Re: [ccp4bb] post-doc and technician position at KULeuven, Belgium - structural biology of ion channel homologs
A position is available for a post-doctoral researcher and technician at the Laboratory for Structural Neurobiology at the KULeuven, Belgium. http://www.xtal.be Our research focus is on ion channel homologs from different classes. Our strategy to identify targets for structural studies relies on a GFP-based pipeline for pre-crystallization screening and nanobody technology for purification of GFP fusion proteins. This pipeline has been established for E. coli and insect cells and permit scale-up to 50 liter cultures in Wave bioreactors. Our laboratory hosts an integrated macromolecular crystallization facility with a Mosquito crystallization robot, 2 RockImagers and an in-house rotating anode X- ray source. In combination with electrophysiological recordings of recombinantly expressed ion channels in Xenopus oocytes we aim to understand the relationship between structure, function and pharmacology of ion channels. We have ongoing collaborations with Pharma Inc. to aid the rational design of new drugs and to facilitate treatment of disorders related to dysfunctional ion channels. Our laboratory is also a participant in the FP7 consortium Neurocypres devoted to structure, function and disease of cys-loop receptors. http://www.neurocypres.eu. We have lively interactions with outstanding groups on campus, including the ion channel group of Thomas Voets (TRP channels) and the structural biology group of Sergei Strelkov. Prior experience in cloning, cell culture and protein purification techniques is required. Post-docs have the opportunity to combine research with limited teaching duties and grow into independent research careers if desired. This call is open to citizens of EMBL member states only. Belgian citizens or researchers previously employed at KULeuven are excluded. To apply for this position please send curriculum vitae, a motivation letter and the names of at least two references to: chris.ulens at med.kuleuven.be The position is open immediately and applications will be accepted until the position is filled. Selected publications: Brams M et al. PLoS Biol (2011) in press Brams M et al. J Biol Chem (2011) http://www.ncbi.nlm.nih.gov/pubmed/21115477 Ulens C et al. J Med Chem (2009) http://www.ncbi.nlm.nih.gov/pubmed/19331415 Dutertre S, Ulens C et al. EMBOJ (2007) http://www.ncbi.nlm.nih.gov/pubmed/17660751 Ulens C et al. Proc Natl Acad Sci U S A. (2006). http://www.ncbi.nlm.nih.gov/pubmed/16505382 Ulens C and Siegelbaum SA. Neuron (2003) http://www.ncbi.nlm.nih.gov/pubmed/14659094
[ccp4bb] off-topic: tag removal
Dear all, I have a question concerning removal of a his-tag sequence. We have crystallized a protein with an important feature at the C-terminal part of the protein. Unfortunately, we cannot express it with a N-terminal his-tag, only with a C-terminal his-tag. Therefore we are looking for a protease which cleaves off the sequence without leaving any extra amino acid on the C-terminus of our protein. Meaning we obtain really the wild type protein. Does anyone know about a protease or cleavage site which is completely removed? Many thanks in advance Phil
Re: [ccp4bb] off-topic: tag removal
See the following reference- Shen A, Lupardus PJ, Morell M, Ponder EL, Sadaghiani AM, et al. 2009 Simplified, Enhanced Protein Purification Using an Inducible, Autoprocessing Enzyme Tag. PLoS ONE 4(12): e8119. doi:10.1371/journal.pone.0008119 From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Philipp Ellinger [filu...@gmx.de] Sent: Wednesday, February 23, 2011 3:17 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] off-topic: tag removal Dear all, I have a question concerning removal of a his-tag sequence. We have crystallized a protein with an important feature at the C-terminal part of the protein. Unfortunately, we cannot express it with a N-terminal his-tag, only with a C-terminal his-tag. Therefore we are looking for a protease which cleaves off the sequence without leaving any extra amino acid on the C-terminus of our protein. Meaning we obtain really the wild type protein. Does anyone know about a protease or cleavage site which is completely removed? Many thanks in advance Phil
Re: [ccp4bb] off-topic: tag removal
Do you have the crystal structure ? Then look for a surface loop/exposed area and insert the His6-tag there. Jürgen On Feb 23, 2011, at 4:17 AM, Philipp Ellinger wrote: Dear all, I have a question concerning removal of a his-tag sequence. We have crystallized a protein with an important feature at the C-terminal part of the protein. Unfortunately, we cannot express it with a N-terminal his-tag, only with a C-terminal his-tag. Therefore we are looking for a protease which cleaves off the sequence without leaving any extra amino acid on the C-terminus of our protein. Meaning we obtain really the wild type protein. Does anyone know about a protease or cleavage site which is completely removed? Many thanks in advance Phil .. Jürgen Bosch Johns Hopkins Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Phone: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-3655 http://web.mac.com/bosch_lab/
Re: [ccp4bb] off-topic: tag removal
