Skip to main content
NCBI home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1991 Aug 2;114(4):701–713. doi: 10.1083/jcb.114.4.701

Three-dimensional structure of myosin subfragment-1 from electron microscopy of sectioned crystals

PMCID: PMC2289899  PMID: 1869586

Abstract

Image analysis of electron micrographs of thin-sectioned myosin subfragment-1 (S1) crystals has been used to determine the structure of the myosin head at approximately 25-A resolution. Previous work established that the unit cell of type I crystals of myosin S1 contains eight molecules arranged with orthorhombic space group symmetry P212121 and provided preliminary information on the size and shape of the myosin head (Winkelmann, D. A., H. Mekeel, and I. Rayment. 1985. J. Mol. Biol. 181:487-501). We have applied a systematic method of data collection by electron microscopy to reconstruct the three-dimensional (3D) structure of the S1 crystal lattice. Electron micrographs of thin sections were recorded at angles of up to 50 degrees by tilting the sections about the two orthogonal unit cell axes in sections cut perpendicular to the three major crystallographic axes. The data from six separate tilt series were merged to form a complete data set for 3D reconstruction. This approach has yielded an electron density map of the unit cell of the S1 crystals of sufficient detail. to delineate the molecular envelope of the myosin head. Myosin S1 has a tadpole-shaped molecular envelope that is very similar in appearance to the pear- shaped myosin heads observed by electron microscopy of rotary-shadowed and negatively stained myosin. The molecule is divided into essentially three morphological domains: a large domain on one end of the molecule corresponding to approximately 60% of the total molecular volume, a smaller central domain of approximately 30% of the volume that is separated from the larger domain by a cleft on one side of the molecule, and the smallest domain corresponding to a thin tail-like region containing approximately 10% of the volume. This molecular organization supports models of force generation by myosin which invoke conformational mobility at interdomain junctions within the head.

Full Text

The Full Text of this article is available as a PDF (1.7 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Agard D. A. A least-squares method for determining structure factors in three-dimensional tilted-view reconstructions. J Mol Biol. 1983 Jul 15;167(4):849–852. doi: 10.1016/s0022-2836(83)80114-4. [DOI] [PubMed] [Google Scholar]
  2. Agard D. A., Stroud R. M. Linking regions between helices in bacteriorhodopsin revealed. Biophys J. 1982 Mar;37(3):589–602. [PMC free article] [PubMed] [Google Scholar]
  3. Akey C. W., Moffat K., Wharton D. C., Edelstein S. J. Characterization of crystals of a cytochrome oxidase (nitrite reductase) from Pseudomonas aeruginosa by x-ray diffraction and electron microscopy. J Mol Biol. 1980 Jan 5;136(1):19–43. doi: 10.1016/0022-2836(80)90364-2. [DOI] [PubMed] [Google Scholar]
  4. Amos L. A., Henderson R., Unwin P. N. Three-dimensional structure determination by electron microscopy of two-dimensional crystals. Prog Biophys Mol Biol. 1982;39(3):183–231. doi: 10.1016/0079-6107(83)90017-2. [DOI] [PubMed] [Google Scholar]
