Much of my lab’s activity focuses on the structure and function of tubulin, the main protein in microtubules. Microtubules play a vital role in the life of all eukaryotic cells, as they are involved in organelle movement, separation of chromosomes during cell division, maintenance of cell shape and other critical cellular activities. The assembly and disassembly of microtubules at particular times are essential steps in the cell cycle. These processes are closely regulated, and interference with the regulatory mechanisms can lead to cell death. These properties have made tubulin both a fascinating specimen for biophysical studies and a useful target for anti-cancer drugs. As a first step in understanding microtubule dynamics and their regulation, we have determined the structure of tubulin by electron crystallography. The crystals used in this work contained the anti-cancer drug Taxol, which binds to tubulin and stabilizes microtubules. To complement the crystal structure, we used cryo-EM to determine the structure of intact microtubules at a resolution that allows very precise docking of the tubulin molecule. In further work we are studying the interaction of tubulin with other drugs that stabilize microtubules and the interactions with some of the proteins that utilize and regulate the microtubule cytoskeleton. We have determined the structure of the tubulin/epothilone-A complex and have preliminary data on several other drugs. We have also obtained maps of kinesin-decorated microtubules at a resolution sufficient to see details of the conformational changes involved in kinesin’s binding onto microtubules and in its ATP hydrolysis cycle. Electron tomography has now been used to study the structure of microtubule doublets and to understand the nature of some of their non-tubulin components. This work is aimed at developing a rational understanding of the functional mechanisms of microtubule dynamics and protein interactions and may reveal the underlying mechanism of microtubule stabilization, eventually allowing development of new, more effective drugs targeted to tubulin.
On a larger scale, we are studying the molecular architecture of some simple but intact cells. Our goal is to apply the same frozen-hydrated specimen preparation methodology used for molecular studies to map out the locations of the major protein complexes in small bacteria. Recent advances suggest that whole microbial cells could be imaged by electron tomography to "molecular" resolution, sufficient to locate and identify large macromolecular complexes in the native state within their cellular contexts. We have worked with a number of different bacteria, and are presently focusing on Caulobacter crescentis and Desulfovibrio vulgaris in the context of two DOE-GTL programs. This work has provided new information on cell morphology and the effects of various mutations in genes that regulate aspects of morphology.
We are also developing instrumentation and technology to improve data collection in electron microscopy, especially applied to protein structure determination. One major project is the development of a new charge-coupled device (CCD) camera for intermediate voltage electron microscopes (IVEMs). CCDs have found wide application in certain aspects of electron microscopy. However, the CCD performance is seriously compromised when the microscope is operated much above 100 kV, due to the decrease in efficiency of the scintillator, which decreases the signal level, and increased lateral scattering of the electrons within the scintillator, which decreases the resolution. We overcome the poor performance of CCD cameras on IVEMs by decelerating the electrons before they reach the camera. In our design the CCD is mounted so that it can be floated to a potential of around 250 kV. When the microscope is operated at 300 kV we then obtain the advantages of the IVEM but the camera will perform even better than on a 100 kV microscope. These adaptations will produce substantial benefits in work ranging from high-resolution structural studies of proteins and viruses to the three dimensional study of cell ultrastructure. Other efforts include development of a Zernike-type phase contrast device for electron microscopy and improvement of image quality by mitigation of charging and other beam-induced effects.
Instrumentation for this work includes two JEOL-4000 electron microscopes (one fully equipped for cryo) and a JEOL-3100 with FEG illumination, in-column energy filter, and +/- 70 degree goniometer with liquid nitrogen or helium cooling.
3-D view of a microtubule doublet from sea urchin sperm. The gold surface represents the experimental density derived by electron tomography, which had resolution sufficient to build a pseudo-atomic model of the tubulin component of the doublet, shown in green. The dark blue component is the density computed from this tubulin component; comparison of the model and experimental densities indicates non-tubulin proteins in the doublet. The grey-scale images are sections through the tomographic reconstruction of one specimen that contained a full set of doublets and singlet microtubules from a single axoneme.
