-c by negative stain electron microscopy

S.A. Burgess,a,* M.L. Walker,a H. Sakakibara,b K. Oiwa,b and P.J. Knighta

a Astbury Centre for Structural Molecular Biology and School of Biomedical Sciences, University of Leeds, Leeds LS2 9JT, UKb Kansai Advanced Research Centre, Communications Research Laboratory, Kobe 651-2492, Japan

Received 5 September 2003, and in revised form 1 October 2003

Abstract

Dynein ATPases contain six concatenated AAA modules within the motor region of their heavy chains. Additional regions of

sequence are required to form a functional ATPase, which a previous study suggested forms seven or eight subdomains arranged in

either a ring or hollow sphere. A more recent homology model of the six AAA modules suggests that these form a ring. Therefore

both the number and arrangement of subdomains remain uncertain. We show two-dimensional projection images of dynein-c in

negative stain which reveal new details of its structure. Initial electron cryomicroscopy shows a similar overall morphology. The

molecule consists of three domains: stem, head, and stalk. In the absence of nucleotide the head has seven lobes of density forming

an asymmetric ring. An eighth lobe protrudes from one side of this heptameric ring and appears to join the elongated cargo-binding

stem. The proximal stem is flexible, as is the stalk, suggesting that they act as compliant elements within the motor. A new analysis of

pre- and post-power stroke conformations shows the combined effect of their flexibility on the spatial distribution of the micro-

tubule-binding domain and therefore the potential range of power stroke sizes. We present and compare two alternative models of

the structure of dynein.

� 2003 Elsevier Inc. All rights reserved.

Keywords: Dynein; AAA protein; Molecular motor; Flagella; Cilia; Axoneme; Electron microscopy

1. Introduction

Dynein ATPases are minus-end directed microtubule

motors found widely among eukaryotic species (Gib-

bons, 1995). Cytoplasmic isoforms participate in a va-

riety of roles within the cytoplasm, including the

positioning and trafficking of numerous organelles

(Karki and Holzbaur, 1999). Axonemal isoforms pro-

duce the bending motions of cilia and flagella by driving

sliding between adjacent microtubule doublets (DiBellaand King, 2001). Within the 9+ 2 axoneme, which is the

most common arrangement of microtubules in cilia and

flagella, dyneins are typically arranged in two distinct

rows along each of the nine microtubule doublets,

forming the inner and outer arms. Dynein motors are

large and complex macromolecular assemblies, com-

posed of between one and three heavy chains (each

>500 kDa) together with a number of intermediate and

* Corresponding author. Fax: +44-113-343-4228.

E-mail address: s.a.burgess@leeds.ac.uk (S.A. Burgess).

1047-8477/$ - see front matter � 2003 Elsevier Inc. All rights reserved.

doi:10.1016/j.jsb.2003.10.005

light chains (DiBella and King, 2001; King, 2000b).

Unlike the myosin and kinesin families of linear motorproteins (Vale and Milligan, 2000), no atomic resolution

structures have yet been determined for dynein and

therefore little is known about the structure and mode of

action of this class of motor.

Each heavy chain folds to form three morphological

and functional domains. The N-terminal third forms an

elongated cargo-binding stem to which the intermediate

and most of the light chains bind (Asai and Koonce,2001; King, 2000a; King, 2003). In flagellar dyneins the

stem binds to the A-tubule of a microtubule doublet.

The stem is also the site of heavy chain multimerization.

The C-terminal two-thirds of the heavy chain contains

the motor (Asai and Koonce, 2001; Harrison and King,

2000; King, 2000b). This consists of a head, about 13 nm

in diameter (Samso et al., 1998), from which extends the

stalk domain, 10–15 nm long (depending on isoform), atthe end of which is a small microtubule-binding domain

(Goodenough and Heuser, 1984; Goodenough et al.,

1987; Sale et al., 1985) which interacts with the micro-

tubule track of this motor (Vallee and Gee, 1998).

206 S.A. Burgess et al. / Journal of Structural Biology 146 (2004) 205–216

In flagellar dyneins this is the B-tubule of the adjacentmicrotubule doublet and so the stalk is sometimes called

the B-link.

The head domain contains six AAA modules

(Fig. 1A) (King, 2000a; Neuwald et al., 1999). The N-

terminal one (AAA1) is the most highly conserved

among the dynein family (Gibbons, 1995) and is con-

sidered the probable site of ATP hydrolysis based on

photocleavage experiments (Gibbons et al., 1991; Gib-bons et al., 1987). Little is known about the roles of

AAA2–4 which, despite possessing P-loop motifs (Gib-

bons et al., 1991; Ogawa, 1991), are not thought to have

significant ATPase activity (Mocz and Gibbons, 1996).

However, one or more of these modules binds nucleo-

tide (Mocz and Gibbons, 1996), probably ADP, since

this regulates the activity of the motor (Shiroguchi and

Toyoshima, 2001; Yagi, 2000). Interestingly, blocking ofnucleotide binding to AAA3 was recently identified as

preventing the ATP-induced release of dynein from

microtubules, establishing the importance of nucleotide

binding outside the active site (Silvanovich et al., 2003).

AAA5 and 6 are not thought to bind nucleotide since

they have lost their P-loops during the course of evo-

lution (Gibbons et al., 1991; Mocz and Gibbons, 1996;

Mocz et al., 1998; Neuwald et al., 1999; Ogawa, 1991).Beyond AAA6 is the C-terminal region of 150–450

amino acids with no known homology or structure but

apparently required for functional ATPase activity (Gee

et al., 1997; Koonce and Samso, 1996).

