Re: [ccp4bb] Choosing MR solutions in the case of perfect twinning with P41212?
Points to think about. 1) Do your data statistics indicate twinning - if they dont iyt is mst unlikely to be present Look at output of truncate ( new Ctruncate is better) Get phenix xtriage report.. 2) SAD phasing - I presume you knw it is P41212 and not P43212? 3) Going to a lower symmetry. You dont need to rerun MR - just keep the molecule where it is and generatea second using symmetry operator y,x,-z 4) Are you doing twinned refinement? If so the Rfactors are not really comparable.. Best test is? Do the maps look better. It is easy to compare them using COOT providing you have kept the same origin for the P41 and P41212 solutions. Eleanor Francis E Reyes wrote: It seems like this space group will be the death of me. I'm working on a structure in SG P41212 one molecule per asu that was solved with experimental SAD phases. The resolution is to 2.5 and the refinement is stuck at an R/Rfree of 30 and 33 with bonds rmsd of 0.011 and angles of 1.597 . The unit cell is 73.604 73.604 114.279 90.00 90.00 90.00. I'm considering the case of perfect twinning where the real s.g. is P41 masked under the higher symmetry in P41212. It seems to be the case in perfect twinning that the approach is to molecular replace the refined model into the lower space group. I reindexed my data to the lower space group P41 and molecular replaced into the reindexed data with Phaser. A single was solution was found with 2 mol per asu (39.6% solvent content) related by NCS. I've refined the now two fold ncs related structure in P41 to a much more respectable R/Rfree of 25.2 and 28.6 with rmsd bonds at 0.004 and angles at 0.865 refining with a twin law and NCS as implemented by phenix.refine. However I'm not happy: [1] a simmulated anneal omit map one of the monomers in P41 where 5 residues in a non crystal contact region of the molecule (I wanted to challenge the omit map) shows nearly no density. (the SA OMIT map was generated with phenix.autobuild using the same refine parameters as the final round of refinement) [2] the NCS selection is a little bit troubling. (maybe the phenix developers can chime in on this) reference = chain 'B' and (resseq 243:293 or resseq 310:370 ) selection = chain 'A' and (resseq 243:293 or resseq 310:370 ) seems as if resseq 243:293 is behaving differently than 310:370? [3] the densities of the side chains of a helix (not an xtal contact) are poorly defined, with geometry for the backbone not so good. There's talk about choosing the correct MR solution (see On the molecular-replacement problem in the presence of merohedral twinning: structure of the N-terminal half-molecule of human lactoferrinW. A. Breyer, R. L. Kingston, B. F. Anderson and E. N. Baker ) . I use phaser to pick my MR solution for P41. Could phaser possibly have chosen poorly? Thanks! FR - Francis Reyes M.Sc. 215 UCB University of Colorado at Boulder gpg --keyserver pgp.mit.edu --recv-keys 67BA8D5D 8AE2 F2F4 90F7 9640 28BC 686F 78FD 6669 67BA 8D5D
Re: [ccp4bb] PHIDM AND FOMDM from DM
Xie Jiabao wrote: Dear all, I am using the density modification tool in ccp4 to generate improved phases for/from my model. I find that the electron density map I generate using Fobs, and density modified phases (PHIDM) are not the same as that generated using Fobs, phicalc (original calculated phases) and FOMDM (new improved figure of merit post dm). Can someone please explain to me why this is so? Thanks in advance, Xie The calculated map is a fourier transform of h k l W*F, PHI So if youwant a DM style map you need to input F=Fobs W=WDM PHI=PHIDM . Density modifiction will change phases and weights.. ( I think DM may also put out a term FDM which equals WDM*FOBS so you could use F=FDM, PHI=PHIDM)) I dont know what PHICALC is but if you use that phase you must use its associated weight Eleanor
Re: [ccp4bb] refinment of mir anomalous data in MLPHARE
