Re: [ccp4bb] offtopic: effect of compound impurities on ITC?
Hi Francis I guess it depends on how much residual high-affinity binder you have in the mixture and what the difference in affinity is between Y and deriv-Y. Another issue is of course whether Y and derY compete for the same binding site and have the same stoichiometry. A well designed displacement ITC experiment and comparisons thereof with ITC data for your high-affinity binder should lead to some good answers. Knowing the ratio of Y vs deriv-Y in your starting compound solution will be an advantage. A very useful reference in thinking about and carrying out displacement ITC in our group has been the one by Velazquez-Campoy and Freire. This article was specifically written to address the application of displacement titrations in ITC. We have applied this approach to address several types of questions concerning interactions in the uM-pM range. Velazquez-Campoy A, Freire E. Isothermal titration calorimetry to determine association constants for high-affinity ligands Nat Protoc. 2006;1(1):186-91. Best regards Savvas Savvas Savvides Unit for Structural Biology @ L-ProBE Ghent University K.L. Ledeganckstraat 35, 9000 Ghent, Belgium Ph. +32 (0)472 928 519 http://www.LProBE.ugent.be/xray.html On 24 Aug 2010, at 17:11, Francis E Reyes wrote: Hi All I'm curious the effect of small impurities in commercially synthesized compounds on ITC and its analysis. Say if compound Y is the high affinity binder, but you make a derivative that differs from a single functional group from Y (you used Y to make this new compound) and you never are able to completely get rid of Y. How does this affect the analysis of determining the derivative's affinity by ITC? References or personal experience is appreciated! F - Francis E. 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] homology modeling
Dear Paul! you might want to have a look at http://salilab.org/modeller/ . The stand alone version is free for academia. Christian ___ Dr. Christian Rausch Lehrstuhl für Biologische Chemie Technische Universität München, Germany On Thu, Aug 26, 2010 at 12:03 AM, Paul Kraft haresea...@yahoo.com wrote: Hello, I've been using an the on-line homology modeling program Jigsaw-3D, and would like to run it on my own Linux box, but it requires CHARM, which I am unvailable to get because I am currently looking for a faculty position... does anyone know of an open source energy minimization program that can be substituted for CHARM (will GROMOS from SWISS PROT work?). Also is their any open source homology modeling program that includes solvent interactions in the minimization (it seems they all require $$, at least the good one's). Thanks in advance. Paul Dr. Paul Kraft Structural Biologist cell 586-596-2770 email: haresea...@yahoo.com This communication and any attachments contain information which is confidential and may also be privileged. It is for the exclusive use of the intended recipient(s). If you are not the intended recipient(s) please note that any form of disclosure, distribution, copying or use of this communication or the information in it or in any attachments is strictly prohibited and may be unlawful. If you have received this communication in error, please notify the sender and delete the email and destroy any copies of it. E-mail communications cannot be guaranteed to be secure or error free, as information could be intercepted, corrupted, amended, lost, destroyed, arrive late or incomplete, or contain viruses. We do not accept liability for any such matters or their consequences. Anyone who communicates with us by e-mail is taken to accept the risks in doing so.
Re: [ccp4bb] Can some utilities of CCP4 do the real-space refinement locally with the residue range explicitly specified?
On 25/08/10 23:24, Garib Murshudov wrote: However 1) coot uses 2mFo-DFc maps Typically yes, but it uses whatever Refmac (or other programs) provide (of course) 2) you should be able to feed any map you want to coot Yes. so it is nice place for experimenting this kind of calculation Hopefully. 3) You may try to relax gemetry R/RC - Refinement Weight. (I often change it to 6 (from 60) if I need to tighten things up.) 4) Usually if the model does not fit into electron density and programs do not want to correct it then there may be some fundamental problem in this part of the model Agreed! On 25/08/10 23:13, Hailiang Zhang wrote: Actually I tried coot real space refine zone, but the model seems not sliding into the best density map Can you expand on that? On 25/08/10 23:33, Pavel Afonine wrote: Compare slides #4 and #5: http://cci.lbl.gov/~afonine/rsr.pdf Yes, slide 5 is what Coot uses. Paul.
Re: [ccp4bb] Can some utilities of CCP4 do the real-space refinement locally with the residue range explicitly specified?
