Direct visualization of myosin-bindingprotein C bridging myosin andactin filaments in intact musclePradeep K. Luthera,1, Hanspeter Winklerb, Kenneth Taylorb, Maria E. Zoghbic,2, Roger Craigc, Raúl Padrónd,John M. Squiree, and Jun Liub,3

aMolecular Medicine Section, National Heart and Lung Institute, Faculty of Medicine, Imperial College London, Exhibition Road, London SW7 2AZ, UnitedKingdom; bInstitute of Molecular Biophysics, Florida State University, Chieftain Way, Tallahassee, FL 32306; cDepartment of Cell Biology, University ofMassachusetts Medical School, 55 Lake Avenue North, Worcester, MA 01655; dCentro de Biología Estructural, Instituto Venezolano de InvestigacionesCientíficas (IVIC), Carretera Panamericana Km 11, Caracas 1020A, Venezuela; and eSchool of Physiology and Pharmacology, University of Bristol, UniversityWalk, Bristol BS8 1TD, United Kingdom

Edited* by Hugh E. Huxley, Rosenstiel Basic Medical Sciences Research Center, Brandeis University, Waltham, MA, and approved May 11, 2011 (received forreview February 26, 2011)

Myosin-binding protein C (MyBP-C) is a thick filament protein play-ing an essential role in muscle contraction, and MyBP-C mutationscause heart and skeletal muscle disease in millions worldwide.Despite its discovery 40 y ago, the mechanism of MyBP-C functionremains unknown. In vitro studies suggest that MyBP-C couldregulate contraction in a unique way—by bridging thick and thinfilaments—but there has been no evidence for this in vivo. Hereweuse electron tomography of exceptionally well preservedmuscle todemonstrate that MyBP-C does indeed bind to actin in intact mus-cle. This binding implies a physical mechanism for communicatingthe relative sliding between thick and thin filaments that does notinvolve myosin and which could modulate the contractile process.

C-protein ∣ sarcomere structure ∣ thick filament structure ∣cardiac muscle regulation ∣ cardiac disease

Myosin-binding protein C (MyBP-C; C-protein) is a 130 kDathick filament protein in vertebrate striated muscle located

on seven to nine stripes 43 nm apart in each half A-band (1–4).Skeletal MyBP-C is a rod-shaped molecule, ∼40 nm long andoften bent near its center (3). The molecule is composed of achain of ten globular, 4 nm-diameter, 10-kDa domains (C1 toC10) from the immunoglobulin (Ig) and fibronectin 3 (FN3) fa-milies, with an additional, MyBP-C-specific motif (also called theM-domain) between C1 and C2 (3, 5). The cardiac isoform,cMyBP-C, has an additional Ig domain, C0, at the N terminus(6, 7), and is phosphorylatable at four sites on the M-domain(2, 6, 8). MyBP-C is essential for the normal functioning ofstriated muscles in humans. Phosphorylation of the cardiac iso-form plays a key role in the enhancement of cardiac function thatresults from β-adrenergic stimulation (2, 3, 6), and mutations inthis isoform are a major cause of familial hypertrophic cardio-myopathy, affecting 60 million people worldwide (2, 9). Similarly,mutations in skeletal MyBP-C cause distal arthrogryposis, adisease characterized by joint contractures and abnormal muscledevelopment (10).

The structural arrangement of MyBP-C in the sarcomere ispoorly understood, although it is likely to play a crucial role inMyBP-C’s function. MyBP-C is bound to the thick filament viaits C-terminal region (C8–C10), which interacts with the myosintail and with titin (3); its N-terminal region can also bind rever-sibly to subfragment 2 of myosin (3, 4, 11, 12). Surprisingly, invitro this myosin-binding protein can also bind to actin, via itsN-terminal region (C0–C2) (13–18). However, there has beenno evidence that this binding occurs physiologically. Electronmicroscopy of isolated thick filaments has revealed the likelylocation of the C-terminal domains on the thick filament surface(19), but the putative actin-binding N-terminal region was notvisualized, possibly due to its disordering when thick filaments

are removed from the native filament lattice. Here we use elec-tron tomography of exceptionally well preserved muscle sectionsto demonstrate that MyBP-C binds to actin in intact muscle. Thisphysical bridge between thick and thin filaments, independentof myosin heads, may play a key role in modulating muscle con-traction in vivo.

