Dr. Pawel Penczek, Professor and Director,
Structural Biology Imaging Center

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.

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. We develop, in collaboration with other groups, a single particle software package SPARX (Hohn, et al). SPARX is provided free of charge as a service to the scientific community and its main features include:

1. New generation of 2D and 3D alignment protocols.
2. Ab initio structure determination programs.
3. Resampling methods for investigation of structural heterogeneity (3D variance).
4. Advanced code for multivariate statistical analysis (PCA, Varimax),
5. New generation of interpolation methods relaxing the need for oversampling the data.
6. Wiki-based interactive documentation
7. Extensive C++ library of general and EM-specific image operations with Python bindings, thus accessible to Python programmer.
8. All structure determination applications written as user transparent Python scripts
9. All major scripts parallelized for clusters of workstations using MPI.

Sparx is available for download at http://sparx-em.org

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.

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, 2006). 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.

Penczek, P.A. Fundamentals of three-dimensional reconstruction from projections. Methods Enzymol 482, 1-33, 2010.

Ratje AH, Loerke J, Mikolajka A, Brünner M, Hildebrand PW, Starosta AL, Dönhöfer A, Connell SR, Fucini P, Mielke T, Whitford PC, Onuchic JN, Yu Y, Sanbonmatsu KY, Hartmann RK, Penczek PA, Wilson DN, Spahn CM. Head swivel on the ribosome facilitates translocation by means of intra-subunit tRNA hybrid sites. Nature 468, 713-716, 2010.

Spahn, Ch.M.T., Penczek, P.A. Exploring conformational modes of macromolecular assemblies by multiparticle cryo-EM. Curr Opin Struct Bio 9, 623-631, 2009.

Schuette JC, Murphy FV 4th, Kelley AC, Weir JR, Giesebrecht J, Connell SR, Loerke J, Mielke T, Zhang W, Penczek PA, Ramakrishnan V, Spahn CM. GTPase activation of elongation factor EF-Tu by the ribosome during decoding. EMBO J. 28:755-765, 2009.

Raunser S, Magnani R, Huang Z, Houtz RL, Trievel RC, Penczek PA, Walz T. Rubisco in complex with Rubisco large subunit methyltransferase. Proc Natl Acad Sci U S A. 106:3160-3165, 2009.

Zhang W, Kimmel M, Spahn CM, Penczek PA. Heterogeneity of large macromolecular complexes revealed by 3D cryo-EM variance analysis. Structure. 2008;16:1770-1776.

Yang Z, Penczek PA. Cryo-EM image alignment based on nonuniform fast Fourier transform. Ultramicroscopy. 108:959-969, 2008.

Ohi MD, Feoktistova A, Ren L, Yip C, Cheng Y, Chen JS, Yoon HJ, Wall JS, Huang Z, Penczek PA, Gould KL, Walz T. Structural organization of the anaphase-promoting complex bound to the mitotic activator Slp1. Mol Cell. 28:871-885, 2007.

Hohn, M., Tang, G., Goodyear, G., Baldwin, P.R., Huang, Z., Penczek, P.A., Yang, Ch., Glaeser, R.M., Adams, P., Ludtke, S.J.: SPARX, a new environment for cryo-EM image processing. J. Struct. Biol., 157:47-55, 2007.

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, 14:661-671, 2006.

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., 154:168-183, 2006.

Schüler M, Connell SR, Lescoute A, Giesebrecht J, Dabrowski M, Schroeer B, Mielke T, Penczek PA, Westhof E, Spahn CM. Structure of the ribosome-bound cricket paralysis virus IRES RNA. Nat Struct Mol Biol. 13:1092-1096, 2006.

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.

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.

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

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.

Search PubMed for a complete list of Dr. Penczek's publications.