Well - that is exactly what COOT does isnt it? Eleanor Hailiang Zhang wrote: Hi, Can some utilities of CCP4 do the real-space refinement locally with the residue range explicitly specified? By the way, I have registered phenix bb. Just didn't realize this before, sorry again. Best Regards, Hailiang
[ccp4bb] Problems in purification
Dear all, I have problems in purifying a protein. The protein is 38,000 daltons and has a N-ter His-Tag. The protein expression levels are low and as a result I have a limit for the purification steps. Initially I used NiNTA columns with 50 mM sodium phosphate buffer pH8, 300 mM NaCl, 20 to 250 mM Immidazole for the affinity purification, but it contains lot of impurities. I varied the salt concentrations out of which I could get optimal results at 20 mM NaCl concentration but still the amount of impurities was more. After affinity purifications I used Ion exchange chromatography using MonoQ column (25 mM tris pH 7.5, NaCl 0 to 1M) which could not seperate the protein from the impurities. I also tried using Hydrophobic interaction chromatography (Resource Ether, Phenyl sepharose, Resource Isopropyl) instead of ionexchange chromatography, which resulted in better purification of the protein, but the problem is I get very less protein after this step and there are still two major impurities. The buffer conditions for HIC was (1.5 M ammonium sulphate, 25 mM Phosphate buffer pH 7). I would be very greatful if someone could help me in this concern. Thanks in advance. Regards, Ganesh
Re: [ccp4bb] offtopic: effect of compound impurities on ITC?
Hi Francis, I might save you some time by telling you up front you should just go back and purify your compound to remove the impurity, you dont even need to read the rest of this, just go. Along the lines of what Savvas was saying, with any equilibrium binding assay between two direct competitors (Y is the impurity and Z is your analyte), if you are working at concentrations above the KD then the resultant complexes (XY and XZ) will partition according to their relative association strengths (dG) and concentrations. So, if Y and Z have equivalent dG values, then the concentration of XY ([XY]) and [XZ] will be a function of [Y] and [Z], if [Y]=[Z] in this circumstance then [XY]=[XZ]. If dGy dGz or [Y] [Z], then you are in the clear. This is why going back to purify Z from Y is a good idea. Now,the great thing about ITC is of course that you can get dG, dH and -TdS in one experiment, but this is also going to bite you in the butt here since you will simultaneously be determining dG, dH and -TdS for both Y and Z, which leaves you will more unknowns that you have data to solve for unless you independently know [X], [Y], [Z] and dG, dH and -TdS for XY or XZ. In fact, the circumstance where you know [X], [Y], [Z] and dG, dH and -TdS for XY or XZ is what Savvas is describing with displacement assays, and unless I am misunderstanding your situation it sounds like you dont know these parameters. For that reason I would not qualify this as a displacement assay, but instead just as a poorly controlled experiment . Now, you might be able to do the experiment with pure Y binding to X to determine dG, dH and TdS, then perform the proposed experiment with impure Y and Z as a displacement binding, but this is going to still be a headache because your uncertainty will be greater, you will not have as accurate a measure of [Y] and [Z] as when they are pure, and since your your direct signal (dH) is going to be from the formation of both XY and XZ (dHtotal = dHy + dHz) S/N will be equal to or less than the experiment with pure Y or pure Z (my nanny used to say 'dont do good experiments with bad reagents, youll just waste time', she was very wise). Hope that helps, cheers~ ~Justin Quoting Savvas Savvides savvas.savvi...@ugent.be: Hi Francis I guess it depends on how much residual high-affinity binder you have in the mixture and what the difference in affinity is between Y and deriv-Y. Another issue is of course whether Y and derY compete for the same binding site and have the same stoichiometry. A well designed displacement ITC experiment and comparisons thereof with ITC data for your high-affinity binder should lead to some good answers. Knowing the ratio of Y vs deriv-Y in your starting compound solution will be an advantage. A very useful reference in thinking about and carrying out displacement ITC in our group has been the one by Velazquez-Campoy and Freire. This article was specifically written to address the application of displacement titrations in ITC. We have applied this approach to address several types of questions concerning interactions in the uM-pM range. Velazquez-Campoy A, Freire E. Isothermal titration calorimetry to determine association constants for high-affinity ligands Nat Protoc. 