ResultsElectron Tomography of Rapidly-Frozen/Freeze-Substituted FrogMuscle. Thin (∼100 nm) longitudinal sections (containing about2.5 unit cells of the A-band lattice in depth) were cut fromrapidly-frozen, freeze-substituted frog sartorius muscle em-bedded in epoxy resin (Fig. 1A). This preparative technique isknown to provide the best preservation of muscle ultrastructureavailable (20). Clear transverse stripes (numbered 1 to 11) areseen at 43 nm intervals on each side of the M-band (M), givingrise to corresponding peaks in the profile plot of these images(Fig. 1B). These stripes are known to represent nonmyosin pro-teins, primarily MyBP-C on stripes 5–11 (the C-zone) (21, 22),and other proteins on stripes 1–4 in the proximal (P-) zone (23).There are no nonmyosin stripes in the distal (D-) zone (23, 24).Two finer lines are seen between each pair of stripes. These linesarise from the arrangement of crowns of myosin heads at a meanaxial spacing of 14.3 nm (25). The averaged Fourier transformof 23 half A-bands (Fig. 1A, inset) shows layer lines at ordersof 43 nm, implying good preservation of the near-helical arrange-ment of the heads (20), with intensity visible as far out as thesixth order, corresponding to an axial resolution of 7 nm (see alsoFig. S1). “Forbidden” meridional reflections, at the first, second,and fourth orders of 43 nm come from the nonmyosin stripes andperturbations in the myosin helix (24–26).

To obtain three-dimensional information on the organizationof MyBP-C, three dual-axis tomographic tilt series were obtainedfrom these sections. The images in each series were aligned, and

Author contributions: P.K.L. designed research; P.K.L., R.C., R.P., and J.L. performedresearch; H.W. and K.T. contributed new reagents/analytic tools; P.K.L., H.W., K.T.,M.E.Z., R.C., R.P., J.M.S., and J.L. analyzed data; and P.K.L. and R.C. wrote the paper.

The authors declare no conflict of interest.

*This Direct Submission article had a prearranged editor.

Data deposition: The density map of the thick filament tomogram has been deposited inthe ElectronMicroscopy Data Bank, http://www.ebi.ac.uk/pdbe/emdb/ (Accession number:EMD-9809).1To whom correspondence should be addressed. E-mail: p.luther@imperial.ac.uk.2Present address: Cell Physiology and Molecular Biophysics, Texas Tech University HealthSciences Center, 3601 4th Street, Lubbock, TX 79430.

3Present address: Department of Pathology and Laboratory Medicine, University of TexasMedical School, 6431 Fannin Street, Houston, TX 77030.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1103216108/-/DCSupplemental.

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three-dimensional tomograms were computed by back-projection(Movie S1 shows one of the aligned tilt series, and Movie S2shows two-dimensional slices of a tomogram). From theseindividual tomograms, 207 thick filaments were extracted andused to calculate a single, averaged in situ thick filament three-dimensional reconstruction. The zone used for averagingextended to a radius of about 45 nm, which includes nearestneighbor thin and thick filaments.

