Article

Flexibility within the Heads of Muscle
Myosin-2 Molecules

Neil Billington 1, † , Derek J. Revill 1 , Stan A. Burgess 1 ,Peter D. Chantler 2 and Peter J. Knight 1
1 - School of Molecular and Cellular Biology and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds LS2 9JT, UK
2 - Unit of Molecular and Cellular Biology, Royal Veterinary College, University of London, Royal College Street, London NW1 0TU, UK

Correspondence to Peter J. Knight: School of Molecular and Cellular Biology, LC Miall Building, University of Leeds,
Leeds LS2 9JT, UK. p.j.knight@leeds.ac.uk
http://dx.doi.org/10.1016/j.jmb.2013.11.028
Edited by R. Craig

Abstract
We show that negative-stain electron microscopy and image processing of nucleotide-free (apo) striatedmuscle 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 viewsof two different crystal structures of apo S1 shows that all types most commonly closely resemble theappearance of the scallop S1 structure rather than the methylated chicken S1 structure. Methylation ofchicken 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/rad 2 isestimated 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.
© 2013 The Authors. Published by Elsevier Ltd. All rights reserved.

Introduction
The mechanical properties of muscle crossbridges
underlie muscle function but are controversial [1–4].
The swinging lever hypothesis of muscle contraction
envisages that the relative movements of the thickand thin filaments originate from the part of theactomyosin crossbridge that is furthest from the actin
filament, acting as a lever. This lever amplifies theconformational change driven by ATP hydrolysis
within the core of the myosin motor leading to mutual
filament sliding [5]. In muscle shortening against
a load, force is produced in addition to shortening;
the characteristics of the work produced depend on
the mechanical properties of the active crossbridge.
These properties are still disputed despite decadesof ingenious study and despite their importancefor understanding the general principles of motor

protein function. In part, this is because the speed
and dynamic nature of the crossbridge cycle andthe complexity of the contractile apparatus makeunambiguous interpretation of ensemble properties
difficult. We have therefore sought to understandthe mechanical properties of the crossbridge by direct
observation of individual myosin heads.
Striated muscle myosins are members of the
myosin-2 branch of the myosin phylogenetic tree
[6,7] and share a common structural plan. Twin
230-kDa heavy chains each form a motor domain(MD), then continue as an extended α-helix originallynamed the "lever arm" [8], but here named moreconcisely the lever before uniting to form a parallel,
in-register, α-helical, coiled-coil tail. The MD plus leveris generally referred to as the myosin head or
subfragment-1 (S1). The lever helix is stabilised by
two calmodulin-like, bi-lobed light chains: an essential

0022-2836/$ - see front matter © 2013 The Authors. Published by Elsevier Ltd. All rights reserved.

J. Mol. Biol. (2014) 426, 894–907

Flexibility in Myosin-2 Heads

light chain (ELC) adjacent to the MD followed by aregulatory light chain (RLC). The ELC binds because
it recognises a canonical IQ motif [9] in the leverhelix, while the RLC recognises a modified IQ motif
in which a distal WxWW sequence produces a
sharp kink in the lever helix [10]. All myosin-2s
have the same length of lever, with the IQ motifs
always the same distance from the MD and the tailand the same distance apart (the I residues of the
IQ motifs are 26 amino acid residues apart). It istherefore expected that all myosin-2 heads share acommon structure, though their enzymatic andmechanical properties will depend on the specificamino acid sequences of the heavy and light chains.
Electron microscopy (EM) has advantages over
X-ray crystallography in defining molecular flexibility.
Although crystallography has produced a wealth ofstructural detail regarding the myosin-2 head, it has
limitations: progress can be slow because crystal
formation is capricious, and desired conformers
may not crystallise; more fundamentally, flexibility isnecessarily suppressed within a crystal. By contrast,
EM allows rapid throughput of samples and can
indicate the range of conformations present in solution
at the point of fixation, albeit at lower resolution. EM,
when combined with image processing to improve thesignal-to-noise ratio of the data, provides a link
between high-resolution structural studies of individual
conformations and data from solution studies regarding the structural dynamics of the protein [11]. Thisapproach has been used to interpret tomograms ofrigor insect flight muscle and has shown that
distortions in the lever region of the S1 crystal structure
have to be made to fit it into the EM density map,
especially when the muscle is stretched [12].
X-ray crystallography has revealed some diversityof structures of the lever of myosin-2 heads. Throughcomparisons between different crystal structures,some sites of flexibility have been discerned [13,14].
In particular, a site in the heavy chain at the junction
between the MD and the lever is a straight helix inmany structures but distorted in others, leading to its
description as a pliant region and the proposal that it
could play a role in crossbridge elasticity [14]. Also,
the shape and position of the lever appear to differ
between heads from different myosins in equivalent
nucleotide states [10,14–16]. It is not known whether
such differences represent true structural differences between the myosins in solution or whether
they arise simply because of the different crystallisation conditions used, which may select particular structures from a dynamic range ofconformations that are similarly populated bythese different myosins in solution. In the compactly folded and shutdown conformer of scallop
myosin, the closer resemblance of its lever to thatseen in the crystal structure of chicken skeletal
myosin rather than scallop striated muscle myosin[17] argues for the latter alternative.

