* Institute of Molecular Biophysics, Florida State University, Tallahassee, Florida 32306-4380; Department of Cell Biology,
Duke University Medical Center, Durham, North Carolina 27710; and § MRC Laboratory of Molecular Biology, Hills Road,
Cambridge CB2 2QH, United Kingdom
Rigor insect flight muscle (IFM) can be
relaxed without ATP by increasing ethylene glycol
concentration in the presence of adenosine 5-[
-
imido]triphosphate (AMPPNP). Fibers poised at a
critical glycol concentration retain rigor stiffness but support no sustained tension ("glycol-stiff state"). This
suggests that many crossbridges are weakly attached to
actin, possibly at the beginning of the power stroke.
Unaveraged three-dimensional tomograms of "glycol-stiff" sarcomeres show crossbridges large enough to
contain only a single myosin head, originating from dense collars every 14.5 nm. Crossbridges with an average 90° axial angle contact actin midway between
troponin subunits, which identifies the actin azimuth in
each 38.7-nm period, in the same region as the actin target zone of the 45° angled rigor lead bridges. These 90°
"target zone" bridges originate from the thick filament and approach actin at azimuthal angles similar to rigor
lead bridges. Another class of glycol-PNP crossbridge
binds outside the rigor actin target zone. These "nontarget zone" bridges display irregular forms and vary
widely in axial and azimuthal attachment angles. Fitting
the acto-myosin subfragment 1 atomic structure into
the tomogram reveals that 90° target zone bridges share
with rigor a similar contact interface with actin, while
nontarget crossbridges have variable contact interfaces.
This suggests that target zone bridges interact specifically with actin, while nontarget zone bridges may not.
Target zone bridges constitute only ~25% of the myosin heads, implying that both specific and nonspecific
attachments contribute to the high stiffness. The 90°
target zone bridges may represent a preforce attachment that produces force by rotation of the motor domain over actin, possibly independent of the regulatory domain movements.
FORCE production by myosin heads during muscle
contraction has long been modeled as a transition
of attached crossbridges from a 90° to a 45° axial
angle. Efforts to image crossbridge forms and angles intermediate between 90° heads in ATP-relaxed insect flight
muscle (IFM)1 and the 45° angled bridges in rigor have
used nucleotide analogs such as adenosine 5 Crossbridges in this partially relaxed, glycol-PNP state
are important because they may represent the form of the
initial attachment of myosin with bound nucleotide preceding force generation (Marston and Tregear, 1984 IFM is superb for structural study because the symmetry
and spatial arrangement of filaments results in paired
crossbridges on opposite sides of the actin filament. This in
turn has given rise to a unique shorthand terminology. The
individual crossbridge forms are not unique to IFM, only
their symmetrical placement about the thin filament. The
filament arrangement also facilitates the microtomy of a
type of thin section with coplanar filaments that provide
views of the entire crossbridge. The best of these, the myac
layer, is a 25-nm-thick longitudinal section containing alternating myosin and actin filaments. In rigor, the maximum number of myosin heads attach to actin, forming
doublet pairs every 38.7 nm, the "double chevrons"
(Reedy, 1968 Target zone is the name given to the region of the thin
filament where crossbridges bind (Reedy, 1968 When aqueous AMPPNP was added to rigor IFM, the
tension dropped by two thirds, but the stiffness remained
as high as rigor. This initially suggested a reversal of the
power stroke, but 3-D reconstructions revealed that the
lead bridges remained attached, midway between troponin
densities, at axial and azimuthal angles close to rigor. The
drop in tension without a large change in axial angle
seemed to contradict the lever arm hypothesis for motion
producing force. However, a cause for the loss of tension was found in tomograms, which showed that rear bridges
detached and were replaced by nonrigor bridges bound to
actins outside of the rigor target zone, to sites not selected
by crossbridges even under the high-affinity conditions of
rigor. These nontarget bridges in aqueous-PNP had variable axial and azimuthal angles and appeared to bind actin
with variable contact interfaces. This suggested that they
were nonspecifically bound to actin. Moreover, their variable structure did not suggest how a simple axial angle change could convert them to a familiar form, such as an
angled rigor bridge.
However, an intriguing doublet crossbridge group with a
consistent structure was recognized in aqueous-PNP. Immediately M-ward of the "lead" rigor-like bridge was a
"nonrigor" bridge bound at a 90° or antirigor angle. In this
doublet, called a mask motif, both lead and M-ward nonrigor bridge pairs had similar azimuths and contact interfaces with actin and bound within the lead bridge target
zone. A simple angle change could convert the M-ward,
nonrigor bridge in a mask motif to a single headed lead
bridge. Thus, in the mask motif, the lead bridge could be at
the end of the power stroke, with the M-ward, nonrigor
bridge near the beginning. The pairing of rigor and antirigor angled crossbridges bound to the same target zone
suggests that crossbridges might act as a relay during muscle contraction (Schmitz et al., 1996 The affinity of myosin for actin in aqueous-PNP is high
compared with weak binding intermediates thought to
represent the beginning of the power stroke (Green and
Eisenberg, 1980 Specimen Preparation and Electron Microscopy
The embedded fibers used to collect sections and images for these 3-D reconstructions were the same fibers of glycerinated dorsal longitudinal
flight muscle of Lethocerus prepared in the experiments of Reedy et al.
(1988) Tomography
We reconstructed two myac layers using a protocol for 3-D tomography
derived from the same methods used in 2-D crystal image processing
(Amos et al., 1982 Attachment Parameters for Crossbridges
The most important parameters that we can derive to define a particular
crossbridge attachment are its angle relative to the filament axis (axial tilt)
and its angle of attachment relative to the actin monomer in a plane perpendicular to the filament axis (azimuthal angle). Our reconstructions do
not resolve actin monomers, but the information on the azimuth can be inferred from the 3-D maps using the troponin marker to establish the orientation at one axial coordinate and the periodicity of the actin filament to define the expected rotation as the axial coordinate changed. Troponin
could be identified clearly in averaged images of glycol-PNP, thereby defining one point in orientation space independent of crossbridges. Previous work with rigor IFM showed that troponin epitopes were positioned
close to the rigor rear crossbridge (Reedy et al., 1994 3-D reconstructions of rigor IFM revealed that the part of troponin that
formed a bump or bead was positioned on a rigor myosin binding site
(Taylor et al., 1989a,b). Moreover, averaged 3-D reconstructions of both
rigor and AMPPNP-treated muscle show that troponin position does not
change between these two states (Winkler et al., 1996 Fitting of Atomic Coordinates and
Surface Representation
Modeling of the acto-S1 atomic structure into the envelope of the 3-D reconstructions was done starting with the C Our model atomic coordinates were those of fully decorated acto-S1.