2006;1(1):186-91. Best regards Savvas Savvas Savvides Unit for Structural Biology @ L-ProBE Ghent University K.L. Ledeganckstraat 35, 9000 Ghent, Belgium Ph. +32 (0)472 928 519 http://www.LProBE.ugent.be/xray.html On 24 Aug 2010, at 17:11, Francis E Reyes wrote: Hi All I'm curious the effect of small impurities in commercially synthesized compounds on ITC and its analysis. Say if compound Y is the high affinity binder, but you make a derivative that differs from a single functional group from Y (you used Y to make this new compound) and you never are able to completely get rid of Y. How does this affect the analysis of determining the derivative's affinity by ITC? References or personal experience is appreciated! F - Francis E. 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
[ccp4bb] CALL FOR PROPOSALS FOR ESRF BEAMTIME WITH ONLINE MICROSPEC (22nd to 25th of October 2010]
- CALL FOR PROPOSALS FOR ESRF BEAM TIME WITH ONLINE MICROSPEC Proposal Deadline *9th September 2010* There will be beam time available at the ESRF for MX data collection with a setup that allows online monitoring of UV/VIS absorbance or fluorescence spectral changes of the crystal during the X-ray diffraction experiment. Users who are interested in using this beam time (including those who are members of BAG Groups) should using the following mechanism: _http://www.esrf.fr/UsersAndScience/UserGuide/Applying/ProposalGuidelines/MXnon-BAGproposal _ and *it must be clearly indicated in the title of the proposal form that the online monitoring of spectral changes is necessary for the project*. A brief description of the device is given below however users are encouraged to consult the web pages for detailed information: _http://www.esrf.fr/UsersAndScience/Experiments/MX/How_to_use_our_beamlines/Run_Your_Experiment/Microspectrophotometer_User_Guide_ As this is not a standard setup, it might take a significant amount of time to train users, align the device, and analyze the data in order to derive relevant data collection schemes. *We will therefore schedule 24 hours for each project*. The deadline for this specific application is *Thursday 9th September 2010*. It is strongly recommended to record beforehand an absorption (fluorescence) spectrum of the crystal on a home microspectrophotometer such as the 4dx one, or at an off-line facility such as the ESRF Cryobench, and to provide it in the application form. Such a spectrum would greatly help determining the feasibility of the experiment. For optimal experimental conditions, crystals should be frozen in minimal amounts of cryosolution, especially when the crystals are small. Finally, please note that *the ESRF sample changer cannot be operated at the same time as the on-line microspec*._ _ *Dates of beam-time:* 22nd - 25th October 2010* *Storage Ring: 7/8 +1 (200mA) Beamline: ID14-1 Energy: 13.27 keV (not tunable) Specifications: UV/VIS-range: 250-1100 nm Light source: Mikropack DH-2000-BAL (Deuterium/Halogen) Fluorescence excitation wavelength: 266, 355, 405, 440, 561 nm ODmax for UV-vis absorbance spectra: 2-2.5 Monitoring light size: 0.03 (min) - 0.15mm(max) Sampling freq (to disk): 10Hz or lower --- -- Dr David FLOT Beam-Line Operation Manager Tel : (+33) 4 76 88 17 63 Structural Biology GroupFax : (+33) 4 76 88 26 24 ESRF B.P. 220, 6 rue Jules Horowitz e-mail : david.f...@esrf.fr F-38043 GRENOBLE CEDEX http://www.esrf.eu
[ccp4bb] Protein X-Ray Crystallographers, Oxford
Senior Scientist X-Ray Crystallographers within our Structural Biology Department Salary: £26,000 - £39,000 + benefits Location: Oxfordshire, UK Full time; Permanent and Temporary (12 month contract) Evotec (UK) Ltd is currently seeking X-Ray Crystallographers for our Structural Biology Department based in Oxfordshire. X-Ray Crystallography works closely with our Discovery Chemistry Department and with clients to develop novel small molecule drugs. The group is at the forefront of new science and technology, and is seeking to expand as business need grows. The successful candidates will be part of the Structural Biology group responsible for expression, purification, crystallisation and structure determination of proteins and protein-ligand complexes. Knowledge of state of the art crystallographic methods is a must, along with skills in molecular biology and protein purification. Expertise in protein-ligand complex crystallisation problems would be an advantage though not essential. You will have excellent written and verbal communication skills and will be a strong team player. Dynamic and innovative, you will be a self-starter with a flexible approach who enjoys a challenge. You will be PhD qualified in Chemistry, Biochemistry, Molecular Biology or Biophysics, with experience in protein X-ray crystallography. Candidates will also be considered who have a Masters degree with extensive experience. We offer competitive salaries plus extensive benefits including annual bonus, pension plan, private medical and dental cover. If you feel that your skills and experience match what we are looking for, please apply by emailing your CV, a brief statement of research accomplishments and interests to humanresources-abing...