The stalk emerges from between AAA 4 and 5 (King,

2000a). The microtubule-binding domain at its distal

end changes its affinity for the microtubule depending

upon the nucleotide in the active site (Koonce andTikhonenko, 2000). Based on structure prediction from

the primary sequence (Gibbons et al., 1991) and on its

dimensions in electron micrographs (Gee et al., 1997),

the stalk most probably consists of an antiparallel

coiled-coil (Gee and Vallee, 1998; Vallee and Gee, 1998).

In the absence of high resolution experimental data,

homology modelling of the dynein AAA modules has

produced a useful hypothetical atomic structure (MoczandGibbons, 2001) inwhich the six AAAmodules form a

planar six-membered ring, possessing quasi-6-fold sym-

metry about an axis running through a central channel

(Fig. 1C). However, the model does not predict the out-

come of truncation studies on cytoplasmic dynein which

have shown that in addition to the six AAAmodules and

the stalk, two other regions of the heavy chain sequence

are required to produce a functional ATPase (Gee et al.,1997; Samso et al., 1998). These are the entire sequence

C-terminal to AAA6 and about 450 residues N-terminal

of AAA1 (Fig 1A). Negative stain electron microscopy

followed by single-particle image processing has sug-

gested that such a functional ATPase fragment possesses

a number of subdomains arranged in a ring about a cen-

tral region of dense staining, presumed to be a channel or

cavity (Samso et al., 1998). The clearest class averagesshowed seven similarly sized subdomains arranged fairly

evenly about the central channel, suggesting that one of

the non-AAA regions folds to form a subdomain intrinsic

to the ring (King, 2000a). However, the lack of structural

information about the non-AAA regions of the dynein

head has precluded the building of a more complete

homology model.

Recently, we described the structure of dynein-c inpre- and post-power stroke conformations by negative

stain electron microscopy and single-particle image

processing (Burgess et al., 2003). Dynein-c is a proces-

sive, single-headed inner arm species from Chlamydo-

monas reinhardtii flagella (Sakakibara et al., 1999) and

has a single heavy chain plus three light chains: one actin

monomer and two copies of a 28 kDa protein (p28)

which all bind to the N-terminal region of the heavychain (LeDizet and Piperno, 1995; Yanagisawa and

Kamiya, 2001). We found that the structure of the head

is complex. It has an asymmetric ring-like appearance of

subdomains and the two faces are different. Part of the

stem �10 nm long (the linker), directly visible only in

perturbed (undocked) molecules, is thought to lie across

one face of the head, contributing to the complexity of

the head structure. Because the stem is seen to moverelative to the head on product release, we proposed that

a swinging movement of the linker is a major contrib-

utor to the power stroke of dynein. The magnitude of

the power stroke is difficult to determine from these

images because of flexibility in both stem and stalk, but

we estimated it to be about 15 nm (Burgess et al., 2003).

Here, using negatively stained and frozen-hydrated

preparations, we extend the analysis of dynein-c to re-veal new features of its substructure. We also refine our

previous analysis of pre- and post-power stroke dynein-c

(Burgess et al., 2003) by aligning the stems of

ADP.vanadate (Vi) and apo-molecules, respectively

(Johnson, 1985). This produces overlapping distribu-

tions of microtubule-binding domains resulting from the

combined flexibility of the stem and stalk. Finally, we

show two alternative structural models for dyneinderived from these and others� data.

2. Materials and methods

2.1. Protein preparation, electron microscopy, and digiti-

zation

Dynein-c was isolated from an outer armless mutant

(oda1) of C. reinhardtii flagella as described (Sakakibara

et al., 1999) except to improve the quality of negative

staining the elution buffer for the final anion exchange

column was a KCl gradient in 30mM MOPS, 5mM

MgCl2, 1mM EGTA, and 0.1mM dithiothreitol, pH 7.4

(MMED buffer). Fresh stock dynein samples (�0.65 lM

S.A. Burgess et al. / Journal of Structural Biology 146 (2004) 205–216 207

protein and about 200mMKCl), or thawed droplets thathad been stored in liquid nitrogen after drop freezing,

were diluted 40-fold into MMED buffer containing

200mM KCl and applied to the carbon coated grid pre-

treated with ultraviolet light as described (Walker et al.,

2000), before staining with 1% uranyl acetate. These

specimens are referred to as being in high ionic strength

buffer. Other specimens were rinsed on the grid with

MMEDbuffer lackingKCl to improve staining. These arereferred to as being in low ionic strength buffer. Frozen-

hydrated specimens were prepared by diluting stock dy-

nein 3-fold into MMED buffer lacking KCl and applying

this to grids coated with a lacey carbon film that had been

glow-discharged in amyl amine. Grids were frozen by

plunging into liquid ethane after two-sided automatic

blotting for 1 s as described (White et al., 1998).

Microscopy was performed on a JEOL 1200EX oper-ating at 80 kV with a LaB6 electron source and a 30 lmobjective aperture which excluded diffraction beyond

1/1.7 nm�1. Micrographs were taken at a nominal mag-

nification of 40 000 times. Frozen-hydrated specimens

were taken at a nominal underfocus of 2.5 lm. No CTF

correction was applied to the images. Micrographs were

digitized on either a Leafscan 45 (Leaf Systems, South-

borough, MA) or an Imacon Flextight 848 scanner (Im-acon A/S, Copenhagen, Denmark), at 20 lm intervals at

the micrograph, corresponding to a pixel size of 0.514 nm

at the specimen (calibrated using the paramyosin repeat

of 14.4 nm).

