Hanspeter Winkler

  • Institute of Molecular Biophysics
  • Research Associate
  • phone: (850) 644-5606
  • winkler@sb.fsu.edu

Extraction of 3-D information from electron micrographs requires the application of complex 3-D reconstruction algorithms. In many instances new algorithms must be developed to deal with new reconstruction methods as well as improve existing ones or adapt them to new situations. My work involves development and implementation of new reconstruction algorithms.

oblique-ifm

One of these was a method for combining images of thin sections through a crystalline specimen that were cut at varying angles to different unit cell axes. The method, called oblique section 3-D reconstruction, is an adaptation of a method originally developed by R. A. Crowther. It allows the user to cut thin sections to reconstruct a thick object. The image to the right is the result of such a reconstruction. The sections used to make the reconstruction were ~150Å thick whereas the unit cell was 520Å thick.

We now concentrate on electron tomography, which is a reconstruction method that can produce a 3-D image of anything, whether crystalline or not. We have developed a new reconstruction method for processing tomographic data and are now applying methods to classify and average different motifs within a paracrystalline specimen. An outgrowth of this work was a method for 3-D distortion correction, which will not only correct for the distortion in the X-Y plane, as is typically done with crystalline specimens, but will correct for bending in the Z direction as well. Such a correction requires a tomogram of the paracrystalline specimen.

We have also applied our tomographic methods to non-crystalline specimens such as ice-embedded viruses and muscle proteins adsorbed on lipid monolayers (myosin V). For the latter we corrected the focus gradient in the micrographs prior to alignment with our marker-free method. Tomograms computed by weighted backprojection were further analyzed with methods common in single particle analysis of projection data (multireference alignment, multivariate statistical analysis and classification). We adapted these methods for 3D data in order to be able to characterize structural heterogeneity in out tomograms.