@evotec.com or to post it to: Evotec (UK) Ltd Human Resources 114 Milton Park Abingdon Oxfordshire OX14 4SA United Kingdom About Evotec Evotec is a leader in the discovery and development of novel small molecule drugs with operational sites in Europe and Asia. The Company has built substantial drug discovery expertise and an industrialised platform that can drive new innovative small molecule compounds into the clinic. In addition, Evotec has built a deep internal knowledge base in the treatment of diseases related to neuroscience, pain, and inflammation. Leveraging these skills and expertise the Company intends to develop best-in-class differentiated therapeutics and deliver superior science-driven discovery alliances with pharmaceutical and biotechnology companies. Evotec has long-term discovery alliances with partners including Boehringer Ingelheim, CHDI, Novartis, Ono Pharmaceutical and Roche. Evotec has product candidates in clinical development and a series of preclinical compounds and development partnerships, including for example a strategic alliance with Roche for the EVT 100 compound family, subtype selective NMDA receptor antagonists for use in treatment-resistant depression. Evotec (UK) Ltd is a limited company registered in England and Wales. Registration number:2674265. Registered office: 114 Milton Park, Abingdon, Oxfordshire, OX14 4SA, United Kingdom. image003.png
Re: [ccp4bb] Problems in purification
I generally find it is possible to purify most overexpressed proteins, even those at low levels of _expression_, with a combination of IEX and HIC methods. We use step-gradients to do our routine purifications, but may use gradients for polishing. If running gradients to polish partially pure samples, take care to run fairly long ones to achieve decent resolution. We routinely polish protein preps on Q-sepharose (Mono-Q should be even better) with at least 10 CV gradients over a narrower range of NaCl concentrations, maybe 0-0.5 M or even smaller. We favor butylsepharose for HIC, as it is less sticky and more selective in our hands. Again, a long gradient (10 CV or more) over a narrower range of salt concentrations will be most effective. We always use GEC for a final polish and desalting. Cheers. On 8/26/2010 8:24 AM, ganesh pathare wrote: Dear all, I have problems in purifying a protein. The protein is 38,000 daltons and has a N-ter His-Tag.The protein _expression_ levels are low and as a result I have a limit for the purification steps. Initially I used NiNTA columns with 50 mM sodium phosphate buffer pH8, 300 mM NaCl, 20 to 250 mM Immidazole for the affinity purification, but it contains lot of impurities. I varied the salt concentrations out of which I could get optimal results at20 mM NaCl concentration but still the amount of impurities was more. After affinity purifications I used Ion exchange chromatography using MonoQ column (25 mM tris pH 7.5, NaCl 0 to 1M) which could not seperate the protein from the impurities. I also tried using Hydrophobic interaction chromatography (Resource Ether, Phenyl sepharose, Resource Isopropyl)instead of ionexchange chromatography, which resulted in betterpurification of the protein,but the problem is I get very less protein after this step and thereare still two major impurities. The buffer conditions for HIC was (1.5 M ammonium sulphate, 25 mM Phosphate buffer pH 7). I would be very greatful if someonecould help me in this concern. Thanks in advance. Regards, Ganesh -- Roger S. Rowlett 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
Re: [ccp4bb] Problems in purification
On Thu, 2010-08-26 at 11:35 -0400, Roger Rowlett wrote: We routinely polish protein preps on Q-sepharose (Mono-Q should be even better) with at least 10 CV gradients over a narrower range of NaCl concentrations, maybe 0-0.5 M or even smaller. Just wanted to add that in my experience the resolution of ion exchange columns depends hugely on flow rate (in addition to the steepness of the gradient). Some columns (e.g. BioRad's UnoQ/UnoS) can be run at really high flow rate (up to 4 ml/min) which is tempting given the small size of these columns (with 6ml UnoQ you can be done with 10CV gradient in 15 minutes). It's more common to use 0.5-1 ml/min, but I've seen some unbelievable resolution when slowing down about an order of magnitude. You'd have to run overnight, but it's worth it. Cheers, Ed. -- I'd jump in myself, if I weren't so good at whistling. Julian, King of Lemurs
Re: [ccp4bb] Problems in purification
