[ccp4bb] : Post-doctoral in structural biology (Paris, France) position
POST-DOCTORAL POSITION IN STRUCTURAL BIOLOGY (PARIS, FRANCE) . A postdoctoral position is available in the group of Dr. Carine Tisne at the Laboratory of Biological Crystallography and NMR of the University Paris Descartes, jointly with the group of Dr. Ciaran Condon at the Laboratory of Microbial Gene Expression (CNRS UPR9073) of the Institut de Biologie Physico-Chimique. We are studying the structure, function and folding of RNA and their complexes using NMR spectroscopy and X-ray crystallography, together with complementary biophysical methods. The postdoctoral research project focusses on the processing of the 5 and 3 ends of 5S rRNA in Gram-positive bacteria using solution NMR and X-ray crystallography as the main techniques. An integral part of our projects is the use and development of state-of-the-art preparative, biochemical and NMR techniques. . Interested candidates should hold a Ph.D. in Biochemistry, Biophysics or Structural Biology. Expertise in NMR spectroscopy and/or X-ray crystallography is mandatory. The successful candidate should be highly motivated, creative and capable of independent research in a two-team environment (10 minute walk between labs). The position is for a two-year term contract (one year in each team). We offer an exciting and enthusiastic work environment together with a stimulating location at the center of Paris (close to Jardin du Luxembourg). Facilities for structural biology include 600 MHz Bruker spectrometer equipped with cryoprobe. A new 700 MHz spectrometer will be installed in 2014. We also have access to high-field NMR spectrometers through the French TGIR and regular access to synchroton beamtime at either the ESRF or SOLEIL as Beam Time Allocation Group member. The ideal start date for the position would be October 2013. . Applications and informal queries about the labs and research projects should be directed by email to Carine Tisne (carine.ti...@parisdescartes.fr[1]) or Ciaran Condon (con...@ibpc.fr[2]). Applications must include a letter of motivation summarizing current and future research interests, a CV, list of publications and letters of recommendation from at least two references. . - PHILIPPE BENAS, PH.D. X-ray diffraction and computing facilities manager Laboratoire de Cristallographie et RMN Biologiques, UMR 8015 CNRS E-mails: philippe.be...@parisdescartes.fr, philippe_be...@yahoo.fr URLs: http://lcrbw.pharmacie.univ-paris5.fr/[3] , http://lcrbw.pharmacie.univ-paris5.fr/spip.php?article18[4] - We do know nothing ! But tomorrow is another day ! ;-) Liens: -- [1] mailto:carine.ti...@parisdescartes.fr [2] mailto:con...@ibpc.fr [3] http://lcrbw.pharmacie.univ-paris5.fr/ [4] http://lcrbw.pharmacie.univ-paris5.fr/spip.php?article18
[ccp4bb] Use Pointless to prepare XDS files for Aimless, not Combat
If you want to import integrated files from XDS into Pointless and Aimless, you should use Pointless to do the conversion rather than the older program Combat. This is what is done if you use the ccp4i interface task Symmetry, Scale, Merge (Aimless) Combat wrongly fills in the FRACTIONCALC column in the unmerged MTZ file with ITEM_PEAK/100 which is different, and then by default Aimless will reject all observations which have a value of less than 95 in this field (unlike Scala which will accept them, as they are flagged as fully-recorded) If you _must_ use Combat (but why?), then you should add the line partials test 0.1 1.05 # accept data mislabelled by COMBAT to your script for Aimless (also possible in ccp4i) Pointless can read one or more MTZ files, XDS files (XDS_ASCII.HKL or INTEGRATE.HKL), and Saint files (.raw) and write an MTZ file suitable for Aimless Also Scalepack files and ShelX files: these are not normally suitable for scaling, but may be used for statistical analysis in Aimless with SCALES CONSTANT or ONLYMERGE options Combat may be useful for conversion of other file formats not read by Pointless, but caveat emptor Phil
[ccp4bb] CCP4 package manager not available for 32 bit OSX?
