domain of the leading head is angled forwardand has a kink shortly after emerging from thecatalytic domain. This has been observed viaelectron microscopy (29), but is at odds withlater work by the same group (31).Conclusion.The specificity and sensitivityof single molecule fluorescence, combined withthe nanometer spatial localization and enhancedphotostability presented here, have enabled us tosee step sizes of individual myosin V heads. Theimprovement in localization results from collect-ing a sufficiently high number of photons withinthe relevant time period (0.5 s) with high SNR,made possible by the high-efficiency, low-noisedetector, and TIRF excitation minimizing back-ground (14). Extended observation was possiblethrough deoxygenation conditions that led tohighly photostable dyes. Our results lead to theconclusion that myosin V moves in a hand-over-hand fashion, in agreement with the recent workof Forkey et al., who measured tilting of BR-calmodulin in myosin V (6 ). Our conclusion isbased on results at low [ATP] but likely holds atphysiological [ATP] as well. Other tightly cou-pled motors, such as kinesin, may follow a sim-ilar scheme, and we now have the tools to ex-amine this and other molecular motors.Two types of hand-over-hand models havepreviously been proposed: an asymmetric modelwhere each head is not functionally equivalentand where the stalk need not twist, and a sym-metric model where each head is functionallyequivalent and the stalk twists 180° on each step.Hua et al. found that the coiled-coil stalk ofkinesin does not systematically twist as the mo-tor steps (8), a result also reported for myosin V(9). Such asymmetric hand-over-hand modelsare attractive for cargo-carrying motors becausethey do not require a twisting of a large cargo or,conversely, a large torque that would tend totwist the motor. A definitive conclusion, howev-er, between a symmetric and asymmetric hand-over-hand mechanism for myosin V—and per-haps other biomolecular motors—will need toawait further experiments.References and Notes1. A. D. Mehta et al., Nature 400, 590 (1999).2. C. Veigel, F. Wang, M. L. Bartoo, J. R. Sellers, J. E.Molloy, Nature Cell Biol. 4, 59 (2002).3. A. Mehta, J. Cell Sci. 114, 1981 (2001).4.S. L. Reck-Peterson, D. W. Provance Jr., M. S. Mooseker,J. A. Mercer, Biochim. Biophys. Acta 1496, 36 (2000).5. T. J. Purcell, C. Morris, J. A. Spudich, H. L. Sweeney,Proc. Natl. Acad. Sci. U.S.A. 99, 14159 (2002).6. J. N. Forkey, M. E. Quinlan, M. T. Shaw, J. E. Corrie,Y. E. Goldman, Nature 422, 399 (2003).7. E. M. De La Cruz, A. L. Wells, S. S. Rosenfeld, E. M.Ostap, H. L. Sweeney, Proc. Natl. Acad. Sci. U.S.A. 96,13726 (1999).8. W. Hua, J. Chung, J. Gelles, Science 295, 844 (2002).9. M. Y. Ali et al., Nature Struct. Biol. 9, 464 (2002).10. R. S. Rock et al., Proc. Natl. Acad. Sci. U.S.A. 98,13655 (2001).11. D. Axelrod, Methods Cell Biol. 30, 245 (1989).12. T. Funatsu, Y. Harada, M. Tokunaga, K. Saito, T.Yanagida, Nature 374, 555 (1995).13. M. Tokunaga, K. Kitamura, K. Saito, A. H. Iwane, T.Yanagida, Biochem. Biophys. Res. Commun. 