Re: [ccp4bb] Met auxotroph and aa-mix
Hi Eric, If you are trying to express your Se-Met protein, it is typically done in a defined media starting with a minimal media base so a mixture of amino acids, particularly those that are essential, is needed. You likely will not get very high yields of SeMet-labeled protein if you simply add SeMet to a rich media like LB or TB, etc. for expression. The B834 auxotroph is not necessary, however. We typically use standard BL21(DE3) cells and follow the procedure by Studier FW. Protein Expr Purif. 2005 May;41(1):207-34 (http://www.ncbi.nlm.nih.gov/pubmed/15915565) to get high yields of SeMet protein. Still requires a defined media though. good luck, Eric Eric T. Larson, PhD Biomolecular Structure Center Department of Biochemistry Box 357742 University of Washington Seattle, WA 98195 email: larso...@u.washington.edu On Sat, 30 Apr 2011, wrote: Dear all, I want to make a Se-Met-labeled protein using the Met auxotroph strain B834. All the protocols I've found require addition of aa-mix. Has anyone expressed in this strain without the aa-mix? Any ideas and suggestions? Thanks in advance! Eric
[ccp4bb] Dehydration treatments
Hi folks, I am currently impressed by the efficiency of dehydration treatments over the diffraction capacity of our crystals in one particular condition. Without any treatment the crystals seldom diffract to 20-30A but in our last synchrotron trip the very same crystals, after been incubated with increasing concentration of low molecular weight PEGs diffracted to 6A. I was wondering if anyone has studied these effects in a systematic way. Does anyone on the ccp4bb knows references or has any experience/pseudo-religious believes that do not care to share with the community about this particular topic? Thank you very much in advance -- Israel Sanchez Fernandez PhD Ramakrishnan-lab MRC Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 0QH, UK
Re: [ccp4bb] Dehydration treatments
Hi Israel! The phenomena you describe seems to be related to cryoprotection, but you don't say anything about collection temperature or cryoprotection used. Controlled-slow dehydration is one way of achieving optimized cryocooling of specially fragile crystals. I would recommend you to read Garman's works, particularly the one that follows: Cryocooling of macromolecular crystals: optimization methods EF Garman? - Methods in enzymology, 2003 Enjoy Horacio Quoting Israel Sanchez : Hi folks, I am currently impressed by the efficiency of dehydration treatments over the diffraction capacity of our crystals in one particular condition. Without any treatment the crystals seldom diffract to 20-30A but in our last synchrotron trip the very same crystals, after been incubated with increasing concentration of low molecular weight PEGs diffracted to 6A. I was wondering if anyone has studied these effects in a systematic way. Does anyone on the ccp4bb knows references or has any experience/pseudo-religious believes that do not care to share with the community about this particular topic? Thank you very much in advance -- Israel Sanchez Fernandez PhD Ramakrishnan-lab MRC Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 0QH, UK
Re: [ccp4bb] Dehydration treatments
Dear Israel, There is a lot to look up in this field. I believe it started with Schick, B. & Jurnak, F. (1994). Acta Cryst. D50, 563-568. at http://scripts.iucr.org/cgi-bin/paper?gr0289 There is a nice review online at http://www-bio3d-igbmc.u-strasbg.fr/~mgsb/biophys/rx/biblio/heras_2005.pdf and then of course there has been the development of the Free-Mounting System by Proteros: http://www.proteros.com/articles.php?sid=18&lang=de based on a different method of hydration control. Good luck! Gerard. -- On Sun, May 01, 2011 at 06:32:21PM +0100, Israel Sanchez wrote: > Hi folks, > > > I am currently impressed by the efficiency of dehydration treatments over > the diffraction capacity of our crystals in one particular condition. > Without any treatment the crystals seldom diffract to 20-30A but in our last > synchrotron trip the very same crystals, after been incubated with > increasing concentration of low molecular weight PEGs diffracted to 6A. > > I was wondering if anyone has studied these effects in a systematic way. > Does anyone on the ccp4bb knows references or has any > experience/pseudo-religious believes that do not care to share with the > community about this particular topic? > > > Thank you very much in advance > > > -- > Israel Sanchez Fernandez PhD > Ramakrishnan-lab > MRC Laboratory of Molecular Biology, > Hills Road, Cambridge, CB2 0QH, UK -- === * * * Gerard Bricogne g...@globalphasing.com * * * * Global Phasing Ltd. * * Sheraton House, Castle Park Tel: +44-(0)1223-353033 * * Cambridge CB3 0AX, UK Fax: +44-(0)1223-366889 * * * ===
Re: [ccp4bb] Dehydration treatments
Dear Israel, there is also a dedicated humidity device developed at EMBL/ESRF Grenoble that allows for systematic dehydration experiments to be carried out at room temperature and soon at 4°C. Here is the primary reference: Sanchez-Weatherby, J., Bowler, M.W., Huet, J., Gobbo, A., Felisaz, F., Lavault, B., Moya, R., Kadlec, J., Ravelli, R.B. and Cipriani, F. (2009) Improving diffraction by humidity control: a novel device compatible with X-ray beamlines. Acta Crystallogr D Biol Crystallogr., 65, 1237-1246. Cheers, Martin Le 01/05/11 19:32, Israel Sanchez a écrit : Hi folks, I am currently impressed by the efficiency of dehydration treatments over the diffraction capacity of our crystals in one particular condition. Without any treatment the crystals seldom diffract to 20-30A but in our last synchrotron trip the very same crystals, after been incubated with increasing concentration of low molecular weight PEGs diffracted to 6A. I was wondering if anyone has studied these effects in a systematic way. Does anyone on the ccp4bb knows references or has any experience/pseudo-religious believes that do not care to share with the community about this particular topic? Thank you very much in advance -- Israel Sanchez Fernandez PhD Ramakrishnan-lab MRC Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 0QH, UK
