[ccp4bb] Post-doctoral position in Paris
Postdoctoral Research Position Structural studies and inhibitors design of NHEJ pathway A 24 month postdoctoral position is available in the Laboratory of Structural Biology and Radiobiology. The group, led by Jean-Baptiste Charbonnier, is located at the CEA Saclay (South Paris) and is part of the I2BC (Institute of Integrated Biology of the Cell) (http://www.i2bc.paris-saclay.fr/). Our team investigates the structural and functional studies of DNA repair machineries. We produce, crystallize and determine the 3D structure of complexes involved in DNA repair. We reported the crystal structure of the XRCC4-Cernunnos/XLF complex and identified a filament organization of this complex (Ropars, 2011, PNAS; Malivert, 2010, J Biol Chem). We combine in our studies, X-ray crystallography, molecular modeling, protein-protein interaction measurements and SAXS. We recently implemented robust protocols for the production in insect cells, with the MultiBac approach, of 6 core factors of the human NHEJ pathway: Ku70/Ku80, Artemis, Ligase4-XRCC4 and Cernunnos. Our first aim is to crystallize multi-protein complexes involving combination of these NHEJ factors and DNA substrates. To guide our crystallization, we characterize the stability of our multi-protein complexes with a large set-up of biochemical and biophysical methods. Our second major aim is to identify and characterize potent inhibitors of the NHEJ pathway for new anti-cancer treatments. For that, we combine structure-based drug design, small molecules screenings, X-ray structure of protein-inhibitor complexes and tight collaboration with DNA repair biologists. Our group is part of the Institute of Integrative Biology of the Cell (I2BC). The I2BC is a mixed research unit supported by the Paris-Sud University, the CNRS and the CEA. The Institute regroups 80 teams of scientists and 15 technological facilities including state-of-the-art equipment in high-throughput crystallization, NMR, EM, super-resolution microscopy, protein-protein interactions, insect cells expression and mass spectrometry. The Institute is in 3 research campus (Orsay Campus of the Univ Paris Sud University, Gif Sur Yvette Campus of CNRS, and Saclay Campus of CEA) with 14 buildings. All the I2BC activities will be joined on the Gif Sur Yvette Campus in 2018. The I2BC generates an excellent scientific environment for high quality and intensive scientific life. Group meetings, external and internal seminars in the Institute are in English. Saclay is 30min south Paris by public transports. We are seeking highly motivated and skilled researchers with broad and extensive experience within one or more of the following areas: DNA Repair, Crystallization, Crystallography, Protein Expression of proteins in insect cells (or E. Coli), Cellular biology, Protein interaction measurements, SAXS, EM. The position is available immediately. Applications should be sent no later than end of June, including motivation letter, CV, summary of research experience and contacts for references to: jb.charbonn...@cea.frmailto:jb.charbonn...@cea.fr (http://www.i2bc.paris-saclay.fr/spip.php?article168) Ropars V (2011) Structural characterization of filaments formed by human (2011) Xrcc4-Cernunnos/XLF complex involved in nonhomologous DNA end-joining. Proc Natl Acad Sci U S A. 108(31):12663-8 Malivert L (2010) Delineation of the Xrcc4-interacting region in the globular head domain of cernunnos/XLF. (2010) J Biol Chem. 2010 285(34):26475-83 de Villartay JP (2009) A histidine in the beta-CASP domain of Artemis is critical for its full in vitro and in vivo functions. DNA Repair (Amst). 8(2):202-8 Guarné (2015) A Insights from a decade of biophysical studies on MutL: Roles in strand discrimination and mismatch removal (2015) Prog Biophys Mol Biol. 117(2-3):149-156. Meurisse J (2014) Hug1 is an intrinsically disordered protein that inhibits ribonucleotide reductase activity by directly binding Rnr2 subunit. (2014) Nucleic Acids Res. 42(21):13174-85 Bacquin A, (2013) The helicase FBH1 is tightly regulated by PCNA via CRL4(Cdt2)-mediated proteolysis in human cells. Nucleic Acids Res. 2013 41(13):6501-13 Gueneau E, (2013) Structure of the MutLα C-terminal domain reveals how Mlh1 contributes to Pms1 endonuclease site. Nat Struct Mol Biol. 2013 20(4):461-8.