P. Ounjai, K. D. Kim, P. V. Lishko and K. H. Downing. Three-dimensional structure of the bovine sperm connecting piece revealed by electron cryotomography. Biology of Reproduction (in press 2012).
H. Dong, J. Y. Shu, Y. F. Ma, S. Liu, O. Gang, M. V. Tirrell, K. H. Downing, T. Xu. Stabilizing micelles by positioning repulsive forces. J. Am. Chem. Soc. (in press 2012).
D. Murat, V. Falahati, L. Bertinetti, R. Csencsits, A. Körnig, K. H. Downing, D. Faivre and A. Komeili. The magnetosome membrane protein, MmsF, is a major regulator of magnetite biomineralization in Magnetospirillum magneticum AMB-1. Molecular Microbiology (in press 2012).
S. Yakovlev, X. Wang, P. Ercius, N. P. Balsara and K. H. Downing. Observation of ionic clusters in a polyelectrolyte membrane by electron tomography. J Am Chem Soc., 133, 20700-3 (2011).
C. V. Sindelar and K. H. Downing. An atomic-level engine that accounts for kinesin motility and catalysis. Proc. Natl. Acad. Sci. USA, 107, 4111-6 (2010).
Y.-C. Yeh, L. R. Comolli, K. H. Downing, L. Shapiro and H. H. McAdams. The Caulobacter Tol-Pal complex is essential for outer membrane integrity and the positioning of a polar localization factor. J. Bacteriol., 192, 4847–4858 (2010).
K. H. Downing and P. E. Mooney. A charge-coupled device camera with electron decelerator for intermediate voltage electron microscopy. Rev. Sci. Instrum. (online Apr. 2008).
H. Sui and K. H. Downing. Molecular architecture of axonemal microtubule doublets revealed by cryoelectron tomography. Nature, 442 475-478 (2006).
E. M. Judd, L. R. Comolli, J. C. Chen, K. H. Downing, W. E. Moerner, H. H. McAdams. Distinct constrictive processes, separated in time and space, divide Caulobacter inner and outer membranes. J. Bacteriology, 187 6874-82 (2005).
K. H. Downing, M. R. McCartney and R. M. Glaeser. Experimental characterization and mitigation of specimen charging on thin films with one conducting layer. Microsc. Microanalysis, 10, 790-796 (2004).
J. H. Nettles, H. Li, B. Cornett, J. Krahn, J. P. Snyder and K. H. Downing. The electron crystallographic conformation and binding mode of epothilone A on ab-tubulin: A distributed pharmacophore. Science, 305, 866-869 (2004).
H. Li, D. J. DeRosier, W. V. Nicholson, E. Nogales and K. H. Downing. Microtubule structure at 8 Å resolution. Structure, 10, 1317–1328 (2002).
S. Zhong, V. M. Dadarlat, T. Head-Gordon, R. M. Glaeser and K. H. Downing. Modeling chemical bonding effects for protein electron crystallography: The transferable fragmental electrostatic potential (TFESP) method. Acta Cryst. A, 58, 162-170 (2002).
J. Löwe, H. Li, K. H. Downing and E. Nogales. Refined structure of ab-tubulin at 3.5 Å. J. Mol. Biol., 313, 1045-1057 (2001).
N. V. Hud and K. H. Downing. Cryoelectron microscopy of l phage DNA condensates in vitreous ice: The fine Structure of DNA toroids. Proc. Natl. Acad. Sci. USA, 98, 14925-14930 (2001).
E. Nogales, K. H. Downing, L. A. Amos and J. Lowe. Tubulin and FtsZ form a distinct family of GTPases. Nature Struct. Biol., 5, 451-458 (1998).
E. Nogales, S. G. Wolf, and K. H. Downing. Structure of the tubulin dimer by electron crystallography. Nature, 391, 199-203 (1998).