[ccp4bb] Vacancy
*/Laboratory Technician in biochemistry/molecular biology/* A part-time position (50%) for a laboratory technician at FPII level or similar (Grupo Profesional 4 of CSIC) is available to work in the Structural MitoLab of the Molecular Biology Institute of Barcelona (IBMB) from the Spanish Research Council (CSIC) in Barcelona (Spain).The IBMB is located within the premises of the Barcelona Science Park (PCB), one of the two major poles for excellence in biosciences in the Barcelona area. Applications are invited from candidates with proven expertise in all techniques associated with management and support tasks of a molecular biology laboratory. The candidate must be self-motivated and dedicated, be able to work independently but also to synergetically interact with other lab members, and have at least a basic level in English, spoken and written. The tentative starting date would be in January, 2015. Interested persons should apply exclusively per e-mail and send a detailed CV, as well as a covering letter including motivation, details on their expertise in protein production, and contact details of three referees to Maria Solà (msv...@ibmb.csic.es mailto:msv...@ibmb.csic.es). Relevant links: Structural MitoLab:www.ibmb.csic.es/home/msola PCB:www.pcb.ub.es/homePCB/live/en/p1.asp http://www.pcb.ub.es/homePCB/live/en/p1.asp Barcelona city:www.bcn.cat/en/ihome.htm http://www.bcn.cat/en/ihome.htm
Re: [ccp4bb] crystallization with hydrophobic ligands
Using mixed solvents is another approach: Ciccone L., Tepshi L., Nencetti, S. Stura E.A. (2014) Transthyretin complexes with curcumin and bromo-estradiol: Evaluation of solubilizing multicomponent mixtures New Biotech. 32:54–64 http://dx.doi.org/10.1016/j.nbt.2014.09.002 Volatile solvents are a problem when you try to harvest the crystals. Enrico. On Wed, 15 Oct 2014 20:35:33 +0200, Jurgen Bosch jbos...@jhu.edu wrote: An alternative is to dissolve your compound in MeOH and dispense it either manually or via robot, let the plate sit sometime in the hood for faster evaporation and then add your protein + reservoir. Jürgen .. Jürgen Bosch Johns Hopkins University Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Office: +1-410-614-4742tel:%2B1-410-614-4742 Lab: +1-410-614-4894tel:%2B1-410-614-4894 Fax: +1-410-955-2926tel:%2B1-410-955-2926 http://lupo.jhsph.edu On Oct 15, 2014, at 5:18 PM, Keller, Jacob kell...@janelia.hhmi.orgmailto:kell...@janelia.hhmi.org wrote: Since you mentioned EtOH, why not do this: -Make a tray with the appropriate mother liquors -Make a drop for each well containing mother liquor and high-concentration ligand in EtOH (you could vary the ratios here as needed.) -Equilibrate by vapor diffusion until EtOH all goes into the well soln (couple of hours at the most?) -Add protein to these drops You could skip right to the protein step if your protein doesn't mind EtOH at fairly high concentrations, and anyway it will be gone fairly quickly, esp at RT Jacob Keller From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK] on behalf of Monica Mittal [monica.mitta...@gmail.commailto:monica.mitta...@gmail.com] Sent: Wednesday, October 15, 2014 2:13 PM To: CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] crystallization with hydrophobic ligands Dear All Can anyone give suggestions for handling the solubility problem of highly hydrophobic compounds, during co-crystallization or inhibition assays? The ligands I am using are almost insoluble in aquous medium. In DMSO or 95% Ethanol, the solubility is higher. Besides crystallization, this solubility is also a hindrance for in-vitro or in-vivo assays requiring higher conc. of ligand. Thanks in advance ! Monica -- Enrico A. Stura D.Phil. (Oxon) ,Tel: 33 (0)1 69 08 4302 Office Room 19, Bat.152, Tel: 33 (0)1 69 08 9449Lab http://www-dsv.cea.fr/ibitecs/simopro/ltmb/cristallogenese LTMB, SIMOPRO, IBiTec-S, CE Saclay, 91191 Gif-sur-Yvette, FRANCE http://scholar.google.com/citations?hl=enuser=Kvm06WIoPAsCpagesize=100sortby=pubdate http://www.chem.gla.ac.uk/protein/mirror/stura/index2.html e-mail: est...@cea.fr Fax: 33 (0)1 69 08 90 71
[ccp4bb] Strategies to bring out over-expressed protein from inclusion bodies to soluble fraction!!!