However, thin filaments in situ are not fully decorated because there are
insufficient myosin heads to label all the actins. The excess S1 heads in the
model structure that did not superimpose or lie close to the in situ crossbridges were removed from the display, and those remaining heads that
were close to the in situ crossbridges were positioned with minimal axial
and azimuthal changes from the orientation indicated by the initial alignment.
Because crossbridges in the glycol-stiff state depart significantly from
the rigor form, we gave ourselves more flexibility in the fitting process
than used in previous work (Schmitz et al., 1996 Essentially the same procedure was used for nontarget zone crossbridges, except that the S1 molecule had to be shifted azimuthally from
the rigor binding site to overlap the crossbridge envelope. We minimized
torsional movement of S1 for the nontarget zone crossbridges for the reasons given above.
Surface representations of the reconstructed maps were obtained using
the Explorer software package (Silicon Graphics, Mountain View, CA).
All data processing calculations were done on Silicon Graphics Indigo2
computers with Extreme and Solid Impact graphics.
2-D Projections of the Tomogram
The 2-D projection of the glycol-PNP 3-D reconstruction
resembles ATP-relaxed muscle in many ways (Fig. 1 C).
Foremost are the collars around the thick filament spaced
at intervals of 14.5 nm that include regularly arranged myosin heads that are not attached to actin and thin crossbridges projecting toward the thin filament with various
axial angles whose distribution is centered around 90°. Fig.
1 A shows, at higher magnification, that the crossbridges emerge from the collars to attach to the thin filament. The
filtered image of the projection (Fig. 1 C, inset, bottom
right) emphasizes the annular collars every 14.5 nm around
the thick filament.
Unlike relaxed muscle, crossbridges connect to the thin
filament at intervals of 38.7 nm. Often, these 90° crossbridges are arranged in rows at the same transverse level
(Fig. 1 C, white triangles). Note also regions where the actin filament is contacted by crossbridges only along one
side (Fig. 1 C, # on adjacent thick filament), a feature that
is seldom seen in relaxed muscle. In the average, the crossbridges are deemphasized, indicating a relatively large disorder in their structure. Particularly prominent in the average are dots spaced every 38.7 nm along the actin filament. These dots are the troponin complex (Fig. 1 C, T). Thus,
troponin as well as the collars on the thick filament are
much more regular features than the crossbridges.
"Mask motif" doublets, first observed in AMPPNP-treated IFM (Schmitz et al., 1996 The computed diffraction pattern (Fig. 1 B) shows an
important feature of the transform of the glycol-stiff state:
spots on the 19.3-nm layer line. In relaxed muscle, the
19.3-nm layer line is absent or very weak. In those states of
IFM that have significant crossbridge attachment, the
19.3-nm layer line is relatively strong in comparison to relaxed muscle, so that this feature is associated with periodic crossbridge attachment.
Surface Relief of the Glycol-PNP Tomogram
To display details of the reconstruction, we have selected
and enlarged a typical region from the very large area imaged in the tomogram. This selected area is indicated by a
white square in Fig. 1 and is shown in surface view in Fig. 2
A. On the left hand side of Fig. 2 A (dark gray), three thick
filaments (1, 3, and 5) and the two intervening thin filaments (2 and 4) have been averaged only along the filament axis and aligned with the unaveraged tomogram
(light gray). The average facilitates identification of the
14.5- and 38.7-nm repeats, which can be observed by sighting across the figure from the left hand edge. Thick and
thin filaments 1 and 2 on the left are simple averages,
while averaged thick and thin filaments 3, 4, and 5 are superimposed with the unaveraged tomogram of the same
filaments.
Although the overall crossbridge arrangement in the
glycol-PNP state is less ordered than rigor, some regularities, such as the troponin position, are detected and emphasized in the averages (Fig. 2 A, filaments 1 and 2). The
position of the troponin complex on the thin filament (Fig.
2 A, T) can be identified by a lump that coincides with the
dark dot seen in the averaged projection. The importance
of the troponin position is that it allows us to identify the
actin dyad azimuth in the lead bridge target zones. Lead
bridge target zones of rigor and AMPPNP-treated muscle
are located midway between two successive troponin densities (Schmitz et al., 1996 The unaveraged tomogram shows bridging densities
reaching from the thick to the thin filaments that tend to
align in transverse rows spaced axially at 38.7-nm intervals
(Fig. 2 A, arrowheads) and that coincide with the dashed
white lines in the average. These "target zone" crossbridges generally attach actin at angles of ~90°, although
we are able to detect large deviations from this value (Fig.
2 A, arrows indicating attachment angle; see also Fig. 5).
In addition, many crossbridges attach to actin in between
those 38.7-nm intervals, and these bridges attach at various angles and azimuthal positions (Fig 2 A, asterisks).
These intermediate crossbridges we refer to as "nontarget
zone" bridges. The relative distributions of target and nontarget bridges are best seen when the target zone is
marked on an outline image (Fig. 2 B). Target zone crossbridges are sufficiently well ordered that they are retained
in the averages, whereas nontarget zone crossbridges are
generally averaged out. Note that where the average and
unaveraged filaments in the reconstruction are superimposed, much of the mass (light gray) that is averaged out is
associated with these less regularly arranged crossbridges.
Most glycol-PNP bridges are smaller than rigor crossbridges, an observation that would suggest that they consist predominately of a single myosin head. Fairly long
stretches occur where crossbridges attach on only one side
of the thin filament (Fig. 2 A, #). On the myosin filament
surface, the 14.5-nm period of crossbridge collars is obvious and in some locations, long pitch helical tracks on the
thick filament are visible (double arrowheads). The averaged myosin filament surface emphasizes the 14.5-nm collars around the thick filament backbone (Fig. 2 A, upper
left, dashed line).
Selected Crossbridge Motifs
To illustrate the major aspects of the reconstruction, we
selected a gallery of crossbridge structures (Fig. 3). In each
panel, the position of the lead bridge target zone is marked
with an asterisk on the thin filament. In nearly all cases,
the sizes of the crossbridges suggest that they hold only
one myosin head.
Target zone crossbridges are most frequently attached
at an axial angle of 90° (Fig. 3, A, B, and D); however, the
angle can vary from a rigor-like 45° angle (Fig. 3 E) to an
antirigor 120° angle (Fig. 3 C). In some cases, two heads,
apparently from different myosin molecules, are seen attaching to adjacent actin monomers (Fig. 3 D). In cases
such as this, the target zone crossbridge can be identified
using the front-back rule (see below). In nearly all cases,
the attachment of opposite crossbridges are staggered, reflecting the 2.75-nm stagger of actin monomers on opposite
long pitch helical strands of the thin filament. Mask-motif
doublets (Fig. 3, H and N) are occasionally seen in the glycol-PNP state, as are partial mask motifs which have crossbridges on only one side of the thin filament (Fig. 3 G).