895
Previous work in our group used negative-stain
EM and single particle image processing on whole
myosin molecules to show that flexibility exists
within the heads [11,18]. The range of shapes seen
was greater than expected given the high stiffnessthat has been estimated for crossbridges in muscle(see Ref. [19]), raising a question concerning thedegree of distortion that each molecule undergoes
when drying in stain during specimen preparation.
However, measurement of myosin tail stiffness
from the quantitative analysis of the diversity
among such EM images [20] provided a similarvalue to solution studies [21], suggesting thatnegative-stain EM produces images that do represent the situation in solution. The stiffness of amacromolecule can be estimated by quantitative
analysis of a set of images because the breadthof the distribution of shapes is inversely related
to stiffness through the Equipartition theorem [22].
The ergodic hypothesis states that the set ofimages can be either of one molecule changing
shape over time or (as here) single images of many
molecules.
In dissecting the problem of crossbridge stiffness
in muscle, it is important to know what degreeof flexibility and elasticity exists within the heads
themselves, when unconnected to the tail. For
this reason, we have here investigated S1 rather
than whole myosin molecules, using negative-stain
EM and image processing. The use of S1 also
avoids any lingering concerns that other parts of
the whole molecule might create distorting forces
during specimen preparation. It also avoids aproblem associated with processing images ofdouble-headed myosin, namely, that the twoheads do not maintain a fixed relationship to eachother and this added variability can hamper imagealignment.
We have compared the appearances in EM ofseveral S1 species, using negative staining since
myosin-2 S1 is too small (~ 130 kDa) to identify byunstained cryo-EM. Scallop and chicken striatedmuscle S1s were imaged to allow direct comparison
with existing crystal structures (PDB IDs: 1SR6/2OS8
and 2MYS, respectively). We also studied rabbit
skeletal muscle S1, which is enzymatically wellcharacterised but has not yet been crystallised.
Indeed, there is no crystal structure of any mammalian
myosin-2 head to date, making it important todetermine how closely such myosins resemble thosecrystal structures that have been determined. We
also studied the effect of reductive methylation on
the structure of chicken S1 since this chemical
modification was used to crystallise it. In particular,
we wished to determine whether the differences inlever shape between crystal structures of scallopand chicken S1s are a genuine species differenceor are instead a consequence of either methylation
or crystallisation.

896

Flexibility in Myosin-2 Heads

Fig. 1. Fields of view and global image averages of different types of myosin-2 S1.(a–d) Areas of raw micrographs offour different types of full-length myosin-2 S1. The scale bar represents 50 nm. (e–i) Global image averages resulting fromalignment of multiple images of each S1 type. (a and e) RbS1. (b and f) ScS1. (c and g) CkS1. (d and h) mCkS1. (i) rabbit
skeletal chymotryptic S1 (A1 ELC). Numbers in (e)–(i) show the number of particles in each dataset. Side length of image
average panels is 31.2 nm.

Results
Features of four types of myosin-2 S1 in
negative stain
To compare the structures of myosin-2 heads fromseveral sources, we negatively stained each typein the absence of nucleotide then examined it by EM.
We compared S1s containing both ELC and RLC
prepared by papain proteolysis of rabbit skeletal
muscle myosin (RbS1), scallop cross-striatedadductor muscle myosin (ScS1), chicken skeletal
muscle myosin (CkS1) and CkS1 that had been
reductively methylated (mCkS1). We found that, ineach case, the S1 molecules adsorbed to the carbon

film in a similar way to the heads of intact myosin:
the long axis typically lay in the plane of the carbon
and there was a strong preference for a particularside of the molecule to adsorb. Moreover, the motor
and lever domains could often be distinguished in
raw micrographs of the negatively stained S1s, as
found previously for intact myosin (Fig. 1a–d) [18,23].
Consequently, a reliable computational alignment of
individual S1 by single particle methods was readily
accomplished; even in the global averages, some
subdomain features of the MD and the two boundlight chains of the lever (ELC and RLC) are apparent(Fig. 1e–g). In particular, the MD shows the resemblance to a human head in profile previously noted
in myosin-2 and myosin-5a [18,24] in which thehuman is facing left. The close similarity of Fig. 1h toFlexibility in Myosin-2 Heads

897

Fig. 2. Range of appearances of nucleotide-free S1 from different sources.Each row of the montage shows the result of
K-means clustering of all the particles of an aligned dataset into 10 classes. Classes have been rearranged along the row
to show the general similarities between the four datasets when compared down a column. (a) RbS1; (b) ScS1; (c) CkS1;(d) mCkS1. Note in (a) and (b), the rightmost panels show that a small fraction of the S1s have misaligned upside down andhave formed a small separate class. Side length of all image panels is 31.2 nm.

Fig. 1g indicates that methylation of almost all thelysine side chains of CkS1 [25] has no effect on its
behaviour as a specimen for negative staining.
To determine whether the overall shape of S1and its orientation on the carbon film was governedby the RLC, we also imaged and aligned rabbit
skeletal S1 that had been truncated after the ELC
by chymotryptic proteolysis [26]. The overall appearance and orientation of the truncated molecule wassimilar to the corresponding regions in class averagesof the myosin S1 molecule digested by papain (Fig. 1icompared to Fig. 1e). This shows that removal of the
C-terminal part of the lever, including the associated
RLC, does not produce a large structural change
in the remainder of the molecule. It also shows that it
is possible to reliably image and align this small headfragment (~ 110 kDa total mass).
Classification of the particles from the four different
full-length S1s shows that, despite the detail in the
global average, there is nevertheless a (limited) range
of appearances of both MD and light chains present ineach of the datasets (Fig. 2). The different appearances are remarkably similar between the different
S1s. In many cases, individual subdomains canbe recognised within the S1 and in greater detail thanin the global average, as is expected for well-aligned
particles of diverse appearance. The N- and C-lobesof the ELC are clearly resolved in some averages.
The RLC is a strong feature of more classes than was
the case with heads of intact myosin [18], probably
because of the absence of the tail and other headthat complicate the head–tail junction of intact myosin.
Nevertheless, the RLC in S1 appears as an ellipse
oriented along the lever axis rather than as two lobes
obliquely crossing it, which is also the result expectedfrom its different arrangement, grasping the hook in theheavy chain lever helix [10,13]. The RLC is a weakerfeature in the global average of ScS1 (Fig. 1f) than theother S1s, and the classification (Fig. 2) correspondingly shows more classes for ScS1 that have a weak
RLC feature, presumably because the RLC is easily
lost from ScS1 preparations.
It is apparent from Fig. 2 that some S1 molecules
appear straight while others curve to the left or to
the right. Since EM and crystallography have shownthat myosin-2 heads are inherently curved, largely
in a single plane [10,27,28], there are two possibleclasses of explanation for this diversity of shape.
First, the molecules may adsorb to the carbon film
in a variety of orientations, yielding varying shapes
in the projection view recorded by EM; second, they
may be inherently flexible. To distinguish between
these alternatives, we analysed the data further
by grouping together particles displaying the same
MD features, in order to test whether there wasstill the variation in shape that would imply flexibility
within such groups having a given orientation. Suchanalyses work best with large datasets. Given thesimilarities of the four datasets shown in Fig. 2,
we pooled all four datasets, keeping track of which
particles were from each individual dataset so that
we could subsequently determine whether there weresystematic differences between them.
Comparison of different types of full-length S1Pooling all four S1 datasets (RbS1, ScS1, CkS1
and mCkS1) and realigning the images produces
a global average similar to those of each separate
dataset, as expected (Fig. 3a). Classification of the898

