[ccp4bb] How to dissolve a hydrophobic peptide?
Dear All, I want to crystallize a mAb Fab in complex with a hydrophobic cyclic peptide. The peptide contains 1 hydrophilic residue, 2 Cys, and 9 hydrophobic residues. The problem is that I can not dissolve this peptide in common buffers. I think it is need to dissolve it in some organic solvent, such as DMSO. But organic solvent is harmful to protein. Is there any sugestions for this situation? Thanks. -- Jiamu Du State Key Laboratory of Molecular Biology Institute of Biochemistry and Cell Biology Shanghai Institutes for Biological Sciences Chinese Academy of Sciences (CAS)
Re: [ccp4bb] Revise
Dear Sajid It sound as though you are running Revise through CCP4i. If you do this, the first thing you normally select is your mtz file. The buttons labelled 'FPH+1' and 'FPH-1' in the gui then default to the same thing. You need to click on at least one of these buttons and select a different label. You should then no longer get duplicate column labels in your output file. Norman Norman Stein CCP4 Daresbury Laboratory Warrington WA4 4AD UK -Original Message- From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of sajid akthar Sent: 15 April 2008 19:03 To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Revise Dear All I'm trying to locate heavy atoms. SO I started with Revise from CCP4. But it fails with error message #CCP4I TERMINATION STATUS 0 REVISE: Duplicate column labels in output file #CCP4I TERMINATION TIME 15 Apr 2008 14:50:34 #CCP4I MESSAGE Task failed Any help pls Thanks Sajid Check out the all-new face of Yahoo! India. Go to http://in.yahoo.com/ Best Jokes, Best Friends, Best Food and more. Go to http://in.promos.yahoo.com/groups/bestofyahoo/
[ccp4bb] Job offer: beamline scientist at [EMAIL PROTECTED]
Deutsches Elektronen-Synchrotron A Research Centre of the Helmholtz Association DESY is one of the world's leading accelerator centres for investigating the structure of matter. DESY develops, builds and operates large accelerator facilities for photon science and particle physics. DESY looks back on a long tradition in synchrotron radiation experiments in a wide range of applications and is currently developing to one of the leading centres in this field worldwide. At present about 40 beamlines are operated at the storage ring DORIS III and the VUV Free-Electron Laser FLASH. Our new project is the conversion of the PETRA storage ring into one of the most brilliant hard X-ray sources PETRA III (http://petra3.desy.de). For the PETRA III project we are seeking a Beamline scientist (m/f). The position: - Head the design, installation and operation of an undulator beamline dedicated to protein crystallography and biological imaging; operated and build jointly by the Max-Planck-Society and the Helmholtz-Society. - Work in a team oriented environment and establish and cultivate close contacts to the user community. - Take on a leading role in the management of the beamline group and pursue you own line of research. Requirements: - Ph.D. in physics, chemistry or a comparable qualification - Experience at a 3rd generation synchrotron radiation source - Long standing experience in design, operation and development of synchrotron radiation instrumentation is a prerequisite - A convincing research record is expected For further information you may also contact Dr. Franz (+49 40/8998-4534, [EMAIL PROTECTED]) or Prof. Dr. Edgar Weckert (+49 40/8998-4509). Salary and benefits are commensurate with those of public service organizations in Germany. DESY operates flexible work schemes. Handicapped persons will be given preference to other equally qualified applicants. DESY is an equal opportunity, affirmative action employer and encourages applications from women. There is an English-speaking Kindergarten on the DESY site. Please send your application quoting the reference code, also by E-Mail to: Deutsches Elektronen-Synchrotron DESY Human Resources Department Code: 35/2008 Notkestrasse 85 22607 Hamburg Germany E-Mail: [EMAIL PROTECTED] Further information and jobs: http://petra3.desy.de/general/job_offers/index_eng.html -- -- Edgar Weckert urls: hasylab.desy.de or petra3.desy.de HASYLAB at DESY email: [EMAIL PROTECTED] Notkestr. 85 phone: +49 (0)40 8998 4509 (Secr.: -2304) D-22603 Hamburgfax: +49 (0)40 8998 4475 Germany -- PGP Public Key: http://www.desy.de/~weckert/E_Weckert.pgp --
Re: [ccp4bb] Optimization of needle crystals?