Target zone crossbridges are often distinguished from
nearby nontarget zone bridges by their axial position relative to troponin (Fig. 3, F and L). An additional distinguishing characteristic is the front-back rule illustrated in
Fig. 3 M. When viewed toward the Z-disk (as shown here),
target zone crossbridges approach the thin filament from
the front left and the back right. These are the same directions of approach used by lead bridges of rigor and AMPPNP; these strongly bound bridges are considered to be stereospecifically bound to actin. The front-back rule reflects the approximately twofold axial symmetry of the actin filament and the well-defined azimuth associated with
stereospecific binding. Glycol-PNP target zone bridges
also bind at the same axial position (the lead bridge target
zone) and obey the front-back rule. We therefore consider
them to bind actin stereospecifically, even though they are
weakly bound. Such weakly, stereospecifically bound heads
are good candidates for conversion to a strongly bound form via a power stroke. We would suppose that with a
change in orientation accompanying a change in biochemical state, these crossbridges could become strong-binding,
force-bearing crossbridges.
In contrast, the nontarget zone bridges may be nonspecifically bound. Due to the helical structure of the thin filament, the azimuthal position of each actin monomer changes
by 23° every 55Å of axial translation, so crossbridges from
successive 14.5-nm collars that originate from similar azimuth on the thick filament would have to undergo large
deformations to align the actin binding site to the myosin
binding site on their partner actins, which would be rotated to different azimuths in the thin filament. The single
crossbridge positioned Z-wards of the target zone in Fig. 3
M illustrates this topological problem. Like the target zone crossbridge, it also approaches actin from the front left
side. However, this axial position is near that of the stereospecifically bound rear bridge of rigor, which would approach actin from the back left side, not the front. This
nontarget crossbridge therefore interacts with a different
surface of the actin monomer than do stereospecifically
bound rigor rear bridges. Weakly bound nontarget crossbridges may be nonspecifically attached and would be expected to detach and rebind actin in a more favorable location to allow conversion to a strong-binding form.
Each bridge pair in the mask motif of glycol-PNP obeys
the front-back rule (Fig. 3 N). The target zone bridges on
the Z-ward side in the mask motif approach actin from the
same directions (front left, back right) as the lead bridges
of rigor and AMPPNP. In contrast to nontarget bridges,
the M-ward bridges in the mask motif also approach actin
from the same directions as the adjacent, Z-ward target
zone bridges, even though they originate from different 14.5-nm collars on the thick filament.
Fitting of Acto-S1 Coordinates into
3-D Reconstructions
The crystal structure of actin (Kabsch et al., 1990
Many glycol-PNP bridges originate from the dense collars of density every 14.5 nm around the thick filaments.
These collars give the thick filaments a wider appearance
compared with rigor, and the exact position of the regulatory domain can be difficult to determine. However, the
angle of approach of the motor domain and some portion
of the regulatory domain can be determined from the 3-D
envelope and compared with the atomic S1 structure.
To visualize the differences between the positions and
angles of stereospecific rigor S1 compared with those of
glycol-PNP crossbridges in the reconstruction, a marker of
the binding site and angle of rigor S1 have been incorporated into the transverse views (Fig. 4 B). This marker
(red) is kept constant at the rigor binding site and angle
even when the S1 molecule has been moved to best match
the glycol-PNP crossbridge envelope. Overlaying unmodified rigor acto-S1 on the reconstruction (Fig. 4 C) indicates that significant crossbridge mass lies M-ward of the atomic
model. Rotating the S1 atomic coordinates from the ~45°
rigor angle to near 90° angle (shown in stereo in Fig. 4 E)
provides a better fit of the atomic structure to the 3-D envelope of the crossbridges in situ. The transverse view
(Fig. 4 D) shows that the azimuthal position of the glycol-PNP target zone bridges follows that of rigor S1, consistent
with a stereospecific attachment of weakly bound, target
zone bridges. The transverse views also show that target
zone bridge pairs originate from the front left-back right positions, as do rigor lead bridges.
Fig. 4, F-H, shows how target and nontarget zone crossbridges differ as seen in longitudinal and transverse view.
The pair of target zone crossbridges are attached to the actin filament at an axial angle of ~90° (Fig. 4, F and G).
Above and below are two nontarget crossbridges. Even
though in surface view (Fig. 4 F) these bridges appear similarly angled, different adjustments of atomic S1 are needed
to best match the angle of the crossbridge envelope, seen
in Fig. 4 G. In the transverse views to the right (Fig. 4 H),
the target zone crossbridges originate from the left front
and back right (obeying the front-back rule) and bind to the same region of actin at similar azimuthal angles as
rigor (Fig. 4 B). The upper nontarget bridge emerges from
the thick filament at the same azimuth as the left target
zone bridge. However, for the nontarget bridge, the axial
separation and concomitant helical rotation places the myosin binding site on actin out-of-reach around the thin filament. The apparent contact with actin is at a completely
different location from that of rigor crossbridges, whether
rear or lead targets. The lower nontarget zone crossbridge is positioned perhaps one actin monomer from the axial
position of the rigor rear crossbridge and thus has a somewhat more favorable contact with actin. Previous fittings
of rigor rear bridges (Schmitz et al., 1996 Structural Parameters of Glycol-PNP Crossbridges
The azimuth at which crossbridges attach to actin is an important piece of information that is not available from
electron micrographs, which are projections, but that can
be obtained and quantified from transverse views of a 3-D
image. Direct observation suggested that weakly bound
glycol-PNP crossbridges showed a wide variety of azimuthal attachment angles compared with a narrow distribution in rigor. The problem is illustrated in Fig. 5 A, where a rigor S1 is bound to actin according to the Rayment model. In Fig. 5 B, a glycol-PNP crossbridge is bound
to actin at a different azimuth than rigor. To quantify the
azimuthal attachment angles of rigor and glycol-PNP
bridges, we selected one actin filament from the present
reconstruction that spanned a complete half sarcomere (Fig. 1 C, black and white dotted outline) and determined
the azimuthal position of each attached crossbridge (74 total). The exact orientation of the actin azimuth was determined by the position of the troponin complex in each
38.7-nm repeat, and the observation that this coincides
with an S1 binding site (Taylor et al., 1989a In glycol-PNP, the distribution of azimuthal attachment
angles is quite large for all crossbridges and spans a range
of ±135° (Fig. 5 I). Many of these crossbridges in glycol-PNP are nontarget zone crossbridges whose nonspecific
attachment gives rise to the broad angular distribution.
The distribution is much narrower for target zone crossbridges (Fig. 5 J), with most crossbridge attachments falling within a ±30° arc around the average rigor value. This
narrower distribution shows that target zone crossbridges have a more specific actin interaction than the nontarget
zone crossbridges. However, the azimuthal angle distribution is much broader than that obtained by the same
method for target zone crossbridges in rigor (Fig. 5 E) and
AMPPNP (Fig. 5 G) as determined from our previous reconstructions (Schmitz et al., 1996 The axial angle distribution for target zone glycol-PNP
crossbridges is very different from that obtained for rigor
and aqueous-PNP target zone crossbridges. In rigor, the
angles are roughly centered about 45° (Fig. 5 F), and for
aqueous-PNP the distribution skewed, with most angles
being near 45° but a significant number at angles closer to 90°
(Fig. 5 H). The axial angular distribution for glycol-PNP crossbridges is centered around 90° (Fig. 5 K) but is considerably broad and encompasses both rigor and antirigor angles.