Flexibility in Myosin-2 Heads

(b)

(a)

(c)

(d)

(e)

64

36

76

33

84

35

32

67

(f)

(g)

Fig. 3. Co-alignment and co-classification of multiple
myosin S1 datasets.(a) Global average and (b) global
variance images resulting from alignment of 17,307
images of S1 from RbS1, ScS1, CkS1 and mCkS1. In(b), white indicates high variance. (c) Class averages
after further alignment of MD alone in each averageagainst the global average MD. These 10 examples (from400 classes) are selected to show that class averages
with differing appearances became aligned with the longaxis of the MD vertical. (d) Eight classes from classification into 100 classes according to the MD appearance,
using hierarchical ascendant classification. These are
the largest classes, each containing N 500 particles; they
include 57.6% of the combined data and were selected
for further analysis. Arbitrary class number top right;
number of particles at lower right. (e) The semi-annularmask used to classify the levers, superimposed (in palergrey) on the global average resulting from alignmentof the MD. (f) How the lever angle for each class (θ)
was measured relative to the vertical axis of the MD bymarking the tip of the lever (+) and using the MD–leverjunction as marked in the global average of the combined
data (×). Lever tips to the left of the MD–lever junction
were assigned negative angles. (g) The marked positionsof the tips of levers after lever classification (black dots)
superposed on the global average. Side length in (a)–(e)is 41.6 nm; in (g), it is 30.9 nm.

aggregated data into a large number of classes allows
the full diversity of appearances to be apparent withimproved signal to noise compared to raw images.
Images from each S1 dataset are spread across these
averages, rather than segregating into distinct classes,
which indicates that systematic differences between
the datasets are small compared to the variations withineach dataset. These class averages contain improved
MD detail so that the MD features alone of eachclass average could be computationally aligned with
the MD long axis vertical within the viewing window(Fig. 3c), and this realignment caused the levers
to become spread away from the vertical. To group
together molecules with the same MD appearance,
we then reclassified the MD-aligned stack of raw
images using only features within the MD. The eight
most abundant appearances of the MD (among 100
classes) are shown in Fig. 3d, from which it is apparent
that there is close similarity of most members in thisgroup to the global average. Thus, many S1 molecules
are seen in the same MD view, allowing lever position
relative to the MD to be explored.
To quantitate the range of lever positions, weclassified the full stack of MD-aligned images into
200 classes using only features in the lever region(Fig. 3e), and we then measured the lever angleof each class relative to the long axis of the MD
by locating the position of the tip of the lever ineach class average (Fig. 3f). The spread of lever
tip positions (Fig. 3g) is concentrated in an arc thatis densest near its centre. This is the behaviour
expected for a thermally excited elasticity between
MD and lever tip that could arise from bending along
the lever and/or flexibility at the MD–lever junction.
However, as noted above, it could in principle also
arise from a range of molecular orientations on the
carbon film without flexibility. However, as a resultof the MD classification of the same image stack,each individual particle could be assigned to an
MD class and a lever angle class. The distributionsof lever angle for any MD class could therefore be
determined, and these distributions for each of thefour S1 types could also be compared. We focusedfurther analyses on the eight MD classes that werethe most abundant.
Lever angle distributions for the two most highly
populated MD classes are shown in Fig. 4. They aredisplayed using percentage cumulative frequency
plots rather than histograms. We chose this display
because such plots show every individual data
point rather than the arbitrary bins used in histograms and, thus, show the data more objectively,
and they are better able to display multiple datasets
where differences between them are small. In thesepercentage cumulative frequency plots, the fitted
Gaussian curves show as sigmoid lines, which crossthe 50% value at the mean fitted lever angle. It isapparent that all four types of S1 show a very similar
extent of lever angle variation, and it is described

899

Flexibility in Myosin-2 Heads(a)
Cumulative frequency (%)

100

75

50

25

0
-80

-60

-40

-20

0

20

40

60

80

Lever angle (degrees)(b)
Cumulative frequency (%)

100

each average containing 20% of the angle-sorted
data. Because the first and the last averages contain awider range of the lever angle distribution, the levers
are more smeared out than in the central average thatcovers only a narrow angular range. Figure 5a and bshows the same MD classes shown in Fig. 4. The
averages show that the substructures of the S1 types
are similar to one another in the central averages anddisplay altered shapes in similar ways when the leveris angled differently. Importantly, they confirm thatthe change in lever angle is not accompanied by achange in MD orientation or appearance, reinforcing
the conclusion that S1 is flexible. The major conclusion, therefore, is that S1 shows flexibility in its shape.
All four S1 types are very similar to one another, bothin the mean angle displayed between motor and leverand in the amount of variation in lever angle present.
Flexibility of S1