2.2. Image processing: whole-molecule alignment and

classification

Digital micrographs were imported into the SPIDER

suite of programs (Frank, 1996) for all subsequent im-

age processing. Particle picking was performed manu-

ally using the computer mouse to locate the

approximate centre of the head domain. This location

within the molecule affects subsequent alignment steps

and we found this to be the optimal location. From

negatively stained preparations we picked 5060 apo-dynein-c particles from 20 micrographs from the high

ionic strength buffer preparation and 7412 apo-dynein-c

particles from 31 micrographs from the low ionic

strength buffer preparation. These two data sets were

treated separately. Windowed images were brought into

mutual alignment by a reference-free strategy. This

produced an initial alignment in which head and stem

were visible in the global average. These are referred toas �whole molecule-aligned� images. Image classification

was performed using K-means clustering as described

(Burgess et al., 1997), using a mask that enclosed

both head and stem only. This produced the initial

classification.

Micrographs of frozen-hydrated molecules were

treated the same as those from negative stain in high and

low ionic strength buffers. From 15 micrographs, 1411particles were picked.

2.3. Head alignment of molecules in low ionic strength

buffer

Different views of the molecule (left, side, and right)

were selected from among classes after the preceding

whole-molecule alignment and processed separately in asecond round of alignment and classification. This was

necessary because flexibility between the head and stem

causes neither of these domains to be fully in register after

the first round, requiring a second round of alignment to

refine the orientation of the heads, thereby obtaining an

alignment of this part of the molecule. The second round

of alignment was entirely new and performed separately

on each of the selected views. Smaller images were win-dowed to contain just the head domain. These were

masked to remove high radius features, but included the

stem and stalk close towhere they join the head to assist in

rotational alignment. Reference-free alignment was per-

formed and the calculated alignment parameters (rota-

tion and translation) were applied to new, larger, and

unmasked windowed images, sufficiently large to contain

the entire stem and stalk. We refer to these images as�head-aligned.� Because of their flexibility, distal parts ofstalk and stem become smeared out in such head-aligned

images. We found that within individual molecules the

flexibility of their stem and stalk was not correlated,

producing many independent conformations. Too many

different conformations exist for a single classification to

capture all of their details optimally. Therefore we clas-

sified the images several times independently (usingK-means clustering) using a number of different masks to

isolate either the stalk or the stem.Masks sufficiently large

to contain all different positions of stem and stalk were

created to maximize the number of classes capturing their

different positions.However, it cannot be guaranteed that

all conformations are captured, particularly those that are

extreme and/or sparsely populated, since there is a trade-

off between increasingmask size and decreasing signal-to-noise ratio (i.e., particle-to-background) within themask.

Themore classes that are allowed, the finer the gradations

of orientation that are detected. With fewer classes, the

stems become increasingly smeared in the averages be-

cause a wider range of orientations are combined. This

indicates that the flexibility is distributed as a continuum,

like a bending spring, rather than discrete states. We refer

to these conformers that result from flexibility asfleximers.

2.4. Stem alignment of pre- and post-power stroke

molecules

Computational alignment of the stems of pre- and

post-power stroke molecules was performed as follows.

208 S.A. Burgess et al. / Journal of Structural Biology 146 (2004) 205–216

Images of head-aligned molecules in the left view wereused after classification to show their various stem po-

sitions. Class averages (not individual raw images) from

both apo- and ADP.Vi-data sets were combined to

make a new single data set. These class averages were

brought into mutual alignment according to their stems

by reference-free methods after applying a mask to each

image to obscure the head and stalk. This produced a

series of alignment parameters that when applied toeach stem class average brought their stems into align-

ment. As expected this caused their heads and stalks to

become smeared. The next step was to determine the

corresponding change in the position of the stalks as

described below.

First, we identified each individual molecule in which

the positions of both stem and stalk had been identified

in previous classifications of these regions after headalignment. Many molecules are lost during classification

of the stem and stalk because one or other of these

structures fall into a class that shows poor detail. Thus,

from 1454 apo-molecules and 1733 ADP.Vi-molecules

originally classified, 680 and 825, respectively, were

obtained in which both stem and stalk positions were

identified.

Second, the image coordinates of the tip of the stalkof each of these individual molecules had already been

identified (manually) using class averages after head

alignment. To these coordinates we applied the rotation

and translation parameters derived from stem align-

ment, thus transforming them to their new positions.

For every individual molecule we then selected the stem

and stalk class averages to which it had been assigned

and spliced them together to create a noise-reducedversion. These were then rotated and translated to align

their stems using the alignment parameters determined

above, then averaged to create the apo- and ADP.Vi-

global averages shown in Fig. 4. The stalk tip positions

are shown as scatter plots superposed on these.

3. Results

3.1. Head substructure

Dynein-c molecules adsorb to the carbon film over a

broad range of orientations when stained for micros-

copy directly from the higher ionic strength buffer

(Fig. 1B). Although we do not know the precise rela-

tionship between each of these views, the point ofemergence of the stem from the head correlates with

different head morphologies, giving an indication of the

head�s general orientation. The stalk is missing from

these image averages because of its thinness and flexi-

bility. Left, side, and right views are most common, but

views intermediate between these are also seen (Fig. 1B).

Side views clearly show that the head is wider across the

top than the bottom, indicating that the two faces seenin left and right views are inclined relative to one

another.

Improved structural detail is obtained by rinsing the

specimen on the grid with low ionic strength buffer be-

fore staining (Fig. 1D) although the range of views is

then more restricted. Global averages of left, side, and

right views after head alignment reveal substructure

within the head and the emergence point of the stalk(Fig. 1D, white arrowheads).

The left view shows a number of subdomains within

the head. These are clearer in contour plots of subclass

averages (Fig. 1F). Such plots show that one roughly

circular subdomain, �4 nm in diameter, is very promi-

nent (Fig. 1F, arrowhead). It lies close to where the stem

and stalk appear to join the head. In addition to this

subdomain, contour plots indicate at least six moresubdomains of varying sizes arranged irregularly in a

ring (Fig. 1F, small arrows).