The Thick Filament Tomogram and Its Interpretation. The thickfilament reconstruction was made by separately averaging tomo-grams of each distinct region of the A-band: the P-, C-, andD-zones (Fig. 2). This procedure provided a higher quality aver-age of each region than averaging over the thick filament as awhole. The most striking feature of the tomogram is the presenceof clear transverse density every 43 nm in the C-zone (S5–S11 inFig. 2), at the same levels as the stripes observed in the rawimages (Fig. 1). These densities are sharply defined axially(4–8 nm, thus maximally one to two MyBP-C domains wide)and extend to high radius (>25 nm from the thick filament cen-ter). As in the original micrographs, the P-zone shows strongtransverse density only at stripe 3, and nonmyosin densitiesare absent from the D-zone. Between adjacent MyBP-C densitiesare two clearly defined additional features. These features corre-spond to the finer lines seen in the micrographs, and representhelically organized crowns of myosin heads. There are threecrowns in each repeat. We define crown 1 as the crown at thelevel of the stripe, while crowns 2 and 3 (c2 and c3) lie betweenthe stripes. This interpretation of the major densities in the

reconstruction is supported by thin cross-sections of the tomo-gram taken every 1.2 nm along the filament axis (Fig. S2).

While the single contour level used to depict the thick filamentin Fig. 2 clearly reveals the MyBP-C and myosin head densities, itdoes not show their relative strengths. A gray-level depiction ofthe tomogram reveals that the high-radius density (MyBP-C) isweak compared to the c2 and c3 crowns of heads, consistent withthe relative masses of heads (6 heads × 130 kDa) and MyBP-C(3 MyBP-Cs × 130 kDa) at each level, together with the diffuseregion over which MyBP-C density extends (Fig. 2 inset,Movie S3). The gray-level depiction also suggests that the highdensity features at low radius in the stripes represent myosinheads (crown 1), as these appear similar to, and lie on the samehelical path as, crown 2 and 3 heads. This interpretation of thestripes (S5–S11) is supported by fitting crown 1 heads fromregions of the filament where the stripe density is weak or absent(and thus the head density is unambiguous) to the C-zone recon-struction. A segment comprising stripe 3 (crowns 2 and 3) andstripe 4 (crown 1), where the stripe density is very weak, wasextracted from the P-zone and fitted to the C-zone at stripes 8and 9 (Fig. 3 A and B). Crowns 2 and 3 of the segment fitted wellwith the underlying C-zone crowns. Most importantly, using thisfitting, crown 1 heads (yellow, blue, Fig. 3 D and E) fitted wellinto the high density, low radius features concluded above tobe myosin heads. A similar result was obtained by fitting crown1 from the D-zone. We conclude that the high-radius cloud ofdensity in the stripes is MyBP-C (purple, Fig. 3 A–E), whilethe low radius, high-density features are myosin heads.

Fig. 1. Observation ofMyBP-C in sectionedmuscle. (A) Electronmicrograph of frog sartorius muscle A-band prepared by fast-freezing/freeze-substitution (20).Transverse stripes of 43 nm periodicity (numbered 1–11) are due to MyBP-C and other nonmyosin proteins, and fine lines of 14.3 nm repeat are due to myosinheads. For ease of description, the numbering at 43 nm intervals is continued beyond stripe 11, although there are no nonmyosin proteins at these positions.Layer lines in the Fourier transform (inset; third and sixth marked) indicate good preservation of myosin head helical order. See also Fig S1. (B) Mean profileplot of several boxed regions similar to that in (A). M, M-band; stripe 1 to 5, P-zone; stripe 5 to 11, C-zone; and stripe 11 to edge of A-band, D-zone. (Scale bar,200 nm).

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The tomogram reveals in addition that the distal MyBP-Cdensity (furthest from the thick filament backbone) is annularand coincides with the positions of the thin filaments (white inFig. 3). The density appears to curve round to contact eitherthe closest or next-closest thin filament to its origin on the thickfilament, without any particular preference (Fig. 3 C–E). Thesevarying contacts may reflect different accessibility of specificMyBP-C binding sites on actin at different axial levels, due tothe unequal thick and thin filament helical repeats. These differ-ent modes of contact are consistent with the known flexibility oftheMyBP-Cmolecule (3), which may be an essential feature of itsstructure, enabling it to bind to variably placed actin-bindingsites. Assuming that the MyBP-C molecules at each 43 nm leveland their actin-binding partners have specific binding sites foreach other, and that the actin filaments in each half sarcomereare in helical register (27), calculations suggest every MyBP-Cmolecule should be able to bind to actin at each level where thereis filament overlap, independent of sarcomere length (Fig. S3).