The distribution of azimuthal and axial attachment angles for rigor, aqueous-PNP, and glycol-PNP crossbridges
(Fig. 5) reveals an underlying specificity in the actin interaction for target zone crossbridges. For these three states,
the azimuthal angle distribution broadens as actin affinity
is lowered but still remains centered around the rigor angle. However, the axial attachment angles change, with the
center of the distribution approaching 90° as actin affinity
is lowered. The data suggest that the actin interaction
changes with chemical state of the crossbridges. Moreover, it suggests that the interaction between actin and myosin
has specificity to the extent that in all three states, target
zone crossbridges are bound to actin at the same interface,
but the motor domain has varying angles.
The reconstructions reported here are the first 3-D images
of in situ crossbridges that are likely to be in a weakly attached state. Near the critical glycol concentration, IFM fibers in glycol-PNP have high mechanical stiffness but will
not bear sustained tension, thereby suggesting a weak interaction with actin. Thus, 3-D reconstructions of crossbridges in this mechanical state should provide images of
good candidates for preforce crossbridges. The structure
of myosin in weakly attached, preforce states has been difficult to identify in the variable images produced by isolated acto-S1 (Applegate and Flicker, 1987 In Glycol-PNP, Target Zone Crossbridges Attach to
Actin Stereospecifically, While Nontarget Zone Bridges
Attach Nonspecifically
Our previous tomograms of IFM treated with aqueous-PNP (Schmitz et al., 1996 In glycol-PNP, both target and nontarget crossbridges
are also present. However, target zone crossbridges in glycol-PNP have a structure significantly different from both
rigor and AMPPNP target zone bridges. Target zone
bridges in glycol-PNP appear exclusively single headed.
This is consistent with a model of Geeves and Conibear
(1995) In contrast, nontarget zone crossbridges do not follow
an obvious pattern with regard to their thick filament origins. They approach actin from a variety of directions and
they bind to actin on different surfaces from that observed
in rigor. The nontarget bridges are therefore considered to
bind nonstereospecifically to actin.
S1 Fittings Show that Glycol-PNP Bridges
Have Different Conformations of the Motor and
Regulatory Domains
Fitting S1 into the envelope of target zone bridges reveals
that the alignment of the rigor structure required both azimuthal adjustment (rotation about the filament axis) and
tilt angle adjustment (rotation about the perpendicular to
the filament axis) to superimpose S1 with the reconstruction envelope. The binding site on actin served adequately
as the pivot point for these adjustments, although good
alignment of the S1 and crossbridge regulatory domains
would still require changes in the angle between the regulatory and motor domains, which were not attempted. The
motor domains of S1 and target crossbridges were brought
into approximate alignment by our adjustments, but the
need to pivot S1 about the actin binding site to obtain a
reasonable fit implies some alteration in the actomyosin
interface compared with that in rigor (and also AMPPNP).
In the case of nontarget zone crossbridges, simply pivoting the S1 about the binding site on actin could not successfully align the S1 atomic model with the reconstruction
envelope. Even approximately superimposing S1 on a
nontarget bridge required translational movements away
from the rigor binding site, indicating that these myosin
heads are binding to a different interface on actin.
These fittings of S1 to glycol-PNP crossbridges can compare the relative positions of the motor domains of the
bridges to the S1 more easily than the relative positions of
the regulatory domains. This is because the light chain domain of a glycol-PNP bridge often merges with the 14.5-nm
collars, and its position relative to the S1 regulatory domain cannot be judged. In contrast, the fittings in rigor
showed the light chain domain free of the thick filament.
However, the fact that glycol-PNP crossbridges extend
from the 14.5-nm collars on the thick filament establishes that the regulatory domains must be differently configured
than rigor or AMPPNP crossbridges. This also demonstrates that the collars themselves, and not an intermediate
point between them, represent the crossbridge origin.
The Maintenance of High Stiffness in Glycol-PNP
Our 3-D reconstructions of rigor, AMPPNP, and glycol-PNP-treated IFM show approximately the same number
of attachments from crossbridges to actin despite the
rather large structural differences between the states. In
rigor, strongly bound target zone attachments dominate,
and over 50% of these involve two-headed lead bridges. Virtually all myosin heads in rigor interact with the same
region of actin, and this is defined as stereospecific binding. After addition of AMPPNP, actin-myosin affinity is
reduced, specifically attached target zone and nonspecific,
nontarget zone crossbridges occur, and fewer than 50% of
the attachments arise from two-headed crossbridges. In
the glycol-PNP state, affinity is further reduced, all crossbridges are one-headed, and only ~50% are target zone bridges. Despite these differences, the isotonic stiffness is the same in all three states (Tregear et al., 1990 Relationship to X-ray Diagrams of IFM
The 3-D reconstruction reveals a distribution of crossbridges in glycol-PNP that can explain the appearance of
intensity on the 19.3-nm layer line (10.12 reflection) in
x-ray and optical diffraction patterns (Tregear et al., 1990 The Transition from Rigor to Stiff Glycol IFM
With these reconstructions of IFM in glycol-PNP, we have
3-D information on a sequence of stable equilibrium states
that have a range of crossbridge structure from strongly
bound bridges at the end of the power stroke in rigor to
weakly attached bridges that may represent the form of
myosin heads at the beginning of a working stroke. The
three states are shown together for comparison in Fig. 6.
Rigor IFM is characterized mechanically by high stiffness and the ability to retain a large imposed tension. In rigor
(Fig. 6, A-C), there are two crossbridge classes. One class,
the lead bridge, contains both heads of a myosin molecule.