75

50

25

0
-80

-60

-40

-20

0

20

40

60

80

Lever angle (degrees)Fig. 4. Lever angle distributions of MD classes.(a) MD
class 64 (2,099 particles); (b) MD class 36 (1,980
particles). Panels show data points for every image within
an MD class, segregated by S1 type and arranged in order ofascending lever angle. RbS1, red; ScS1, blue; CkS1, green;mCkS1, black. Smooth lines of the same colours are
Gaussian best fits to each set of data.

well by a Gaussian spread. The mean lever angles
for the four types are all close to 0°, with ScS1
tending to angle a few degrees more to the left.
Averaged over all eight MD classes, the mean
angles are as follows: RbS1, − 1.1°; ScS1, − 5.2°;
CkS1, − 0.8°; mCkS1, + 1.1° (see detailed breakdown in Table 1). MD class 35 looks the least similar
to the typical head-in-profile appearance (Fig. 3d),
indicating that the S1 is viewed from a different
angle. Correspondingly, the mean lever angles are
generally shifted to lower values but the differences
between the S1 types remain similar (Table 1).
Because of the large numbers of particles measured,
the differences in lever angle distributions between S1
types in many MD classes reach statistical significance, but the differences are small.
To observe the variations in structure of S1 between
and within the major MD classes, we made a set of fiveimage averages for each type of S1 in each MD class,To determine the sites of flexibility within S1, we
subdivided the 2,099 images in the most abundant
MD class (Fig. 3d; head-in-profile appearance)
into 10 or 20 classes based on lever angle (Fig. 5cor SI Movie 1, respectively). Inspection of the classes
shows that the levers change little in shape or
substructure; for instance, the two-lobed appearanceof the ELC can be resolved in several classes. This
indicates that the lever itself is relatively stiff. Instead,
SI Movie 1 indicates that a major site of flexibility isat the junction between the MD and the lever. To
determine the location of this site more objectively, foreach class, we drew a straight line through the leverfrom its tip to its junction with the MD and extrapolated
it across the MD. The coordinates were calculatedof every intersection of each line with every other line
(shown as a scatter plot in Fig. 5d), and the position of
the median x and y values of the n(n − 1)/2 = 190
expected intersections was thus obtained. The great
majority of intersections are tightly grouped into the
MD–lever junction, reinforcing the observation thatflexibility in the head is mainly a hinge at this site.
Superposition of the ScS1 apo crystal structure showsthat the location where bending is focused is indeed at
the junction between the MD and the lever (Fig. 5e)
at about heavy chain residue 772. This indicates that,within the error of our estimate, the lever flexes in thepliant region (residues 774–781) previously describedin myosin-2 between the converter and ELC [15,17,29]
and predicted to be a source of elasticity in the crossbridge [14].
Measurement and comparison of lever stiffness
For a thermally excited torsion spring, such as
the hinge between the MD and the lever, the torsion
spring constant, κ, is inversely related to the variancein lever angle, σ 2, through the Equipartition theoremas κ = kBT/σ 2, where kB is Boltzmann's constant and900

Flexibility in Myosin-2 Heads

Table 1. Lever angle and bending stiffness of the four S1 types
Lever angle SD(°)

Torsion spring constant(pN·nm/rad2)

Cantilever-type stiffness(pN/nm)− 2.74− 3.40− 2.72− 2.312.90− 0.65− 0.54
0.82

21.82
23.58
22.73
22.94
21.95
24.12
23.07
24.42

27.85
23.85
25.67
25.20
27.53
22.79
24.92
22.23

0.435
0.373
0.401
0.394
0.430
0.356
0.389
0.347

32
33
35
36
64
67
76
84

− 7.66− 6.11−12.61− 5.36− 2.12−10.76
0.87
2.26

25.99
24.64
26.27
25.75
26.21
25.14
24.37
23.00

19.64
21.84
19.21
20.00
19.31
20.99
22.34
25.06

0.307
0.341
0.300
0.313
0.302
0.328
0.349
0.392

CkS1
CkS1
CkS1
CkS1
CkS1
CkS1
CkS1
CkS1

32
33
35
36
64
67
76
84

− 2.86− 4.46− 6.76− 1.664.02− 5.36
4.58
6.35

25.22
28.26
26.78
26.45
26.21
26.47
25.25
22.66

20.86
16.60
18.49
18.96
19.30
18.93
20.80
25.82

0.326
0.259
0.289
0.296
0.302
0.296
0.325
0.403

mCkS1
mCkS1
mCkS1
mCkS1
mCkS1
mCkS1
mCkS1
mCkS1

32
33
35
36
64
67
76
84

2.84
− 3.74− 6.01− 1.11
6.00
1.81
4.68
4.22

19.72
23.02
25.17
22.37
21.19
22.09
21.33
20.87

34.12
25.04
20.93
26.50
29.53
27.18
29.14
30.46

0.533
0.391
0.327
0.414
0.461
0.425
0.455
0.476

S1 type

MD class

RbS1
RbS1
RbS1
RbS1
RbS1
RbS1
RbS1
RbS1

32
33
35
36
64
67
76
84

ScS1
ScS1
ScS1
ScS1
ScS1
ScS1
ScS1
ScS1

Mean lever angle(°)

MD class corresponds to the major classes shown in Fig. 3d. Mean lever angle and standard deviation (SD) are obtained from Gaussian fits toeach group of particles. Torsion spring constant and cantilever-type stiffness are calculated as described in Experimental Procedures.