You didn't mention which detergent in the conditions. From the picture it looks like crystals of detergent. -- Hubing Lou Centre for Biomolecular Sciences University of St Andrews North Haugh St Andrews Fife KY16 9ST Scotland Quoting Ngo Duc Tri [EMAIL PROTECTED]: Dear ccp4 users, I crystallized a membrane protein and got some conditions showing the small needle crystals. All condition contained Mg2+ and high buffer pH. I learn that it is difficult to optimize the needle crystal. However I would like to ask your experience how to optimize in this case. Here is the attached picture of my crystal. Thank you very much for your advices! My best regards, TriNgo PhD Student - Sungkyunkwan University, Korea -- University of St Andrews Webmail: https://webmail.st-andrews.ac.uk
Re: [ccp4bb] Optimization of needle crystals?
You may be able to check whether the cyrstals are protein or detergent this using powder diffraction. (please excuse the shameless plug): Acta Cryst. (2008). A64, 169-180 Powder crystallography on macromolecules I. Margiolaki and J. P. Wright http://journals.iucr.org/a/issues/2008/01/00/sc5011/index.html Good luck, Jon Hubing Lou wrote: You didn't mention which detergent in the conditions. From the picture it looks like crystals of detergent. Quoting Ngo Duc Tri [EMAIL PROTECTED]: Dear ccp4 users, I crystallized a membrane protein and got some conditions showing the small needle crystals. All condition contained Mg2+ and high buffer pH. I learn that it is difficult to optimize the needle crystal. However I would like to ask your experience how to optimize in this case. Here is the attached picture of my crystal. Thank you very much for your advices! My best regards, TriNgo PhD Student - Sungkyunkwan University, Korea
Re: [ccp4bb] Optimization of needle crystals?
Hi Ngo, your needles actually look like very thin plates. They seem promising to me. From appearance its impossible to tell whether the crystals are detergent or not. DDM is apparently known to form crystals, but I've never really gotten any (DDM is very soluble). What temperature have you used? 4C would favor forming DDM crystals. The round crystals are often seen in membrane protein crystallization. They generally don't diffract, contain a lot of detergent (maybe all detergent) and are very hard to get better (ie get sharp edges and/or diffraction). What I would do at this point is trying to get the needle/plate crystals better (by using techniques/tricks that are used for soluble proteins, such as vary Mg2+ salt conc and identity, temperature, volume/reservoir ratio, protein conc. and even trying different detergents and/or mixtures including DDM). For now I would go with the assumption that the crystals are protein. Good luck! 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: [EMAIL PROTECTED] http://www.umassmed.edu/pmm/faculty/vandenberg.cfm From: CCP4 bulletin board on behalf of Ngo Duc Tri Sent: Wed 4/16/2008 9:48 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Optimization of needle crystals? Dear experts, You didn't mention which detergent in the conditions. From the picture it looks like crystals of detergent. The initial buffer contains 50mM Tris 7.7, 100mM NaCl and 0.015% DDM. I got two forms of crystal: needle and round shape. The needle crystals grow from conditions containing Mg2+ salt and high pH. The round crystal grow from conditions containing Mg2+, high pH and small PEG. I already fail to optimize the round shape (by changing the PEG concentration as well as pH or protein concentration). So that's the reason why I think about optimization of the needle form. Here I attach more pictures to show you the needle and the round crystal. I never applied powder diffraction before so maybe it's hard for me to confirm it's the crystal of detergent or not. Thank you very much for all of your helpful suggestion! My best regards, TriNgo PhD Student- Sungkyunkwan University, Korea
Re: [ccp4bb] Optimization of needle crystals?