In side view a lateral subdomain, about �3� 5 nm in

size, protrudes from the left side of the head at an angle

of about 30� to the long axis of the molecule (Fig. 1D,

black arrowhead). Examination of class averages inter-

mediate between left and side views, indicate that the

prominent subdomain of the left view is part of thelateral subdomain (Fig. 1E, arrowheads). It is clear that

the stalk does not emerge from the lateral subdomain

(see also Fig. 3). By contrast, the stem, though not

clearly imaged in side views close to where it joins the

head, does emerge from the lateral subdomain (see

Fig. 3). The fact that the stem is clearly imaged joining

the head in left views (Fig. 1D, arrow) argues that it is in

contact with the carbon film. Its absence in side viewssuggests it is elevated above the stain. Thus the side view

is related to the left view by a right-handed rotation.

This also indicates that the lateral subdomain lies be-

tween the head and the carbon film in left views, ac-

counting for the marked deposition of stain around it.

In low ionic strength buffer we do not see the view that

shows the lateral subdomain protruding from the right-

hand side of the head, but occasionally it is seen inpreparations in high ionic strength buffer (Fig. 1B,

arrowheads).

In right views the subdomain organization of the

head is clearest. Three well-defined peripheral subdo-

mains of similar size (�3–4 nm in diameter), appear

distinct from the mass encircling the central stain de-

posit (Figs. 1D and G, asterisks). In addition to these

three peripheral subdomains, a further four can beidentified in contour plots of subclass averages (Fig. 1G,

small arrows). Their sizes and shapes vary, as do their

distances from the accumulation of stain near the centre

of the head. Right views lack the prominent subdomain

seen in left views, consistent with the lateral subdomain

now lying on top of the head and therefore above the

stain layer, as expected from our interpretation of left

Fig. 1. Dynein heavy chain topology, overview of apo-dynein-c

structure and homology model of dynein. (A) Arrangement of AAA

modules (numbered 1–6) and the ATP-sensitive microtubule-binding

domain (M, shaded) within the heavy chain sequence of dynein.

Horizontal distances are to scale. Black bar indicates the minimal re-

gion required to produce a functional ATPase. (B) Overview of dynein-

c morphology from specimens prepared in high ionic strength buffer.

Selected orientations, including left, side, and right views, of the

molecule on the carbon film. Arrowheads show the lateral subdomain.

(C) Homology model of dynein (PDB code:1hn5) shown in three ori-

entations at the same scale as (D). AAA modules are coloured as in

(A), and numbered. (D) Selected subclasses from head-aligned dynein-

c molecules stained after a low salt rinse. White arrowhead indicates

the base of the stalk; black arrow indicates bend in the neck of the

stem; black arrowhead indicates the lateral subdomain and is aligned

with its long axis; asterisks indicate peripheral subdomains. (E) In-

termediate views between left and side views show that the prominent

subdomain in left views is the lateral subdomain in side views (ar-

rowheads). Contour plots of selected subclass averages of left (F) and

right (G) views showing a number of additional subdomains (arrows)

surrounding the central stain deposit. Together with the prominent

subdomain of left views (arrowhead) and the three subdomains seen in

right views (asterisks), seven subdomains in all are seen in each view.

Large arrows in (F) and (G) indicate an indentation in the periphery of

the head. Number of images per class range between (B) 27 and 43, (E)

38 and 45, (F) 192 and 306, (G) 72 and 95. Number of images in (D)

left¼ 3148; side¼ 416; and right¼ 1335. Scale bars in (B) 20 nm,

(C–G) 10 nm.

S.A. Burgess et al. / Journal of Structural Biology 146 (2004) 205–216 209

and side views. An indentation in the outer perimeter ofthe head is visible adjacent to the peripheral subdomain

furthest from the stalk (Fig. 1G, big arrow). The posi-

tion of this indentation relative to the stalk is equivalent

to that of a similar indentation seen in left views

(Fig. 1F, big arrow), but otherwise the left and right

faces are dissimilar and their subdomain morphologies

are not mirror images. We conclude that right views

show that seven subdomains form an asymmetric ring inthe head of dynein-c.

3.2. Stem substructure

The stem consists of three distinct subdomains: neck,

shaft, and base (Figs. 2A and B) in addition to the linker

described previously (Burgess et al., 2003). The neck

emerges from the head tangentially and is a narrowstructure about 2 nm wide and 8 nm long. Where it joins

the head the neck bends sharply. Distal to the neck, the

shaft subdomain is composed of two roughly parallel

structures, each about 2 nm wide and 8 nm long. One of

these is contiguous with the neck and appears to make

contact with the other at two discrete points. In speci-

mens rinsed in low ionic strength buffer before staining

these two parallel structures are not seen clearly, sug-gesting a systematic change in the orientation of the

stem under these conditions. At its distal end the base of

the stem is resolved into two subdomains of different

sizes, totalling �8 nm long. The more proximal of these

appears to contact both elongated structures of the

shaft. A summary of our interpretation of these sub-

domains is shown in Fig. 2B. A similar morphology is

seen in favourable right views (Fig. 2A, lower row) re-lated to left views (Fig. 2A, upper row) by mirror sym-

metry, except that the neck is usually less clear in right

views, consistent with our interpretation that the stem

emerges from one face of the head.

