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Application of novel approaches of crystallization and X-FEL serial crystallography to the structural characterization of enzyme: tRNA complexes

Research unit

UPR 9002 - Architecture et réactivité de l'ARN (IBMC)
15, rue Rene Descartes 67084 - Strasbourg Cedex

Group

Name: Traduction mitochondriale et pathologies

Group leader: CATHERINE FLORENTZ ET MARIE SISSLER - c.florentz@ibmc-cnrs.unistra.fr, m.sissler@ibmc-cnrs.unistra.fr

Group leader's phone:

Website: Visit website

Group organization:
- Chercheurs: 5
- ITA: 1
- Doctorants: 2
- Post-Docs: 2
- Autres: 0

Publications of the team linked to the topic (3 last years):
1) Thermodynamic properties distinguish human mitochondrial aspartyl-tRNA synthetase from bacterial homolog with same 3D architecture. A. Neuenfeldt, B. Lorber, E. Ennifar, A. Gaudry, C. Sauter, C. Florentz & M. Sissler. Nucleic Acids Res. (2013), 41, 2698-2708.
2) Structural insights into protein-only RNase P complexed with tRNA. A. Gobert, P. Pinker, O. Fuchsbauer, B. Gutmann, R. Boutin, P. Roblin, C. Sauter & P. Giegé. Nature communications (2013), 4, 1353.
3)

About PhD

PhD Director: SAUTER Claude - c.sauter@unistra.fr

Phone: 03 88 41 71 02

Junior advisor: non

Co-tutely: non

Co-Director: non

About PhD topic :

Title: Application of novel approaches of crystallization and X-FEL serial crystallography to the structural characterization of enzyme: tRNA complexes

Project: This doctoral project is part of functional and structural studies of complexes involving enzyme / mitochondrial nucleic acids conducted by the team "Translation and mitochondrial pathologies" (UPR 9002, CNRS, IBMC). Among these tRNA binding enzymes, the aspartyl-tRNA synthetase (AspRS) found in human mitochondria and whose point mutants are associated with a leukoencephalopathy. Their characterization by X-ray crystallography would enable the comparison of their 3D structures with that of the wild-type enzyme to better understand the origin of related disorders [1]. There is also a new family of tRNA processing enzymes discovered in 2008, RNase P protein of PRORP family. [2] In both cases, the first crystals have been obtained but, as very often in this type of study, they require optimization to be usable in X-ray diffraction.

The optimization of the quality of biological crystals and their packaging for analysis using a synchrotron X-ray source can be long and tedious. Thanks to its experience in biological crystal growth, the host team recently turned to innovative technology and a more rational approach for the preparation of crystals. With the support of LabEx MitoCross, it has acquired a new instrument, the Xtal Controller 900 (Xtal-Concepts GmbH, Hamburg); it can handle a crystallization drop of a few microliters playing on concentrations of biomolecule and crystallizing agent (through microinjectors) while following the evolution of the system live by light scattering (allowing the detection of aggregation and nucleation events, of the formation of nanocrystals) and video microscopy (to follow crystal growth), all in a temperature and humidity controlled chamber. For the first time, the search and optimization of crystallization conditions are feasible in a rational and controlled manner, with a real-time monitoring of the experiments. This PhD project will contribute to the development of this technology and explore the possibilities it offers in terms of crystal growth and structural biology studies, in collaboration with the laboratory of Professor Christian Betzel (University of Hamburg) who designed this instrument and the Xtal-Concepts society. Direct application is the production of nanocrystals required for analyses by ultra rapid and intense pulses of X-rays provided by the free electron lasers (XFEL). The European XFEL source (http://www.xfel.eu/en/) is under construction in Hamburg near the laboratory German partner. Since the demonstration of its feasibility in 2011, serial femtosecond crystallography (SFC) based on sequential analysis of nanocrystals by XFEL revealed its potential for samples difficult to obtain in the form of micrometric crystals. The perspective of the opening of the European source in 2017 is a very important driver of this project can be divided into three main objectives:

- Exploiting and developing the Controller Xtal Technology along with ongoing work in the team on the crystallisation of eukaryotic mitochondrial enzymes described above, alone and in complex with their RNA substrates.

- Developing production protocols of nanocrystals required for the analysis by ultra rapid and intense pulses of X-rays provided by the free electron lasers (XFEL).

- Using these breakthrough technologies to enable a detailed description of the structure and the dynamics of the systems studied, their dysfunction and related events of molecular recognition or catalysis.

Wished skills: The candidate will at least have some experience in biomolecule purification, crystallization or structural biology (preferably in biocrystallography). Complementary skills will be acquired in the host team during the PhD training.

Expertises which will be acquired during the training: This PhD project is intended for a candidate who is strongly motivated by interdisciplinary research and will enable her/him to gain a solid experience at the interface of biology, chemistry and physics, with skills ranging from preparative biochemistry (purification and crystallisation of biomolecules) to structural biology (biophysical characterization of biomolecules in solution, biocomputing analysis and crystal structure determination).