hi Ngo, Reduce such showering by increasing the reservoir volume, or by increasing the protein concentration. On Wed, Apr 16, 2008 at 4:08 PM, Van Den Berg, Bert [EMAIL PROTECTED] wrote: Hi Ngo, your needles actually look like very thin plates. They seem promising to me. From appearance its impossible to tell whether the crystals are detergent or not. DDM is apparently known to form crystals, but I've never really gotten any (DDM is very soluble). What temperature have you used? 4C would favor forming DDM crystals. The round crystals are often seen in membrane protein crystallization. They generally don't diffract, contain a lot of detergent (maybe all detergent) and are very hard to get better (ie get sharp edges and/or diffraction). What I would do at this point is trying to get the needle/plate crystals better (by using techniques/tricks that are used for soluble proteins, such as vary Mg2+ salt conc and identity, temperature, volume/reservoir ratio, protein conc. and even trying different detergents and/or mixtures including DDM). For now I would go with the assumption that the crystals are protein. Good luck! 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: [EMAIL PROTECTED]://www.umassmed.edu/pmm/faculty/vandenberg.cfm -- *From:* CCP4 bulletin board on behalf of Ngo Duc Tri *Sent:* Wed 4/16/2008 9:48 AM *To:* CCP4BB@JISCMAIL.AC.UK *Subject:* Re: [ccp4bb] Optimization of needle crystals? Dear experts, You didn't mention which detergent in the conditions. From the picture it looks like crystals of detergent. The initial buffer contains 50mM Tris 7.7, 100mM NaCl and 0.015% DDM. I got two forms of crystal: needle and round shape. The needle crystals grow from conditions containing Mg2+ salt and high pH. The round crystal grow from conditions containing Mg2+, high pH and small PEG. I already fail to optimize the round shape (by changing the PEG concentration as well as pH or protein concentration). So that's the reason why I think about optimization of the needle form. Here I attach more pictures to show you the needle and the round crystal. I never applied powder diffraction before so maybe it's hard for me to confirm it's the crystal of detergent or not. Thank you very much for all of your helpful suggestion! My best regards, TriNgo PhD Student- Sungkyunkwan University, Korea -- S.Jayashankar Research Student Institute for Biophysical Chemistry Hannover Medical School Germany
Re: [ccp4bb] Optimization of needle crystals?
DDM does not form crystals in aqueous solution. The published phase diagrams show this. In fact, few of the alkyl glycoside detergents crystallize if the sugar group is a beta anomer, even in organic solvents. This is one of the reasons why they are so difficult to purify by recrystallization. Addressing your questions: 1. Whenever I see crystals only in conditions containing Mg++ conditions, I think phosphate contamination. Did you use a phosphate buffer in any of your purification steps. What happens in conditions with Zn or Cd? Even a hint of phosphate in the presence of Mg++ and a bit of ammonium sulfate will produce struvite (a type of kidney stone). These tend to be needle crystals. 2. Dissolve up a couple of drops containing the most crystals in SDS- PAGE sample buffer and see if the protein is intact. Slow proteolysis of your protein by contaminating proteases can trim it to a crystallizable form. If it is, just consider truncating your protein for expression. 3. You seem to have a lot of precipitate around with the crystals. Is this always so? Your DDM concentration seems a bit low. Remember that even though you are at ~ 2XCMC, you must have enough detergent to solubilize the protein. At ~ 2XCMC (0.015% or ~0.3 mM), half of the detergent is not in micelles. If your protein needs to bind 40 molecules of detergent to stay soluble, you need 4 mM DDM to solubilize a 100KD protein at 10 mg/mL (~.1mM protein concentration). Hope this helps and 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: [EMAIL PROTECTED] On Apr 16, 2008, at 10:08 AM, Van Den Berg, Bert wrote: Hi Ngo, your needles actually look like very thin plates. They seem promising to me. From appearance its impossible to tell whether the crystals are detergent or not. DDM is apparently known to form crystals, but I've never really gotten any (DDM is very soluble). What temperature have you used? 4C would favor forming DDM crystals. The round crystals are often seen in membrane protein crystallization. They generally don't diffract, contain a lot of detergent (maybe all detergent) and are very hard to get better (ie get sharp edges and/or diffraction). What I would do at this point is trying to get the needle/plate crystals better (by using techniques/tricks that are used for soluble proteins, such as vary Mg2+ salt conc and identity, temperature, volume/reservoir ratio, protein conc. and even trying different detergents and/or mixtures including DDM). For now I would go with the assumption that the crystals are protein. Good luck! 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: [EMAIL PROTECTED] http://www.umassmed.edu/pmm/faculty/vandenberg.cfm From: CCP4 bulletin board on behalf of Ngo Duc Tri Sent: Wed 4/16/2008 9:48 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Optimization of needle crystals? Dear experts, You didn't mention which detergent in the conditions. From the picture it looks like crystals of detergent. The initial buffer contains 50mM Tris 7.7, 100mM NaCl and 0.015% DDM. I got two forms of crystal: needle and round shape. The needle crystals grow from conditions containing Mg2+ salt and high pH. The round crystal grow from conditions containing Mg2+, high pH and small PEG. I already fail to optimize the round shape (by changing the PEG concentration as well as pH or protein concentration). So that's the reason why I think about optimization of the needle form. Here I attach more pictures to show you the needle and the round crystal. I never applied powder diffraction before so maybe it's hard for me to confirm it's the crystal of detergent or not. Thank you very much for all of your helpful suggestion! My best regards, TriNgo PhD Student- Sungkyunkwan University, Korea
[ccp4bb] S-Met replacement
Dear All what are the experiences in the community in terms of physico-chemical property changes if one replace a S-Methionine by a Se-Methionine? Are there structural a/o functional changes known, e.g. the wild type protein binds a ligand whereas the Se-Met version does not? thank you in advance Jörg Stetefeld, Ph.D.