The myosin heads in the two-headed lead bridge have different structures imposed by their common origin and separate target actins. One head generally appears more angled than the other, giving the crossbridge an overall
triangular shape. The single-headed crossbridge class, the
rear bridges, have variable angles and are bent azimuthally considerably more than lead bridges. Modeling of the
atomic acto-S1 structure into the 3-D images of rigor muscle suggests that rear bridges have a highly strained conformation (Schmitz et al., 1996
On addition of AMPPNP to rigor IFM, tension drops by
70%, but stiffness remains high as in rigor (Reedy et al.,
1988 It is ironic that relative to the 90° to 45° lever arm model,
the greatest mechanical change occurs on addition of
AMPPNP to rigor IFM, while the angle changes in the target zone crossbridges are minimal. On glycol addition to
AMPPNP, the mechanical changes are relatively small,
while the structural changes are far more pronounced. The
angled, rigor-like lead bridges in AMPPNP are replaced in
glycol-PNP by a single-headed 90° crossbridge in the lead
target zone. The change in angle from 45° to 90° that occurs with minimal sarcomere lengthening indicates that
significant crossbridge detachment and redistribution has
occurred. These stable states were originally hoped to
show a reversal of the power stroke. However, in order for
these stages to be viewed as a reverse power stroke, the
structural changes must occur in attached crossbridges. As
our 3-D reconstructions show, significant crossbridge detachment and redistribution occurs during the transition from rigor to glycol-PNP. The crossbridge redistribution
that is a significant part of the maintenance of stiffness is
due to the occurrence of nonspecifically attached, nontarget crossbridges, which must detach and rebind actin in a
more favorable location to convert to a strongly bound 45°
angle. The importance of such nonspecific, myosin-actin
interactions may be the recruitment of crossbridges to the
vicinity of the actin filament during active contraction,
where they can bind quickly to target zone actin sites when
these are brought into favorable alignment by filament sliding.
The sequence from rigor to glycol-PNP shows overall a
45° to a 90° angle change, but the crossbridge forms seen in
situ reveal relatively independent changes in axial angle of
the motor domain and reconfiguration of the regulatory
domain of myosin. The 90° glycol-PNP target zone bridges
establish that myosin heads originating from 14.5-nm collars can attach specifically to actin with a nonrigor form
and angle. Conversion of the bridges to 45° associated with strong binding could occur by rotation of the motor domain while bound to actin. Such a rotation independent of
the regulatory domain could allow the motor domain to
develop a strong-binding configuration on actin while
building strain in the crossbridge. This possibility provides
a mechanism to "cock" the regulatory domain lever arm
for the eventual working stroke while at the same time allowing the strong-binding interface to form. The strain could then be released, and a working stroke produced,
through independent rotation of the regulatory domain.
This aspect distinguishes our interpretation from models
that require only a single binding configuration on actin
(Rayment et al., 1993) and models that allow rotation of
the entire crossbridge on the actin interface (Huxley and
Simmons, 1971-[
-imido]
triphosphate (AMPPNP) in stable equilibrium states to
drive the crossbridges backwards from the 45° angle in
rigor to an attached 90° preforce form, otherwise similar to
myosin heads in ATP-relaxed fibers (Reedy et al., 1988
; Tregear et al., 1990
). However, AMPPNP alone will not
fully relax IFM, and crossbridges binding AMPPNP retain
many rigor-like features (Schmitz et al., 1996
; Winkler et
al., 1996
). On the other hand, AMPPNP in combination
with ethylene glycol will relax IFM. When poised at a critical glycol concentration, muscle stiffness is as high as rigor,
suggesting crossbridge attachment, but fibers will not bear
sustained tension (Clarke et al., 1984
; Tregear et al., 1984
).
Two-dimensional (2-D) analysis of electron micrographs showed that this stiff glycol-PNP state resembled ATP-relaxed fibers in having dense collars every 14.5 nm along
the thick filament and thin crossbridges originating from
these collars at various axial angles around 90°. However,
unlike relaxed muscle, stiff glycol-PNP fibers showed both
90° angled bridges that were regularly spaced every 38.7 nm and more intensity on the 19.3-nm layer line in optical
and x-ray diffraction patterns (Reedy et al., 1988
; Tregear
et al., 1990
).
;
Tregear et al., 1984
; Reedy et al., 1988
). This putative preforce 90° crossbridge could not be characterized in 3-D because its variable form and lattice arrangement precluded imaging by averaging methods of 3-D reconstruction. Recently, nonaveraging tomographic methods have been developed and successfully applied to rigor and aqueous-PNP, facilitating characterization of variable crossbridge
forms that occur in situ (Taylor and Winkler, 1995
, 1996
;
Schmitz et al., 1996
; Winkler and Taylor, 1996
).
). "Lead bridges," which form the pair proximal to the M-band, consist of both heads of a myosin molecule and show an overall axial angle of 45° (Taylor et al.,
1984
). "Rear bridges," which form the pair proximal to the
Z-disk, consist of a single myosin head angled closer to
90°. Crossbridges originate from the thick filament along
helical tracks so the azimuths of their origins follow a regular pattern. Relative to the thin filament in the myac
layer, the lead bridges originate from the left-front and
back-right of the adjacent thick filaments, while rear
bridges originate from the left-back and right-front. At
their actin ends, the crossbridge attachments follow the
changing rotation of the actin protomers along the actin
helix. The combination of the azimuth of the origin and
the azimuth of the crossbridge contact to actin define the
azimuthal angle of the crossbridge.
); by implication this is the region of the thin filament where actin
monomers are most favorably placed for actomyosin interaction. In our previous 3-D reconstructions of rigor and
aqueous-PNP (Schmitz et al., 1996
; Winkler et al., 1996
), it
was recognized that troponin maintained a constant position with respect to the most regularly positioned crossbridges, the lead bridges, and could thus be used as a landmark to determine the actin dyad orientation in the lead
bridge target zone. The most sterically favorable actin position for crossbridge binding in the IFM lattice is midway
between troponin densities, where lead bridges bind. The
strained structure of the rigor rear bridges suggests that
they bind at the very edge of the target zone (Schmitz et
al., 1996
; Winkler et al., 1996
). The target zone defined by
lead bridges alone is narrower than target zones previously considered for rigor muscle (Reedy, 1968
) because it does not include rear bridge targets.
).
; Biosca et al., 1990
). Therefore, the M-ward
crossbridge in the mask motif may not represent the best
candidate for a preforce crossbridge. Thus, it is important
to characterize crossbridge structure in a state with lower
actomyosin affinity, such as the stiff glycol-PNP state,
where earlier 2-D analysis indicated that weakly attached 90° bridges are prevalent (Reedy et al., 1988
). In this work, we have used two spatially invariant features, troponin
position and lead crossbridge origins, to identify distinct
classes of crossbridges. The invariant position of troponin
recognized in 3-D reconstructions allows us to identify the
lead bridge target zone and the actin dyad orientation relative to the bound crossbridges. In addition, the "front-back" rule for the azimuth of the origins of the lead target
zone bridges distinguishes crossbridges that bind actin
with the correct azimuth for specific binding from those
that bind nonspecifically. By fitting the myosin subfragment 1 (S1) atomic structure to the in situ bridges, we can
compare the positions of the motor and regulatory domains. Previous results and models have introduced the
idea that during a power stroke, the crossbridge rotates
over the actin binding site while acting as a long, relatively
rigid lever arm (Huxley and Simmons, 1971
), while others
propose that the motor domain position remains constant and light chain domain movements provide a shorter lever
arm (Rayment et al., 1993b
; Whittaker et al., 1995
). Our
previous results (Reedy et al., 1987
, 1988
; Schmitz et al.,
1996
; Winkler et al., 1996
) and the present work show (a)
that regulatory domain position can vary significantly
while motor domain position remains constant and (b)
that the motor domain can bind actin with varying orientations. This work supports the possibility that both rotation
of the motor domain on actin and movements of the regulatory domain could contribute to the power stroke.