Re: [ccp4bb] Dehydration treatments
Hi Israel, Just to add to that, and as I know you use the Diamond synchrotron, the device mentioned is available (on advance request) on the beamline I02. Cheers -- David On 1 May 2011 19:50, Martin Weik wrote: > Dear Israel, > > there is also a dedicated humidity device developed at EMBL/ESRF Grenoble > that allows for systematic dehydration experiments to be carried out at room > temperature and soon at 4°C. Here is the primary reference: > > Sanchez-Weatherby, J., Bowler, M.W., Huet, J., Gobbo, A., Felisaz, F., > Lavault, B., Moya, R., Kadlec, J., Ravelli, R.B. and Cipriani, F. (2009) > Improving diffraction by humidity control: a novel device compatible with > X-ray beamlines. Acta Crystallogr D Biol Crystallogr., 65, 1237-1246. > > Cheers, > > Martin > > > Le 01/05/11 19:32, Israel Sanchez a écrit : > > Hi folks, >> >> >> I am currently impressed by the efficiency of dehydration treatments over >> the diffraction capacity of our crystals in one particular condition. >> Without any treatment the crystals seldom diffract to 20-30A but in our last >> synchrotron trip the very same crystals, after been incubated with >> increasing concentration of low molecular weight PEGs diffracted to 6A. >> >> I was wondering if anyone has studied these effects in a systematic way. >> Does anyone on the ccp4bb knows references or has any >> experience/pseudo-religious believes that do not care to share with the >> community about this particular topic? >> >> >> Thank you very much in advance >> >> >> -- >> Israel Sanchez Fernandez PhD >> Ramakrishnan-lab >> MRC Laboratory of Molecular Biology, >> Hills Road, Cambridge, CB2 0QH, UK >> >> >>
Re: [ccp4bb] Dehydration treatments
Dear Israel, This paper describes a procedure to crystal desiccation that allowed the improvement of diffraction: C. Abergel Acta Cryst. (2004). D60, 1413-1416 http://scripts.iucr.org/cgi-bin/paper?S0907444904013678 HTH Good luck, Catarina -- Catarina Rodrigues, PhD Student Molecular Transport & Signalling AFMB UMR 6098 CNRS/UI/UII Case 932 163 Avenue de Luminy 13288 Marseille cedex 9 (France) Tel : +33 04 91 82 55 60 Fax : 04 91 26 67 20 e-mail:catarina.rodrig...@afmb.univ-mrs.fr http://www.afmb.univ-mrs.fr/ Le 1 mai 2011 à 19:32, Israel Sanchez a écrit : > Hi folks, > > > I am currently impressed by the efficiency of dehydration treatments over the > diffraction capacity of our crystals in one particular condition. Without any > treatment the crystals seldom diffract to 20-30A but in our last synchrotron > trip the very same crystals, after been incubated with increasing > concentration of low molecular weight PEGs diffracted to 6A. > > I was wondering if anyone has studied these effects in a systematic way. Does > anyone on the ccp4bb knows references or has any experience/pseudo-religious > believes that do not care to share with the community about this particular > topic? > > > Thank you very much in advance > > > -- > Israel Sanchez Fernandez PhD > Ramakrishnan-lab > MRC Laboratory of Molecular Biology, > Hills Road, Cambridge, CB2 0QH, UK > > > > -- > This message has been scanned for viruses and > dangerous content by MailScanner, and is > believed to be clean. -- This message has been scanned for viruses and dangerous content by MailScanner, and is believed to be clean.
Re: [ccp4bb] Dehydration treatments
Dear Israel, as Martin pointed out we have a device here at the ESRF/EMBL, the HC1b, that produces a stream of air with a precisely controlled RH at the sample position that we have used with some success to monitor the effects dehydration has on diffraction quality. The same device is also available at Diamond, Max-Lab and, I believe, BESSY. The example you describe is a classic example of the sort of system that will usually benefit from controlled dehydration. Depending on the size and concentration of the LMW PEG you are using you have probably reduced the "RH" surrounding your crystal by ~10%. The best thing to do now is repeat these experiments using the HC1b to really define the changes in the lattice of your crystals and find the optimum dehydration conditions for your crystals. At the ESRF the device can be requested for any experimental session (just click the check box on the A form) and I presume that this will be similar at the other synchrotrons. As well as the reference describing the device we have recently published a further description of typical experimental conditions and some successful applications: http://dx.doi.org/10.1016/j.jsb.2011.03.002 And the ESRF webpage is here: http://www.esrf.fr/UsersAndScience/Experiments/MX/About_our_beamlines/ID14-2/HC1b Good luck! Matt On 01/05/2011 19:32, Israel Sanchez wrote: Hi folks, I am currently impressed by the efficiency of dehydration treatments over the diffraction capacity of our crystals in one particular condition. Without any treatment the crystals seldom diffract to 20-30A but in our last synchrotron trip the very same crystals, after been incubated with increasing concentration of low molecular weight PEGs diffracted to 6A. I was wondering if anyone has studied these effects in a systematic way. Does anyone on the ccp4bb knows references or has any experience/pseudo-religious believes that do not care to share with the community about this particular topic? Thank you very much in advance -- Israel Sanchez Fernandez PhD Ramakrishnan-lab MRC Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 0QH, UK -- Matthew Bowler Structural Biology Group European Synchrotron Radiation Facility B.P. 220, 6 rue Jules Horowitz F-38043 GRENOBLE CEDEX FRANCE === Tel: +33 (0) 4.76.88.29.28 Fax: +33 (0) 4.76.88.29.04 http://go.esrf.eu/MX http://go.esrf.eu/Bowler ===