  5. Applegate D., Reisler E. Nucleotide-induced changes in the proteolytically sensitive regions of myosin subfragment 1. Biochemistry. 1984 Sep 25;23(20):4779–4784. doi: 10.1021/bi00315a038. [DOI] [PubMed] [Google Scholar]
  6. Applegate D., Reisler E. Protease-sensitive regions in myosin subfragment 1. Proc Natl Acad Sci U S A. 1983 Dec;80(23):7109–7112. doi: 10.1073/pnas.80.23.7109. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Baker T. S., Amos L. A. Structure of the tubulin dimer in zinc-induced sheets. J Mol Biol. 1978 Jul 25;123(1):89–106. doi: 10.1016/0022-2836(78)90378-9. [DOI] [PubMed] [Google Scholar]
  8. Baker T. S., Caspar D. L., Hollingshead C. J., Goodenough D. A. Gap junction structures. IV. Asymmetric features revealed by low-irradiation microscopy. J Cell Biol. 1983 Jan;96(1):204–216. doi: 10.1083/jcb.96.1.204. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Berger J. E. Optical diffraction studies of crystalline structures in electron micrographs. I. Theoretical considerations. J Cell Biol. 1969 Dec;43(3):442–447. doi: 10.1083/jcb.43.3.442. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Bálint M., Wolf I., Tarcsafalvi A., Gergely J., Sréter F. A. Location of SH-1 and SH-2 in the heavy chain segment of heavy meromyosin. Arch Biochem Biophys. 1978 Oct;190(2):793–799. doi: 10.1016/0003-9861(78)90339-9. [DOI] [PubMed] [Google Scholar]
  11. Craig R., Szent-Györgyi A. G., Beese L., Flicker P., Vibert P., Cohen C. Electron microscopy of thin filaments decorated with a Ca2+-regulated myosin. J Mol Biol. 1980 Jun 15;140(1):35–55. doi: 10.1016/0022-2836(80)90355-1. [DOI] [PubMed] [Google Scholar]
  12. DeRosier D. J., Moore P. B. Reconstruction of three-dimensional images from electron micrographs of structures with helical symmetry. J Mol Biol. 1970 Sep 14;52(2):355–369. doi: 10.1016/0022-2836(70)90036-7. [DOI] [PubMed] [Google Scholar]
  13. Egelman E. H., Francis N., DeRosier D. J. Helical disorder and the filament structure of F-actin are elucidated by the angle-layered aggregate. J Mol Biol. 1983 Jun 5;166(4):605–629. doi: 10.1016/s0022-2836(83)80286-1. [DOI] [PubMed] [Google Scholar]
  14. Eisenberg E., Greene L. E. The relation of muscle biochemistry to muscle physiology. Annu Rev Physiol. 1980;42:293–309. doi: 10.1146/annurev.ph.42.030180.001453. [DOI] [PubMed] [Google Scholar]
  15. Eisenberg E., Hill T. L. Muscle contraction and free energy transduction in biological systems. Science. 1985 Mar 1;227(4690):999–1006. doi: 10.1126/science.3156404. [DOI] [PubMed] [Google Scholar]
  16. Elliott A., Offer G. Shape and flexibility of the myosin molecule. J Mol Biol. 1978 Aug 25;123(4):505–519. doi: 10.1016/0022-2836(78)90204-8. [DOI] [PubMed] [Google Scholar]
  17. Flicker P. F., Wallimann T., Vibert P. Electron microscopy of scallop myosin. Location of regulatory light chains. J Mol Biol. 1983 Sep 25;169(3):723–741. doi: 10.1016/s0022-2836(83)80167-3. [DOI] [PubMed] [Google Scholar]
  18. Highsmith S., Eden D. Myosin subfragment 1 has tertiary structural domains. Biochemistry. 1986 Apr 22;25(8):2237–2242. doi: 10.1021/bi00356a058. [DOI] [PubMed] [Google Scholar]
  19. Huxley A. F., Simmons R. M. Proposed mechanism of force generation in striated muscle. Nature. 1971 Oct 22;233(5321):533–538. doi: 10.1038/233533a0. [DOI] [PubMed] [Google Scholar]
