The conversion of the chemical energy of ATP into mechanical work by myosin involves coordinated changes in the actomyosin affinity and the orientation of myosin cross-bridges relative to the fiber axis 
. Generally, one or more weak binding intermediates, referred to as A-states, precede strong binding states, referred to as R-states, which produce filament sliding 
. A leading model describing these conformational changes evolved initially from spectroscopic evidence combined with structural information available at the time 
with later support from the atomic structures of myosin subfragment 1 (S1) 
and of the actin filament 
. This model incorporates the concept that the actin-binding motor domain (MD) of myosin maintains a single, stereospecific orientation when actin-bound in the strong binding configuration. The second major domain of myosin is the lever arm, which consists of the myosin converter domain, essential and regulatory light chains, and their bound α-helical heavy chain segment. The working stroke is produced by lever arm rotation about a pivot point near the ATP binding site 
Differences in A-states and R-states predominately involve the configuration of a long cleft that divides the myosin MD into upper and lower 50 kDa subdomains, the so-called actin binding cleft. A-states, which have weak actin affinity, have an open cleft while R-states, which bind strongly to actin, have a closed cleft 
. The lever arm orientation of A-states can be either “up” in a pre-working-stroke position or “down” in a post-working-stroke position; R-states are lever-arm-down states. This model is supported by crystal structures of various isoforms and types of myosin S1 with different nucleotides bound 
and when strongly bound to actin in vitro 
The structural changes that occur in the A- to R-state transition are poorly defined, especially for myosin heads operating in situ. In the model described above, A-state myosin heads search for the myosin binding site on actin through a rapid equilibrium between attached and detached states until the MD alights on the myosin binding site on actin in the correct orientation for cleft closure. An alternative model involves diffusion of the myosin head on actin to the correct location and orientation for strong binding 
. The two models produce differences in the potential size of the working stroke.
Working strokes of 10–12 nm are about the maximum that can be achieved by a purely axial motion of the myosin lever arm and have been observed for single myosin S1 molecules in vitro 
. However, working strokes much larger have also been reported 
. To achieve longer working strokes requires either axial rotation of the MD during the working stroke 
, which can produce a small increase in working stroke, or diffusion of the MD along the actin filament by one or more subunits which can produce much larger working strokes 
. Several observations suggest that the initial weak binding actin-myosin interaction changes in structure or orientation on actin during tension development. X-ray diffraction of frog muscle 
indicates that tension development involves a stabilization of the MD from a disordered actin attachment to an ordered one. A disordered to ordered transition of attached cross-bridges is suggested by electron paramagnetic resonance of spin labelled MDs 
. However, diffusion of the myosin head along actin as a means to increase the working stroke remains controversial, especially if it is considered that filament movement might require a strongly bound myosin attachment.
Visualizing active cross-bridges, including those bound to actin in the weak binding states thought to precede strongly bound force producing states, is essential for defining the structural transitions that constitute the working stroke of myosin. Because of their low actin affinity and possible heterogeneous structure when attached to actin, weakly-bound states are difficult to trap in vitro in numbers with sufficient homogeneity to be amenable to direct visualization by any of the powerful averaging techniques of cryoEM. However, 3-D visualization can be achieved using the technique of electron tomography (ET) which is capable of imaging individual molecules within a highly heterogeneous ensemble 
. ET has produced 3-D images of insect flight muscle (IFM), including the variable conformations of in situ cross-bridges in rigor 
, in a weakly-bound equilibrium state produced by adenylyl-imidodiphosphate (AMPPNP) and ethylene glycol 
as well as in snapshots of actively contracting IFM fibers 
IFM displays two levels of contraction depending on [Ca2+
]. Stretch activation, which is characterized by rapid alternating contractions of antagonist muscles during flight, is the contraction mode most often studied 
. Stretch activation can be induced in skinned fibers at pCa <6.0 
. IFM also produces sustained isometric contractions at pCa <4.5, which we refer to as isometric high static tension or iso-HST, that correspond to an isometric tetanus in vertebrate muscle. In vivo
, iso-HST occurs during the thermogenic “shivering” of preflight warmup 
, when opposing flight muscles contract simultaneously and isometrically to raise the muscle temperature to 40°C where flight can be sustained.
Active myosin heads interact with actin independently of each other so a snapshot of contracting muscle reveals the structure of multiple acto-myosin states within the context of the muscle lattice. Snapshots previously obtained from isometrically activated vertebrate striated muscle revealed a wide range of attachment angles in projections 
, but these cross-bridges were not visualized in 3-D where detailed interpretation in terms of atomic structure would be possible.
Like the results obtained from vertebrate muscle, iso-HST cross-bridges visualized for the first time by ET also showed a wide range of attachment angles which could be ordered into a sequence compatible with a progressive 13 nm working stroke 
. Averages computed along axial columns equivalent to the 116 nm long lattice repeat common to both the actin and myosin filaments revealed that actin binding of active cross-bridges was restricted to limited thin filament segments termed “actin target zones” as previously recognized in rigor 
. Target zones of IFM are positioned midway between successive regulatory complexes which are composed of the three troponin (Tn) peptides. Subsequently, the distribution and orientation of attached cross-bridges from these same tomograms suggested that, in the absence of filament sliding, the variably angled cross-bridge attachments become locally stabilized in each target zone 
. This observation in turn suggested that individual tension-generating cross-bridges can cycle with little axial translocation or change in axial lever arm angle.
Here we report a more detailed view of the rich variety of myosin head forms in the iso-HST state resulting from improvements in both data collection and analysis that have increased the resolution by 2.5× over the earlier work. The helical arrangement of actin subunits is now resolved, facilitating assignment of particular cross-bridge forms to specific actin subunits within the 38.7 nm repeat that spans from one Tn complex to the next. Multivariate data analysis (MDA) and classification of 3-D repeats is used to quantify individual cross-bridge forms from the number of repeats within each class 
. Identification of strong and weak binding cross-bridge forms is greatly improved revealing some novel thin filament attachments not previously detected. This leads to a more sharply defined set of cross-bridge structures and interactions in a tension generating muscle than has been possible previously.