Dear cc4bb enthusiasts, This is slightly off topic but many protein crystallographers might be familiar with this problem. I have been trying to over-express a bacterial (non-E.Coli) protein in E.Coli and more than 80% goest to inclusion bodies. I tried the following Lowering the IPTG concentration to 0.1mM (range test from 0.075 mM - 0.5 mM) Cold shock for 30 minutes in ice before induction Slow rotation speed at 18 degrees O/N after induction While these steps helped a bit I still get about 50-60% of my protein in inclusion bodies. I would like to know what other steps do you suggest to enhance the yield in the soluble fraction (without changing the host strain or manipulating the DNA) Thanks in advance Ivan
Re: [ccp4bb] Strategies to bring out over-expressed protein from inclusion bodies to soluble fraction!!!
Have you tried adding a detergent such as SDS to your cells when you lyse them? This in my experience helps improve the soluble fraction significantly although it may lead to downstream problems with removal of the detergent. From: xaravich ivan [mailto:xaravich.i...@gmail.com] Sent: 17 October 2014 02:52 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Strategies to bring out over-expressed protein from inclusion bodies to soluble fraction!!! Dear cc4bb enthusiasts, This is slightly off topic but many protein crystallographers might be familiar with this problem. I have been trying to over-express a bacterial (non-E.Coli) protein in E.Coli and more than 80% goest to inclusion bodies. I tried the following Lowering the IPTG concentration to 0.1mM (range test from 0.075 mM - 0.5 mM) Cold shock for 30 minutes in ice before induction Slow rotation speed at 18 degrees O/N after induction While these steps helped a bit I still get about 50-60% of my protein in inclusion bodies. I would like to know what other steps do you suggest to enhance the yield in the soluble fraction (without changing the host strain or manipulating the DNA) Thanks in advance Ivan table width=100% border=0 cellspacing=0 cellpadding=0 style=width:100%; tr td align=left style=text-align:justify;font face=arial,sans-serif size=1 color=#99span style=font-size:11px;This communication is intended for the addressee only. It is confidential. If you have received this communication in error, please notify us immediately and destroy the original message. You may not copy or disseminate this communication without the permission of the University. Only authorised signatories are competent to enter into agreements on behalf of the University and recipients are thus advised that the content of this message may not be legally binding on the University and may contain the personal views and opinions of the author, which are not necessarily the views and opinions of The University of the Witwatersrand, Johannesburg. All agreements between the University and outsiders are subject to South African Law unless the University agrees in writing to the contrary. /span/font/td /tr /table
Re: [ccp4bb] Strategies to bring out over-expressed protein from inclusion bodies to soluble fraction!!!
Hi Ivan, We've had good luck with the addition of chloramphenicol ~1 hour prior to harvest, as described in: Carrió, M. M., and Villaverde, A. (2001) Protein aggregation as bacterial inclusion bodies is reversible. FEBS Lett. 489, 2933. We usually combine this with lower temperature growth (20 C). Best regards, Mark Mark A. Wilson Associate Professor Department of Biochemistry/Redox Biology Center University of Nebraska N118 Beadle Center 1901 Vine Street Lincoln, NE 68588 (402) 472-3626 mwilso...@unl.edu On 10/17/14 7:51 AM, xaravich ivan xaravich.i...@gmail.com wrote: Dear cc4bb enthusiasts, This is slightly off topic but many protein crystallographers might be familiar with this problem. I have been trying to over-express a bacterial (non-E.Coli) protein in E.Coli and more than 80% goest to inclusion bodies. I tried the following Lowering the IPTG concentration to 0.1mM (range test from 0.075 mM - 0.5 mM) Cold shock for 30 minutes in ice before induction Slow rotation speed at 18 degrees O/N after induction While these steps helped a bit I still get about 50-60% of my protein in inclusion bodies. I would like to know what other steps do you suggest to enhance the yield in the soluble fraction (without changing the host strain or manipulating the DNA) Thanks in advance Ivan
Re: [ccp4bb] Strategies to bring out over-expressed protein from inclusion bodies to soluble fraction!!!