The distribution of actin filaments around the myosin filamentsuggests that at any level there would generally be a mixture ofMyBP-C molecules moving to the left or the right to find the mostaccessible actin-binding site. However, the resolution of thetomogram and the averaging procedures used have not allowedus to test this prediction, and the paths shown (white, Fig. 3 B–D)and the schematic drawing in Fig. 3F represent only a guide as topossible conformations.

Fitting the Tomogram to a Reconstruction of Isolated Thick Filaments.While the tomogram shows broadly how MyBP-C is organized inthe filament lattice of intact muscle, the resolution is limited to∼7 nm, and details of MyBP-C’s binding to the thick filament areunclear. Conversely, reconstruction of isolated, negatively stainedcardiac thick filaments has revealed clear density that is thoughtto represent MyBP-C C-terminal domains on the thick filamentsurface; however, projecting regions are absent from the recon-struction, probably due to disorder in the absence of the filament

Fig. 2. Tomographic reconstruction of thick filament. (A, B) Interleaved stereo images of averaged, surface-rendered frog muscle thick filament tomogram;(A) face view, (B) tilted 20°. The density threshold was selected to optimize visualization of MyBP-C (present at stripes S5–S11, corresponding to the stripenumbers in Fig. 1). Between these stripe levels are two layers of density due to crowns of myosin heads, with a periodicity of about 14.3 nm (labeled c2 and c3).(Inset) Density representation (in stereo, tilted forward 20°) of averaged tomogram of stripes 7–9, showing that MyBP-C density is weak compared to themyosin head crowns (see also Movie S3).

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lattice (19). To obtain a more complete picture of MyBP-C orga-nization, we have combined these complementary approaches.The higher resolution (3–4 nm) isolated filament reconstructionwas fitted into the tomogram of the in situ filament by matchingthe positions of the highest density regions of each map, whichcorrespond to the myosin heads. Although the two reconstruc-tions are based on very different methods of specimen prepara-tion and on different muscle types, there was good agreement inthe position of the myosin heads in the two maps (Fig. 4, Fig. S4;Movie S4). Crucially, the fitting shows that the axial position ofthe MyBP-C stripe of the tomogram coincides with that of theputative MyBP-C domains observed in the isolated filament(orange in Fig. 4); this coincidence is a result of matching headdensities and was not a criterion used for the fitting. This fittingsuggests that MyBP-C both binds to and projects from the fila-ment at crown 1.

DiscussionVisualization of MyBP-C in Intact Muscle. The three-dimensionalorganization of MyBP-C in the sarcomere has remained obscureever since it was first shown to be responsible for the 43 nm-spaced stripes in vertebrate muscle A-bands over 30 yr ago (21).While MyBP-C’s narrow axial distribution was inferred from thenarrowness of the stripes, its radial extent was unknown, and thepossibility that it might interact with actin in vivo (as suggested byin vitro observations) remained an unproven hypothesis. Ouruse of fast-freezing, freeze-substitution, and electron tomographyhas enabled us to reveal the three-dimensional arrangement ofMyBP-C in situ. The averaged tomogram confirms that MyBP-Clies in narrow planes perpendicular to the thick filament and, cru-cially, that a major portion is at high radius, beyond the myosincrowns and in contact with actin. We conclude that MyBP-Cinteracts with actin in vivo, and further, because our studiesare of relaxed muscle, that it can do so when tropomyosin is

in the blocking position on the thin filament (28). While thisfinding contrasts with an early in vitro finding suggesting thatMyBP-C could only bind to activated thin filaments (29), it is con-sistent with subsequent studies showing binding at low as well ashigh Ca2þ (16, 30). Although the tomogram clearly demonstratesMyBP-C binding to actin, we cannot entirely rule out the possi-bility that binding to neighboring thick filaments might also occur.However, MyBP-C is only just long enough to span between thick

