- Institute of Molecular Biophysics
- Research Associate
- phone: (850) 644-5606
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.
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
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
- H. Winkler & K. A. Taylor. 3D reconstruction by combining data
from sections cut oblique to different unit cell axes. Ultramicroscopy 55,
- 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,
- 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.
- 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).