  20. Huxley H. E., Faruqi A. R., Kress M., Bordas J., Koch M. H. Time-resolved X-ray diffraction studies of the myosin layer-line reflections during muscle contraction. J Mol Biol. 1982 Jul 15;158(4):637–684. doi: 10.1016/0022-2836(82)90253-4. [DOI] [PubMed] [Google Scholar]
  21. Huxley H. E., Kress M. Crossbridge behaviour during muscle contraction. J Muscle Res Cell Motil. 1985 Apr;6(2):153–161. doi: 10.1007/BF00713057. [DOI] [PubMed] [Google Scholar]
  22. Kabsch W., Mannherz H. G., Suck D., Pai E. F., Holmes K. C. Atomic structure of the actin:DNase I complex. Nature. 1990 Sep 6;347(6288):37–44. doi: 10.1038/347037a0. [DOI] [PubMed] [Google Scholar]
  23. Katoh T., Lowey S. Mapping myosin light chains by immunoelectron microscopy. Use of anti-fluorescyl antibodies as structural probes. J Cell Biol. 1989 Oct;109(4 Pt 1):1549–1560. doi: 10.1083/jcb.109.4.1549. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Knight P., Trinick J. Structure of the myosin projections on native thick filaments from vertebrate skeletal muscle. J Mol Biol. 1984 Aug 15;177(3):461–482. doi: 10.1016/0022-2836(84)90295-x. [DOI] [PubMed] [Google Scholar]
  25. Lowey S., Slayter H. S., Weeds A. G., Baker H. Substructure of the myosin molecule. I. Subfragments of myosin by enzymic degradation. J Mol Biol. 1969 May 28;42(1):1–29. doi: 10.1016/0022-2836(69)90483-5. [DOI] [PubMed] [Google Scholar]
  26. Lu R. C., Moo L., Wong A. G. Both the 25-kDa and 50-kDa domains in myosin subfragment 1 are close to the reactive thiols. Proc Natl Acad Sci U S A. 1986 Sep;83(17):6392–6396. doi: 10.1073/pnas.83.17.6392. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Mahmood R., Yount R. G. Photochemical probes of the active site of myosin. Irradiation of trapped 3'-O-(4-benzoyl)benzoyladenosine 5'-triphosphate labels the 50-kilodalton heavy chain tryptic peptide. J Biol Chem. 1984 Nov 10;259(21):12956–12959. [PubMed] [Google Scholar]
  28. Margossian S. S., Lowey S. Substructure of the myosin molecule. IV. Interactions of myosin and its subfragments with adenosine triphosphate and F-actin. J Mol Biol. 1973 Mar 5;74(3):313–330. doi: 10.1016/0022-2836(73)90376-8. [DOI] [PubMed] [Google Scholar]
  29. Margossian S. S., Stafford W. F., 3rd, Lowey S. Homogeneity of myosin subfragments by equilibrium centrifugation. Biochemistry. 1981 Apr 14;20(8):2151–2155. doi: 10.1021/bi00511a012. [DOI] [PubMed] [Google Scholar]
  30. Milligan R. A., Flicker P. F. Structural relationships of actin, myosin, and tropomyosin revealed by cryo-electron microscopy. J Cell Biol. 1987 Jul;105(1):29–39. doi: 10.1083/jcb.105.1.29. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Milligan R. A., Whittaker M., Safer D. Molecular structure of F-actin and location of surface binding sites. Nature. 1990 Nov 15;348(6298):217–221. doi: 10.1038/348217a0. [DOI] [PubMed] [Google Scholar]
  32. Mornet D., Bertrand R. U., Pantel P., Audemard E., Kassab R. Proteolytic approach to structure and function of actin recognition site in myosin heads. Biochemistry. 1981 Apr 14;20(8):2110–2120. doi: 10.1021/bi00511a007. [DOI] [PubMed] [Google Scholar]
  33. Mornet D., Bertrand R., Pantel P., Audemard E., Kassab R. Structure of the actin-myosin interface. Nature. 1981 Jul 23;292(5821):301–306. doi: 10.1038/292301a0. [DOI] [PubMed] [Google Scholar]