Hi. What size are the impurities? If they are smaller than your protein, then they could actually be truncation products, which will be difficult to purify away since they maintain some of the same characteristics as the full length protein. You can check for C-terminal truncations using a His-antibody, but N-terminal ones will be harder to detect. If the impurities are larger (particularly if they are around 70 kDa), you could be looking at E. coli chaperones. To improve, the purity of the first Ni-NTA step, I would include a more stringent wash. How many column volumes do you wash with now, and how high of imidazole concentration? You can go up to 20 mM Imidazole in your wash. You could also include some glycerol in your buffer (up to 10%) and betamercaptoethanol (around 5 mM) to break non-specific protein interactions. For ion exchange, run a shallow gradient and include more column volumes of wash before elution. For the third step purification, I would recommend using size exclusion chromatography. Either Superdex 200 or Sephacryl S-100 would probably work to remove some impurities as long as the impurities are a different size than your protein of interest. I would use between 150 mM - 1 M NaCl in the buffer, depending on how strong the non-specific interaction is, and 1 - 2 mM DTT. Make sure to collect small fractions (0.3 - 1.5 mL) to reduce contamination from nearby peaks. Matt On Thu, Aug 26, 2010 at 8:24 AM, ganesh pathare ganeshpath...@gmail.comwrote: Dear all, I have problems in purifying a protein. The protein is 38,000 daltons and has a N-ter His-Tag. The protein expression levels are low and as a result I have a limit for the purification steps. Initially I used NiNTA columns with 50 mM sodium phosphate buffer pH8, 300 mM NaCl, 20 to 250 mM Immidazole for the affinity purification, but it contains lot of impurities. I varied the salt concentrations out of which I could get optimal results at 20 mM NaCl concentration but still the amount of impurities was more. After affinity purifications I used Ion exchange chromatography using MonoQ column (25 mM tris pH 7.5, NaCl 0 to 1M) which could not seperate the protein from the impurities. I also tried using Hydrophobic interaction chromatography (Resource Ether, Phenyl sepharose, Resource Isopropyl) instead of ionexchange chromatography, which resulted in better purification of the protein, but the problem is I get very less protein after this step and there are still two major impurities. The buffer conditions for HIC was (1.5 M ammonium sulphate, 25 mM Phosphate buffer pH 7). I would be very greatful if someone could help me in this concern. Thanks in advance. Regards, Ganesh
Re: [ccp4bb] LIGPLOT or similar (quick summary)
Dear All, people have pointed me to: LigPlot+ (http://www.ebi.ac.uk/thornton-srv/software/LigPlus/) MOE from ChemComp (not free) PDBSUM (http://www.ebi.ac.uk/pdbsum/): prerun LIGPLOT results, only for released pdb coordinates. looks like I am stuck to Windows for now - at least it runs. Mark On 25 Aug 2010, at 19:51, Mark J van Raaij wrote: Dear All, Having just installed LIGPLOT under Windows, I find it rather convoluted to run. It has to be run via de command line window, and I try to avoid Windows as much as I can anyway. I also tried to install the Unix version on MacOSX, but was not able to get it running properly, probably at least partly due to my relative lack of informatics skills... Is there an alternative program that does the same (pref. with MacOSX version)? For the LIGPLOT developers, ideal would be a MacOSX installer (dmg) - I think it would lead to more use of your program. Greetings, Mark van Raaij
Re: [ccp4bb] LIGPLOT or similar (quick summary)
Hi, PDBsum can be used for not released co-ordinates as well- by using the generate option (found on the left panel on the webpage). Priyamvada -Original Message- From: Mark J van Raaij [mailto:mjvanra...@cnb.csic.es] Sent: Thursday, August 26, 2010 12:46 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] LIGPLOT or similar (quick summary) Dear All, people have pointed me to: LigPlot+ (http://www.ebi.ac.uk/thornton-srv/software/LigPlus/) MOE from ChemComp (not free) PDBSUM (http://www.ebi.ac.uk/pdbsum/): prerun LIGPLOT results, only for released pdb coordinates. looks like I am stuck to Windows for now - at least it runs. Mark On 25 Aug 2010, at 19:51, Mark J van Raaij wrote: Dear All, Having just installed LIGPLOT under Windows, I find it rather convoluted to run. It has to be run via de command line window, and I try to avoid Windows as much as I can anyway. I also tried to install the Unix version on MacOSX, but was not able to get it running properly, probably at least partly due to my relative lack of informatics skills... Is there an alternative program that does the same (pref. with MacOSX version)? For the LIGPLOT developers, ideal would be a MacOSX installer (dmg) - I think it would lead to more use of your program. Greetings, Mark van Raaij
[ccp4bb] Heme Proteins