What are you assigning as FP and FPH? Can you send the complete command script? You have set a real occupacy to 1.0 which is not appropriate if FP and FPH are equal - it should be 0.000 But I await you complete script.. Eleanor Alpharyun Ni wrote: Hi everyone! I have a problem when I use mlphare to refine against the anomalous data of mir set. The following lines are what I set for one of my heavy atom sites: deriv AU wave 1.0391 fpr -15 fdp 10 dcycle phase all refcyc all kbov all exclude sigfph 3.0 exclude diso 402.0 atom AU 0.318 0.671 0.097 1.0 1.0 bfac 20.0 atref AX ALL AY ALL AZ ALL AOCC 1 3 5 7 9 AB 2 4 6 8 10 In my log file, I found I always have a very high Cullis R-factor for the anomalous data, which means my anomalous data are useless. But as shown in the scaleit output log file, the anomalous data should not be this kind of bad. Here are some parts of the log file, 1/resol^2 Nref_a DANO_obs DANO_calc LOC_ano CullR_ano $$ 0.0075315.9 2.9 16.321.03 0.015 19519.4 1.9 19.401.00 0.026 41015.2 0.9 15.131.00 0.040 68118.5 0.6 18.491.00 0.057100125.6 0.5 25.551.00 0.077140217.4 0.3 17.431.00 0.101187312.2 0.2 12.171.00 0.128154212.2 0.1 12.231.00 $$ TOTAL715716.1 0.4 16.081.00 Could someone tell me what's the problem here? Did I set something wrong for refining against anomalous data or did I do any mistakes here? Any comments would be helpful! Thanks! Regards, Alphar Ni
[ccp4bb] Off topic: 182 kDa protein is too large to crystallize
Dear all, I´m new in the field of crystallography. So please excuse my first question is off topic. I´m trying to purify a 182 kDa his-tagged bacterial enzyme. The expression in E. coli works quite well. After Ni-NTA the protein isn´t pure enough at all, so I tried several gelfiltration columns, but my enzyme interacts with the carbohydrate matrix, iex didn´t work either. So does anyone have suggestions? Perhaps a 182 kDa protein is too large to crystallize anyway? Cheers, Marek
Re: [ccp4bb] Off topic: 182 kDa protein is too large to crystallize
Hi, 182kDa is large I'd say, but not too large for crystallisation. Since you are using a Ni-column anyway you could re-clone it with a His-tag that is cleavable by an enzyme (TeV, FactorX,... - I am a little out of date about which enzyme is the most favoured on nowadays). This would add one extra purification step, one with and an withouth His-tag. Since these are complementary, this ought to yield already pretty pure protein. What buffer did you choose for the Gel filtration - did you have enough salt to reduce interaction with the column? And what exactly do you mean with 'interacts' - does the protein not elute at all? Same question holds for the Ion exchange, what was the gradient you drove? A polishing GF-step at the end of purification is definitely something you want aim at. Tim -- Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A On Mon, 9 Feb 2009, Marek Frischerkase wrote: Dear all, I´m new in the field of crystallography. So please excuse my first question is off topic. I´m trying to purify a 182 kDa his-tagged bacterial enzyme. The expression in E. coli works quite well. After Ni-NTA the protein isn´t pure enough at all, so I tried several gelfiltration columns, but my enzyme interacts with the carbohydrate matrix, iex didn´t work either. So does anyone have suggestions? Perhaps a 182 kDa protein is too large to crystallize anyway? Cheers, Marek
[ccp4bb] Getting Molprobity to run using WinCoot
Dear all, I am trying to use the molprobity server with WinCoot 0.6-pre-1. I have followed the instructions here http://www.ysbl.york.ac.uk/~lohkamp/coot/wincoot-faq.html and believe I have put probe and reduce into the right places and pointed to them in the coot.py script. however, when I load up the protonated molecule generated in molprobity, Probe clashes is still greyed out under the validate tab. Further, I tried to run teh .scm file produced by the server under Calculate - Run Script, but that does nothing at all. I am in a muddle here, any help please? Cheers Andy.