  34. Okamoto Y., Yount R. G. Identification of an active site peptide of skeletal myosin after photoaffinity labeling with N-(4-azido-2-nitrophenyl)-2-aminoethyl diphosphate. Proc Natl Acad Sci U S A. 1985 Mar;82(6):1575–1579. doi: 10.1073/pnas.82.6.1575. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Pollard T. D., Doberstein S. K., Zot H. G. Myosin-I. Annu Rev Physiol. 1991;53:653–681. doi: 10.1146/annurev.ph.53.030191.003253. [DOI] [PubMed] [Google Scholar]
  36. Prince H. P., Trayer H. R., Henry G. D., Trayer I. P., Dalgarno D. C., Levine B. A., Cary P. D., Turner C. Proton nuclear-magnetic-resonance spectroscopy of myosin subfragment 1 isoenzymes. Eur J Biochem. 1981 Dec;121(1):213–219. doi: 10.1111/j.1432-1033.1981.tb06451.x. [DOI] [PubMed] [Google Scholar]
  37. Rayment I., Winkelmann D. A. Crystallization of myosin subfragment 1. Proc Natl Acad Sci U S A. 1984 Jul;81(14):4378–4380. doi: 10.1073/pnas.81.14.4378. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Reedy M. C., Beall C., Fyrberg E. Formation of reverse rigor chevrons by myosin heads. Nature. 1989 Jun 8;339(6224):481–483. doi: 10.1038/339481a0. [DOI] [PubMed] [Google Scholar]
  39. Rimm D. L., Sinard J. H., Pollard T. D. Location of the head-tail junction of myosin. J Cell Biol. 1989 May;108(5):1783–1789. doi: 10.1083/jcb.108.5.1783. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Salmon E. D., DeRosier D. A surveying optical diffractometer. J Microsc. 1981 Sep;123(Pt 3):239–247. doi: 10.1111/j.1365-2818.1981.tb02468.x. [DOI] [PubMed] [Google Scholar]
  41. Sutoh K. Mapping of actin-binding sites on the heavy chain of myosin subfragment 1. Biochemistry. 1983 Mar 29;22(7):1579–1585. doi: 10.1021/bi00276a009. [DOI] [PubMed] [Google Scholar]
  42. Sutoh K., Yamamoto K., Wakabayashi T. Electron microscopic visualization of the ATPase site of myosin by photoaffinity labeling with a biotinylated photoreactive ADP analog. Proc Natl Acad Sci U S A. 1986 Jan;83(2):212–216. doi: 10.1073/pnas.83.2.212. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Sutoh K., Yamamoto K., Wakabayashi T. Electron microscopic visualization of the SH1 thiol of myosin by the use of an avidin-biotin system. J Mol Biol. 1984 Sep 15;178(2):323–339. doi: 10.1016/0022-2836(84)90147-5. [DOI] [PubMed] [Google Scholar]
  44. Taylor K. A., Reedy M. C., Córdova L., Reedy M. K. Three-dimensional image reconstruction of insect flight muscle. I. The rigor myac layer. J Cell Biol. 1989 Sep;109(3):1085–1102. doi: 10.1083/jcb.109.3.1085. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Taylor K. A., Reedy M. C., Córdova L., Reedy M. K. Three-dimensional image reconstruction of insect flight muscle. II. The rigor actin layer. J Cell Biol. 1989 Sep;109(3):1103–1123. doi: 10.1083/jcb.109.3.1103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Tokunaga M., Sutoh K., Toyoshima C., Wakabayashi T. Location of the ATPase site of myosin determined by three-dimensional electron microscopy. Nature. 1987 Oct 15;329(6140):635–638. doi: 10.1038/329635a0. [DOI] [PubMed] [Google Scholar]
  47. Tokunaga M., Suzuki M., Saeki K., Wakabayashi T. Position of the amino terminus of myosin light chain 1 and light chain 2 determined by electron microscopy with monoclonal antibody. J Mol Biol. 1987 Mar 20;194(2):245–255. doi: 10.1016/0022-2836(87)90372-x. [DOI] [PubMed] [Google Scholar]