Fig. 3. Identification of MyBP-C. (A, B) Interleaved stereo images showing part of the averaged thick filament tomogram between stripes 7 and 9 (purplecolored mesh, with density thresholded to optimize visualization of MyBP-C). The location of actin filaments has been drawn in (white) as they are suppressedby the averaging protocol (which is centered on the myosin filament), due to disorder in the filament lattice presumably caused by variable amounts ofcompression occurring during the specimen preparation procedures (including freezing, freeze-substitution, dehydration, and sectioning), and also by shrink-age effects due to electron radiation. Actin filaments are clearly visualized at these positions when a lower threshold is used (Fig. 4). Blue and yellow showfitting of heads (crowns 2 and 3 from stripe 3 and crown 1 from stripe 4) into the reconstruction (see text). (C–E) Transverse views at stripes 7 to 9, with actinpositions depicted in white circles; contact between MyBP-C and actin is seen at each level. In (D and E), stripe 4, crown 1 density is shown in yellow and blue[cf. (A, B) above]. In (B), (C) and (D) curved, white lines depict possible paths of MyBP-C, which appear to approach and make contact with actin filamentsanticlockwise (stripe 7), clockwise (stripe 8), and in both directions (stripe 9). (F) Model of possible MyBP-C arrangement. Amyosin filament is shown surroundedby six actin filaments, with two possible paths of MyBP-C connecting them via the N terminus. C-terminal MyBP-C domains, which may run axially along thefilament, are not shown as they would be obscured (see Fig. S5).

Fig. 4. Fitting of isolated filament reconstruction into in situ tomogram.Mouse isolated filament reconstruction (gray surface rendering) (19) isdocked into the frog tomogram (purple mesh). Four crowns of myosin headsare shown, including crown 1 lying under MyBP-C in stripe 7. Asymmetricmyosin head dimer atomic structures are fitted into crowns 1 and 3 (circled)of the isolated filament reconstruction (19). The MyBP-C disk at stripe 7coincides axially with three globular domains. (orange) in the isolated fila-ment reconstruction, thought to be the C-terminal myosin/titin-bindingdomains (19). The mesh densities in the tomogram lying at high radius(outside the isolated filament reconstruction) at c2 and c3 are actin filaments(discontinuous at the threshold used). Fig. S4 provides additional details.

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filaments and could do so only by projecting radially out towardsits thick filament neighbor (Fig. S5). A straight, radial path to-wards neighboring thick filaments is inconsistent with the bent/curved path leading to actin that we see.

Relation to In Vitro Observations. Our tomogram strongly supportsand complements previous observations of MyBP-C binding toactin. These earlier studies, based on centrifugation assays(14–16, 30–32), light and electron microscopy (14, 16, 29, 30,33–35), neutron scattering data (36), and motility assays(15, 32, 37, 38), studied binding to F-actin (14–16, 31, 32) andto regulated thin filaments (14, 15, 29–32), and used bothtissue-prepared protein (14, 29, 30, 32) and expressed wholeMyBP-C or N-terminal fragments (15, 16, 31). While these invitro studies clearly demonstrated MyBP-C’s ability to interactwith F-actin and thin filaments, and the consequent functionaleffects (14, 15, 18, 32, 37–39), our work demonstrates that thisbinding is relevant in vivo. The in vitro studies show that bothskeletal (14, 29, 30) and cardiac (15, 16, 18, 31, 32) MyBP-Ccan bind to actin. For our experiments we used frog skeletalmuscle, whose excellent order and orientation made it possibleto obtain the accurate longitudinal sections needed in this work(20). MyBP-C is similar in overall domain structure in skeletaland cardiac muscles, the main differences being the presence incardiac MyBP-C of an additional N-terminal domain [C0, whichmay (18) or may not (16) contribute significantly to actin binding]and of phosphorylation sites on the M-domain (2, 3, 5–8).It therefore appears likely that in situ binding of MyBP-C to thinfilaments in cardiac muscle could be similar to that describedhere for skeletal muscle. While our tomogram nicely revealsthe extension of MyBP-C from the thick filament to the vicinityof the thin filaments, it does not provide molecular detail on theinteraction with actin. Such details are now emerging fromneutron scattering and EM studies of F-actin decorated withMyBP-C (33–36).

