COMMUNICATION Troponin Organization on Relaxed and Activated Thin Filaments Revealed by Electron Microscopy and Three-dimensional Reconstruction William Lehman 1 *, Michael Rosol 1 , Larry S. Tobacman 2 and Roger Craig 3 1 Department of Physiology and Biophysics, Boston University School of Medicine, 715 Albany Street, Boston MA 02118, USA 2 Departments of Biochemistry and Internal Medicine, College of Medicine, University of Iowa, 200 Hawkins Drive, Iowa City, IA 52242, USA 3 Department of Cell Biology University of Massachusetts Medical School, 55 Lake Avenue North, Worcester MA 01655, USA The steric model of muscle regulation holds that at low Ca 2 concen- tration, tropomyosin strands, running along thin ®laments, are con- strained by troponin in an inhibitory position that blocks myosin-binding sites on actin. Ca 2 activation, releasing this constraint, allows tropo- myosin movement, initiating actin-myosin interaction and contraction. Although the different positions of tropomyosin on the thin ®lament are well documented, corresponding information on troponin has been lack- ing and it has therefore not been possible to test the model structurally. Here, we show that troponin can be detected on thin ®laments and demonstrate how its changing association with actin can control tropo- myosin position in response to Ca 2 . To accomplish this, thin ®laments were reconstituted with an engineered short tropomyosin, creating a favorable troponin stoichiomtery and symmetry for three-dimensional analysis. We demonstrate that in the absence of Ca 2 , troponin bound to both tropomyosin and actin can act as a latch to constrain tropomyosin in a position on actin that inhibits actomyosin ATPase. In addition, we ®nd that on Ca 2 activation the actin-troponin connection is broken, allowing tropomyosin to assume a second position, initiating actomyosin ATPase and thus permitting contraction to proceed. # 2001 Academic Press Keywords: actin; electron microscopy; muscle regulation; tropomyosin; troponin *Corresponding author The steric mechanism of regulation, conceived over 25 years ago on the basis of modeling X-ray diffraction data 1-3 , has never been directly demon- strated. Electron microscopy and three-dimensional (3D) reconstruction have been used to visualize steric blocking and unblocking of myosin-binding sites on actin by tropomyosin, 4-7 con®rming one essential aspect of the model. However, 3D recon- struction based on actin helical symmetry has failed to reveal troponin density along the thin ®la- ment because troponin occurs on only every seventh actin monomer. 8 It has therefore not been possible to directly determine the structural basis of troponin's function. To circumvent this problem, we have reconstituted thin ®laments with a short tropomyosin, 9 interacting with four instead of the normal seven actin monomers, creating a more favorable stoichiometry and symmetry for 3D anal- ysis. This has permitted 3D reconstruction of both troponin and tropomyosin in low and high Ca 2 - concentration states and thus direct testing of the steric model. Consistent with the model, we observe that troponin directly links tropomyosin and actin at low [Ca 2 ]. The connection to actin is broken at high [Ca 2 ], allowing tropomyosin movement and consequent cross-bridge cycling. An advantage in studying macromolecular struc- tures with helically distributed subunits is that many views of the subunits can be seen in a single electron micrograph; 10,11 3D reconstruction of heli- cally arranged ®laments such as F-actin is therefore possible. 12 The 3D reconstruction of tropomyo- sin on F-actin is also possible, since tropomyosin strands and F-actin follow the same long-pitch helical paths, with tropomyosin making continu- ous, approximately equivalent, contacts with successive actin monomers along F-actin. 4-7,13,14 In E-mail address of corresponding author: wlehman@bu.edu doi:10.1006/jmbi.2001.4514 available online at http://www.idealibrary.com on J. Mol. Biol. (2001) 307, 739±744 0022-2836/01/030739±6 $35.00/0 # 2001 Academic Press