Dr. Pawel Penczek

Department of Biochemistry and Molecular Biology
Director Structural Biology Research Center
Program in Biochemistry and Molecular Biology

University of Texas-Houston Medical School
P.O. Box 20708 - Houston, Texas 77225
(713) 500-5416: fax (713) 500-0652
email: Pawel.A.Penczek@uth.tmc.edu

Ph.D., Warsaw University
Postdoctoral Fellow, Wadsworth Center, Albany NY

Structural Determination of Proteins and Molecular Assemblies

We are interested in the determination of three-dimensional structures of large macromolecular complexes with low or non-existing symmetry in single-particle form using stain and cryo-electron microscopy (cryo-EM), and computer image processing techniques.

One area of focus is determination of the structure of a newly identified protein called HRS (hepatocyte growth factor regulated tyrosine kinase substrate) (Pullan et al., Structure, in press). HRS is phosphorylated by various kinases in response to a variety of growth factors and is expressed in the cytoplasm of all cells. In addition, HRS was also implicated in exocytosis of synaptic vesicles. The main interest is in its regulatory functions. The determination by cryo-EM of the tertiary structure of HRS to 16Å together with knowledge of its amino acid sequence has enabled us to propose a model of hexameric HRS insertion into the endosomal membrane. With the knowledge of the native structure, we will continue the structural determination of HRS complexes, including HRS bound to ubiquitinated cargo and other known binding proteins involved in the process of cargo sorting and vesicular trafficking.

Our main area of interest is high-resolution cryo-EM and the development of efficient and largely automatic tools for single particle analysis and three-dimensional reconstruction. Due to a very low Signal-To-Noise Ratio of EM data, there is a necessity to collect and merge large numbers (in excess of 100,000) of individual particle images. Thus, in our approach we focus on automation of the main steps of EM structure determination: particle picking, alignment of images, and structure refinement. We design utilities that should be able to cope with a large number of particle images under various noise conditions.

Unconstrained by crystal packing, the molecule in a single particle specimen can be thought to exhibit the entire range of native conformations. The ability to find the structure of each conformer is one of the most important potential assets of 3D cryo-EM of single particles. On the other hand, the realization of high resolution for each of these conformers poses daunting problems of data collection and processing, considering the statistical requirements stated before. Therefore, we are also interested in the development of efficient and robust computational methods of structure refinement and structure validation in the presence of multiple conformational or binding states.

Three-dimensional structure of HRS protein determined by cryo-electron microscopy and computer image-processing techniques.

The structure is hexameric with six subunits arranged in an anti-parallel fashion. The background shows a cryo micrograph with side and end views of single particles of HRS from which the structure was computed.

Selected References

Pullan, L., Mullapudi, S., Huang, Z., Baldwin, P.R., Chin, Ch., Sun, W., Tsujimoto, S., Kolodziej, S., Stoops, J.K., Lee, J.C., Waxham, M.N., Bean, A.J., Penczek, P.A.: The endosome-associated protein Hrs is hexameric and controls cargo sorting as a “master molecule”. Structure, in press.

Penczek, P.A., Frank, J., Spahn, Ch.M.T.: A method of focused classification, based on the bootstrap 3-D variance analysis, and its application to EF-G-dependent translocation. J. Struct. Biol., in press.

Penczek, P.A., Chao, Y., Frank, J., Spahn, Ch.M.T.: Estimation of variance in single particle reconstruction using the bootstrap technique. J. Struct. Biol., in press.

Blau, M., Mullapudi, S., Becker, T., Dudek, J., Zimmermann, R., Penczek, P.A. and Beckmann, R.: ERj1p uses a universal ribosomal adaptor site to coordinate the 80S ribosome at the membrane. Nature Structural and Molecular Biology, 12:1015-1016, 2005.

Baldwin, P.R. and Penczek P.A.: Estimating alignment errors in sets of 2-D images. J. Struct. Biol., 150:211-225, 2005.

Mullapudi, S., Pullan, L., Bishop, O. T., Khalil, H., Stoops, J. K., Beckmann, R., Kloetzel, P. M., Kruger, E. and Penczek, P. A.: Rearrangement of the 16S precursor subunits is essential for the formation of the active 20S proteasome. Biophysical J., 87:4098-4105, 2004.

Penczek, P.A., Renka, R. and Schomberg, H.: Gridding-based direct Fourier inversion of the three-dimensional ray transform. J. Opt. Soc. Am. A. 21:499-509, 2004.

Huang, Z. and Penczek, P.A.: Application of template matching technique to particle detection in electron micrographs. J. Struct. Biol. 145:29-40, 2004.

Joyeux, L. and Penczek, P.A.: Efficiency of 2D alignment methods. Ultramicroscopy 92:33-46, 2002.

Huang, Z., Baldwin, P.R., Mullapudi, S.R. and Penczek, P.A.: Automated determination of parameters describing power spectra of micrograph images in electron microscopy. J. Struct. Biol. 144:79-94, 2003.

Penczek, P.A.: Three-dimensional Spectral Signal-to-Noise Ratio for a class of reconstruction algorithms. J. Struct. Biol. 138:34-46, 2002.

Beckmann, R., Spahn, C. M. T., Eswar, N., Helmers, J., Penczek, P.A., Sali, A., Frank, J. and Blobel, G.: Architecture of the protein-conducting channel associated with the translating 80S ribosome. Cell 107:361-372, 2001.

Mouche, F., Boisset, N. and Penczek, P.A.: Lumbricus terrestris hemoglobin - the architecture of linker chains and structural variation of the central toroid. J. Struct. Biol. 133:176-192, 2001.

Spahn, Ch.M.T., Kieft, J.S., Grassucci, R.A., Penczek, P.A., Zhou, K., Doudna, J.A. and Frank, J.: Hepatitis C virus IRES RNA induced changes in the conformation of the 40S subunit. Science 291:1959-1962, 2001.

Yang, C., Ng, E.G. and Penczek P.A.: Unified 3-D structure and projection orientation refinement using quasi-Newton algorithm. J. Struct. Biol., 149:53-64, 2005.