Molecular Cell, Vol. 19, 595–605, September 2, 2005, Copyright ©2005 by Elsevier Inc.DOI 10.1016/j.molcel.2005.07.015
The Structural Basis of Myosin V Processive
Movement as Revealed by Electron Cryomicroscopy
Niels Volkmann,1HongJun Liu,1
Larnele Hazelwood,1Elena B. Krementsova,2
Susan Lowey,2Kathleen M. Trybus,2,*
and Dorit Hanein1,*
1The Program of Cell Adhesion
The Burnham Institute
La Jolla, California 92037
2Department of Molecular Physiology and Biophysics
University of Vermont
Burlington, Vermont 05405
The processive motor myosin V has a relatively high
affinity for actin in the presence of ATP and, thus, of-
fers the unique opportunity to visualize some of the
weaker, hitherto inaccessible, actin bound states of
the ATPase cycle. Here, electron cryomicroscopy to-
gether with computer-based docking of crystal struc-
tures into three-dimensional (3D) reconstructions
provide the atomic models of myosin V in both weak
and strong actin bound states. One structure shows
that ATP binding opens the long cleft dividing the ac-
tin binding region of the motor domain, thus destroy-
ing the strong binding actomyosin interface while
rearranging loop 2 as a tether. Nucleotide analogs
showed a second new state in which the lever arm
points upward, in a prepower-stroke configuration (le-
ver arm up) bound to actin before phosphate release.
Our findings reveal how the structural elements of
myosin V work together to allow myosin V to step
along actin for multiple ATPase cycles without disso-
The basic mechanism by which all myosins interact
with actin is generally conserved, but different myosins
have tuned their structural, kinetic, and mechanical
properties to optimize performance for their particular
cellular role. Myosin V has evolved to spend most of its
cycle time attached to actin (i.e., a high-duty cycle), a
requirement for it to move processively along an actin
filament as it transports cargo (reviewed in Vale, 2003).
Myosin V has also undergone structural adaptations to
its lever arm, which is elongated and contains six IQ
motifs that each binds one calmodulin. This large span
between motor domains allows the two heads to bind
approximately 36 nm apart from each other as the mole-
cule strides along an actin track for multiple ATPase
cycles before detachment. However, the detailed struc-
tural mechanism that underlies such processive move-
ment is still not understood.
The only actin bound myosin states that have been
amenable to investigation by 3D electron cryomicros-
*Correspondence: email@example.com (K.M.T.); dorit@
copy to date are “strongly bound” states, i.e., heads
with bound ADP or heads without nucleotide (apo),
which represent the final steps in the ATPase cycle.
These studies showed that the lever arm is in the post-
power-stroke position (lever arm down) when myosin is
strongly bound to actin and that additional downward
movement can occur for some myosins upon ADP re-
lease (Volkmann and Hanein, 2000; Whittaker et al.,
1995). We previously showed that the large cleft that
divides the actin binding region of the motor domain is
tightly closed in the rigor state for both smooth and
skeletal myosins (Volkmann et al., 2000, 2003). In addi-
tion, we have shown that loops at the actin interface
(loop 2) and at the nucleotide binding pocket (loop 1)
are both flexible and therefore not visible in the crystal
structures and become stabilized by actin binding
(Volkmann et al., 2000, 2003). Recently, Holmes et al.
(2003) have confirmed our observation for a closed ac-
tin binding cleft by using a rigor map of skeletal myosin.
Prior electron microscopy studies on myosin V, using
negative staining and two-dimensional image averag-
ing, have shown the molecule spanning the w36 nm
actin helical repeat during ATP hydrolysis (Burgess et
al., 2002; Walker et al., 2000). Two types of lever arm
positions that resemble the crystallographic pre- and
postpower-stroke conformations were observed in pro-
jection, but no high-resolution information on changes
within the motor domain could be obtained.
The only atomic-resolution myosin V structures avail-
able to date are in detached states, either without nu-
cleotide (Coureux et al., 2003) or in the presence of ADP
or ADP.BeFx(Coureux et al., 2004). The nucleotide-free
structure captures a unique conformation in which the
lever arm is in a downward position, the actin binding
cleft is in a closed conformation, and specific interac-
tions between elements at the active site prevent high-
affinity nucleotide binding. The structure in the pres-
ence of ADP was obtained by soaking preformed
nucleotide-free crystals with ADP, resulting in minimal
alterations of the nucleotide-free structure. Thus, this
structure is believed to represent an intermediate state
in which ADP is bound weakly to myosin and confirms
that a closed cleft geometry is compatible with ADP
binding (Coureux et al., 2004). The structure of myosin
V in the presence of ADP.BeFxis similar to the post-
power-stroke structures of myosin II (Fisher et al., 1995;
Gulick et al., 1997; Rayment et al., 1993b) that are char-
acterized by an open cleft and a downward lever arm
position. These atomic myosin V structures provide de-
tailed information about changes within the detached
myosin head, but corresponding changes in actin and
at the actomyosin interface can only be deduced by
studying actin bound myosin.