Hi , I am trying to use the CCP4 package manager on my 32 bit OSX 10.6.8 running laptop and it complains /bin/sh: Volume/setup/.pm/Setup.app/Contents/MacOS/Setup: Bad CPU type in executable. Is there a 32 bit package manager available... Thanks Hari attachment: Screen shot 2013-07-10 at 10.57.53 AM.png
Re: [ccp4bb] CCP4 package manager not available for 32 bit OSX?
Sadly not, we are dropping support for 32-bit Macs. You can still install 32-bit CCP4 6.3.0 from dmg. Sorry -- Eugene On 10 Jul 2013, at 16:04, hari jayaram wrote: Hi , I am trying to use the CCP4 package manager on my 32 bit OSX 10.6.8 running laptop and it complains /bin/sh: Volume/setup/.pm/Setup.app/Contents/MacOS/Setup: Bad CPU type in executable. Is there a 32 bit package manager available... Thanks Hari Screen shot 2013-07-10 at 10.57.53 AM.png -- Scanned by iCritical.
[ccp4bb] NIH Funded Postdoc Position in Protein Crystallography at Scripps Florida
Dear All, We are currently seeking one postdoctoral fellow to work in the Guo laboratory in the Department of Cancer Biology at the Scripps Florida campus of TSRI. The lab is focused on the structural and cell biology of aminoacyl-tRNA syntheses and their related pathways. The position is fully funded by NIH for at least 3 years for projects focusing on structural analysis of human aminoacyl-tRNA synthetase related novel protein complexes. Successful experience of PhD in a relevant scientific discipline and proficiency in structural biology and molecular biology is required. An appropriate post doctoral fellow candidate would be highly motivated with strong interest in Science, and willing to work on challenging but significant protein complexes in a multidisciplinary setting. Applicants please send your CV, a list of 3 professional references and your personal interest to the lab's research to guo...@scripps.edu. More information about the Scripps Research Institute can be found here: http://www.scripps.edu/about/index.html Min Guo, Ph.D. Assistant Professor Department of Cancer Biology The Scripps Research Institute - Florida 130 Scripps Way 2C1 Jupiter, Florida 33458 (P) +1.561.228.3201 (F) +1.561.228.2917 guo...@scripps.edu
[ccp4bb] Detergent solubilization step (membrane proteins)
Dear BBers, Sorry for the non-ccp4 post. I'd like to hear tips and suggestions from the membrane protein folks in the community. I am currently purifying two different membrane proteins expressed in E. coli. While I am able to extract practically all of my first protein from the cell membrane into buffer containing 1% DDM in 1hr at 4C, it doesn't seem as though that works very well for my second protein. I understand every protein is a unique beast; I am just trying to increase the yields for my second protein as much as possible. For my second protein, I have tried solubilizing for 1hr at 4C in 1% DDM as well as in 1-2% NM for 1hr at 4C but am only able to solubilize about half of total protein in the membrane. Have folks seen substantial increase in % solubility with longer incubations with detergent? Or should I consider the issue that the fraction that doesn't solubilize may be misfolded, just cut my losses and grow tons more bacterial cultures. Many thanks for sharing your successes and heartaches on this matter! Raji -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University
[ccp4bb] refmac-linux-I'm out of touch question
Hi folks, I am wanting to ask for some linux help as I want to try a newer version of refmac (5.8) to test it against a refinement problem I have. My problem is that I rarely use linux these days and I'm at a loss as to how to run the latest version as my knowledge of linux has disappeared. I have downloaded the binaries for refmac 5.8 which gives me refmacgfortran and libcheckgfortran. And now I am stuck. Would someone be so kind as to instruct me on where to put these, if they need compiling etc? Many thanks Joel _ Joel Tyndall, PhD Senior Lecturer in Medicinal Chemistry National School of Pharmacy University of Otago PO Box 56 Dunedin 9054 New Zealand Skype: jtyndall Ph: +64 3 479 7293
Re: [ccp4bb] Detergent solubilization step (membrane proteins)