PUBLICATIONS

  • H. Winkler & K. A. Taylor. 3D reconstruction by combining data from sections cut oblique to different unit cell axes. Ultramicroscopy 55, 357-371. (1994)
  • K. A. Taylor and H. Winkler. 3-D reconstruction of paracrystalline biological specimens by tomography. Proc. Microscopy and Microanalysis, G. W. Bailey, M. H. Ellisman, R. A. Hennigar and N. J. Zaluzec, eds. Jones and Begell Pub., New York. pp 734-735. (1995)
  • H. Winkler and K. A. Taylor. Software for 3-D reconstruction from images of oblique sections through 3-D crystals. J. Struct. Biol. 116, 241-247. (1996)
  • H. Winkler and K. A. Taylor Three-dimensional distortion correction applied to tomographic reconstructions of sectioned crystals. Ultramicroscopy 63, 125-132. (1996)
  • H. Winkler, Mary C. Reedy, Michael K. Reedy, Richard Tregear and Kenneth A. Taylor. 3-D structure of nucleotide bearing crossbridges in situ: oblique section reconstruction of insect flight muscle in AMPPNP at 23°C. J. Mol. Biol. 264, 302-322. (1996)
  • Holger Schmitz, Mary C. Reedy, Michael K. Reedy, Richard T. Tregear, H. Winkler, Kenneth A. Taylor. Electron tomography of Insect Flight Muscle in Rigor and AMPPNP at 23°C. J. Mol. Biol. 264, 279-301. (1996)
  • H. Schmitz, M. C. Reedy, M. K. Reedy, R. T. Tregear, H. Winkler, K. A. Taylor. Tomographic 3-D reconstruction of insect flight muscle partially relaxed by AMPPNP and ethylene glycol. J. Cell Biol. 139, 695-707. (1997)
  • Kenneth A. Taylor, Jinghua Tang, Yifan Cheng , and Hanspeter Winkler. The use of electron tomography for structural analysis of disordered protein arrays. J. Struct. Biol. 120(3), 372-386. (1997)
  • H. Schmitz, M. C. Reedy, M. K. Reedy, R. T. Tregear, H. Winkler, K. A. Taylor. Tomographic 3-D Reconstruction of Insect Flight Muscle Partially Relaxed by AMPPNP and Ethylene Glycol. J. Cell Biol. 139, 695-707. (1997)
  • Hanspeter Winkler & Kenneth A. Taylor. Multivariate statistical analysis of three-dimensional cross-bridge motifs in insect flight muscle. Ultramicroscopy 77, 141-152. (1999)
  • Kenneth A. Taylor, Holger Schmitz, Mary C. Reedy, Yale E. Goldman, Clara Franzini-Armstrong, Hiro Sasaki, Richard T. Tregear, Kate Poole, Carmen Lucaveche, Robert J. Edwards, Li Fan Chen, Hanspeter Winkler, and Michael K. Reedy. Tomographic 3-D reconstruction of quick frozen, Ca++-activated contracting insect flight muscle. Cell 99, 421-431. (1999)
  • Kenneth A. Taylor, Jinghua Tang, Yifan Cheng & Hanspeter Winkler. The use of electron tomography for structural analysis of disordered protein arrays. J. Struct. Biol. 120, 372-386. (1997)
  • Pascual-Montano, A., Taylor, K.A., Winkler, H., Pascual-Marqui, R.D., Carazo, J.M. Quantitative Self Organizing Maps for clustering electron tomograms. J. Struct. Biol. 138, 114-122. (2002)
  • Chen, Li Fan, Winkler, Hanspeter, Reedy, Michael K., Reedy, Mary C. & Taylor ,Kenneth A. Molecular Modeling of Averaged Rigor Crossbridges from Tomograms of Insect Flight Muscle. J. Struct. Biol. 138(2) 92-104. (2002)
  • Hanspeter Winkler and Kenneth A. Taylor. Focus gradient correction applied to tilt series image data used in electron tomography. J. Struct. Biol. 143(1), 24-32. (2003)
  • Jun Liu, Mary C. Reedy, Yale E. Goldman, Clara Franzini-Armstrong, Hiroyuki Sasaki, Richard T. Tregear, Carmen Lucaveche, Hanspeter Winkler, Bruce A. J. Baumann, John M. Squire, Thomas C. Irving, Michael K. Reedy, and Kenneth A. Taylor. Electron tomography of fast frozen, stretched rigor fibers reveals elastic distortions in the myosin crossbridges. J. Struct. Biol. 147, 268-282. (2004)
  • Richard Tregear, Kenneth Taylor, Hanspeter Winkler, Mary Reedy, Yale Goldman, Clara Franzini-Armstrong, Hiro Sasaki, Carmen Lucaveche and Michael Reedy. Number and orientation of crossbridges in target zones of active insect flight muscle. Biophys. J. 86, 3009-3019. (2004)
  • Liu, Jun and Wu, Shenping and Reedy, Mary C., Winkler, Hanspeter, Lucaveche, Carmen, Cheng, Yifan, Reedy, Michael K., Taylor, Kenneth A. Electron tomography of swollen rigor fibers of insect flight muscle reveals a short and variably angled S2 domain. J. Mol. Biol.. 362, 844-860. (2006)
  • Winkler, Hanspeter, Taylor, Kenneth A. Accurate marker-free alignment with simultaneous geometry determination and reconstruction of tilt series in electron tomography. Ultramicroscopy106, 240-254. (2006)
  • K. A. Taylor, J. Liu & H. Winkler. Localization and classification of repetitive structures in electron tomograms of paracrystalline assemblies. In: Electron Tomography: Methods for Three-dimensional Visualization of Structures in the Cell, 2nd edition, Joachim Frank, Ed. Springer-Verlag. pp 417-439 (2006).
  • Winkler, Hanspeter. 3D reconstruction and processing of volumetric data in cryo-electron tomography. J. Struct. Biol 157(1), 126-137. (2007)
  • Hanspeter Winkler, Jun Liu, Kenneth A. Taylor, Ping Zhu, Kenneth H. Roux. Electron tomography of macromolecular assemblies. In: Proceedings of the 4th IEEE International Symposium on Biomedical Imaging: From Nano to Macro, pg 240-243 (2007).
  • Feng Ye, Jun Liu, Hanspeter Winkler and Kenneth A. Taylor. Integrin αIIb β3 in a membrane environment remains the same height after Mn2+ activation when observed by cryo-electron tomography. J. Mol. Biol. 378(5), 976-986 (2008).
  • Ping Zhu, Hanspeter Winkler, Kenneth A. Taylor, and Kenneth H. Roux. Cryoelectron tomography of HIV-1 envelope spikes: further evidence for tripod-like legs. PLoS Pathogens 4(11) (2008).
  • Hanspeter Winkler, Ping Zhu, Jun Liu, Feng Ye, Kenneth H. Roux, and Kenneth A. Taylor. Tomographic subvolume alignment and subvolume classification applied to myosinV and SIV envelope spikes. J. Struct. Biol., 165(2), 64-77 (2009).
  • Shenping Wu, Jun Liu, Mary C. Reedy, Hanspeter Winkler, Michael K. Reedy & Kenneth A. Taylor. Methods for identifying and averaging variable molecular conformations in tomograms of actively contracting insect flight muscle. J. Struct. Biol. 168, 485-502 (2009). PMCID: PMC2805068.
  • Shenping Wu, Jun Liu, Mary C. Reedy, Richard T. Tregear, Hanspeter Winkler, Clara Franzini-Armstrong, Hiroyuki Sasaki, Carmen Lucaveche, Yale E. Goldman, Michael K. Reedy, and Kenneth A. Taylor. Electron tomography of cryofixed, isometrically contracting insect flight muscle reveals novel actin-myosin interactions. PLoS ONE 5(9): e12643 (2010).
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