T is absolute temperature [30]. This constant wascalculated for each type of S1 for each of the eight
MD classes (Fig. 3d) using the standard deviation of
the distribution of lever angles assigned to individual
particles (e.g., see Fig. 4). For the most abundant
MD class, κ is in the range 19–29 pN·nm/rad 2
for the four types of S1. The values for the other
MD classes also lie close to this range (Table 1),
including for MD class 35 that shows S1 in a different
orientation, indicating that stiffness at the pliant region
may be isotropic. The overall mean ± SD value for κ
is 23.4 ± 4.2 pN·nm/rad 2.
A more useful representation of the torsion
spring stiffness for comparison to the stiffness ofcrossbridges in muscle, or to single molecule studies
such as those performed using the optical trap, is theapparent stiffness measured at the tip of the lever,considering it as a bending beam. This cantilever-typelever stiffness was calculated for each type of S1for each of the eight MD classes (Fig. 3d) as describedin Experimental Procedures (Table 1). There is no
systematic difference in lever stiffness among theeight MD classes. The mean ± SD stiffness values

across these classes are 0.39 ± 0.032 pN/nm
for RbS1, 0.33 ± 0.031 pN/nm for ScS1, 0.31 ±0.042 pN/nm for CkS1 and 0.44 ± 0.062 pN/nm for
mCkS1. Although some of these mean values are
statistically different from one another, the differences are small. They could derive from differences
between the S1 types in the amino acid sequencein the pliant region that could affect its stability
or could arise merely from small systematic differences in specimen preparation. The overall averagelever stiffness estimate is 0.37 ± 0.065 pN/nm.
Comparison of crystal structures of S1 to EMclass averages
It is clear from Fig. 5e that the crystal structureof apo ScS1 can be oriented to fit well into a classaverage of the combined S1 data that is in the central(unstrained) region of the spread of lever angle. To
test whether this was true for each S1 type taken
separately, we compared a set of density projections
covering all orientations of both ScS1 and mCkS1
crystal structures by cross-correlation against class901

Flexibility in Myosin-2 Heads

(a) i

(b) i
104

104

104

104

106

ii

102

102

102

102

105

92

92

92

92

95

120

120

120

120

122

80

80

80

80

82

ii
92

92

92

92

92

iii

iii
120

120

120

120

123

iv

iv
102

102

102

102

106

(c)

(d)

(e)

Fig. 5. Lever angle variation within a single MD orientation.(a and b) MD classes 64 and 36, respectively, as shown in
Fig. 3d. Within each panel, an MD class is separated into four rows comprising the four types of S1 [(i), RbS1; (ii), ScS1;(iii), CkS1; (iv), mCkS1], and each row contains five image averages, each made using one-fifth of the particles after
sorting them into order of increasing lever angle (i.e., the leftmost panel of a row contains the 20% of the particles with thelever angled most to the left). Number of particles in each average is shown at lower right. Side length of all image panels is
31.2 nm. (c) Data from MD class 64 (all S1 types combined) regrouped into 10 equal-sized classes of ascending leverangle. (d) Scatter plot of the intersections between lines drawn through the lever long axis of the MD class 64 after
regrouping particles into 20 equal-sized classes of ascending lever angle (there are n(n–1)/2 = 190 such intersections), as
also used to make SI Movie 1. The scatter plot is superposed on the global average of class 64. (e) The median point of thescatter plot (black cross) superposed on apo ScS1 crystal structure (PDB ID: 1SR6; grey ribbons) itself oriented to best
match the appearance of the fifth class in (c) (faded, in background).

averages of the four S1 types in each of the eight
populous MD appearances (Fig. 3d). Such a comparison cannot be rigorous because the EM data
are dominated by negative stain that is absent
from the crystal structures (discussed in Ref. [18]).
Nevertheless, for the averages that include the centreof the angular spread (e.g., the central columns in

Fig. 5a and b), all 32 comparisons showed that the
ScS1 structure was the closer fit, even to the mCkS1
data. This is principally due to the smaller angle
between motor and lever long axes in the ScS1
structure yielding a closer match to the rather straight
S1 shape of all S1 types at the centre of their leverangle distributions (Fig. 6).

902

Flexibility in Myosin-2 Heads

Fig. 6. Example of projection matching of S1 crystal structures to EM class averages.(a) Image average of RbS1 from
MD class 64, using 20% of images closest to the mean lever angle (central panel from Fig. 5a-i). (b) 2D projection of apo
ScS1 crystal structure density that best matches (a); cross-correlation coefficient, 0.681. (c) 2D projection of apo mCkS1
crystal structure density that best matches (a); cross-correlation coefficient, 0.636. 2D projection densities were inverted
so that high density is pale, zero density is black, to better match the EM image. Side length of all panels is 31.2 nm.

Discussion
We have extended earlier work on intact muscle
myosin molecules [18] by finding that isolated
myosin-2 heads retaining either one light chain
or both light chains (total mass, 110–130 kDa) are
amenable to quantitative analysis through negativestain EM and single particle image processing. These
combined techniques produce class averages inwhich individual subdomains of the myosin motor
and light chains can be identified and sites and extentsof flexibility can be identified. S1s from myosin-6[31] and myosin-18A [32] have also been studied inthis way. Interestingly, myosin-6 typically adsorbed tothe carbon film also in the head-in-profile view we see,but facing the opposite way; that is, the other sideof the head is adsorbed as compared with myosin-2
and myosin-18A (and also myosin-5 heads in a twoheaded construct [24]).
All S1 types investigated display a variety of
appearances when the images are grouped andaveraged. From the appearance of the MDs, thisvariety is owing to some variation in the orientationby which each S1 molecule binds to the carbon
substrate but, principally, to conformational variability within a given population of molecules. The typicalappearance of all types was similar at this resolution
to crystal structures of nucleotide-free ScS1, in
which the long axis of the MD is approximately
parallel to the long axis of the lever, rather than to the
crystal structure of mCkS1, which is more bent.
Interestingly, while this was true in the case of classaverages from unmodified CkS1, it remained true
even when the protein had been methylated so as toreproduce the material used to determine the first S1
crystal structure [10]. This suggests that the mCkS1
crystal structure represents just one of a range ofconformations available to the molecule and that
other conformations, more closely resembling thoseseen in the ScS1 crystal structures, are more common
in solution. Conversely, some class averages of every
S1 type are as bent as the mCkS1 crystal structure,