Thanks Mark, this is a good tip what would you use for Rosetta cells though? Tetracyclin? On Fri, Oct 17, 2014 at 8:31 AM, Mark Wilson mwilso...@unl.edu wrote: Hi Ivan, We've had good luck with the addition of chloramphenicol ~1 hour prior to harvest, as described in: Carrió, M. M., and Villaverde, A. (2001) Protein aggregation as bacterial inclusion bodies is reversible. FEBS Lett. 489, 2933. We usually combine this with lower temperature growth (20 C). Best regards, Mark Mark A. Wilson Associate Professor Department of Biochemistry/Redox Biology Center University of Nebraska N118 Beadle Center 1901 Vine Street Lincoln, NE 68588 (402) 472-3626 mwilso...@unl.edu On 10/17/14 7:51 AM, xaravich ivan xaravich.i...@gmail.com wrote: Dear cc4bb enthusiasts, This is slightly off topic but many protein crystallographers might be familiar with this problem. I have been trying to over-express a bacterial (non-E.Coli) protein in E.Coli and more than 80% goest to inclusion bodies. I tried the following Lowering the IPTG concentration to 0.1mM (range test from 0.075 mM - 0.5 mM) Cold shock for 30 minutes in ice before induction Slow rotation speed at 18 degrees O/N after induction While these steps helped a bit I still get about 50-60% of my protein in inclusion bodies. I would like to know what other steps do you suggest to enhance the yield in the soluble fraction (without changing the host strain or manipulating the DNA) Thanks in advance Ivan
Re: [ccp4bb] Command line tool for RMSD calculation
Dear all, Thanks a lot for your input. Later I found a R package called bio3d that is very suitable for my purpose. Best, Chen On Wed, Oct 15, 2014 at 5:53 PM, Steven Chou stevezc...@gmail.com wrote: Hi Chen, I used SUPPOSE previously. It worked very well. The compositions of the structures have to be the same! It works on Linux. http://structbio.vanderbilt.edu/~jsmith/suppose/ If the compositions of your structures are different, you may first load them to PyMOL, then align them use its commandline. http://pldserver1.biochem.queensu.ca/~rlc/work/teaching/BCHM823/pymol/alignment/ If the compositions/sequences of your structures are very very different, but their overall structure are similar, you can do the alignment using a PyMOL plugin called 'cealign'. It works on both Mac and Linux. http://pymolwiki.org/index.php/Cealign Here is my note on how to run SUPPOSE. remove the outputs from the previous runs rm -rf *.rot.pdb mean.pdb all.pdb # if the input file names are XXX.pdb, those of the output files are XXX.rot.pdb and a mean.pdb fit the structrue models based on the CA atoms, and then calculate the RMSD of the backbone atoms suppose -mat -rot -mean -fit :11-52,85-97:CA -calc :11-52,85-97:C,CA,N restrt_*.pdb # 0.639 fit the structrue models based on the CA atoms, and then calculte the RMSD of the heavy atoms suppose -mat -rot -mean -fit :11-52,85-97:CA -calc :11-52,85-97:C*,N*,O*,S* restrt_*.pdb # 1.23 # Do not visulize the rotated pdb files with molmol at this step, because molmol will shift the individual files. # That is, the output files have already been perfectly supperimposed, # but molmol gives an illusion that they have not been rotated correctly. join the pdb with the AWK script ./joinpdb -o all.pdb *.rot.pdb # input files: *.rot.pdb # output file: all.pdb display the rotated pdb with molmol now. molmol all.pdb # molmol can only display the combined pdb (i.e., 20 models in one file) correctly!!! HTH, Steven On Wed, Oct 15, 2014 at 4:05 PM, Chen Zhao c.z...@yale.edu wrote: Dear all, I am just wondering whether there is a simple command line tool for RMSD calculation between two aligned structures? I just cannot find a good answer online, and ccp4bb can always impress me. Thank you so much in advance, Chen -- Steven Chou
Re: [ccp4bb] Strategies to bring out over-expressed protein from inclusion bodies to soluble fraction!!!
