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:
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.
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.
UTHealth Medical School
Department of Biochemistry and Molecular Biology
6431 Fannin Street, MSB 6. 220
Houston, Texas 77030
713-500-5416 Direct 713-500-0652 Fax
Ph.D. - Warsaw University
Postdoctoral Fellow - Wadsworth Center, Albany NY
Cryo-electron microscopy, Single particle reconstruction
Yang Z, Fang J, Chittuluru J, Asturias FJ, Penczek PA.
Structure. 2012 Feb 8;20(2):237-47. doi: 10.1016/j.str.2011.12.007.
Penczek PA, Fang J, Li X, Cheng Y, Loerke J, Spahn CM
Ultramicroscopy. 2014 May;140:9-19. doi: 10.1016/j.ultramic.2014.01.009. Epub 2014 Feb 7.
Wu B, Peisley A, Tetrault D, Li Z, Egelman EH, Magor KE, Walz T, Penczek PA, Hur S.
Mol Cell. 2014 Aug 21;55(4):511-23. doi: 10.1016/j.molcel.2014.06.010. Epub 2014 Jul 10.
Shukla AK, Westfield GH, Xiao K, Reis RI, Huang LY, Tripathi-Shukla P, Qian J, Li S, Blanc A, Oleskie AN, Dosey AM, Su M, Liang CR, Gu LL, Shan JM, Chen X, Hanna R, Choi M, Yao XJ, Klink BU, Kahsai AW, Sidhu SS, Koide S, Penczek PA, Kossiakoff AA, Woods VL Jr, Kobilka BK, Skiniotis G, Lefkowitz RJ.
Nature. 2014 Aug 14;512(7513):218-22