Re: [ccp4bb] Off topic: 182 kDa protein is too large to crystallize
Hi Marek, I agree with Tim. 182kDa is certainly not too large for crystallization. As for purification . Have you tried different buffer conditions for your Ni-NTA purification ? Adjusting the pH or salt component may improve the purification sufficiently. You could try changing the salt completely (NaBr rather than NaCl seems to help for some of my proteins) or playing with concentration. I have had success with buffers containing 5mM up to 500mM salts. Also, Tris might not be the best buffer to use with Ni columns if weak binding is an issue, so try HEPES or something else. Do you include some (5-10mM) imidazole in your lysis buffer ? This would prevent some of the contaminants binding to your column initially which may affect how your protein is interacting with the matrix. You could try charging your metal affinity column with something other than Ni. Strip off the Ni with EDTA and recharge with Co2+ or Zn2+, for example. As for ion exchange, again adjusting pH can give good results. Also, you can try (from GE) Heparin or Blue columns. Even if your protein does not bind, perhaps your contaminants will, and their 5ml HiTrap columns certainly won't break the bank. Gel Filtration would be a good polishing step, as Tim suggests, but I guess if you are losing all your prep on that column, and you can achieve a pure homogeneous sample by the other methods, then its not necessarily essential. Or worst case, re-clone with a different tag, or no tag at all. We recently worked with a no-tag construct which purified (~95%) on a single Q sepharose column. But maybe we were just plain lucky ! All the best. Magnus Dr. Magnus S. Alphey Wellcome Trust Biocentre College of Life Sciences University of Dundee Dundee DD1 5EH Tel: +44 1382 385762 Fax: +44 1382 345764 The University of Dundee is a registered Scottish charity, No: SC015096
Re: [ccp4bb] Getting Molprobity to run using WinCoot
Hi Andy, where shall I start? 1.) Where is your probe.exe and reduce.exe? These should either be in your global PATH or in driveletter:\YourWinCootDirectory\bin. 2.) if you want to point to specific probe/reduce.exe files. These should be given in the .(dot)coot.py file (which in WinCoot is read from $COOT_HOME [driveletter:\YourWinCootDirectory]). 3.) Current WinCoot does not read .scm files. However the molprobity web page should provide a python (py) file too. Which you can read. Hope this helps, Bernhard P.S. There is a Coot BB (http://www.jiscmail.ac.uk/lists/coot.html) were you can ask Coot specific questions (even WinCoot ones ;-) ) Dear all, I am trying to use the molprobity server with WinCoot 0.6-pre-1. I have followed the instructions here http://www.ysbl.york.ac.uk/~lohkamp/coot/wincoot-faq.html and believe I have put probe and reduce into the right places and pointed to them in the coot.py script. however, when I load up the protonated molecule generated in molprobity, Probe clashes is still greyed out under the validate tab. Further, I tried to run teh .scm file produced by the server under Calculate - Run Script, but that does nothing at all. I am in a muddle here, any help please? Cheers Andy. This message was sent using IMP, the Internet Messaging Program.