3.3. Stem flexibility

The neck of the stem is flexible (Fig. 3A). The sharpbend in the neck close to where it joins the head is

preserved in the global average of left views (Fig. 1D,

arrow), indicating that this structure does not move as

the stem flexes. This implies that stem distortion occurs

either by pivoting within this bend or by bending more

distally along the neck. We examined this in detail in

left, side, and right views from molecules rinsed with low

ionic strength buffer before staining. Classification of thestem region of head-aligned molecules produces classes

that differ principally in the orientation of the long axis

of the stem (Fig. 3A). We analysed the location of stem

flexibility by extrapolating straight lines drawn manually

through the shaft and base of each stem fleximer where

these were seen clearly. The distribution of their inter-

cepts with one another was determined by applying a

Fig. 2. Stem substructure. (A) Selected class averages of left (upper

row) and right (lower row) views of the molecule in high ionic strength

buffer. The stem has three subdomains, neck (N), shaft (S), and base

(B) indicated in the cartoon in (B). (C) Molecules in low ionic strength

buffer show a range of morphologies, displaying identical appearances

in left (upper row) and right (lower row) orientations of the head.

Number of images per class range between (A) 28 and 60, (C) 33 and

65. Scale bar: 20 nm.

210 S.A. Burgess et al. / Journal of Structural Biology 146 (2004) 205–216

low-pass (Gaussian) filter to an image containing these

lines superposed. Contour plots of these are shown for

left, side, and right views (Fig. 3B). In each case the

centre of gravity of the intercepts is located to a position

Fig. 3. Stem flexibility is located in the neck subdomain. (A) Selected class av

to create composite images in which both these structures are visible. In each

is seen clearly only in left views. (B) Contour plots showing the density distrib

shaft and base of each stem class average, superposed on class averages show

stem in left and right views. Stem angles were measured relative to the mean

Number of images per class in (A) left stalks: 37, 33, 31; left stems: 22, 59, 13;

right stems: 20, 24, 26. Scale bar: (A) 30 nm; (B) 15 nm.

outside the head, and 20 nm from the distal end of thestem, placing it within the neck.

The extent of stem flexibility is altered by the orien-

tation of the head on the carbon substrate (Fig. 3C). The

distributions of stem angles indicate that in right views it

is considerably more flexible than in left views. As a

result, the contour plot of the right view is more nearly

circular in shape. The added range of flexibility occurs in

a direction towards the stalk. Some poorly populatedclass averages showing very smeared stems (see above)

suggest that the stem approaches very close to the stalk,

and possibly crosses over it (data not shown).

Stem flexibility also has a torsional as well as a planar

component. In left and right views a variety of stem

morphologies is seen in different class averages after

alignment of their stems (Fig. 2C). We interpret these as

different orientations of the stem on the carbon sub-strate. Essentially identical appearances are found in left

and right views after co-alignment of their stems. If

these different appearances do represent different

erages showing different stem and stalk positions were spliced together

row the stem is identical, in each column the stalk is identical. The neck

ution of the intercepts of extrapolated straight lines drawn through the

n also in Fig. 1D. (C) Histograms showing the range of flexibility of the

position of the stalk for each orientation, as indicated in the cartoons.

side stalks: 20, 21, 13; side stems: 22, 20, 19; right stalks: 24, 23, 32; and

Fig. 4. Power stroke of dynein-c. (A) Mean conformations of ADP.Vi-

and apo-molecules produced by splicing mean stem and mean stalk

images together. (B) Distribution of stalk tip positions from all indi-

vidual molecules in which both stem and stalk positions were captured

by classification (n ¼ 825 for ADP.Vi, n ¼ 680 for apo) after compu-

tational alignment of their stems. The scatter plots are superposed on

the global averages after this alignment, which shows, as expected,

smearing of the head and stalk. Scale bar: 15 nm.

S.A. Burgess et al. / Journal of Structural Biology 146 (2004) 205–216 211

orientations of the stem on the carbon substrate, whichseems the most probable explanation, their equivalence

in left and right orientations of the head demonstrate a

torsional component of flexibility of at least 180� withinthe stem. The role of such torsional flexibility in dynein

function is not clear.

3.4. Stalk substructure

Analysis of left views of the molecule showed the

stalk to be flexible and �2 nm wide by 15.5 nm long

(Burgess et al., 2003). In side views it looks very similar

to this, but in right views it appears shorter (12.3 nm)

suggesting that part of it is hidden in right views. In all

three views of the head, stalk fleximers are often curved

or kinked counter clockwise from base to tip (Fig. 3A).

Thus, contrary to expectation, the stalk in right views isnot curved in the opposite sense to that in left views.

3.5. Power stroke

Previously we reported the structure of ADP.Vi-dy-

nein-c as well as its structure in the absence of nucleotide

(apo-dynein-c). These biochemical states represent,

respectively, the pre- and post-power stroke conforma-tions of the motor (Johnson, 1985). As with the apo-

dynein-c molecules shown here, we found abundant left

views of ADP.Vi-dynein-c molecules showing flexibility

in the stalk and stem. Class averages showing mean

conformations of the stem and stalk relative to the head

(Fig. 4A) were used to estimate a mean size of the power

stroke of 15 nm (Burgess et al., 2003). We generated

these mean conformations of the whole molecule bysplicing together class averages in which stem and stalk

conformations were closest to their calculated mean

positions. The resulting images were then manually ro-

tated and translated to bring their stems into alignment.

Finally we measured the displacement of the tip of the

stalk (the microtubule-binding domain) to estimate the

mean size of the power stroke. However, the consider-

able length of the molecule (40–45 nm) makes the resulthighly sensitive to the rotational alignment between

ADP.Vi- and apo-molecules. An angle error of 5�changes the apparent power stroke by about 4 nm.

We have now refined this analysis by computationally

aligning the stems of all fleximers from both nucleotide

conditions. We then reconstructed all individual mole-

cules in which both stem and stalk position were cap-

tured by classification. This allowed us to plot thedistribution of stalk tip positions from all individual

molecules (Fig. 4B). This analysis produces a mean

displacement of the tip of the stalk of 15.6 nm, in close

agreement with our previous estimate. The new analysis

reveals the region explored by the tip of the stalk as a

result of the combined flexibility of stalk and stem. Their

partially overlapping distributions have both angular

and radial components, both of which are reduced inapo-molecules, as a result of the stiffer apo-stalk

(Burgess et al., 2003).