Materials and Methods
at 5°C (cold glycol-stiff state). These were bundles of two or three
fibers mounted on a tension transducer to measure stiffness and tension
during the exposure of the fibers to glycol-PNP. Primary fixation was carried out while the fibers were mounted on the transducer, and then they were removed and processed for EM. Parallel experiments under similar
conditions were monitored by x-ray diffraction (Tregear et al., 1990
). Tilt
series micrographs were obtained at magnification of 18,500 on a Phillips
EM 420 (Mahwah, NJ) operated at 100 kV. Images were digitized on a microdensitometer (model PDS 1010M; Perkin-Elmer Corp., Norwalk, CT)
at a step size corresponding to 1.25 nm with respect to the original object.
). The reconstruction steps were the same as described
earlier (Schmitz et al., 1996
). The reconstruction protocol produces a 3-D
reconstruction with a resolution of ~4 nm. Each data set comprised a single uniaxis tilt series of 37 images covering the tilt range from 69° to
76°.
).
). Thus, troponin can
be used as a marker, independent of the crossbridges to define the actin
dyad orientation and the azimuth of the rigor myosin binding site on actin
within the reconstruction. With this position thus defined, it is then possible to determine the long pitch helical azimuth at any place on a thin filament. The azimuthal angle of attachment in glycol-PNP was determined mostly for the motor domain part of attached crossbridges since thick filament origins are obscured by additional mass from crossbridges that are
not attached to actin and lie close to the thick filament.
coordinates for a five-subunit
actin filament with one S1 molecule attached (kindly provided by Dr. I. Rayment, University of Wisconsin, Madison, WI). From these coordinates, we built a 14 monomer actin filament saturated with S1 molecules
and placed it within the reconstruction envelope using the O x-ray crystallography program (Jones et al., 1991
). For model construction, the helical
operator for a 28/13 helix was used. The atomic coordinates are those for a
rigor actomyosin complex, with a uniform angle between S1 and actin and
a rigor conformation of the S1 head. However, rigor is significantly different from glycol-PNP, necessitating a fitting procedure in the present work
that is somewhat different from that used previously (Schmitz et al., 1996
).
The first step in the fitting procedure was to select a contour threshold for
the reconstruction that would just envelope the atomic coordinates of the
actin filament in crossbridge free regions of the thin filament between the
troponin complexes. The second step was to obtain an initial alignment of
the model structure with the reconstruction. Because of the large structural differences between rigor and glycol-PNP, initially we matched the
alignment of the actin dyad orientation and an S1 in the atomic model
with the actin dyad orientation and troponin bump in the reconstruction.
This defined the actin dyad orientation and azimuth of S1 binding even
though the atomic model and glycol-PNP structures were different. After
defining the actin dyad orientation based on troponin, we aligned other
S1s with the envelopes of the crossbridges.
; Winkler et al., 1996
). The
rigor structure of the S1 molecule was never changed. However, we did
change the rigor relationship between actin and S1 using a somewhat constrained movement of the S1 molecule. We allowed the azimuth of contact
between S1 and actin and the attachment angle to vary while preventing,
as much as possible, a rotation of the S1 about an axis through the motor
domain (torsional movement). We minimized torsional movement in the
fittings, but in some cases a small amount occurred. At this stage in our
analysis, we do not feel that our data are of sufficient resolution to either
support or rule out torsional movement of S1 on actin so that the small
amount seen in the figures should be considered incidental to the analysis
and not significant.
Results
Fig. 1.
(A and C) 2-D-projection of a 3-D tomogram of
IFM fixed in glycol-PNP.
Both the image and its transform (B) appear quite similar to those of original EMs
taken at 0° specimen tilt. The
full size of the actual reconstruction is about 0.8 x 0.8 µm. The pattern of attached
crossbridges shows many
variations but nevertheless expresses two periodicities.
The 14.5-nm repeat marked
by dense collars along thick
filaments is the most regular
feature. The enlargement in
A makes clear that most
crossbridges originate from
these collars. The more regular crossbridge attachments
favor thin filament sites laterally aligned in transverse
rows and axially spaced every 38.7 nm, as marked by arrowheads. These more regular bridges favor angles near
90°. Regions of an actin filament where crossbridges attach on only one side are
marked by #. The white-framed area in C is the region chosen for the surface representation in Fig. 2. The dotted frame indicates where
azimuths of crossbridge origin and attachment were determined for Fig. 5. The small insert at the lower right is a 2-D average of the reconstruction. T, troponin complex. Z-line is at the bottom.
[View Larger Version of this Image (180K GIF file)]
Fig. 2.
(A) Surface rendering of the white-framed
area in Fig. 1. Light gray, unaveraged tomogram; dark
gray, averaged filaments replacing or superimposed on
the same filaments of the
original tomogram averaged
only along the y-axis. In the
superimposed region, wherever the unaveraged tomogram displays mass that does
not appear in the average,
the extra mass is shown in
light gray. M, myosin filament; A, actin filament; T,
troponin complex. A 14.5-nm
repeat of crossbridge collars
is visible on the thick filament even without averaging. The troponin landmark,
best detected in the averages, is identified every 38.7 nm
along thin filaments. Target
zones in glycol-PNP fall midway between troponin sites.
These are the same levels (A,
dotted lines at lower left; single arrowheads at lower center and right) occupied by
the regular crossbridge attachments noted in Fig 1.
The narrow zone of regular
attachment indicates that target zones in glycol-PNP are
more axially restricted than
rigor, apparently because
rear bridge targets are not
favored. Target zone bridges
vary in angle around 90°
(reflected by the angle of
white arrows). Nontarget zone
bridges (*) attach between target zone levels and their
structure is highly variable.
Extended stretches that lack
bridges are fairly common
(#). Actin filaments are often
off center where asymmetry
of crossbridge attachments is
pronounced. Z-line is at the
bottom. (B) Target zone
crossbridge attachments mapped onto white silhouette of A. Horizontal black bars mark levels where semiregular near-90° crossbridges
attach to target zones, whose locations were determined by the position of troponin in the average. Horizontal gray stripes mark levels of target zones, positioned midway between troponin bumps, in axial average on left. Black lines represent the actual attachments at or
near those actin target zones. Most of the time, these lines fall into the gray regions, showing that the rows of 38.7-nm crossbridges attach in the rigor lead bridge target zone. However, a few attachments fall outside the target zones (rows 4 and 8). Registration of axial
average with underlying and adjacent filaments is computed, not manual. The gray stripes fit well to levels of individual target zones
across the adjacent tomogram, but for best reading of troponin repeat and actin azimuth on single filaments (as in Fig. 5), the local average of that filament is used.