which suggests that this bent conformation is part of
the range of conformations naturally available to themolecule but that only a small proportion of
the molecules adopt this conformation at any given
time (Fig. 5). The earlier study of the heads of intact
myosin-2 [18] also found the typical shape was lessbent than the mCkS1 crystal structure, but at that time,
the ScS1 structure had not been determined.
The distributions of lever angles in the four S1types investigated were remarkably similar, including the mean lever angle (Fig. 4 and Table 1). The
differences between them are small compared to
the large differences seen in the lever angle whencomparing the crystal structures of ScS1 and mCkS1[10,28]. This contrasts with data from S1s bound toF-actin, for which differences in S1 shape between
S1 types have been detected, and the CkS1 lever
was more curved than that of insect flight muscle S1[33,34].
Overall, the range of appearances of the S1
molecule seen in this study includes conformations
similar to those seen by X-ray crystallography while
also suggesting a continuum of conformations arounda single, optimum conformation (similar to that
represented by the global average). This variation ismost likely to be due to thermally excited flexibility
within the molecule rather than to large rearrangements of the subdomains of the motor relative to eachother to produce a series of discrete conformations.
This conclusion is supported by the fact that leverangles are distributed as a Gaussian around a single
peak rather than over multiple peaks, as would be thecase if the molecule were adopting distinct conformations. It is possible that diversity of appearances canalso have contributions from adsorption of the S1 onto
the carbon film and the surface tension forces duringdrying of the film of stain. By working with just theheads rather than whole myosin molecules, we haveminimised the shearing forces that could arise whendifferent parts of a single molecule adsorb at different
times or the distortion of a large-scale threedimensional (3D) structure when it dries down

Flexibility in Myosin-2 Heads

onto the two-dimensional (2D) film. In general, drying
forces are expected to act perpendicular to the planeof the carbon film, whereas the flexibility we observe isin the plane of the film. The good match between the
global image average of apo ScS1 and the apo ScS1
crystal structure suggests that there is little systematic
distortion, while the similarity of the torsional stiffness
to that of molecules in solution (see below) suggeststhat the flexibility observed is not induced by specimen
preparation.
By classifying images based on features in theMD, and then reclassifying within each MD classbased on features in the lever, we have identified
the major site of flexibility to be the pliant region of
the myosin heavy chain between the converter ofthe MD and the ELC of the lever [14], rather than,for instance, a curvature along the whole lever orthe fulcrum within the MD that is associated withthe lever swing of the power stroke [15,35]. All
myosin-2s that have been studied, including scallop
striated muscle myosin, show pronounced bendingat this pliant region in forming the specific headsdown relaxed conformation [17,20,36–38], eventhough in ScS1 the crystal structures in various
states all have little flexion at this site. The fact thatthe switch between heads-down and active conformations is controlled merely by relatively weakbinding of Ca 2 + to scallop ELC [39] or by phosphorylation of the RLC in many other myosins indicatesthat this distortion requires little energy. The specificheads-down conformation of myosin-2 is formed invivo in the presence of ATP, which favours adoptionof the pre-power stroke conformation of the MD (alsoknown as the post-recovery stroke conformation)[24], in which the converter domain is in a markedly
different location with respect to the rest of the MD. It
will be of interest to determine whether flexion in thepliant region has different characteristics in the
pre-power stroke MD conformation.
S1 from several sources crystallised in the absenceof nucleotide has shown two distinct conformations
known as the rigor-like and post-rigor states (seeRef. [16] and references therein); thus, it is of interest
to examine whether our data distinguish which
conformation of S1 predominates under our solution
conditions. In vivo, the post-rigor conformation would
require binding of ATP to S1 to force the structuralchanges that weaken the strong rigor actomyosinbond, but for isolated S1, a sulfate ion in the nucleotide
pocket can suffice to induce a similar change.
For ScS1, the rigor-like and post-rigor states
are represented by PDB IDs 2OS8 and 1SR6,
respectively. Comparisons between the two scallop conformers show that there are no large-scale
shape differences that would allow us to discriminate between the conformers. However, normal
mode analysis of the transition between the two
structures in myosin-5 S1 has suggested thatflexibility in the converter region differs markedly,