Raji, First, I would suggest increasing the solubilization time by attempting solubilization overnight at 4 degrees. I would also suggest a few more detergent choices. In addition to DDM, my other favories are OG and Elugent (a mixture of several detergents) for extraction from *E. coli* membranes. If one of these still doesn't extract your protein of interest, you can move onto other more exotic options. I know it's a bit self-plugging, but the products division of Emerald Bio sells a 96-well detergent screen you can use to go through a wide variety of solubilization detergent options at small scale. We've used it in-house in our services division to get some nice results for several of our membrane protein projects. However, you may be fighting a losing battle as many times protein that doesn't extract from the membranes can be misfolded or aggregated, remaining strongly associated with the membranes. Cheers, Jim On Wed, Jul 10, 2013 at 2:23 PM, Raji Edayathumangalam r...@brandeis.eduwrote: Dear BBers, Sorry for the non-ccp4 post. I'd like to hear tips and suggestions from the membrane protein folks in the community. I am currently purifying two different membrane proteins expressed in E. coli. While I am able to extract practically all of my first protein from the cell membrane into buffer containing 1% DDM in 1hr at 4C, it doesn't seem as though that works very well for my second protein. I understand every protein is a unique beast; I am just trying to increase the yields for my second protein as much as possible. For my second protein, I have tried solubilizing for 1hr at 4C in 1% DDM as well as in 1-2% NM for 1hr at 4C but am only able to solubilize about half of total protein in the membrane. Have folks seen substantial increase in % solubility with longer incubations with detergent? Or should I consider the issue that the fraction that doesn't solubilize may be misfolded, just cut my losses and grow tons more bacterial cultures. Many thanks for sharing your successes and heartaches on this matter! Raji -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University -- Jim Fairman, Ph D. Crystal Core Leader I Emerald BioStructures http://www.emeraldbiostructures.com/ Tel: 206-780-8914 Cell: 240-479-6575 E-mail: fairman@gmail.com jfair...@embios.com
Re: [ccp4bb] Detergent solubilization step (membrane proteins)
Hi Raji, the amount of detergent used is more important than the concentration. The more cells you have, the more detergent is required. To give you an idea, we tend to use 100 ml of 1% detergent (=1 g) for 12 liters of cells (Ecoli) of OD600 1. This is probably on the low side; if cost weren't an issue I'd prefer using at least 2% (2 g) for this amount of cells. So, the lower extraction yield for your second protein could simply be due to higher ODs. To increase yields you can either (as Jim said) increase extraction time and/or increase the amount of detergent. Also, the milder the detergent (like DDM), the less efficient it tends to be in extraction. So, 1 g of DM will be more efficient than 1 g of DDM, but of course the DM may not keep your protein happy.For very stable proteins, the detergents to use are LDAO and Elugent (a mixture of alkylglucosides); these are also very cheap compared to DDM. A final possibility is indeed that some of the protein may be misfolded and unsolubilizable. Good luck, Bert From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Raji Edayathumangalam [r...@brandeis.edu] Sent: Wednesday, July 10, 2013 10:23 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Detergent solubilization step (membrane proteins) Dear BBers, Sorry for the non-ccp4 post. I'd like to hear tips and suggestions from the membrane protein folks in the community. I am currently purifying two different membrane proteins expressed in E. coli. While I am able to extract practically all of my first protein from the cell membrane into buffer containing 1% DDM in 1hr at 4C, it doesn't seem as though that works very well for my second protein. I understand every protein is a unique beast; I am just trying to increase the yields for my second protein as much as possible. For my second protein, I have tried solubilizing for 1hr at 4C in 1% DDM as well as in 1-2% NM for 1hr at 4C but am only able to solubilize about half of total protein in the membrane. Have folks seen substantial increase in % solubility with longer incubations with detergent? Or should I consider the issue that the fraction that doesn't solubilize may be misfolded, just cut my losses and grow tons more bacterial cultures. Many thanks for sharing your successes and heartaches on this matter! Raji -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University
Re: [ccp4bb] Detergent solubilization step (membrane proteins)