4. Discussion

4.1. Structure of the head

The negative-stained images of dynein-c that we have

analysed are the most detailed so far obtained of any

dynein. In particular, the stalk as well as the stem is

usually visible in raw images. In an earlier study of a

stemless construct of a cytoplasmic dynein the stalk was

not visible (Samso et al., 1998). Since the stem was also

absent there was a lack of landmarks for establishing

correct alignment during subsequent processing. Wesuspect that a consequent misalignment of such particles

may underlie the discrepancy between the roughly

symmetrical, spherical model that resulted from the

earlier work and the asymmetric, ring-like structure we

now find.

The 3D structure of the head of dynein remains un-

known, but the two-dimensional projection images

presented here provide important clues about some as-pects of it. Right views suggest that the head is con-

structed around a heptameric ring structure with the

seven lobes surrounding either a central channel, in-

dentation or cavity. Side views show an eighth lobe, the

lateral subdomain, protruding from one side of the head

and which joins the stem. It might be thought that

212 S.A. Burgess et al. / Journal of Structural Biology 146 (2004) 205–216

adsorption onto carbon, followed by staining and dry-ing have grossly distorted the molecule, but our initial

images of frozen-hydrated dynein-c by electron cry-

omicroscopy have yielded averages entirely compatible

with views seen in negative stain (Fig. 5). Therefore, we

conclude that the structure we have described is likely to

be real. The head of dynein is therefore more complex

than either earlier imaging or the homology model

suggested.The most unexpected feature of the head is its inter-

action with the linker portion of the stem described

previously (Burgess et al., 2003). The linker docks onto

the upper face of the head (when seen in the right view)

in two different positions governed by the nucleotide

bound in the active site (see also Fig. 6). When un-

docked, the linker is revealed as a relatively large

structure about 2 nm wide and 10 nm long. It is not yetclear whether the lateral subdomain is a new view of the

docked linker (and hence a component of the stem) or

whether this structure derives from the head (e.g., the

sequence N-terminal to AAA1, C-terminal to AAA6, or

between adjacent AAA modules) or has contributions

from both domains.

4.2. Comparisons with the homology model

In the homology model of dynein (Mocz and Gib-

bons, 2001), a channel runs through the head roughly

perpendicular to the plane of the six AAA modules and

along the axis of pseudo-6-fold symmetry (Fig. 1C,

upper and lower panels). In contrast, dynein-c has seven

lobes of density surrounding a central region of stain

accumulation. Uncertainty about whether the lateralsubdomain derives from the linker or the head means we

are unable to compare this feature to the model, but

such a feature cannot be reproduced by any orientation

of the model. Therefore, the number of subdomains in

the data and the model do not agree. However, the data

and model appear to share the same general architecture

of a ring-like arrangement of subdomains. Whether

Fig. 5. Compatible class averages from (A) frozen-hydrated and (B)

negatively stained molecules of dynein-c. Number of particles per class

range between (A) 15 and 20 and (B) 33 and 52. Scale bar 20 nm.

these form a simple planar ring structure or split washertype arrangement, is not yet clear.

The origin of the seventh subdomain of the head re-

mains a mystery. The authors of the homology model

pointed out the discrepancy between their pseudo-6-fold

symmetric structure and the first reported dynein

structure showing seven lobes (Samso et al., 1998), but

were unable to include additional subdomains in their

model because of a lack of structural data on the pre-sumed candidate sequences. The C-terminal sequence

has been proposed to form the seventh subdomain

(King, 2000a), since heavy chain constructs devoid of

this sequence lack ATPase activity (Gee et al., 1997;

Koonce and Samso, 1996). This deficiency is interpreted

as a failure of the construct to fold correctly, although it

should be noted that this interpretation does not ex-

plicitly require that the sequence forms a distinct sub-domain within the ring. The sequence N-terminal to

AAA1, whose presence is also essential for ATP hy-

drolysis (Gee et al., 1997), has also been proposed as the

potential seventh module (Fan and Amos, 2001).

Fig. 6. Two alternative models of dynein-c structure and power stroke

(see text). The head is composed of seven subdomains, including the six

AAA modules plus the C-terminus, arranged either counter clockwise

(A) or clockwise (C). The conformational change on product release,

inferred from the mean conformations (B), shows a swinging move-

ment of the linker. The conformational change in and between the

AAA modules is unknown. Right views of the apo-model (lower row)

illustrate the interaction of the linker with one face of the head.

S.A. Burgess et al. / Journal of Structural Biology 146 (2004) 205–216 213

Indeed, this region has been tentatively identified aspossessing a highly degenerate seventh AAA module

(Fan and Amos, 2001). A third possibility is that stret-

ches of sequence between one or more adjacent AAA

modules may form the seventh subdomain within the

ring. The sequence between AAA1 and 2 is relatively

short (�50 amino acids), but between AAA2 and 3 and

between AAA3 and 4 it is longer (�100 residues). Be-

tween AAA5 and 6 it is still longer (�220 residues). Atleast one of these intervening sequences, 100 amino acids

long, if folded compactly, would produce a structure

about 3 nm in diameter, sufficient to account for the

smallest subdomain seen in right views.

Whatever the origin of the seventh lobe, it is not yet

known whether the true structure of the dynein head is a

planar ring-like arrangement of AAA modules. The

homology model has a ring structure because it wasbased on hexameric assemblies of AAA oligomers

(Mocz and Gibbons, 2001). However, the model�s pre-

diction that the stalk and stem should emerge from the

head diametrically opposite one another is supported by

images of dynein-c in which the linker is undocked from

the head (Burgess et al., 2003). Therefore, in the absence

of high resolution experimental 3D data, it seems rea-

sonable to assume that the head of dynein contains aroughly planar ring of seven subdomains together with

additional mass that may either derive from the head or

from the linker portion of the stem (Fig. 6).