Implications of MyBP-C Linking Myosin and Actin Filaments. The invitro studies suggest that binding to actin occurs primarilythrough the C1 and M-domains (near the N terminus), the latterbeing phosphorylation-dependent in cardiac muscle (16). Arecent report concludes that such N-terminal binding is weak andoccurs through nonspecific, electrostatic interactions; surpris-ingly, it also demonstrates that in vitro, specific binding can occurvia the C-terminal (myosin-binding) half of the molecule (31).How the myosin-binding end could also bind to actin in intactmuscle is not clear, and the in vivo relevance of this findingremains in question (31) (but see Fig. S5). Combination of ourtomographic data from in situ thick filaments with our earlierreconstruction of isolated filaments (19) suggests a simple modelfor MyBP-C organization in the relaxed state, in which theC-terminal three or four domains bind longitudinally along thethick filament surface (19) while the N-terminal half extends outtowards the thin filaments (Figs. 3 and 4). This arrangementargues against a collar model for MyBP-C (3, 40). With the Nterminus close to actin, even weak, nonspecific interactions maybecome significant and have profound physiological conse-quences, by modulating filament lattice stability and sensing andmodulating filament sliding. Such interactions could be strongenough to stabilize the filament lattice (possibly enhancing theefficiency of contraction) but weak enough to permit slidingduring stretch or contraction (15). Weakening of actin bindingby phosphorylation of the M-domain that occurs in cardiacMyBP-C (16) could contribute to the enhanced contractility ofthe heart that occurs in response to β-adrenergic stimulation(2, 3). Binding to actin could further explain a long-standingenigma: how MyBP-C, present in a restricted zone of the thickfilament, and only at every third level of myosin heads within thatzone, can have an effect on thick-thin filament sliding as a whole

(41). Clearly MyBP-C cannot interact with all myosin heads.Direct connection to actin (16, 41) could explain effects at thewhole filament level straightforwardly.

Our structural observations support the view that theN-terminal end of MyBP-C can modulate contraction of striatedmuscle. The N terminus can interact in vitro with both myosin S2(11, 12) and actin (14, 16). While our tomogram reveals clear in-teraction with actin in situ, detail is not sufficient to reveal anyputative binding to S2. However, the tomogram shows clear myo-sin head density close to the filament backbone in relaxed muscle,and it is difficult to envisage how the N-terminal domains thatbind to actin could simultaneously attach to S2 on either the sameor a neighboring thick filament (Fig. S5). Our studies do notaddress what happens during contraction. Weakening of the44.2 nm MyBP-C X-ray reflection on contraction suggests thatMyBP-C may become much more mobile in this state (26).One possibility is that binding to S2 occurs transiently by switch-ing with actin during filament sliding.

Materials and MethodsSpecimen Preparation. Frog sartorius muscle was skinned in 1% triton ina relaxing solution, rapidly-frozen/freeze-substituted, embedded, and sec-tioned on a diamond knife. The preparation of this sample is described inour earlier study (20) in which we demonstrate the exceptional preservationof fine structure made possible by these techniques.