Hi Gloria, Yes, chloramphenicol-resistant cells are presumably a problem for this method, so other ribosome-targeting antibiotics (such as tetracycline, as you mention) may work in those cases. We've not tried this, but it would be interesting to see if it works. It also would serve to test the mechanism a bit, as similar behavior would indicate that halting protein translation is the critical aspect. I will add that, in my experience, this method works best when there is some small amount of soluble material detectable on SDS-PAGE. For totally insoluble material, we've not much success, but we also have not explored all of the options discussed in the FEBS Letters reference. Best regards, Mark Mark A. Wilson Associate Professor Department of Biochemistry/Redox Biology Center University of Nebraska N118 Beadle Center 1901 Vine Street Lincoln, NE 68588 (402) 472-3626 mwilso...@unl.edu On 10/17/14 10:05 AM, Gloria Borgstahl gborgst...@gmail.com wrote: Thanks Mark, this is a good tip what would you use for Rosetta cells though? Tetracyclin? On Fri, Oct 17, 2014 at 8:31 AM, Mark Wilson mwilso...@unl.edu wrote: Hi Ivan, We've had good luck with the addition of chloramphenicol ~1 hour prior to harvest, as described in: Carrió, M. M., and Villaverde, A. (2001) Protein aggregation as bacterial inclusion bodies is reversible. FEBS Lett. 489, 2933. We usually combine this with lower temperature growth (20 C). Best regards, Mark Mark A. Wilson Associate Professor Department of Biochemistry/Redox Biology Center University of Nebraska N118 Beadle Center 1901 Vine Street Lincoln, NE 68588 (402) 472-3626 mwilso...@unl.edu On 10/17/14 7:51 AM, xaravich ivan xaravich.i...@gmail.com wrote: Dear cc4bb enthusiasts, This is slightly off topic but many protein crystallographers might be familiar with this problem. I have been trying to over-express a bacterial (non-E.Coli) protein in E.Coli and more than 80% goest to inclusion bodies. I tried the following Lowering the IPTG concentration to 0.1mM (range test from 0.075 mM - 0.5 mM) Cold shock for 30 minutes in ice before induction Slow rotation speed at 18 degrees O/N after induction While these steps helped a bit I still get about 50-60% of my protein in inclusion bodies. I would like to know what other steps do you suggest to enhance the yield in the soluble fraction (without changing the host strain or manipulating the DNA) Thanks in advance Ivan
[ccp4bb] CCP4-6.4.0 Update 022
Dear CCP4 Users An update for the CCP4-6.4.0 series has just been released, consisting of the following changes: • BLEND: – update to version 0.5.9 Note that auto-updates work only with CCP4 6.4.0 series, therefore please upgrade if necessary. The Update Manager is now included in the package so you do not need to install it separately. In addition, all available updates will be installed automatically if you are using Setup Manager for CCP4 installation. Please report any bugs to c...@stfc.ac.uk Many thanks for using CCP4. The CCP4 Core Team
Re: [ccp4bb] Strategies to bring out over-expressed protein from inclusion bodies to soluble fraction!!!
You've tried the obvious things. Lowering the IPTG concentration doesn't really work all that well, at least in my hands. We find we can still get maximal expression from many promoters like trc with only 0.2 mM IPTG, and the response doesn't modulate well: it's either on or off. Growing at lower temperature, like 18-22 deg C sometimes works, but sometimes not. Here are a few more approaches: 1. Use leaky expression in a pET vector system. Just don't induce it at all and grow for 4-18 hours. The slow expression may allow for protein folding before kinetic trapping in protein tangles. You may want to combine this with lower temperatures and less rich media (e.g., LB instead of TB) to slow growth and protein synthesis. 2. Put multiple, tandem copies of your gene behind the promoter. I'm not sure how this works, but it may tie up enzymes transcribing the message from the plasmid to slow mRNA transcription and thus translation. I never really understood the chemistry behind this, and no one could explain it to me in a cohereht way. However it works, it can slow protein expression enough to allow for soluble, instead of precipitated protein. This works like a charm to express the alpha subunit of trp synthase. The vector has 4 tandem copies of the gene after a trc promoter. You can make 1000 mg of soluble protein per 100 mL of culture (!) (I didn't mess up my zeros.) 3. Express your protein with a solubilizing tag on the N-terminus. I think there are some commercial vectors that put NusA in front of your gene. This may allow for soluble expression. NusA is supposedly better than 6X His tagging for making soluble protein. (Hasn't worked for me yet, but the literature suggests some successes.) 4. Try a different promoter. We use a modified version of pTrc99 that has at trc promoter instead of the T7 promoter. It is tightly controlled by IPTG, with no leaky expression. It can be a fierce promoter, but it may not be as fierce as T7, and can give you different results. Have fun! And good luck! ___ Roger S. Rowlett Gordon Dorothy Kline Professor Department of Chemistry Colgate University 13 Oak Drive Hamilton, NY 13346 tel: (315)-228-7245 ofc: (315)-228-7395 fax: (315)-228-7935 email: rrowl...