Ruprecht-Karls-Universität Heidelberg
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Pfeffer0218 - Scientist (f/m) / PhD position
Project no:
Pfeffer0218

Project leader:

Project supervisor:
Pfeffer, Stefan
Application deadline:
31. May 2018
Start of PhD project:
1. Jul 2018

Project description:

Title:
Cryo-EM analysis of the co-translational machinery for protein folding and maturation in eukaryotes
Summary:
Co-translational folding and maturation of proteins require an intricate network of folding chaperones and processing enzymes that act on the growing nascent protein in a co-translational manner. Structural information on ribosome-nascent chain-chaperone complexes is sparse, because the involved interactions are mostly transient, labile and possibly highly flexible. This renders the involved assemblies inaccessible to classical reductionist structural biology approaches that rely on extensive biochemical purification and require conformationally homogenous particle populations for averaging. We consequently pursue a different approach and image these processes using cryo electron tomography (cryo-ET)-based strategies, which can reveal the three-dimensional arrangement of individual macromolecules even in crowded native microenvironments at molecular resolution and therefore render extensive biochemical purification unnecessary. This approach allows us to analyze the three-dimensional spatial distribution of ribosomes, chaperones and processing enzymes for individual native polysomal assemblies under conditions that preserve the labile and transient interactions governing co-translational protein folding and maturation.

The central objective of this project will be to study defined polysomal assemblies engaged in the synthesis of model substrates in eukaryotic organisms. Cryo-ET approaches will be used to visualize these assemblies in both a non-cellular context and in sections of vitrified unperturbed cells obtained using focused ion beam (FIB) milling.
References:
1) Pfeffer, S., Burbaum, L., Unverdorben, P., Pech, M., Chen, Y., Zimmermann, R., Beckmann, R., Förster, F. Structure of the native Sec61 protein-conducting channel. Nat Commun. 2015

2) Mahamid, J., Pfeffer, S., Schaffer, M., Villa, E., Danev, R., Cuellar, L.K., Förster, F., Hyman, A.A., Plitzko, J.M., Baumeister, W. Visualizing the molecular sociology at the HeLa cell nuclear periphery. Science. 2016.

3) Hrabe, T., Chen, Y., Pfeffer, S., Cuellar, L.K., Mangold, A.V., Förster, F. PyTom: a python-based toolbox for localization of macromolecules in cryo-electron tomograms and subtomogram analysis. J. Struct. Biol. 2012.

4) Brandt, F., Etchells, S.A., Ortiz, J.O., Elcock, A.H., Hartl, F.U., and Baumeister, W. The native 3D organization of bacterial polysomes. Cell. 2009.
Methods that will be used:
High-resolution cryo electron microscopy and tomography approaches; advanced image processing; standard biochemical, molecular and cell biology methods
Cooperation partners:
Various groups at the ZMBH.
Personal qualifications:
We are looking for a highly motivated PhD student holding a Master’s degree in life sciences with a strong background in computational biology. Experience in electron microscopy is of advantage, but not required.
Keywords:
Structural biology, cryo-EM, cryo-ET, cryo electron microscopy, ribosome, co-translational protein folding, protein maturation, chaperones