Hi CCPers Can anyone provide insights about expressing heme containing proteins in E.col? Does E.coli need any porphyrin precursor during expression or you need special E.coli strains. I have references mentioning delta-aminolevulinic acid for one ortholog but none for another. The enzyme is CYP51. Thanks Hari
Re: [ccp4bb] Heme Proteins
Dear Hari, You might look at Lucy Waskell's experiences with full length mammalian Cytochrome P450 2B4. While she got 50 mg of purified protein per liter of culture, many other heme proteins are much harder to express in E. coli with heme assembly proteins, chaperones, etc. You just have to try it. However, for cytochrome P450s, you might want to resort to some molecular engineering to increase expression (a la Eric Johnson and co.; look of cytochrome P450 2C9 structure for example). Saribas, Gruenke, and Waskell (2001) Overexpression and Purification of the Membrane-Bound Cytochrome P450 2B4. Protein Expression and Purification 21, 303–309. Good luck, Michael R. Michael Garavito, Ph.D. Professor of Biochemistry Molecular Biology 513 Biochemistry Bldg. Michigan State University East Lansing, MI 48824-1319 Office: (517) 355-9724 Lab: (517) 353-9125 FAX: (517) 353-9334Email: rmgarav...@gmail.com On Aug 26, 2010, at 12:56 PM, Hari Namboodiri wrote: Hi CCPers Can anyone provide insights about expressing heme containing proteins in E.col? Does E.coli need any porphyrin precursor during expression or you need special E.coli strains. I have references mentioning delta-aminolevulinic acid for one ortholog but none for another. The enzyme is CYP51. Thanks Hari
Re: [ccp4bb] Heme Proteins
I think you need a special trick to express C-type heme proteins. For B hemes (protoheme, protoporphryn 9), which I think is what the P450's have, E.coli is always expressing several proteins which contain it. But I guess there may be some way to up the production in case of an overexpresser. Hari Namboodiri wrote: Hi CCPers Can anyone provide insights about expressing heme containing proteins in E.col? Does E.coli need any porphyrin precursor during expression or you need special E.coli strains. I have references mentioning delta-aminolevulinic acid for one ortholog but none for another. The enzyme is CYP51. Thanks Hari
[ccp4bb] Can CCP4 refine B factors for several residues only?
Hi, I want to refine B factors for several residues only (all the other B factors and all coordinates fixed, I know it sounds weird but there is a reason to try that). Is there anyway CCP4 can do this? Thanks for any suggestions! Best Regards, Hailiang
Re: [ccp4bb] Problems in purification
Have you tried expression tricks like Rosetta cells? Testing different colonies and/or starting from fresh transformants? Sometimes that matters. If your protein is an oligomer and your contaminants are degradation products, you might try adding some urea. If desparate, you could spike the fraction collector tubes with EDTA when you run the Ni column, as well as using the usual protease inhibitors. Phoebe = Phoebe A. Rice Dept. of Biochemistry Molecular Biology The University of Chicago phone 773 834 1723 http://bmb.bsd.uchicago.edu/Faculty_and_Research/01_Faculty/01_Faculty_Alphabetically.php?faculty_id=123 http://www.rsc.org/shop/books/2008/9780854042722.asp Original message Date: Thu, 26 Aug 2010 12:07:45 -0400 From: CCP4 bulletin board CCP4BB@JISCMAIL.AC.UK (on behalf of Matthew Bratkowski mab...@cornell.edu) Subject: Re: [ccp4bb] Problems in purification To: CCP4BB@JISCMAIL.AC.UK Hi. What size are the impurities? If they are smaller than your protein, then they could actually be truncation products, which will be difficult to purify away since they maintain some of the same characteristics as the full length protein. You can check for C-terminal truncations using a His-antibody, but N-terminal ones will be harder to detect. If the impurities are larger (particularly if they are around 70 kDa), you could be looking at E. coli chaperones. To improve, the purity of the first Ni-NTA step, I would include a more stringent wash. How many column volumes do you wash with now, and how high of imidazole concentration? You can go up to 20 mM Imidazole in your wash. You could also include some glycerol in your buffer (up to 10%) and betamercaptoethanol (around 5 mM) to break non-specific protein interactions. For ion exchange, run a shallow gradient and include more column volumes of wash before elution. For the third step purification, I would recommend using size exclusion chromatography. Either Superdex 200 or Sephacryl S-100 would probably work to remove some impurities as long as the impurities are a different size than your protein of interest. I would use between 150 mM - 1 M NaCl in the buffer, depending on how strong the non-specific interaction is, and 1 - 2 mM DTT. Make sure to collect small fractions (0.3 - 1.5 mL) to reduce contamination from nearby peaks. Matt On Thu, Aug 26, 2010 at 8:24 AM, ganesh pathare ganeshpath...