[ccp4bb] small crystallisation incubator
Dear all, sorry for the off-topic question. I am looking for an affordable+small crystallisation incubator (16-21 deg Celsius). Ideally, it would fit neatly under my bench (in a genetics lab) and hold a few dozen 96 and some 24 well plates. A friend already suggested to get a wine-cooler?! I would be grateful for some advice. Thanks! Michael
Re: [ccp4bb] Optimization of needle crystals?
Dear Ngo, You could try additive screens, such as those sold by Hampton Research. Using an additive identified during screening, I was able to optimize conditions giving thin, fiber-like needles to get chunky plates. This was with a soluble protein, though. I don't know whether this approach will also work with a membrane protein. Good luck, Katya On Wed, Apr 16, 2008 at 3:53 AM, Ngo Duc Tri [EMAIL PROTECTED] wrote: Dear ccp4 users, I crystallized a membrane protein and got some conditions showing the small needle crystals. All condition contained Mg2+ and high buffer pH. I learn that it is difficult to optimize the needle crystal. However I would like to ask your experience how to optimize in this case. Here is the attached picture of my crystal. Thank you very much for your advices! My best regards, TriNgo PhD Student - Sungkyunkwan University, Korea
[ccp4bb] refmac5
Dear All Thanks for your suggestions to run revise. Suggestions from Mike Latchem helped me to overcome the problem. I have another question. I am trying to run REFMAC5. It is giving some warning message. # There is no file name for parameter MAPOUT1 The CCP4 program is lable to fail. Please enter a file name and Run again # After dismissing the warning message the program is not running. I do not know about this parameter file. Any suugestions please Thank you Syed Bollywood, fun, friendship, sports and more. You name it, we have it on http://in.promos.yahoo.com/groups/bestofyahoo/
Re: [ccp4bb] How to dissolve a hydrophobic peptide?
This might help... Hassell AM, An G, Bledsoe RK, Bynum JM, Carter HL 3rd, Deng SJ, Gampe RT, Grisard TE, Madauss KP, Nolte RT, Rocque WJ, Wang L, Weaver KL, Williams SP, Wisely GB, Xu R, Shewchuk LM. Abstract Crystallization of protein-ligand complexes. Acta Crystallogr D Biol Crystallogr. 2007 Jan;63(Pt 1):72-9. Epub 2006 Dec 13. Review. PMID: 17164529 [PubMed - indexed for MEDLINE] hth Dave On 16/04/2008, Jiamu Du [EMAIL PROTECTED] wrote: Dear All, I want to crystallize a mAb Fab in complex with a hydrophobic cyclic peptide. The peptide contains 1 hydrophilic residue, 2 Cys, and 9 hydrophobic residues. The problem is that I can not dissolve this peptide in common buffers. I think it is need to dissolve it in some organic solvent, such as DMSO. But organic solvent is harmful to protein. Is there any sugestions for this situation? Thanks. -- Jiamu Du State Key Laboratory of Molecular Biology Institute of Biochemistry and Cell Biology Shanghai Institutes for Biological Sciences Chinese Academy of Sciences (CAS) -- David C. Briggs PhD Father Crystallographer http://www.dbriggs.talktalk.net AIM ID: dbassophile