903
such that flexion occurs at the pliant region in the
rigor-like structure whereas flexion is instead at
the converter–SH1 helix junction within the MD
in the post-rigor structure [40]. Since the EM data
show that the fulcrum of lever flexibility lies in the pliant
region, and not within the MD, it appears that, under ourconditions, muscle S1 is predominantly in the rigor-like
conformation, as should perhaps be expected since
our solution lacks sulfate.
Our calculation of the torsion spring constant at the
MD–lever junction in apo S1 (~ 23 pN·nm/rad 2) isremarkably similar to the value (≥ 20 pN·nm/rad 2)
found for apo myosin-5 [41]. That value wasobtained by tethering the lever to a surface andobserving through the light microscope the timevarying position of beads attached to the MD, andit was hardly changed when ATP was present.
Our value is about 2 orders of magnitude higher thanthat derived from measuring the spread of angles
between fluorescent actin filaments attached tothe two heads of scallop heavy meromyosin
molecules in solution (0.52 pN·nm/rad 2 at low
calcium conditions and 0.17 pN·nm/rad 2 at highcalcium) [42], though the stiffness being observed,
in that case, may be that of the junction of eachhead with the tail, which is expected to be low[43].
The majority of our data on lever flexibility refers
to molecules in which the MD is seen in the headin-profile view; thus, it is natural to wonder howthat would translate into axial and azimuthal lever
movement if the MD were strongly attached to theactin filament. Inspection of such S1-decorated actin[44] shows that the head-in-profile view is obtainedby viewing the actin filament obliquely from thebarbed end at an angle of ~ 45°. Therefore, if flexionof the lever occurred only in that plane, it would be
divided roughly equally between azimuthal and axial
components, and thus, the apparent axial stiffness
would be about 1.4 × higher than that we report.
However, we find the same lever stiffness for an MDclass that is differently oriented, which suggeststhat flexion at the pliant region is isotropic, and thus,that the axial stiffness is likely to be the same as wehave measured.
The torsional spring at the MD–lever junction
produces a stiffness for displacement of the tipof the myosin-2 lever of 0.37 pN/nm. The value
obtained is much lower than recent determinations of
the stiffness of myosin-2 heads attached to actin,
either as single molecules in the optical trap or as
crossbridges in muscle [45]. Because this stiffness,when derived from a torsional stiffness, is inversely
proportional to the square of lever length then, otherthings being equal, the stiffness of myosin-5 withits 3-fold longer lever is expected to be one-ninth of
this value, that is, 0.041 pN/nm, which is much lower
than the estimate obtained from heads bound toactin (0.2 pN/nm) [46] and would seem to suggest an904
impossibly large distortion (~ 60 nm) near the 2.5 pN
stall force of myosin-5.
How might the low stiffness of the pliant region
be reconciled with the higher stiffness of myosin
heads moving along their actin tracks? Evidence
suggests that the answer may be that strong binding
to actin stiffens the head. 3D reconstructions of actin
filaments saturated with myosin heads from various
sources [33,47–49] consistently show strong featuresin the levers and a lack of contact between them, out tohigh radius from the actin, implying that the lever
maintains a rather fixed geometric relationship to theactin helix; that is, it is not flexing at the MD–leverjunction. NMR studies of myosin heads found over
20% of mobile structure and all this became immobile
upon binding actin [50]. A high proportion of rapid
backbone amide hydrogen exchange in S1 isquenched by actin binding [51]. In contrast, flexibility
is implied by formation of the specific heads-down
structure in shutdown myosin-2; furthermore, ourmeasurement of flexibility in isolated myosin heads
implies a standard deviation of the tip of the myosin-2
lever of about 3.5 nm, which would greatly smearout the density in 3D reconstructions. An attractive
hypothesis is that easy flexibility at the MD–leverjunction allows the myosin head great freedom tolocate an actin binding site, after which this junction
stiffens in an allosteric response to the formationof a strong interface between the MD and actin, andthus contributes to force generation.

Flexibility in Myosin-2 Heads

had actin-binding capability. The actomyosin pellet
was then resuspended in ATP-containing buffer andcentrifuged again. The supernatant was collected
and all S1 in the supernatant that had been released
from actin by the presence of ATP was considered to
be fully active. ATP was then removed by dialysis
against an apyrase-containing buffer [at least 100 ml
containing 2.5 units apyrase (Sigma A6535)] or using
a NAP-5 desalting column (Amersham Biosciences).
Samples were also mixed with 0.5 units/ml apyrase(Sigma A6535) on ice for 15 min prior to use to ensurecomplete removal of ATP and ADP. The particularpreparation of apyrase used had equal rates of conversion for ATP to ADP and for ADP to AMP. One unitof the enzyme is defined as the amount that liberates
1.0 μmol of inorganic phosphate from ATP or ADP per
minute at pH 6.5 and 30 °C, and this rate drops
10-fold in ice (H. D. White, personal communication).
Electron microscopy
Thawed proteins were diluted with 25 mM KCl,
10 mM 3-(N-morpholino)propanesulfonic acid, 2 mM
MgCl2 and 1 mM ethylene glycol bis(β-aminoethyl
ether) N,N,N',N'′-tetraacetic acid (pH 7.0 at 20 °C)
then applied to grids with a thin carbon film andnegatively stained with 1% aqueous uranyl acetate as
described previously [60] but without using a buffer
rinse prior to staining. Final protein concentrations
were 50–100 nM. Micrographs were recorded on filmusing a Jeol 1200EX microscope at moderate dose(~ 100 e/Å 2)

Experimental Procedures
Image processing of individual S1 types
Proteins
Rabbit skeletal myosin was prepared from rabbit
dorsal back and hind leg muscle using a method
based on Ref. [52]. Myosin was digested with papain
to produce RbS1 with both ELC and RLC [53]. RbS1
containing only the A1 ELC was prepared by chymotryptic digestion [26]. Scallop myosin was preparedfrom the striated adductor muscle of Pecten maximus
by ammonium sulfate fractionation (Ref. [54] as
modified in Ref. [55]). ScS1 was prepared from thepurified myosin by papain digestion and further
purified by ammonium sulfate fractionation (Ref. [56]with modifications described in Ref. [57]). Purified
CkS1, prepared using papain, was a kind gift of
Dr. Ivan Rayment. It was reductively methylated[58], and amino acid analysis (Alta Bioscience)
showed that 97.4% of lysines had been methylated,
as expected [25].
In order to remove inactive heads from eachprotein preparation (apart from mCkS1, which is all
inactive [25]), we used a method based on Ref. [59].
The S1 was first centrifuged with actin, and thesupernatant was removed, to select for S1 that

Micrographs were digitised at a pixel size corresponding to 0.52 nm in the specimen and imported
into the SPIDER software suite, as described previously [11]. Individual heads were selected manually
by a mouse click midway along the molecule andwindowed out of the micrographs to form a series
of individual images. These were then aligned byreference-free methods. K-means (rather than hierarchical) clustering was used to divide the dataset
into groups for averaging in the initial classification
as this method is most useful for revealing diversity
within the dataset by subdividing a dataset into groups
of approximately equal size [61]. A mask was drawn
around the whole molecule (using the global varianceimage as a guide) to reduce the effect of backgroundnoise in the classification, as described previously [11].
Image processing of the pooled datasets
To allow a comparison between different S1s, wepooled datasets from four different S1s. To reducebias, we truncated the RbS1 and ScS1 datasets to be
the same size (4,619 particles) as the CkS1 dataset,giving a combined dataset of 17,307 particles. The