Re: [ccp4bb] offtopic: related to protein purification
Dear Sonia Your value of 0,1ucal/sec is weak but if you can determine the intial and final plateau and the slope at the transition, it may be ok. Triplicate experiments will give you an idea of the quality of your measurements. Similarly, the control experiments with only the ligand injected in the dialysis buffer and/or with a mutant of your GTPase will help you to evaluate the quality of your preliminary data Otherwise, you can vary the following parameters 1- Protein concentration in cell: Increasing the protein concentration should increase the signal and improve the determination of the slope (classical parameter changed in ITC and called the c factor, see Ropars, V et al 2011 PNAS for an example) 2- Temperature: you can have higher heat exchange at a lower temperature (see Czarny, B 2013 J Med Chem) 3- pH and buffer: Similarly,measurements varying these parameters may change the thermograms and will give energetics information (see Czarny, B 2013 J Med Chem) For the n value of 0.33 Is your enzyme a monomer? Such n value may be related to stoichiometries of 1 ligand to a trimer of protein (or a dimer if n is not well defined) It can also be due to a weak fraction of protein active due to the purification protocol. It is interesting to follow the n value with different preparation of proteins. N can vary with proteins just issued from last purification step or with older proteins Hope it can help Regards JB De : CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] De la part de Sonia Majumdar Envoyé : lundi 4 août 2014 08:45 À : CCP4BB@JISCMAIL.AC.UK Objet : [ccp4bb] offtopic: related to protein purification Dear all, I am working on a GTPase with two tandem G-domains. While doing ITC experiments the heat change obtained is very poor 0.1ucal/sec, however the ITC profile is showing gradual saturation. The n value is very poor 0.33. I presumed the protein has bound GDP and tried to separate the nucleotide bound and unbound forms using Mono-Q. 3 peaks were obtained containing the same protein. However, ITC done with the proteins corresponding to the 3 peaks gave the same results. Please suggest what could the three peaks possibly mean and how to modify the ITC experiments. Thanks in advance. Regards.
Re: [ccp4bb] ITC with heterogeneous protein
Dear Sajid If the binding site of your ligand is remote from the dimerization interface, it should normally not be a problem. You will bind two ligands for a dimer and one ligand for a monomer and you should be able to fit the isotherm with one unique site even in presence of a mix of monomers and dimers. If the binding site is close to the dimerization interface or partially dissociate the dimer, this may give more complicate signals difficult to analyzed You can also try to evaluate with ITC or other biophysical technic, the Kd of your dimer. With ITC, the Kd of your dimer may be evaluated by injecting a concentrated solution of your protein via the serynge against a cell containing your dialysis buffer. You may observe an heat exchange due to dimer dissociation (see for example Li, J, Weis RM, 1996, with CheA) Knowing the Kd may help to design the ITC experiment. For example, you can use a concentration of protein 10 fold higher (if possible) than the Kd to have mainly dimers Cheers JB JB Charbonnier Laboratory of Structural Biology and Radiobiology iBiTec-S (Institute of Biology and Technologies of Saclay), CEA , FRANCE jb.charbonn...@cea.fr -Message d'origine- De : CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] De la part de sajid akthar Envoyé : vendredi 18 juillet 2014 11:24 À : CCP4BB@JISCMAIL.AC.UK Objet : [ccp4bb] ITC with heterogeneous protein Dear All, This is an off-topic question. I have protein solution of heterogeneous (contains both monomer and dimer). I want to perform ITC with this protein. I doubt whether this heterogeneity will interfere the binding study. Any advice please. Thank you Sajid
[ccp4bb] Postdoctoral position – DNA repa ir - Paris
Postdoctoral position – Structural Biology of DNA repair A postdoctoral position is available to study DNA repair proteins involved in double-strand break repair. This post-doc is founded by Institut National du Cancer (INCa). All candidates must have a broad experience ranging from biochemistry (protein expression and purification) to structural studies (NMR or crytallography). Our laboratory is well equipped for all steps from molecular biology to NMR or crystallography. We have regular access to ESRF synchrotron in Grenoble and to SOLEIL synchrotron that is very close to the laboratory. We are equipped with three NMR spectrometers (700MHz Bruker, 600MHz and 500MHz). The laboratory is 40 minutes drive from Paris near Orsay University and CNRS center of Gif-s-Yvette. The salary ranges from 25 000€/year to 29 000€/year according to experience. Please send your CV and a covering letter with the names of three referees to Dr JB Charbonnier ([EMAIL PROTECTED]). Further information can be obtain about the laboratory and the institute on our website at: http://www-dsv.cea.fr/ibitecs/5