Re: [ccp4bb] Off topic: 182 kDa protein is too large to crystallize
Hello Marek, 182 kDa protein is nothing special - unless it has huge areas of disorder, membrane-association domains, coiled coils, or something like that. Much larger proteins and protein complexes have been successfully crystallized. With respect to purification - this is where you may want to diversify. There are numerous avenues open to you: you can explore other forms of distributive chromatography (HIC, IE, etc.); you can opt to use other affinity matrices (dye resins, etc.) or you can try sizing using a non-carbohydrate matrix. Not to mention that there are numerous options for IMAC - Ni-NTA is just one of many. I would be glad to help you further off the main list if you want to share more details. Cheers, Artem
[ccp4bb] Off-topic: Open Source / Freeware Binding Curve Fitting Software
Hello All, can anyone recommend a nice, free software package for analyzing binding curves? The application I have in mind is fluorescence polarization studies between a FITC-peptide and a protein, for which I already seem to have good data. Thanks in advance, Jacob Keller *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program Dallos Laboratory F. Searle 1-240 2240 Campus Drive Evanston IL 60208 lab: 847.491.2438 cel: 773.608.9185 email: j-kell...@northwestern.edu *** - Original Message - From: Michael Colaneri To: CCP4BB@JISCMAIL.AC.UK Sent: Friday, February 06, 2009 4:26 PM Subject: [ccp4bb] Hi prep column from GE We have a hi prep SP column from GE. We try to load ~14 mg protein but all goes to FT. We lowered the pH and changed the buffer but no luck. We would appreciate all suggestions. Mike Colaneri
Re: [ccp4bb] TLS Create/Edit module error erases project folder
The folder unfortunately is gone. I played with this more and it seems that not only does the TLS Create/Edit module have this error, the ccp4mg module does as well. If I abort the process to remove the directory the project file and contents then are removed regardless of input but the folder is saved from removal (thankfully). I cannot get my ccp4mg to work on the Fedora 9 OS I have installed. There appears to be a problem with the OpenGL interface and that Fedora 9 is using a newer X.org interface type. I can get the molecule sketcher to work though. I do not know if this is related or not and again there are no log or trace files to account for what this problem may be. Thank You --- On Sat, 2/7/09, Winn, MD (Martyn) martyn.w...@stfc.ac.uk wrote: From: Winn, MD (Martyn) martyn.w...@stfc.ac.uk Subject: Re: [ccp4bb] TLS Create/Edit module error erases project folder To: CCP4BB@JISCMAIL.AC.UK Date: Saturday, February 7, 2009, 10:21 PM Well, I am sure this is not meant to happen. It doesn't seem to happen with my 6.1 version First question: are you sure that the project directory really has gone? Rather than it no longer showing in ccp4i. Check for the directory in a terminal window. If it has really gone, then you have to rely on your disk backup. If it is still there, then you should be able to reload it via DirectoriesProjectDir Cheers Martyn -Original Message- From: CCP4 bulletin board on behalf of Michael Jackson Sent: Fri 2/6/2009 3:30 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] TLS Create/Edit module error erases project folder Hello, I was using the TLS Create/Edit module to create a TLS file from scratch. In the module I ran it once but wanted to change some parameters. While I had the module interface still open, I selected to delete the TLS project entry just made in the list of jobs. When I did this I did not realize it was going to erase the entire project directory I was currently in. There was a warning that it was going to erase a directory but I thought it was some created subdirectory in the project folder when I had ran the TLS module intially. I did not have any trace back or log file to recover to send with this message. I am using the ccp4 6.0.2 with the interface 1.4.4.2 versions on a linux intel dual core PC using Fedora 9 OS.
Re: [ccp4bb] Off-topic: Open Source / Freeware Binding Curve Fitting Software
