Abstract

The long-term objective of this program of research is to develop methods of structural and functional studies for macromolecular assemblies in single-particle (noncrystalline) form, using low-dose electron microscopy of frozen hydrated samples. The recent publication of a 25 angstrom 3D cryo map of the ribosome has been one of the most successful application of these methods to date. Subsequently we were able to use 3D cryo EM difference mapping to locate tRNA in a translating ribosome. The proposed program of research seeks to seize the new opportunities of 3D difference mapping. Specifically, this proposal focuses on the study of the elongation cycle, subunit-subunit association and the path of the nascent polypeptide in the prokaryotic ribosome. We will also begin to use structural and functional probes along with 3D cryo-EM difference mapping in the study of the yeast ribosome. Hand in hand with these investigations, we will continue to make efforts to improve the resolution. To this end we will develop a spot scan data collection procedure, streamline particle selection and processing to enable collection of increased numbers of particles, and work towards an improved understanding of image formation for biological particles in ice. We will also implement time-resolved techniques to study transient states of tRNA, factor binding, and possible conformational changes of the ribosome during the elongation cycle.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
5R37GM029169-18
Application #
2900544
Study Section
Biophysical Chemistry Study Section (BBCB)
Project Start
1982-04-01
Project End
2002-03-31
Budget Start
1999-04-01
Budget End
2000-03-31
Support Year
18
Fiscal Year
1999
Total Cost
Indirect Cost

  Institution

Name
Wadsworth Center
Department
Type
DUNS #
110521739
City
Menands
State
NY
Country
United States
Zip Code
12204

  Publications

Malyutin, Andrey G; Musalgaonkar, Sharmishtha; Patchett, Stephanie et al. (2017) Nmd3 is a structural mimic of eIF5A, and activates the cpGTPase Lsg1 during 60S ribosome biogenesis. EMBO J 36:854-868
Chen, Bo; Kaledhonkar, Sandip; Sun, Ming et al. (2015) Structural dynamics of ribosome subunit association studied by mixing-spraying time-resolved cryogenic electron microscopy. Structure 23:1097-105
Langlois, Robert; Pallesen, Jesper; Ash, Jordan T et al. (2014) Automated particle picking for low-contrast macromolecules in cryo-electron microscopy. J Struct Biol 186:1-7
Boël, Grégory; Smith, Paul C; Ning, Wei et al. (2014) The ABC-F protein EttA gates ribosome entry into the translation elongation cycle. Nat Struct Mol Biol 21:143-51
Agirrezabala, Xabier; Liao, Hstau Y; Schreiner, Eduard et al. (2012) Structural characterization of mRNA-tRNA translocation intermediates. Proc Natl Acad Sci U S A 109:6094-9
Langlois, Robert; Frank, Joachim (2011) A clarification of the terms used in comparing semi-automated particle selection algorithms in cryo-EM. J Struct Biol 175:348-52
Langlois, Robert; Pallesen, Jesper; Frank, Joachim (2011) Reference-free particle selection enhanced with semi-supervised machine learning for cryo-electron microscopy. J Struct Biol 175:353-61
Agirrezabala, Xabier; Schreiner, Eduard; Trabuco, Leonardo G et al. (2011) Structural insights into cognate versus near-cognate discrimination during decoding. EMBO J 30:1497-507
Li, Wen; Trabuco, Leonardo G; Schulten, Klaus et al. (2011) Molecular dynamics of EF-G during translocation. Proteins 79:1478-86
Sengupta, Jayati; Bussiere, Cyril; Pallesen, Jesper et al. (2010) Characterization of the nuclear export adaptor protein Nmd3 in association with the 60S ribosomal subunit. J Cell Biol 189:1079-86

Showing the most recent 10 out of 117 publications