4.3. Stem structure and composition

Our images provide the most detailed structure of the

stem of any dynein to date and show it to be a com-plicated structure comprising linker, neck, shaft, and

base. In dynein-c the stem consists of some or all of the

heavy chain sequence N-terminal to AAA1, together

with two copies of a 28 kDa light chain (p28) and a

single molecule of actin (Yanagisawa and Kamiya,

2001). Dyneins have an N-terminal region upstream of

AAA1 of between 1600 and 1930 amino acid residues,

i.e., between 1/3 and 1/4 of the entire heavy chain se-quence (Mocz and Gibbons, 2001). Whether or not an

AAA-like fold exists in this region, the sequence here is

likely to be important in all dyneins because multiple

heavy chain sequence alignments indicate 13 completely

conserved residues and 27 with high consensus within

�350 residues upstream of the BoxII motif of AAA1

(Mocz and Gibbons, 2001). If instead this region forms

the linker, given its central role in the mechanism of thepower stroke (Burgess et al., 2003), it might be expected

to be highly conserved. It is not known how much, if

any, of this is incorporated into the head and how much

into the stem, nor how the part in the stem is folded. The

only clue so far from sequence analysis is that there are

multiple short stretches with predicted coiled-coil se-

quence. If the whole stem, �35 nm long, were to be built

of coiled-coil it would require �240 residues per coiled-coil strand. Therefore, in principle, there is enough

polypeptide available to build up to a six-stranded

coiled-coil. A simple two-, four- or six-stranded coiled-

coil model would place the N-terminus close to, or

within the head of dynein. A simple three- or five-

stranded one would place it near or in the base. More

complex permutations are also possible. The observed

similarity in width of the linker and neck to the coiled-coil stalk (Burgess et al., 2003) suggests that these parts

of the stem may indeed be coiled-coil structure but the

limited resolution does not allow us to discriminate be-

tween different stranded forms. It is relevant to note that

a concatenated three-stranded coiled-coil a-actinin motif

has been shown to form a functional substitute lever for

a myosin motor (Kliche et al., 2001). This shows first

that a coiled-coil can transmit a force that tends to bendit, as might be required in dynein (see Section 4.6).

Second, the a-actinin motif is built up by alternate long

and short a-helices separated by hairpin loops, which

enables a three-stranded structure to be produced by

local, rather than global folding of the polypeptide.

Antibody labelling to map the sequence within the stem

may be one approach to solve the folding topology

within this domain.Our finding that two parallel structures exist within

the shaft of the stem is intriguing. The two strands are

�8 nm long and similar in width to the coiled-coil stalk

shown in Fig. 3A. One is contiguous with the neck and

base, the other appears to make at least two contacts

with it and a further one with the base. Chemical cross-

linking studies (Yanagisawa and Kamiya, 2001) indicate

that the N-terminal region of dynein-c heavy chainbinds two copies of p28, which in turn bind the actin

subunit. Structure prediction suggests that the p28 light

chain is capable of forming a coiled-coil dimer (LeDizet

and Piperno, 1995) with a length we estimate to be

11 nm, although whether they actually dimerize is not

yet established. The natural interpretation of our images

is therefore that the strand in the shaft that is contiguous

with the neck is the heavy chain and the other is a coiled-coil dimer of p28. The known dimensions of actin imply

that it is too large to be part of the linker, neck or shaft,

and that it must therefore form part of the base of the

stem together with the presumed non-coiled-coil part of

p28 and possibly some of the dynein heavy chain. An-

tibody labelling will be required to test this hypothesis.

4.4. Stem flexibility

Flexibility within the neck of the stem is one of the

most obvious features of dynein-c prepared for electron

microscopy. The focal point of stem flexibility is lo-

cated within the neck subdomain a few nanometres

further from the head than the bend in the neck. This

indicates that a hinge at the bend cannot alone account

214 S.A. Burgess et al. / Journal of Structural Biology 146 (2004) 205–216

for the flexibility. Instead, it implies distortion moredistally in the neck. However, we cannot distinguish

between continuous flexibility distributed along the neck

and, for example, two discrete hinge points, one at each

end.

Flexibility in solution may become amplified during

adsorption, staining, and drying. That is, the extent of

flexibility seen in our images may be greater than occurs

in solution. Support for such preparation-induced dis-tortion is provided by the observed change in the extent

of stem flexibility when the molecule adsorbs in different

orientations. With the head oriented in the right view,

the extent of stem flexibility is greater (Fig. 3). Increased

flexibility between the head and the distal part of the

stem implies destabilization of the intervening struc-

tures, i.e., the neck. How this occurs is unknown, but

consistent with this idea, it is noteworthy that undock-ing of the linker from the head appears to be seen only

when the head adsorbs in the right view (Burgess et al.,

2003). Thus, undocking may be a more extreme conse-

quence of the destabilization phenomenon that increases

neck flexibility in this orientation. Electron cryomi-

croscopy of dynein-c frozen in the absence of a support

film will be required to examine flexibility in solution in

more detail.If the extent of preparation-induced distortion also

depends on the nucleotide state of the molecule this

would have an impact on our determination of the mean

size of the power stroke. In our previous report, no

change in stem flexibility was observed between apo-

dynein-c and ADP.Vi-dynein-c. However, stalk flexi-

bility was reduced in apo-molecules, indicating greater

stiffness of this domain (Burgess et al., 2003). Such achange in the stalk was not unexpected, since this

structure must communicate between the active site in

the head and the microtubule-binding domain at its

distal end (Gee and Vallee, 1998; King, 2000a; Vallee

and Gee, 1998). Moreover it is consistent with the in-

creased stiffness of nucleotide-free flagella over those in

ADP and vanadate (Okuno, 1980). It is nevertheless

possible that a systematic difference in distortion of themolecule could contribute to our estimate of the mag-

nitude of the power stroke (see below).