I have used qtiplot to fit nmr titration curves. http://soft.proindependent.com/qtiplot.html Another open-source app for data analysis and plotting http://scigraphica.sourceforge.net/index.html I run ubuntu linux and they are both available in the ubuntu repositories. Luke Kontogiannis MRC Laboratory of Molecular Biology Hills Road Cambridge CB2 0QH UK Hello All, can anyone recommend a nice, free software package for analyzing binding curves? The application I have in mind is fluorescence polarization studies between a FITC-peptide and a protein, for which I already seem to have good data. Thanks in advance, Jacob Keller *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program Dallos Laboratory F. Searle 1-240 2240 Campus Drive Evanston IL 60208 lab: 847.491.2438 cel: 773.608.9185 email: j-kell...@northwestern.edu *** - Original Message - From: Michael Colaneri To: CCP4BB@JISCMAIL.AC.UK Sent: Friday, February 06, 2009 4:26 PM Subject: [ccp4bb] Hi prep column from GE We have a hi prep SP column from GE. We try to load ~14 mg protein but all goes to FT. We lowered the pH and changed the buffer but no luck. We would appreciate all suggestions. Mike Colaneri
Re: [ccp4bb] Off topic: Crystal degredation with age
Hi Ed, A bit late on the subject I have collected atomic resolution data (around 0.97A) on both bovine and porcine phospholipase A2 crystals which at the time of data collection were between 10-16 years old. Crystallization setup was liquid-liquid diffusion in glass capillaries. Differently from the other stories, here there's no degradation involved...just rock-solid Xtals. In the paper we stated: 'Crystals are stable in the crystallization solution...' which I guess well reflects their behavior. Best wishes, Roberto On 5 Feb 2009, at 19:11, Edward Snell wrote: /lurk_mode_off /dumb_question_on Dear All, I was recently trying to find references on how age may degrade a crystal, i.e. grow them and use them or preserve them as fresh as possible. I seem to remember seeing a couple of papers on this but my memory is fading and I have been unable to locate them. Can anyone jog my memory or tell me if I'm imagining things? I've found plenty on the protein prep etc. but nothing on the crystal. Thanks, Eddie. Edward Snell Ph.D. Assistant Prof. Department of Structural Biology, SUNY Buffalo, Hauptman-Woodward Medical Research Institute 700 Ellicott Street, Buffalo, NY 14203-1102 Phone: (716) 898 8631 Fax: (716) 898 8660 Email: esn...@hwi.buffalo.edu Telepathy: 42.2 GHz Heisenberg was probably here!Crystallization, how quaint! /dumb_question_off /lurk_mode_on --- Roberto Steiner Randall Division of Cell and Molecular Biophysics New Hunt's House King's College London Guy's Campus London, SE1 1UL Phone +44 (0)20-7848-8216 Fax +44 (0)20-7848-6435 e-mail roberto.stei...@kcl.ac.uk
[ccp4bb] protocol for harvesting proteins from bacterial periplasmic space
Dear all, I plan to prepare a protein fused to pelB signal peptide and secreted to bacterial periplasmic space. Does anyone have a detailed protocol for harvesting the protein from bacterial periplasmic space? The protein needs to be in the native state and will be purified on a nickel column. I would appreciate your help very much! Matt
Re: [ccp4bb] Off-topic: Open Source / Freeware Binding Curve Fitting Software
On 10:43 Mon 09 Feb , Jacob Keller wrote: can anyone recommend a nice, free software package for analyzing binding curves? The application I have in mind is fluorescence polarization studies between a FITC-peptide and a protein, for which I already seem to have good data. Try fityk http://www.unipress.waw.pl/fityk/ or extrema http://exsitewebware.com/extrema/. -- Thanks, Donnie Donnie Berkholz Developer, Gentoo Linux Blog: http://dberkholz.wordpress.com pgpJ2XljtpW4n.pgp Description: PGP signature
[ccp4bb] postdoctoral fellow in membrane protein structure
Applications are invited for a postdoctoral position to work on the structure determination of rhomboid intramembrane proteases critical in cell signaling pathways. These proteases have been linked to human disorders such as hereditary blindness and breast cancer. Our aim is to understand the molecular interactions with their substrates, setting the stage for rational design of novel inhibitors. We seek an enthusiastic candidate experienced in macromolecular crystallography Experience with membrane protein biochemistry would be an asset. In addition, the successful candidate should be well-versed in one or more of the following areas: molecular biology, protein purification, crystallization, or membrane protein lipid biochemistry. This is an opportunity to join a lab experienced in the expression and crystallization of membrane proteins (See PNAS (2007) 104(3):750-4, and Science, (2003). 