I could add that even for membrane proteins that may not be stable in eg DM after purification, DM may still be a viable option for extraction since there will be large amounts of stabilising lipids present. Just make sure you change to a milder detergent for the post-extraction steps. Bert From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Raji Edayathumangalam [r...@brandeis.edu] Sent: Wednesday, July 10, 2013 10:23 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Detergent solubilization step (membrane proteins) Dear BBers, Sorry for the non-ccp4 post. I'd like to hear tips and suggestions from the membrane protein folks in the community. I am currently purifying two different membrane proteins expressed in E. coli. While I am able to extract practically all of my first protein from the cell membrane into buffer containing 1% DDM in 1hr at 4C, it doesn't seem as though that works very well for my second protein. I understand every protein is a unique beast; I am just trying to increase the yields for my second protein as much as possible. For my second protein, I have tried solubilizing for 1hr at 4C in 1% DDM as well as in 1-2% NM for 1hr at 4C but am only able to solubilize about half of total protein in the membrane. Have folks seen substantial increase in % solubility with longer incubations with detergent? Or should I consider the issue that the fraction that doesn't solubilize may be misfolded, just cut my losses and grow tons more bacterial cultures. Many thanks for sharing your successes and heartaches on this matter! Raji -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University
Re: [ccp4bb] Detergent solubilization step (membrane proteins)
Thanks Jim and Bert for all your suggestions. I plan to increase time and/or amount of DDM and see what happens before switching detergents. I'll keep the suggestions about other detergents and 96-detergent screen in the back of my mind. Bert, I noted your suggestion of also using a larger amount of total detergent and will definitely try that. Jim, thanks for suggesting Elugent. Never heard about it before so great to know. Many thanks and cheers, Raji On Wed, Jul 10, 2013 at 5:23 PM, Raji Edayathumangalam r...@brandeis.eduwrote: Dear BBers, Sorry for the non-ccp4 post. I'd like to hear tips and suggestions from the membrane protein folks in the community. I am currently purifying two different membrane proteins expressed in E. coli. While I am able to extract practically all of my first protein from the cell membrane into buffer containing 1% DDM in 1hr at 4C, it doesn't seem as though that works very well for my second protein. I understand every protein is a unique beast; I am just trying to increase the yields for my second protein as much as possible. For my second protein, I have tried solubilizing for 1hr at 4C in 1% DDM as well as in 1-2% NM for 1hr at 4C but am only able to solubilize about half of total protein in the membrane. Have folks seen substantial increase in % solubility with longer incubations with detergent? Or should I consider the issue that the fraction that doesn't solubilize may be misfolded, just cut my losses and grow tons more bacterial cultures. Many thanks for sharing your successes and heartaches on this matter! Raji -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University
Re: [ccp4bb] refmac-linux-I'm out of touch question
Hi Joel, You can just replace your current 'refmac5' and 'libcheck' files. Perhaps after making a backup. They are in the 'bin' directory of your CCP4 installation. The easiest way to find them is using 'which refmac5'. HtH, Robbie Verzonden met mijn Windows Phone Van: Joel Tyndall Verzonden: 10-7-2013 23:49 Aan: CCP4BB@JISCMAIL.AC.UK Onderwerp: [ccp4bb] refmac-linux-I'm out of touch question Hi folks, I am wanting to ask for some linux help as I want to try a newer version of refmac (5.8) to test it against a refinement problem I have. My problem is that I rarely use linux these days and I'm at a loss as to how to run the latest version as my knowledge of linux has disappeared. I have downloaded the binaries for refmac 5.8 which gives me refmacgfortran and libcheckgfortran. And now I am stuck. Would someone be so kind as to instruct me on where to put these, if they need compiling etc? Many thanks Joel _ Joel Tyndall, PhD Senior Lecturer in Medicinal Chemistry National School of Pharmacy University of Otago PO Box 56 Dunedin 9054 New Zealand Skype: jtyndall Ph: +64 3 479 7293
