An Atomic Model of the Thin Filament in the Relaxed and Ca 2C -Activated States Alnoor Pirani 1 †, Maia V. Vinogradova 2 †, Paul M. G. Curmi 3 , William A. King 3 , Robert J. Fletterick 2 , Roger Craig 4 , Larry S. Tobacman 5 Chen Xu 6 , Victoria Hatch 1 and William Lehman 1 * 1 Department of Physiology & Biophysics, Boston University School of Medicine, Boston MA 02118, USA 2 Department of Biochemistry & Biophysics, University of California, San Francisco CA 94143, USA 3 School of Physics, University of New South Wales, Sydney NSW 2052, and Centre for Immunology, St. Vincent’s Hospital, Sydney NSW 2010 Australia 4 Department of Cell Biology University of Massachusetts Medical School, Worcester MA 01655, USA 5 Departments of Medicine and Physiology & Biophysics University of Illinois at Chicago Chicago, IL 60612, USA 6 Rosenstiel Basic Medical Sciences Research Center Brandeis University, Waltham MA 02454, USA Contraction of striated muscles is regulated by tropomyosin strands that run continuously along actin-containing thin filaments. Tropomyosin blocks myosin-binding sites on actin in resting muscle and unblocks them during Ca 2C -activation. This steric effect controls myosin-crossbridge cycling on actin that drives contraction. Troponin, bound to the thin filaments, couples Ca 2C -concentration changes to the movement of tropomyosin. Ca 2C -free troponin is thought to trap tropomyosin in the myosin-blocking position, while this constraint is released after Ca 2C - binding. Although the location and movements of tropomyosin are well known, the structural organization of troponin on thin filaments is not. Its mechanism of action therefore remains uncertain. To determine the organization of troponin on the thin filament, we have constructed atomic models of low and high-Ca 2C states based on crystal structures of actin, tropomyosin and the “core domain” of troponin, and constrained by distances between filament components and by their location in electron microscopy (EM) reconstructions. Alternative models were also built where troponin was systematically repositioned or reoriented on actin. The accuracy of the different models was evaluated by determining how well they corresponded to EM images. While the initial low and high-Ca 2C models fitted the data precisely, the alternatives did not, suggesting that the starting models best represented the correct structures. Thin filament reconstructions were generated from the EM data using these starting models as references. In addition to showing the core domain of troponin, the reconstructions showed additional detail not present in the starting models. We attribute this to an extension of TnI linking the troponin core domain to actin at low (but not at high) Ca 2C , thereby trapping tropomyosin in the OFF-state. The bulk of the core domain of troponin appears not to move significantly on actin, regardless of Ca 2C level. Our observations suggest a simple model for muscle regulation in which troponin affects the charge balance on actin and hence tropomyosin position. q 2005 Elsevier Ltd. All rights reserved. Keywords: actin; calcium; muscle regulation; tropomyosin; troponin *Corresponding author Introduction Muscle contraction results from the ATP- dependent interactions of myosin-crossbridges that project from thick filaments and cyclically attach, translate and detach from actin monomers of the thin filament. The thin filament proteins, troponin and tropomyosin, act to inhibit contraction in resting striated muscle, and Ca 2C reverses this inhibition. 1 Ca 2C -binding to troponin followed by myosin-binding to actin results in movement of tropomyosin away from its low-Ca 2C position (where it sterically interferes with actin-myosin 0022-2836/$ - see front matter q 2005 Elsevier Ltd. All rights reserved. † A.P. and M.V.V contributed equally to the work. Abbreviation used: EM, electron microscopy. E-mail address of the corresponding author: wlehman@bu.edu doi:10.1016/j.jmb.2005.12.050 J. Mol. Biol. (2006) 357, 707–717