Here, we use electron cryomicroscopy (cryoEM) to
visualize actin bound structures of an expressed mono-
meric murine myosin V construct containing the motor
domain and two calmodulin binding IQ motifs (MD2IQ).
Myosin V’s relatively high affinity for actin in the pres-
ence of ATP and triphosphate analogs allows us to vi-
sualize previously inaccessible states. Thus, 3D recon-
Table 1. Reconstruction Quality Indicators
ADPApo ATP AMPPNPd
Units in averagea
Units per turn
2.157 ± 0.002
2.158 ± 0.002
2.159 ± 0.003
2.156 ± 0.005
4.22 ± 0.31
2.161 ± 0.007
5.43 ± 0.42
aUnits for the nucleotide states are corrected for repeated use of the same units.
bCalculated from the contributing filaments, there is no analogy for iterative helical real space refinement.
cNo docking was performed for the ADP.AlF4state.
dAMMPNP refers to prepower-stroke map after sorting.
structions were obtained of the actin-MD2IQ complex
in the presence of ATP, AMPPNP, ADP.AlF4, ADP, and in
the nucleotide-free state, structures that should mimic
the normal progression through the hydrolysis cycle.
We show that ATP weakens actin binding by opening
the long cleft dividing the motor domain while rearrang-
ing loop 2 as a tether. The presence of AMPPNP or
ADP.AlF4induces a conformational state with the lever
arm pointing in a prepower-stroke upwards configura-
tion. The actin bound nucleotide-free and ADP struc-
tures, similar to the detached crystal structures, have
their cleft tightly closed with the lever arm pointing
Reconstructions of Actin Decorated with Myosin V
in the Strong Binding States (ADP, apo) and in the
Presence of ATP Show Postpower-Stroke
Electron cryomicroscopy and helical reconstruction
techniques were used to generate 3D maps for actin
filaments as well as for myosin V MD2IQ-decorated ac-
tin filaments in the absence of nucleotide and in the
presence of ADP or ATP (see Table 1 for quality indica-
tors). We also processed each data set by using the
iterative helical real space refinement method (Egel-
man, 2000). The resulting reconstructions were very
similar (correlation >95%) to the respective helical re-
constructions. All three nucleotide states show the le-
ver arm in postpower-stroke position, similar to those
obtained for actin bound myosin II (Holmes et al., 2003;
Rayment et al., 1993a; Volkmann et al., 2000, 2003;
Whittaker et al., 1995) (Figure 1).
Acto-myosin V incubated with ATP could result in a
mixed population of the true ATP state and various in-
termediate states. To ensure that our reconstructions of
actin bound myosin V in the presence of ATP represent
homogeneous populations, we carefully analyzed the
appearance and optical diffraction patterns of filaments
before we selected them for averaging. Inhomogeneity
would be immediately apparent in the images as well
as the diffraction patterns and would also affect the
averaging statistics adversely. All filaments included in
the average appeared homogenous, and the averaging
statistics of the selected filaments support this notion
in that its quality is comparable to those of the strongly
bound states (see also Table 1).
To further test for mixed conformations in the result-
ing reconstruction, we determined the Absolute Values
of Individual Differences (AVID) for these maps. The
AVID procedure was specifically developed to identify
mixtures of states and conformations in helical recon-
structions (Rost et al., 1998). Because the AVID maps
of the ATP state are virtually featureless (all values were
less than one standard deviation away from the mean),
there is no significant structural variability or mixture
present in the ATP reconstructions. We also checked
the density distribution of the reconstructions for signs
of mixed populations, which would change the relative
strength of features in the maps. Regions of the un-
derlying structure that are not well locked into space
would spread their density over a larger area and would
appear weaker than well-determined entities (see the
Supplemental Data available with this article online). In
the reconstructions done in the presence of ATP, the
relative strength of actin, the motor domain, and the
light chains are comparable to those of the apo and
ADP maps. We conclude that the reconstructions ob-
tained in the presence of ATP represent homogenous
populations of a state that is significantly different from
the ADP state (see below). We will refer to this state as
the “ATP state.”
Docking Analysis Indicates a 2.4 nm Lever-Arm
Movement upon ADP Release
An atomic model for filamentous actin (see Experimen-
tal Procedures for details) was docked into maps of un-
decorated actin. The accuracy of the docking in this
region was estimated at 0.18 nm. Modular docking
(Volkmann and Hanein, 1999) of multiple crystal struc-
tures was performed on single asymmetric units of the
respective actin bound myosin-V densities to obtain
atomic models for the apo, ADP, and ATP actin bound
myosin V (254 docking experiments). To compile the sta-
tistics, the available crystal structures were divided into
three different groups corresponding to the prestroke (le-
ver arm up), poststroke (open cleft and lever arm
down), and closed-cleft (lever arm down) conforma-
tions (see Supplemental Data for details). The correla-
tion statistics (Table S1) show that for all three recon-
structions, the prepower-stroke group fits significantly
less well than the other two groups when the converter
is present. If the converter is deleted, there is no differ-
ence among the three groups. The analysis of the solu-
tion sets results in estimated docking accuracies for
the MD region of 0.24 nm, 0.26 nm, and 0.34 nm for the
apo, ADP, and ATP maps, respectively.
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