301:616-620). The University of Alberta, located in Edmonton, Alberta (Canada), is home to a large and interactive community of biomedical scientists http://www.med.ualberta.ca/ ; support and facilities for structural biology are excellent. Our department provides interaction with numerous crystallographers. A newly commissioned synchrotron facility is located a short drive away. In addition, the membrane protein research group provides a dedicated forum for membrane protein research at the University of Alberta (http://www.mprg.med.ualberta.ca/index.php ). Edmonton, having a population of over 1 million, offers a cosmopolitan environment with world class performing arts, sports, culinary and recreational opportunities. In addition, the citys proximity to the Rocky Mountains including the towns of Jasper and Banff is an additional bonus. Salary is commensurate with training and experience at the CIHR standard rates. A medical and dental benefit package is included with salary. The University of Alberta hires on the basis of merit. We are committed to the principle of equity in employment. We welcome diversity and encourage applications from all qualified women and men, including persons with disabilities, members of visible minorities, and Aboriginal persons. The records arising from this competition will be managed in accordance with provisions of the Alberta Freedom of Information and Protection of Privacy Act (FOIPP). Please direct cv's or inquiries to Dr. M. Joanne Lemieux at the following email address: joanne.lemi...@ualberta.ca. ** M. Joanne Lemieux, Ph.D. Assistant Professor Department of Biochemistry Medical Science Bldg. Office 4-53 Lab 4-51 University of Alberta Edmonton AB, T6G 2H7 office phone: 780-492-3586 lab phone: 780-492-3465 fax: 780-492-0886 Lab web page: http://xray.biochem.ualberta.ca/~joanne/ Membrane protein research group: http://www.mprg.med.ualberta.ca/index.php
[ccp4bb] Se oxidation
Dear AllI would like to know whether oxidation of Se entails any problem for SAD or MAD experiments and/ or how to resolve it. Cannot use DTT or reducing agents in my protein (extracellular and disulphide bonds are important).ThanksDr. R.DepetrisWeill Cornell Medical College _ Get 5 GB of storage with Windows Live Hotmail. http://windowslive.com/Explore/Hotmail?ocid=TXT_TAGLM_WL_hotmail_acq_5gb_112008
Re: [ccp4bb] Se oxidation
Hi, I wouldn't worry about Se oxidation. In principle having a mix of oxidized/reduced seleniums is unfavorable, as you'll have less signal at the edge (broadening). However, all-oxidized Se apparently makes things better (sharper and more intense peak; I forgot the reference, i think it may have been an Acta Cryst. D paper). We never bother with adding EDTA and/or reducing agents and haven't had problems determining structures by SAD. You should be fine. Cheers, Bert Bert van den Berg University of Massachusetts Medical School Program in Molecular Medicine Biotech II, 373 Plantation Street, Suite 115 Worcester MA 01605 Phone: 508 856 1201 (office); 508 856 1211 (lab) e-mail: bert.vandenb...@umassmed.edu http://www.umassmed.edu/pmm/faculty/vandenberg.cfm From: CCP4 bulletin board on behalf of aka akaka Sent: Mon 2/9/2009 1:27 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Se oxidation Dear All I would like to know whether oxidation of Se entails any problem for SAD or MAD experiments and/ or how to resolve it. Cannot use DTT or reducing agents in my protein (extracellular and disulphide bonds are important). Thanks Dr. R.Depetris Weill Cornell Medical College Get 5 GB of storage with Windows Live Hotmail. Sign up today. http://windowslive.com/Explore/Hotmail?ocid=TXT_TAGLM_WL_hotmail_acq_5gb_112008
Re: [ccp4bb] Se oxidation
Hi, I wouldn't bother either. I once phased a structure based on about 160 oxidized Se in the AU (Hothorn et al., JBC, 2006). Just make sure that most of them are oxidized and do a proper absorption scan. best Michael aka akaka wrote: Dear All I would like to know whether oxidation of Se entails any problem for SAD or MAD experiments and/ or how to resolve it. Cannot use DTT or reducing agents in my protein (extracellular and disulphide bonds are important). Thanks Dr. R.Depetris Weill Cornell Medical College Get 5 GB of storage with Windows Live Hotmail. Sign up today. http://windowslive.com/Explore/Hotmail?ocid=TXT_TAGLM_WL_hotmail_acq_5gb_112008