905

Flexibility in Myosin-2 Heads

data were aligned by reference-free methods. The
aligned images were classified into 400 classes using
K-means clustering and a whole molecule mask. Fromthe 400 classes, 309 class averages that showeda clearly identifiable MD and lever were selected(containing 13,176 particles), and 91 classes werediscarded (generally having poor staining) in which
both MD and lever could not easily be identified. Using
the 309 class average images, we then aligned the
motors in the images such that the long axis of the
motor was approximately vertical within the window.
This was performed using a model image createdusing the global average from the previous alignment,
shifted such that the MD was centrally located within
the window and rotated so that its long axis wasvertical with respect to the window. Class averages
rather than individual images were aligned in this way
since we found that the high noise in individual images
precluded the alignment of such a relatively small
and roughly circular region (the MD alone is only
~ 80 kDa). The parameters from the whole moleculealignment and the MD alignment were combined into asingle set of shift and rotation vectors, which were thenapplied to the original windowed images to produce
a stack of images in which all molecules were alignedwith their MD approximately vertical within the window.
The global variance image from the MD-aligned
image stack was used to draw a mask around the MD.
The stack of individual images was classified into 100
classes based on the pixels within the mask, using
hierarchical ascendant classification to create large
classes of the most abundant MD appearances. A
second mask was created using the global varianceimage to draw around the region encompassing all
possible lever positions and excluding the MD. Thiswas performed iteratively to find the tightest possible
lever mask that did not exclude any lever positions.
The stack of individual images was classified into 200
lever classes based on the pixels within the mask,using K-means clustering to obtain classes of similarsize.
For each of the 200 lever classes, to calculate theangle of the lever relative to the vertical axis of theimage window, and thus relative to the verticallyaligned MD, a point deemed to best represent the tipof the lever was selected. Using this pixel location
together with the pixel location of the point where thelever joins the MD (marked in the global average imageof MD-aligned images), we calculated the lever angle(Fig. 3f).
The particles in each MD class were separated
into four subclasses, one for each S1 type. Each ofthese subclasses was then divided into five bins,
based on ascending lever angles assigned to theparticles such that the first bin contained the 20%of images from that type of S1 with the lowest leverangles and the next bin contained the next 20% of
lowest lever angles and so on. The average image
was then calculated from the contents of each bin.

Calculation of lever stiffness
The torsion spring constant of the lever of eachspecies of S1 was calculated using κ = kBT/σ 2,
where κ is the torsion spring constant (N·m/rad 2), kB
is the Boltzmann constant (1.381 × 10 −23 N·m/K), T is
the absolute temperature (293 K in all cases) and σ 2is the variance of the lever displacement (rad 2) [30].
To convert torsion spring constant to apparent
cantilever stiffness (k), we took the energy for rotationof the lever through angle θ to be equal to the energy
required to bend the lever such that its tip is displacedby the same distance (x) as produced by the angularrotation. Thus, kx 2/2 = κθ 2/2. For a lever of length
L, x = Lsinθ. Hence, for small displacements, for whichsinθ ≈ θ, k = κ/L 2. L was taken to be 8 nm for all S1s.
Comparison to crystal structures
To allow comparison of EM averages with crystal
structures of myosin-2 S1, we made low-resolution 2D
projections of nucleotide-free structures of ScS1 (PDB
ID: 1DFK) and mCkS1 (PDB ID: 2MYS). The structure
file 1DFK contains only β-carbons for the side chains;these were completed using Swiss PDB Viewer.
2MYS.pdb contains only α-carbons of the light chains;
we used a structure file containing all atoms for thesechains, kindly supplied by Dr. Ivan Rayment.
PDB files were converted to 3D SPIDER volumes.
An angular document file containing three Euler angles
defining quasi-evenly spaced projection directions wascreated with an angular step of 5º in θ and was used to
produce a set of 799 2D projections from each of thetwo 3D structure files that represented one hemisphereof projections. Each EM class average was thencompared to each of the two sets of projection images,
as well as to their mirror images that represent theother hemisphere. Cross-correlation score was used
to determine the optimum rotation and translationof each projection, and the projection with thehighest score was taken to be the best match ofeach crystal structure to that EM class average.
Although the EM data were collected in theabsence of calcium and the ScS1 crystal structure
data to which they were compared were collectedin the presence of calcium, crystallographic data
indicate that the effect of calcium on the overall
conformation of ScS1 is likely to be small [16,62].
Supplementary data to this article can be foundonline at http://dx.doi.org/10.1016/j.jmb.2013.11.
028.

Acknowledgements
We thank Drs. Hitesh Patel, Howard White andIvan Rayment for supplying protein and for helpful

906

Flexibility in Myosin-2 Heads

discussions. This work was funded in part by a Biotechnology and Biological Sciences Research Council
postgraduate studentship to N.B., a Wellcome Truststudentship to DJR (086778/Z/08/Z) and by NHLBI
intramural funds (HL001786) to James Sellers.
Received 1 October 2013;
Received in revised form 25 November 2013;
Accepted 29 November 2013
Available online 9 December 2013
Keywords:
electron microscopy;
image processing;
stiffness;pliant region;
crossbridge† Present address: N. Billington, Laboratory of Molecular Physiology, NHLBI, National Institutes of Health,Bethesda, MD 20892-8015, USA.

This is an open-access article distributed under the termsof the Creative Commons Attribution-NonCommercial-No
Derivative Works License, which permits non-commercial
use, distribution, and reproduction in any medium,provided the original author and source are credited.
Abbreviations used:
S1, myosin subfragment-1; CkS1, chicken skeletal muscle
myosin subfragment-1; mCkS1, methylated chicken skeletal muscle myosin subfragment-1; RbS1, rabbit skeletal
muscle myosin subfragment-1; ScS1, scallop crossstriated adductor muscle myosin subfragment-1; MD,
motor domain; ELC, essential light chain; RLC, regulatory
light chain; EM, electron microscopy; 3D, threedimensional; 2D, two-dimensional.

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