[View Larger Version of this Image (93K GIF file)]
Fig. 5.
Comparison of the
azimuthal and axial orientation of crossbridges in rigor,
aqueous-PNP, and glycol-PNP. In A, the rigor acto-S1 coordinates are shown in longitudinal and in transverse
view in their normal orientation. At the bottom, the
shaded rectangle next to the
filled circle shows the attachment area, the center of
which we give the value of
0.0° as shown in C. (B) A
nontarget zone crossbridge
in a position often seen in the
glycol-PNP reconstruction, two actin monomers below
the lead target zone. The
transverse view of B shows
that the nontarget bridge attaches to a different part of
the actin monomer than the
rigor bridge in A. This azimuthal position would be assigned an angle of 70°. C illustrates the narrow range of
azimuthal attachment areas
on the actin monomer seen
in rigor. D illustrates the
broad range of azimuthal attachment areas when all glycol-PNP crossbridges are included. (E) Plot of the
narrow distribution of the azimuthal contact of rigor lead bridges to actin. (F) Plot of
the range of axial attachment
angles seen in rigor. (G)
Aqueous AMPPNP lead
bridges also show a narrow azimuthal contact with actin. (H) Axial angles of aqueous AMPPNP lead bridges show a spread comparable to rigor but are slightly displaced toward 90°. (I) Plot of the distribution of azimuthal angles for all glycol-PNP bridges compared with (J) the distribution of target
zone bridges only. (K) Plot of the range of axial attachment angles for glycol-PNP target zone bridges shows that 90° is the most frequent angle. Data for (E-H) were taken from reconstructions previously reported (Schmitz et al., 1996).
[View Larger Version of this Image (46K GIF file)]
), are rare in the glycol-stiff state, but a few can be seen in the 2-D projection (Fig.
1 C,
). Rather more frequent are partial mask motifs consisting of rigor and nonrigor crossbridges positioned on
only one side of the thin filament.
; Winkler et al., 1996
). In between the troponin densities are regularly placed bridging
bars (Fig. 2 A, dashed white line) that represent the "target
zone" crossbridges.
Fig. 3.
Gallery of glycol-PNP crossbridges. Asterisks
mark the axial positions of
target zones. A, B, and D
typify the most prevalent 90°
axial attachment angle of target zone bridges. C, E, F, and G show the large axial angle
variation that can be detected in target zone bridges.
G also shows a partial mask
motif and an irregular nontarget bridge. (H) Complete
mask motif. (I) 90° target bridge pair and one mask
bridge. (J-L) show target
bridges and variably structured nontarget bridges. (M)
Target and nontarget crossbridges with transverse view.
The azimuth of the origin of the lower nontarget crossbridge in (M) and its approach to actin are the same
as the upper target zone
crossbridge, although the azimuths of their binding sites
have rotated 81° and their
predicted origins also differ.
(N) Mask motif in horizontal
and transverse view, showing that both bridge pairs
originate from the thick filament and approach actin at
similar azimuths. Transverse
views look toward the Z-line.
In longitudinal view, Z-line is
at the bottom.
[View Larger Version of this Image (91K GIF file)]
), myosin
S1 (Rayment et al., 1993a
), and the derived model of acto-S1 (Rayment et al., 1993b
) allows us to compare the
atomic structures with the in situ crossbridges of our tomographic reconstructions. For the fittings, we selected typical crossbridges from the 3-D map and displayed them in
three different representations: a 2-D projection of the
crossbridge motif, its surface rendering and a wireframe
representation overlaid with the acto-S1 coordinates. Fig.
4, A-E, shows a target zone crossbridge pair fitted with the
acto-S1 model. In surface view (Fig. 4 A), the two crossbridges attach with an axial angle of ~80°. Note that these
bridges have a cylindrical shape along the full length, unlike the triangular shape of rigor bridges, whose origin at
the thick filament is narrow compared with the broader
actin end. The fittings confirm that the cylindrical profiles
of glycol-PNP crossbridges are large enough to contain
only one S1 head.
Fig. 4.
Fittings of the
acto-S1 atomic model into
target zone and nontarget
zone crossbridges of IFM in
glycol-PNP. (A) Projection image in which the particular
motif is boxed and enlarged
in the surface view. The asterisk marks the level of the
actin target zone. B is a view
down the filament axis of the
rigor atomic model. The orientation of the red ball and
stick identify the binding site
and azimuthal angle of rigor
S1. (C) Unmodified rigor
acto-S1 model superimposed
onto target zone crossbridges
shows that the 45° angle of
rigor is not matched by the
90° angle of glycol-PNP crossbridges. (D) Transverse view
of target zone crossbridge
seen in E, fitted with S1
whose axial angle has been
changed to 90°. (E) Stereo
longitudinal view (ocular divergence) of target zone bridges showing the good fit
achieved when the axial angle of S1 is pivoted to 90°. In
longitudinal view, the motor
domain of the S1 (C-E, yellow) and the regulatory domain (orange and white) fit
into the crossbridge envelope
(red). (F) Projection and surface image of a region containing two target zone and
two nontarget zone crossbridges. (G) Acto-S1 model
superimposed on the reconstruction. The orientation of
S1 has been changed to align
with each crossbridge envelope. (H) Transverse view of
the same fitting of each of
the three crossbridge levels in G, showing that while the target zone bridges have similar azimuthal orientation as rigor S1 and bind to
the same place on actin, the nontarget bridges do not. In particular, to align S1 with the upper nontarget bridge envelope required moving the actin-S1 interface to a different part of actin.
[View Larger Version of this Image (109K GIF file)]
; Winkler et al.,
1996
) indicate that considerable distortion of a rigor acto-S1
is needed to position it onto the rear crossbridge. That
same kind of distortion is implied here but with a weak-binding glycol-PNP crossbridge. In aqueous-PNP, rigor-like rear bridges are absent. The weak actin affinity of glycol-PNP crossbridges, which tolerate more variation in angle and azimuth (see below) than rigor or aqueous-PNP,
may facilitate an actin interaction that is otherwise unfavorable.
,b). In our analysis, we define the binding azimuth in rigor as 0.0°.
). Most of the measurements for those states fall within ±20° of the 0.0° value defined for rigor. When rigor rear bridges are added to the
rigor distribution (data not shown), the azimuthal distribution shifts very slightly toward a more positive angle.
Discussion
; Frado and
Craig, 1992
; Walker et al., 1995
). Even if myosin head
form in a weakly bound, preforce state could be preserved
and identified in acto-S1, under in vitro conditions the isolated molecules do not produce force, they are not under
the constraints enforced by their integration in a lattice of
filaments, and stiffness cannot be measured as an assay for attachment. These nonaveraging tomographic reconstructions allow us to observe variable myosin head form in situ
and provide a means to analyze them and distinguish the
most likely candidates for preforce crossbridge form.