@colgate.edu On 10/17/2014 8:51 AM, xaravich ivan wrote: Dear cc4bb enthusiasts, This is slightly off topic but many protein crystallographers might be familiar with this problem. I have been trying to over-express a bacterial (non-E.Coli) protein in E.Coli and more than 80% goest to inclusion bodies. I tried the following Lowering the IPTG concentration to 0.1mM (range test from 0.075 mM - 0.5 mM) Cold shock for 30 minutes in ice before induction Slow rotation speed at 18 degrees O/N after induction While these steps helped a bit I still get about 50-60% of my protein in inclusion bodies. I would like to know what other steps do you suggest to enhance the yield in the soluble fraction (without changing the host strain or manipulating the DNA) Thanks in advance Ivan
Re: [ccp4bb] comparisons - views
I've had a Gryphon for 2+ years and use it in an undergraduate environment. It's been trouble-free, and there are no instrument consumables, just blocks and trays. OK, I do have to feed it deionized water and a PCR tube of diluted protein for each set. It can set a 96-well tray in under 2 minutes. The basic protocol I use is 200+200 nL drops. The software is easy enough to use that my undergrads know how to program it to do 1 or 2 drop screens or partial plates. I don't have the LCP module but you can get that installed or retro-fitted. I'm pretty sure the acquisition cost of the Gryphon is much less than the Mosquito and NT8. I squeezed mine on a NSF-RUI grant as project research equipment. Cheers, ___ Roger S. Rowlett Gordon Dorothy Kline Professor Department of Chemistry Colgate University 13 Oak Drive Hamilton, NY 13346 On 10/17/2014 1:56 AM, Dean Derbyshire wrote: Apologies for the slightly off topic… thought this was the best way to get views from a wide (relevant) audience. Any views on differences – pros and cons – and experiences with: Mosquito; Gryphon and NT8? And similarly with Minstrel and Rock imager. Related to that last ‘comparison’ what’s the prevailing thoughts on SONICC vs standard UV laser technology… any experiences with coping phase separation or condensation ? Thanks Dean * Dean Derbyshire* Box 1086 SE-141 22 Huddinge SWEDEN Visit: Lunastigen 7 Direct: +46 8 54683219 www.medivir.com http://www.medivir.com -- This transmission is intended for the person to whom or the entity to which it is addressed and may contain information that is privileged, confidential and exempt from disclosure under applicable law. If you are not the intended recipient, please be notified that any dissemination, distribution or copying is strictly prohibited. If you have received this transmission in error, please notify us immediately. Thank you for your cooperation.
Re: [ccp4bb] comparisons - views
Still very happy with our Mosquito. We have had it for 8 years. Minimal maintenance. Significant consumables cost (pipette reel), but very worth the money. Just make sure you train people properly on the instrument and don't let anybody use it, who does not know what they are doing. Klaus === Dr. Klaus Fütterer Deputy Head of School Undergraduate Admissions Room 717, Biosciences Tower School of Biosciences P: +44-(0)-121-414 5895 University of Birmingham E: k.futte...@bham.ac.uk Edgbaston T: @KFbrumbio Birmingham, B15 2TT, UK W: http://tinyurl.com/futterer-lab === On 17 Oct 2014, at 19:41, Roger Rowlett wrote: I've had a Gryphon for 2+ years and use it in an undergraduate environment. It's been trouble-free, and there are no instrument consumables, just blocks and trays. OK, I do have to feed it deionized water and a PCR tube of diluted protein for each set. It can set a 96-well tray in under 2 minutes. The basic protocol I use is 200+200 nL drops. The software is easy enough to use that my undergrads know how to program it to do 1 or 2 drop screens or partial plates. I don't have the LCP module but you can get that installed or retro-fitted. I'm pretty sure the acquisition cost of the Gryphon is much less than the Mosquito and NT8. I squeezed mine on a NSF-RUI grant as project research equipment. Cheers, ___ Roger S. Rowlett Gordon Dorothy Kline Professor Department of Chemistry Colgate University 13 Oak Drive Hamilton, NY 13346 On 10/17/2014 1:56 AM, Dean Derbyshire wrote: Apologies for the slightly off topic… thought this was the best way to get views from a wide (relevant) audience. Any views on differences – pros and cons – and experiences with: Mosquito; Gryphon and NT8? And similarly with Minstrel and Rock imager. Related to that last ‘comparison’ what’s the prevailing thoughts on SONICC vs standard UV laser technology… any experiences with coping phase separation or condensation ? Thanks Dean Dean Derbyshire Mail Attachment.jpeg Box 1086 SE-141 22 Huddinge SWEDEN Visit: Lunastigen 7 Direct: +46 8 54683219 www.medivir.com -- This transmission is intended for the person to whom or the entity to which it is addressed and may contain information that is privileged, confidential and exempt from disclosure under applicable law. If you are not the intended recipient, please be notified that any dissemination, distribution or copying is strictly prohibited. If you have received this transmission in error, please notify us immediately. Thank you for your cooperation.