  48. Toyoshima Y. Y., Kron S. J., McNally E. M., Niebling K. R., Toyoshima C., Spudich J. A. Myosin subfragment-1 is sufficient to move actin filaments in vitro. Nature. 1987 Aug 6;328(6130):536–539. doi: 10.1038/328536a0. [DOI] [PubMed] [Google Scholar]
  49. Vibert P. J. Domain structure of the myosin head in correlation-averaged images of shadowed molecules. J Muscle Res Cell Motil. 1988 Apr;9(2):147–155. doi: 10.1007/BF01773736. [DOI] [PubMed] [Google Scholar]
  50. Vibert P., Cohen C. Domains, motions and regulation in the myosin head. J Muscle Res Cell Motil. 1988 Aug;9(4):296–305. doi: 10.1007/BF01773873. [DOI] [PubMed] [Google Scholar]
  51. Walker J. E., Saraste M., Runswick M. J., Gay N. J. Distantly related sequences in the alpha- and beta-subunits of ATP synthase, myosin, kinases and other ATP-requiring enzymes and a common nucleotide binding fold. EMBO J. 1982;1(8):945–951. doi: 10.1002/j.1460-2075.1982.tb01276.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Walker M., Knight P., Trinick J. Negative staining of myosin molecules. J Mol Biol. 1985 Aug 5;184(3):535–542. doi: 10.1016/0022-2836(85)90300-6. [DOI] [PubMed] [Google Scholar]
  53. Walker M., Trinick J. Electron microscopy of negatively stained scallop myosin molecules. Effect of regulatory light chain removal on head structure. J Mol Biol. 1989 Aug 5;208(3):469–475. doi: 10.1016/0022-2836(89)90510-x. [DOI] [PubMed] [Google Scholar]
  54. Walker M., Trinick J. Visualization of domains in native and nucleotide-trapped myosin heads by negative staining. J Muscle Res Cell Motil. 1988 Aug;9(4):359–366. doi: 10.1007/BF01773879. [DOI] [PubMed] [Google Scholar]
  55. Warrick H. M., Spudich J. A. Myosin structure and function in cell motility. Annu Rev Cell Biol. 1987;3:379–421. doi: 10.1146/annurev.cb.03.110187.002115. [DOI] [PubMed] [Google Scholar]
  56. Weeds A. G., Taylor R. S. Separation of subfragment-1 isoenzymes from rabbit skeletal muscle myosin. Nature. 1975 Sep 4;257(5521):54–56. doi: 10.1038/257054a0. [DOI] [PubMed] [Google Scholar]
  57. Winkelmann D. A., Almeda S., Vibert P., Cohen C. A new myosin fragment: visualization of the regulatory domain. Nature. 1984 Feb 23;307(5953):758–760. doi: 10.1038/307758a0. [DOI] [PubMed] [Google Scholar]
  58. Winkelmann D. A., Lowey S. Probing myosin head structure with monoclonal antibodies. J Mol Biol. 1986 Apr 20;188(4):595–612. doi: 10.1016/s0022-2836(86)80009-2. [DOI] [PubMed] [Google Scholar]
  59. Winkelmann D. A., Mekeel H., Rayment I. Packing analysis of crystalline myosin subfragment-1. Implications for the size and shape of the myosin head. J Mol Biol. 1985 Feb 20;181(4):487–501. doi: 10.1016/0022-2836(85)90422-x. [DOI] [PubMed] [Google Scholar]
  60. Yanagida T. Angle of active site of myosin heads in contracting muscle during sudden length changes. J Muscle Res Cell Motil. 1985 Feb;6(1):43–52. doi: 10.1007/BF00712310. [DOI] [PubMed] [Google Scholar]
  61. Yanagida T. Angles of nucleotides bound to cross-bridges in glycerinated muscle fiber at various concentrations of epsilon-ATP, epsilon-ADP and epsilon-AMPPNP detected by polarized fluorescence. J Mol Biol. 1981 Mar 15;146(4):539–560. doi: 10.1016/0022-2836(81)90046-2. [DOI] [PubMed] [Google Scholar]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press

RESOURCES