Electron Tomography. Images of three dual-axis tilt series of well oriented,thin (∼100 nm), longitudinal sections similar to Fig. 1A were collected asdescribed in Wu, et al (42). Briefly, grids were transferred into a Gatan model670 Ultrahigh tilt analytical holder (Gatan), examined in an FEI CM300-FEGelectron microscope (FEI Company) at 300 kV, and imaged with a TVIPS Tem-Cam F224 2 k × 2 k CCD camera (TVIPS GmbH). EM-MENU software (TVIPS)was used for automated tilt series data collection in low dose mode. Eachtilt series of ∼100 images was collected from −70° to þ70° using cosine ruleincrements. For each dual-axis dataset, one series was collected while tiltingabout an axis parallel to the filaments. The second tilt series was obtainedafter manually rotating the specimen grid ∼90° and relocating the sameregion. Section shrinkage that occurs during electron irradiation (mainlyin the depth of the section), and which could thus cause changes duringcollection of the tomographic series, was minimized by preirradiation(43, 44). Section shrinkage at a set electron dose has a rapid phase and thena slow phase where the section is relatively stable. Following the rapid initialshrinkage induced by the preirradiation, imaging in low dose mode in thestable phase avoided any further significant shrinkage during tomographicdata collection. We noted from the tomograms that the shrinkage observedhad no significant effect on the thick filaments themselves. However, it didaffect the interfilament spacing as judged from the cross-sections of thereconstructions, which showed variable distance between the filaments.

The two orthogonal tilt series from each area were aligned using thePROTOMO marker-free tilt series alignment package (45). The tomogramsfrom the two series were then merged by patch correlation using IMOD(46). In total, three dual-axis tomograms were generated in this way fromthree different areas in the same EM grid.

Subvolume Averaging. Longitudinal sections of striated muscle show trans-verse stripes numbered 1 to 11. These stripes appear different along the thickfilament. Therefore we chose the whole thick filament in half an A-band asthe individual unit for subvolume averaging. To obtain the final thickfilament reconstruction shown, cylindrical volumes that included single thickfilaments and the surrounding six actin filaments and myosin filaments(radius of 45 nm) were extracted from the tomograms and iteratively alignedin three-dimensions and then averaged. For each tomogram, one thickfilament was selected as a reference. All the other thick filaments were iden-tified by computing a cross-correlation map between the reference and theentire tomogram. In total, 207 (67, 72, 68) filaments were selected from thethree tomograms. The initial alignment was based on the center of the cross-correlation peak that identified each filament within each tomogram. Aftertaking into account the azimuthal rotation of filaments, all the filamentsfrom the three tomograms were aligned and averaged to generate athree-dimensional structure of the thick filament, which includes transversestripes numbered 1 to 11. Once the structure of the thick filament was con-vincingly ascertained, 3-fold symmetry was imposed for subsequent roundsof refinement. Three real-space masks were designed separately for local

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refinement in order to improve the structural details of the three distinctregions of the A-band: the P-, C-, and D-zones. Surface renderings werecarried out using UCSF Chimera (47).

ACKNOWLEDGMENTS. We thank Gerald Offer and David Warshaw for criticalreading of the manuscript, Steven Marston for discussions, Cathy Timsonfor expert technical help, and Lorenzo Alamo and Antonio Pinto for helpin preparing Fig. 2 inset and Fig. S1. This work was supported by project

grants from the British Heart Foundation to P.K.L. (PG/06/010), by NationalInstitutes of Health (NIH) grants to R.C. (R01 AR034711), K.T. (R01GM030598), and D. Warshaw (P01 HL059408), and by grants from HowardHughes Medical Institute (HHMI 55005953) and FONACIT (Fondo Nacionalde Ciencia y Tecnología) to R.P. Molecular graphics images were producedusing the UCSF Chimera package from the Resource for Biocomputing,Visualization, and Informatics at the University of California, San Francisco(supported by NIH P41 RR-01081).

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