@gmail.com wrote: Dear all, I have problems in purifying a protein. The protein is 38,000 daltons and has a N-ter His-Tag. The protein expression levels are low and as a result I have a limit for the purification steps. Initially I used NiNTA columns with 50 mM sodium phosphate buffer pH8, 300 mM NaCl, 20 to 250 mM Immidazole for the affinity purification, but it contains lot of impurities. I varied the salt concentrations out of which I could get optimal results at 20 mM NaCl concentration but still the amount of impurities was more. After affinity purifications I used Ion exchange chromatography using MonoQ column (25 mM tris pH 7.5, NaCl 0 to 1M) which could not seperate the protein from the impurities. I also tried using Hydrophobic interaction chromatography (Resource Ether, Phenyl sepharose, Resource Isopropyl) instead of ionexchange chromatography, which resulted in better purification of the protein, but the problem is I get very less protein after this step and there are still two major impurities. The buffer conditions for HIC was (1.5 M ammonium sulphate, 25 mM Phosphate buffer pH 7). I would be very greatful if someone could help me in this concern. Thanks in advance. Regards, Ganesh
Re: [ccp4bb] Heme Proteins
Hari, I have expressed sulfite oxidase (with a N-terminal heme) in E. coli strain TP1000 which resulted in red protein. I did not supplement the media with iron or a porphyrin precursor. Sincerely, James On Aug 26, 2010, at 12:56 PM, Hari Namboodiri wrote: Hi CCPers Can anyone provide insights about expressing heme containing proteins in E.col? Does E.coli need any porphyrin precursor during expression or you need special E.coli strains. I have references mentioning delta-aminolevulinic acid for one ortholog but none for another. The enzyme is CYP51. Thanks Hari
Re: [ccp4bb] Can CCP4 refine B factors for several residues only?
On Thursday 26 August 2010 11:56:39 am Hailiang Zhang wrote: Hi, I want to refine B factors for several residues only (all the other B factors and all coordinates fixed, I know it sounds weird but there is a reason to try that). Maybe you could tell us what this reason is? Is there anyway CCP4 can do this? Thanks for any suggestions! Suggestion 1) Calculate structure factors for the entire rest of the model. Include these as F_partial contributing to the refinement of a model containing only your residues of interest. In this refinement, refine only the B terms. Caveat: I think you will encounter problems with how to handle the bulk solvent correction. Perhaps that must be included in F_partial also, and omitted from the subsequence mini-refinement. Suggestion 2) - Place your residues of interest in a single TLS group. - Do not assign any other atoms or residues to a TLS group. - Refine the entire model using refmac in TLS refinement mode. Choose 5 or 10 cycles of TLS refinement, but 0 cycles of coordinate/Biso refinement. Disregard all output other than the refined TLS description for the B factors in your residues of interest. - Use TLSANL to expand the TLS parameters back to individual Biso if you like. Best Regards, Hailiang -- Ethan A Merritt Biomolecular Structure Center, K-428 Health Sciences Bldg University of Washington, Seattle 98195-7742
Re: [ccp4bb] Can CCP4 refine B factors for several residues only?
Thanks a lot Ethan, I will give it a try. Best Regards, Hailiang On Thursday 26 August 2010 11:56:39 am Hailiang Zhang wrote: Hi, I want to refine B factors for several residues only (all the other B factors and all coordinates fixed, I know it sounds weird but there is a reason to try that). Maybe you could tell us what this reason is? Is there anyway CCP4 can do this? Thanks for any suggestions! Suggestion 1) Calculate structure factors for the entire rest of the model. Include these as F_partial contributing to the refinement of a model containing only your residues of interest. In this refinement, refine only the B terms. Caveat: I think you will encounter problems with how to handle the bulk solvent correction. Perhaps that must be included in F_partial also, and omitted from the subsequence mini-refinement. Suggestion 2) - Place your residues of interest in a single TLS group. - Do not assign any other atoms or residues to a TLS group. - Refine the entire model using refmac in TLS refinement mode. Choose 5 or 10 cycles of TLS refinement, but 0 cycles of coordinate/Biso refinement. Disregard all output other than the refined TLS description for the B factors in your residues of interest. - Use TLSANL to expand the TLS parameters back to individual Biso if you like. Best Regards, Hailiang -- Ethan A Merritt Biomolecular Structure Center, K-428 Health Sciences Bldg University of Washington, Seattle 98195-7742
Re: [ccp4bb] Can CCP4 refine B factors for several residues only?