235,47(1997).14. Materials and Methods are available as supportingonline material on Science Online.15. T. Schmidt, G. J. Schu¨tz, W. Baumgartner, H. J. Gruber,H. Schindler, Proc. Natl. Acad. Sci. U.S.A. 93, 2926(1996).16. U. Kubitscheck, O. Ku¨ckmann, T. Kues, R. Peters,Biophys. J. 78, 2170 (2000).17. T. D. Lacoste et al., Proc. Natl. Acad. Sci. U.S.A. 97,9461 (2000).18. R. E. Thompson, D. R. Larson, W. W. Webb, Biophys. J.82, 2775 (2002).19. J. Gelles, B. J. Schnapp, M. P. Sheetz, Nature 331, 450(1988).20. N. Bobroff, Rev. Sci. Inst. 57, 1152 (1986).21. M. K. Cheezum, W. F. Walker, W. H. Guilford, Biophys.J. 81, 2378 (2001).22. Y. Harada, K. Sakurada, T. Aoki, D. D. Thomas, T.Yanagida, J. Mol. Biol. 216, 49 (1990).23. K. Adachi et al., Proc. Natl. Acad. Sci. U.S.A. 97, 7243(2000).24. Y. Sambongi et al., Science 286, 1722 (1999).25. J. E. T. Corrie et al., Nature 400, 425 (1999).26. T. Sakamoto, I. Amitani, E. Yokota, T. Ando, Biochem.Biophys. Res. Commun. 272, 586 (2000).27. A. P. Bartko, R. M. Dickson, J. Phys. Chem. B 103,3053 (1999).28.Fitting the data to two rate constants does not improvethe fit; however, the curve is fit equally well to a rangeof (k1,k2), where k1and k2can differ by a factor of 2 ormore. Hence, we cannot rule out asymmetry in the ratesof the two heads.29. M. L. Walker et al., Nature 405, 804 (2000).30. M. Rief et al., Proc. Natl. Acad. Sci. U.S.A. 97, 9482(2000).31. S. Burgess et al., J. Cell Biol. 159, 983 (2002).32. J. A. Spudich, Nature Rev. Mol. Cell Biol. 2, 387(2001).33.Supported by NIH grants AR44420 (to P.R.S.), GM65367(to T.H.), and AR26846 (to Y.E.G.); NSF grants DBI-02-15869 (to P.R.S. and T.H.) and 9984841 (to P.R.S.); theCarver Trust Foundation (to P.R.S.); and the DOE, Divi-sion of Materials Sciences (under award no. DEFG02-91ER45439), through the Frederick Seitz Materials Re-search Laboratory at the University of Illinois at Urbana-Champaign (to P.R.S.). S.A.M. was supported by a Na-tional Research Service Award in Molecular Biophysics(through NIH training grant PHS 5 T32 GM08276). Wethank J. E. T. Corrie for gift of bis-rhodamine, E. Grattonfor initial suggestions and discussions, B. C. Stevens forcomputer programming assistance, and I. Rasnik for flu-orescence imaging assistance.Supporting Online Materialwww.sciencemag.org/cgi/content/full/1084398/DC1Materials and Methods Figs. S1 to S3Movie S1References11 March 2003; accepted 21 May 2003Published online 5 June 2003;10.1126/science.1084398Include this information when citing this paper.Antibody Domain Exchange Is anImmunological Solution toCarbohydrate Cluster RecognitionDaniel A. Calarese,1Christopher N. Scanlan,2,5Michael B. Zwick,2Songpon Deechongkit,3Yusuke Mimura,5Renate Kunert,6Ping Zhu,7Mark R. Wormald,5Robyn L. Stanfield,1Kenneth H. Roux,7Jeffery W. Kelly,3,4Pauline M. Rudd,5Raymond A. Dwek,5Hermann Katinger,6Dennis R. Burton,1,2*Ian A. Wilson1,4*Human antibody 2G12 neutralizes a broad range of human immunodeficiency virustype 1 (HIV-1) isolates by binding an unusually dense cluster of carbohydratemoieties on the “silent” face of the gp120 envelope glycoprotein. Crystal structuresof Fab 2G12 and its complexes with the disaccharide Man␣1-2Man and with theoligosaccharide Man9GlcNAc2revealed that two Fabs assemble into an interlockedVHdomain-swapped dimer. Further biochemical, biophysical, and mutagenesis datastrongly support a