Re: [ccp4bb] Se oxidation
Hi,I believe it is not important as long as you run a proper scan of the crystal. Both forms will allow proper phasing. This is very well described in a paper from Thomazeau et al. here is the reference MAD on threonine synthase: the phasing power of oxidized selenomethionine. Acta Crystallogr D Biol Crystallogr.javascript:AL_get(this,%20'jour',%20'Acta%20Crystallogr%20D%20Biol%20Crystallogr.'); 2001 Sep;57(Pt 9):1337-40. Epub 2001 Aug 23. Cheers, Pascal Egea, PhD Post Doctoral Researcher UCSF Department of Biochemistry and Biophysics Stroud laboratory On Mon, Feb 9, 2009 at 10:27 AM, aka akaka druida...@hotmail.com wrote: Dear All I would like to know whether oxidation of Se entails any problem for SAD or MAD experiments and/ or how to resolve it. Cannot use DTT or reducing agents in my protein (extracellular and disulphide bonds are important). Thanks Dr. R.Depetris Weill Cornell Medical College -- Get 5 GB of storage with Windows Live Hotmail. Sign up today.http://windowslive.com/Explore/Hotmail?ocid=TXT_TAGLM_WL_hotmail_acq_5gb_112008
Re: [ccp4bb] protocol for harvesting proteins from bacterial periplasmic space
Hi Matt, Why not check the NEB pMal system manual (http://www.neb.com/nebecomm/ManualFiles/manualE8000.pdf). It has a good cold osmotic shock protocol that I've been using for my periplasmic MBP fusions. Zhijie - Original Message - From: Matt Colins matt.colins2...@yahoo.com To: CCP4BB@JISCMAIL.AC.UK Sent: Monday, February 09, 2009 12:52 PM Subject: [ccp4bb] protocol for harvesting proteins from bacterial periplasmic space Dear all, I plan to prepare a protein fused to pelB signal peptide and secreted to bacterial periplasmic space. Does anyone have a detailed protocol for harvesting the protein from bacterial periplasmic space? The protein needs to be in the native state and will be purified on a nickel column. I would appreciate your help very much! Matt
Re: [ccp4bb] protocol for harvesting proteins from bacterial periplasmic space
http://www.sciencedirect.com/science?_ob=ArticleURL_udi=B6V5N-4C8PFBS-2_us er=10_rdoc=1_fmt=_orig=search_sort=dview=c_acct=C50221_version=1 _urlVersion=0_userid=10md5=aee9aceae8dfa17b363ee2ec634debb0 Osmotic shock is OK, or you can try chlorophorm disruption. You can Google for either to find them. http://www.sciencedirect.com/science?_ob=ArticleURL_udi=B6T30-476TXJW-CK_u ser=10_rdoc=1_fmt=_orig=search_sort=dview=c_acct=C50221_version=1 _urlVersion=0_userid=10md5=836b012efa98a0fe0259708135fc1144 Please note that osmotic shock extraction typically employs EDTA which is obviously bad for IMAC. If you're not worried about also extracting cytoplasmic proteins, then a combination of 2:1 I-PER with B-PER (Pierce) works fine and is compatible with IMAC. Please note that just because you have pelB signal on your protein does not guarrantee that your protein finds its way to the periplasm. Secretion in general and secretion in Gram-negative hosts in particular can be very tricky and sometimes impossible. If you have time/effort available I would heartily recommend Bacillus megaterium as an alternative host - it has good secretory capacity and some of its strains have been selected not to secrete appreciable quantities of proteases which (as people who work with B. subtilis will tell you) can be a right pain in the tuches. There's even a commercially available B. megaterium system that does not require chromosomal integration (again, unlike B. subtilis). Artem -Original Message- From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Matt Colins Sent: Monday, February 09, 2009 12:53 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] protocol for harvesting proteins from bacterial periplasmic space Dear all, I plan to prepare a protein fused to pelB signal peptide and secreted to bacterial periplasmic space. Does anyone have a detailed protocol for harvesting the protein from bacterial periplasmic space? The protein needs to be in the native state and will be purified on a nickel column. I would appreciate your help very much! Matt