One more additional way is to apply harmonic restraint on all coordinates and all B values apart from those you want to refine. If harmonic restraints are strong enough then B values will not move much. But I do not like this option. Regards Garib On 26 Aug 2010, at 19:56, Hailiang Zhang wrote: Hi, I want to refine B factors for several residues only (all the other B factors and all coordinates fixed, I know it sounds weird but there is a reason to try that). Is there anyway CCP4 can do this? Thanks for any suggestions! Best Regards, Hailiang
[ccp4bb] AW: [ccp4bb] Heme Proteins
Hary, Delta-aminolevulinic acid is the classical heme (heme b) precursor for your experiments. As far as I know, the synthesis of porphyrin goes very quickly in E. coli, since I tried it once with a bacterial P450 and got a fat overexpression band after ~2hrs in the lysate. Also the photometric experiments worked well afterwards, means the protein also contained the porphyrin. However, I was also working on a CYP51 and important in this case was to make sure that the cells do NOT get so much air during shaking. That means e.g. low shaker speed, no buffles etc. Temperature should be low, but this also depends on your vector. Do you use e.g. pCWori+ - pET vectors do often not work for CYP51s - ? Sometimes, there are other additives rather than dALA used like thymine etc. Moreover, for these P450 expressions, often Fernbach flasks are taken (they are a little bit wider on the bottom than the Erlenmeyer flasks). I wonder if this really makes sense for the CYP51 expressions though, because the idea of these bottles is to INCREASE the interface of bacterial medium and air HTH Jan --- Hari Namboodiri hari_nambood...@epistemebiolabs.com schrieb am Do, 26.8.2010: Von: Hari Namboodiri hari_nambood...@epistemebiolabs.com Betreff: [ccp4bb] Heme Proteins An: CCP4BB@JISCMAIL.AC.UK Datum: Donnerstag, 26. August, 2010 18:56 Uhr Hi CCPers Can anyone provide insights about expressing heme containing proteins in E.col? Does E.coli need any porphyrin precursor during expression or you need special E.coli strains. I have references mentioning delta-aminolevulinic acid for one ortholog but none for another. The enzyme is CYP51. Thanks Hari
Re: [ccp4bb] turn granular to crystal
Hi, A little danger this condition. I suggest you should firstly make sure they are protein crystals. I know it is very difficult. I suggest you can add proteases to the drops. If crystals dissolve, no problem, they are protein crystals. Best Yibin 2010-08-27 yybbll 发件人： rui 发送时间： 2010-08-27 02:04:54 收件人： yybbll 抄送： 主题： Re: [ccp4bb] turn granular to crystal hi, no , I didn't use detergent. THe composition of the well buffer is just PEG4000 and MgCl2 and NaAc. On Wed, Aug 25, 2010 at 9:52 AM, yybbll yyb...@gmail.com wrote: Hi, Did you use detergent? It seems look like detergent crystals. 2010-08-25 yybbll 发件人： rui 发送时间： 2010-08-25 20:37:18 收件人： CCP4BB 抄送： 主题： [ccp4bb] turn granular to crystal Hi, All, I'm trying to crystallize a soluble protein and got something like granular, they are rounded shaped and not so regular and also don't have sharp edges. See the attached pic. The current condition is PEG4000 and pH around 5. How can I improve this condition? Thanks a lot.
Re: [ccp4bb] Problems in purification
I assume you are trying to do a non-denaturing prep. Have you run your sample on a size exclusion column to see if it is aggregated? If it is aggregated, it can stick to a lot of contaminating proteins which will be difficult if not impossible to separate. ho On Thu, Aug 26, 2010 at 8:24 AM, ganesh pathare ganeshpath...@gmail.comwrote: Dear all, I have problems in purifying a protein. The protein is 38,000 daltons and has a N-ter His-Tag. The protein expression levels are low and as a result I have a limit for the purification steps. Initially I used NiNTA columns with 50 mM sodium phosphate buffer pH8, 300 mM NaCl, 20 to 250 mM Immidazole for the affinity purification, but it contains lot of impurities. I varied the salt concentrations out of which I could get optimal results at 20 mM NaCl concentration but still the amount of impurities was more. After affinity purifications I used Ion exchange chromatography using MonoQ column (25 mM tris pH 7.5, NaCl 0 to 1M) which could not seperate the protein from the impurities. I also tried using Hydrophobic interaction chromatography (Resource Ether, Phenyl sepharose, Resource Isopropyl) instead of ionexchange chromatography, which resulted in better purification of the protein, but the problem is I get very less protein after this step and there are still two major impurities. The buffer conditions for HIC was (1.5 M ammonium sulphate, 25 mM Phosphate buffer pH 7). I would be very greatful if someone could help me in this concern. Thanks in advance. Regards, Ganesh