I have a question concerning removal of a his-tag sequence. We have crystallized a protein with an important feature at the C-terminal part of the protein. Unfortunately, we cannot express it with a N-terminal his-tag, only with a C-terminal his-tag. Therefore we are looking for a protease which cleaves off the sequence without leaving any extra amino acid on the C-terminus of our protein. Meaning we obtain really the wild type protein. Does anyone know about a protease or cleavage site which is completely removed? C-terminal intein fusion is a self-cleavable tag that cuts cleanly. NEB sells kit for intein-chitin binding domain fusion for affinity purification but of course you can add any other tag of your liking. - Dima
Re: [ccp4bb] off-topic: tag removal
Hi Phil, Must you tag your protein? Have you tried to express it without a tag? Perhaps you can purify the un-tagged protein with a series of chromatographic (or other) steps - particularly if it expresses well. Eric Eric T. Larson, PhD Biomolecular Structure Center Department of Biochemistry Box 357742 University of Washington Seattle, WA 98195 email: larso...@u.washington.edu On Wed, 23 Feb 2011, Philipp Ellinger wrote: Dear all, I have a question concerning removal of a his-tag sequence. We have crystallized a protein with an important feature at the C-terminal part of the protein. Unfortunately, we cannot express it with a N-terminal his-tag, only with a C-terminal his-tag. Therefore we are looking for a protease which cleaves off the sequence without leaving any extra amino acid on the C-terminus of our protein. Meaning we obtain really the wild type protein. Does anyone know about a protease or cleavage site which is completely removed? Many thanks in advance Phil
[ccp4bb] PISA ligand handling
Hi all, I have sort of an oddball question regarding how PISA reads the ligand file into the program for calculations. Does the element symbol of the PDB file take precedence over the atom name or residue name? Specifically, if a ligand contains either atom names or residue names that are inconsistent with those in the CCP4 monomer library will the resulting energetic calculation be valid? Thanks, Katherine
[ccp4bb] scala : cornercorrect file
if anyone knows of a cornercorrect file I might be able to use in scala, perhaps for an adsc q[any], please let me know off-list if you don't mind. -Bryan
Re: [ccp4bb] scala : cornercorrect file
I'm not sure that I would recommend this Phil On 23 Feb 2011, at 17:03, Bryan Lepore wrote: if anyone knows of a cornercorrect file I might be able to use in scala, perhaps for an adsc q[any], please let me know off-list if you don't mind. -Bryan
[ccp4bb] Postdoc and PhD student positions, Biozentrum Basel
Postdoc and PhD student positions, Biozentrum Basel, Maier Lab Our new lab at the Biozentrum is part of the core program Structural Biology Biophysics, which gathers excellence in molecular and nanoscience research including structural studies by electron microscopy, biological NMR and X-ray crystallography. We are striving to better understand eukaryotic lipid metabolism and its regulation through structural and functional studies on large multifunctional enzymes and membrane proteins (cf. Science, 321, 1315; Nature, 459, 726; Q. Rev. Biophys., 43, 373). Our approach builds on X-ray crystallography as a key method to obtain insights at atomic resolution in combination with strategies for the stabilization, labeling and trapping of macromolecules, aided by biophysical characterization of macromolecular interactions and functional analysis. We are looking for enthusiastic scientists, who share our dedication to solve challenging problems in biology, to join our lab at Postdoc or PhD student level. PhD candidates are expected to have a master¹s degree in biological sciences and practical experience in molecular biology or protein chemistry. Postdoc candidates should have recently obtained their PhD (or obtain it soon) with practical experience in structural studies and the production of difficult protein targets in eukaryotic expression systems and are expected to have demonstrated their scientific excellence by at least one significant publication as first author. Both positions are available immediately. Basel is an international center for biomedical research, with major academic institutions and research departments of leading life science companies. Situated in Switzerland on the borders to France and Germany along the river Rhine, Basel offers a high quality of living. The Biozentrum is an internationally renowned institute for life science research. With over 400 employees from 30 countries, it is the largest natural science department of the University of Basel. If you are interested in joining our team please send a short C.V. including publication record and a concise outline of prior research experience together with addresses of up to three references to timm.ma...@unibas.ch. Informal enquiries via e-mail are welcome.
Re: [ccp4bb] off-topic: tag removal
Shen A, Lupardus PJ, Morell M, Ponder EL, Sadaghiani AM, et al. 2009 Simplified, Enhanced Protein Purification Using an Inducible, Autoprocessing Enzyme Tag. PLoS ONE 4(12): e8119. doi:10.1371/journal.pone.0008119 Unless the protein in question happen to be Leu as a C-terminal residue, this tag won't give wild type C-terminus after cleavage. - Dima