) revealed two classes of crossbridges. One class, the lead bridge, was bound to the actin
targets of rigor lead bridges. The structure of lead bridges
in aqueous-PNP was slightly different from rigor lead
bridges. However, lead crossbridges in AMPPNP were
bound to the same part of the actin monomer as their rigor
counterparts, suggesting that these target zone crossbridges bound specifically to actin. Crossbridges of the
second class bound in regions of the thin filament where
rigor crossbridges are not observed. Fitting the atomic
structure of rigor acto-S1 into the reconstructions revealed
that AMPPNP crossbridges in this second class bound to
different parts of the actin monomer than rigor bridges
suggesting that these nontarget zone crossbridges bound
nonspecifically to actin.
, in which weak binding of both myosin heads is unlikely because of strain effects. The distribution of tilt angles in glycol-PNP is centered around 90° rather than 45°.
However, the change in the tilt angle of glycol-PNP target zone bridges is not accompanied by a change in the azimuth of the thick filament origin; target zone bridges in
glycol-PNP originate from similar azimuths on the thick
filament as those in rigor and aqueous-PNP. Glycol-PNP
target zone bridges also bind to the same region of the actin monomer as rigor bridges. For these reasons, we consider the glycol-PNP target zone bridges to be bound to actin at the interface associated with strong-binding, force-bearing crossbridges. However, the interaction of the target zone crossbridges differs in some details. For example,
the azimuthal and axial angular distribution for glycol-PNP target zone crossbridges is broader than for rigor and
aqueous-PNP, particularly so for the axial angle. Since the
axial angle can take on values both less than 90° (rigor-like) and greater than 90° (anti-rigor-like), it seems unlikely that the motor domain of S1 can be binding actin in
a single orientation.
). This establishes that weakly bound, single-headed crossbridges
must contribute significantly to isotonic stiffness. Furthermore, it is possible that, despite the considerable difference in crossbridge structure, the stiffness per crossbridge
is approximately the same and independent of whether the
crossbridges are classified target zone or nontarget zone.
To be otherwise would require that the stiffness per crossbridge increase while its affinity for actin decreased. Our
observations are consistent with the theory that stiffness
per crossbridge is independent of its state (Schoenberg
and Eisenberg, 1987
) and would seem to suggest that nonspecifically attached crossbridges make a similar contribution to stiffness as those stereospecifically bound.
;
Reedy et al., 1988
). In ATP-relaxed IFM, intensity on the
19.3-nm layer line is not observed (Reedy et al., 1988
,
1992). The location of the troponin complex every 38.7 nm
along the thin filament gives rise to much of the intensity
observed on the 38.7-nm layer line in relaxed IFM. Because the lead bridge target zones are positioned midway between troponin complexes, the additional mass following the actin helix contributed by the regular binding of
glycol-PNP crossbridges in target zones will halve the 38.7-nm
period, thereby increasing intensity on the 19.3-nm layer
line, rather than on the 38.7-nm layer line. On the other
hand, the mass of the nonspecifically attached nontarget
crossbridges does not follow the actin helix and, therefore,
will not contribute significantly to intensity on the 38.7- or
19.3-nm layer lines. X-ray diagrams of contracting IFM
show a pronounced increase in the intensity of the 19.3 nm (10.12 reflection) during stretch activation (Tregear, R.T.,
R.J. Edwards, T.C. Irving, K.C. Poole, M.C. Reedy, H. Schmitz, E. Towns-Andrews, and M.K. Reedy, manuscript
submitted for publication). The increase in 19.3 intensity
of contracting IFM can also be explained by the regular
binding of crossbridges to the lead bridge target zone.
; Winkler et al., 1996
).
Fig. 6.
Comparison of 3-D
tomographic reconstructions
of IFM in three equilibrium
states: Rigor (A-C) and
AMPPNP (D-F) (from
Schmitz et al., 1996) and the
glycol-PNP reconstruction
described here (G-I). Surface renderings of attached
crossbridges at the lead bridge
target zone on top, surface
rendering of a larger area in
the middle and 2-D projections on bottom. The orientation with respect to the muscle is with Z-line on the
bottom. In B, E, and H, the
axially averaged filament is
shown in red to the right of
the unaveraged tomogram on
the left. In the tomograms,
target zone crossbridges are
painted orange for ease of
identification. The comparison
clarifies the main differences
among the three states: the
rigor state shows well-
ordered, double-headed crossbridges arranged in double
chevrons consisting of lead
and rear bridge pairs every
38.7 nm. The lead bridges,
which hold two myosin
heads, are attached at a 45°
angle, while the smaller, single-headed rear bridges attach on average at a 90° angle.
There is no 14.5-nm periodicity visible on the myosin filament surface. In aqueous-PNP, the 45° attached lead
bridge motif every 38.7 nm is
retained, while rear bridges
redistribute to random-like
positions. A weak 14.5-nm
periodicity can be seen on
the myosin filament surface. Lead bridges often appear to
be single headed. The glycol-PNP state shows single-headed attached crossbridges
every 38.7 nm, but their
shape, size, and attachment
angle differs from the lead bridges. In addition, many
random-like attached bridges
can be seen. The myosin filament surface reveals a strong
14.5-nm repeat of crossbridge
shelves.
[View Larger Version of this Image (99K GIF file)]
). AMPPNP causes detachment of rear bridges and
their redistribution to other actin monomers not normally
occupied in rigor (Fig. 6, D-F). Lead bridges are retained,
but their structure is slightly altered from the rigor lead
bridges. AMPPNP lead bridges have a rigor tilt angle, except that the regulatory domain region shows a small 0.5-
1.0-nm M-ward shift, and the bridges are less azimuthally bent in transverse view (Winkler et al., 1996
). Both one-
and two-headed lead bridges are present. The glycol-PNP
state is characterized by the loss of the remaining tension
but little change in stiffness, which remains at or near the
rigor level. In the glycol-PNP state (Fig. 6, G-I), both the
45° attachment angle and the triangular shape of crossbridges in the lead target zone are lost. Instead, 90° single-headed crossbridges predominate, and the only ordered class of bridges is found bound at the lead bridge target
zones.
).
Received for publication 13 March 1997 and in revised form 10 July 1997.
The PDS 1010M densitometer was purchased with funds from National Science Foundation grant PCM-8400167. This work was also supported by DFG grant Schm 1035/1 (H. Schmitz) and National Institutes of Health grants GM-30598 (K.A. Taylor) and AR-14317 (M.K. Reedy).
2-D and 3-D, two- and three-dimensional;
AMPPNP, adenosine 5-[
-imido]triphosphate;
aqueous-PNP, aqueous solution of AMPPNP (without ethylene glycol);
glycol-PNP, mixtures of ethylene glycol and AMPPNP;
IFM, insect flight muscle;
S1, myosin subfragment 1.
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