4.5. Implications of stem and stalk flexibility

The physiological relevance of flexibility is not pres-

ently understood. Flexibility within the neck and stalk

shows that these elements deform either as a result ofthermal (Brownian) fluctuations in solution or when

external load is applied to the molecule. In optical trap

experiments, dynein-c takes multiple steps along the

microtubule, exerting increasingly higher forces on the

trapped bead, until the motor releases from the micro-

tubule (slippage) and the bead returns to the trap centre

(Sakakibara et al., 1999). Associated with these excur-

sions away from the trap centre is a reduction in thermalnoise, indicative of a stiffer motor at higher forces,

suggesting the existence of compliant elements within

the functional motor. This implies that within a working

dynein motor, exerting force on a microtubule against a

load, the stalk and neck are likely to deform first before

slippage of the motor occurs.

Evidence for flexibility within outer arm dyneins in situ

has been obtained from electron microscopy of freeze-etch replicas of cilia and flagella. Stalks in adjacent outer

arm complexes in the same nucleotide state adopt differ-

ent angles, especially in the absence of nucleotide (Bur-

gess, 1995; Burgess et al., 1991a; Burgess et al., 1991b;

Goodenough and Heuser, 1982; Goodenough and He-

user, 1985a; Goodenough and Heuser, 1985b), indicating

that the stalk does indeed deform in situ. Stems have also

been seen to deform but only in flagella disrupted bysurface tension (Goodenough and Heuser, 1984; Sale

et al., 1985), so the relevance of this to the normal function

of dynein is not known. It is not yet possible to decide

whether there is flexibility in dyneins in the inner row of

arms (Burgess et al., 1991b; Goodenough and Heuser,

1985b; Woolley, 1997) because they are less extensively

revealed in freeze-etch replicas and their arrangement

along themicrotubule doublet ismore complex, repeatingat intervals of 96 nm compared to 24 nm in the outer arms.

However, disorder observed between successive repeats

of the inner arm pattern suggests that the flexibility we

have observed in dynein-c may occur also in situ.

The combined flexibility of stalk and stem seen in pu-

rified dynein-c produces a wide range of conformations in

both ADP.Vi- and apo- conditions. If these fleximers of

dynein-c were to be representative of the range of con-formations in a (motile) flagellum, their broad overlap-

ping distributions imply that loss of products may

occasionally be accompanied either by amuch larger than

average displacement, a zero net displacement or even to a

small reverse one.

4.6. Model

Based on our current understanding of the available

sequences of dynein heavy chains, and assuming that the

as yet unknown sequence of dynein-c follows the same

topology, we consider it reasonable to construct a

working model in which the six AAA modules and the

C-terminus form a planar heptameric ring. We favour

the C-terminus as the more likely seventh subdomain

over the sequence N-terminal to AAA1 since the latter ismore likely to be required to form the linker (see above).

The origin of the lateral subdomain remains unknown

but is illustrated here as deriving entirely from the lin-

ker. This forms the basis of our current structural model

for dynein-c (Fig. 6). However, depending upon a

clockwise or counter clockwise arrangement of AAA

modules, two different models can be constructed

S.A. Burgess et al. / Journal of Structural Biology 146 (2004) 205–216 215

(Figs. 6A and C). At present we cannot discriminatedecisively between these two alternatives.

During the power stroke, the stem moves relative to

the head and stalk (Fig. 6B). We illustrate the unseen

movement of the linker as a rigid-body rotation of the

entire stem (linker–neck–shaft–base), which sweeps the

linker across the face of the head. The origin of this

movement presumably lies within the active site of

AAA1, but the nature of the structural change there iscompletely unknown. So too are the presumed struc-

tural changes within and between AAA2–4 which lie on

the communication pathway between the active site and

the ATP-sensitive microtubule-binding domain at the

tip of the stalk. Our previous analysis comparing head

substructure between ADP.Vi- and apo-dynein-c was

unable to reveal the structural rearrangements within

the head subdomains, partly because only left viewswere available in sufficient numbers and the linker ob-

scures details of the head substructure. Right views,

which show head subdomains most clearly, were rela-

tively rare among ADP.Vi-molecules and probably not

related directly to left views by a 180� rotation. There-

fore, in the model we do not illustrate structural changes

within the AAA modules. The general similarity of

ADP.Vi- and apo-heads in left views (Burgess et al.,2003) argues against a gross rearrangement of subdo-

mains within the head.

According to the model, movement of the linker al-

ters its potential interactions with subdomains within

the head. In the counter clockwise model, these inter-

actions change from AAA6 in the pre-power stroke

conformation to AAA2-5 in the post-power stroke

conformation. In the clockwise model the correspondingchange is from AAA3 to AAA4-6 plus C-terminus.

Therefore, the counter clockwise model has the attrac-

tive quality of suggesting more extensive interactions

between the linker and the potentially regulatory AAA

modules (AAA2–4). However, this implies that the

C-terminus is one of the three prominent peripheral

subdomains in right views whereas their similar size fits

more naturally with their being AAA2–4, as predictedby the clockwise model. Only further experimentation

will show which, if either, model is correct.

Acknowledgments

This study was partly supported by Special Coordi-

nation Funds for Promoting Science and Technology of

the MEXT (Japan) and by NIH and BBSRC (UK).

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