Re: [ccp4bb] heavy atom derivative choice
David Briggs schrieb: Bart Hazes used to have an excellent set of notes online about heavy atom derivatisation - I can't seem to find the URL right now... I believe the CCP4 wiki has the information that you're looking for (at http://strucbio.biologie.uni-konstanz.de/ccp4wiki/index.php/Soaking_with_heavy_atoms ) Now if people would add their notes to the wiki that would actually become an even better ressource ... Unfortunately it seems that Bart Hazes' webpage cannot currently be accessed, and the "cache results" do no longer seem to appear in my Google searches! best, Kay -- Kay Diederichshttp://strucbio.biologie.uni-konstanz.de email: kay.diederi...@uni-konstanz.deTel +49 7531 88 4049 Fax 3183 Fachbereich Biologie, Universität Konstanz, Box M647, D-78457 Konstanz This e-mail is digitally signed. If your e-mail client does not have the necessary capabilities, just ignore the attached signature "smime.p7s". smime.p7s Description: S/MIME Cryptographic Signature
Re: [ccp4bb] heavy atom derivative choice
>For gel-shift assay, do people normally use a special gel tank for heavy >metal work? We used to use a Pharmacia Phast system, as this is bufferless and made it very easy to contain heavy metal contamination. Ours caught fire and died a couple of years ago, and I have yet to find a good cheap replacement - does anyone know of a native gel equivalent of the Invitrogen E-PAGE for example? In the absence of such niceties, just treat the gels and electrophoresis solutions as heavy metal waste.
Re: [ccp4bb] heavy atom derivative choice
Hi, 1. KSCN will suck up a lot of the Hg and other metals -- can you switch to something else? 2. Hg typically does not enter the protein core. 3. It's generally not necessary to use a special tank, as long as you treat the resulting solutions as heavy-atom waste; but of course each lab's safety precautions are different. Artem > > Does mercury tends to get into the protein core to denature protein or > not? > > For gel-shift assay, do people normally use a special gel tank for heavy > metal work? > > thanks in advance > > Xiaoli Xiong (Alex) > > > --On 15 July 2009 14:33 +0200 Sebastiano Pasqualato > wrote: > >> Hi all, >> I've got crystals of a protein of ca 200 residues, with 2 free >> cysteines, 5 histidines, 2 methionines. >> We have nice diffraction for the native crystals, that grow in 150 mM >> KSCN, 17% PEG 3350, bis tris propane pH 8.8. >> We are crystallising the SeMet derivative, but I'm not completely sure >> I >> will be able to have nice crystals by Saturday, when we have tunable >> time at the ESRF. >> I was thinking of trying with some heavy atom soaks, but only have like >> 30 crystals, so limited trials allowed! >> Which compound would you advice as more likely to work, and thus worth >> testing? >> Thanks in advance for the suggestions, >> ciao >> s >> >> >> >> -- >> Sebastiano Pasqualato, PhD >> IFOM-IEO Campus >> Dipartimento di Oncologia Sperimentale >> Istituto Europeo di Oncologia >> via Adamello, 16 >> 20139 - Milano >> Italy >> >> tel +39 02 9437 5094 > > > > -- > Xiaoli Xiong > PhD Candidate > Department of Cellular and Molecular Medicine > School of Medical Sciences > University of Bristol > University Walk > Bristol BS8 1TD, UK > x.l.xi...@bristol.ac.uk >
Re: [ccp4bb] heavy atom derivative choice
There is a Xenon chamber at 14-1 if I remember it correctly. I never had luck though. How about Iodine or Gadolinium? Jürgen .. Jürgen Bosch Johns Hopkins Bloomberg School of Public Health Department of Biochemistry & Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Phone: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-3655 http://web.me.com/bosch_lab/ On Jul 15, 2009, at 13:13, Jacob Keller kell...@md.northwestern.edu> wrote: Xenon/Krypton, anyone? If you have the equipment, might as well try it. I think I have seen the apparatus at some beamlines, although I did hear recently that xenon is ridiculously expensive now. 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: "Phil Jeffrey" To: Sent: Wednesday, July 15, 2009 11:32 AM Subject: Re: [ccp4bb] heavy atom derivative choice X Xiong, Cellular & Molecular Medicine wrote: My Question is: Does mercury tends to get into the protein core to denature protein or not? This is more likely to happen for a small "bare" Hg like Hg2+ in HgCl2 or Hg(OAc)2 than it is for a large organomercury compound like PCMB, PCMBS etc so if you were especially concerned about that, start with the latter compounds. I'd also probably try Me3Pb(OAc) as an alternative to mercurials. Phil Jeffrey Princeton
Re: [ccp4bb] heavy atom derivative choice
Xenon/Krypton, anyone? If you have the equipment, might as well try it. I think I have seen the apparatus at some beamlines, although I did hear recently that xenon is ridiculously expensive now. 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: "Phil Jeffrey" To: Sent: Wednesday, July 15, 2009 11:32 AM Subject: Re: [ccp4bb] heavy atom derivative choice X Xiong, Cellular & Molecular Medicine wrote: My Question is: Does mercury tends to get into the protein core to denature protein or not? This is more likely to happen for a small "bare" Hg like Hg2+ in HgCl2 or Hg(OAc)2 than it is for a large organomercury compound like PCMB, PCMBS etc so if you were especially concerned about that, start with the latter compounds. I'd also probably try Me3Pb(OAc) as an alternative to mercurials. Phil Jeffrey Princeton
Re: [ccp4bb] heavy atom derivative choice
X Xiong, Cellular & Molecular Medicine wrote: My Question is: Does mercury tends to get into the protein core to denature protein or not? This is more likely to happen for a small "bare" Hg like Hg2+ in HgCl2 or Hg(OAc)2 than it is for a large organomercury compound like PCMB, PCMBS etc so if you were especially concerned about that, start with the latter compounds. I'd also probably try Me3Pb(OAc) as an alternative to mercurials. Phil Jeffrey Princeton
Re: [ccp4bb] heavy atom derivative choice
Dear All, We have a very similar problem, I have got a protein of 200 residues predicted to have two free cys (but 14 internal cys!), 1 free histidine, 2 methionines (all predicted to be free). Native crystals diffracted to 2.2 A was obtained in 200 mM KSCN, 20% PEG 3350, 13% glycerol, bis tris propane pH 7.5, 20mM spermine, 50mM DTT(absolutely needed to obtain crystal). We tried iodide soak and sulphur SAD but phasing was not promising. From all the posts I have read, mercury seems to be the best to try to derivatise it but probably needs to remove DTT. My Question is: Does mercury tends to get into the protein core to denature protein or not? For gel-shift assay, do people normally use a special gel tank for heavy metal work? thanks in advance Xiaoli Xiong (Alex) --On 15 July 2009 14:33 +0200 Sebastiano Pasqualato wrote: Hi all, I've got crystals of a protein of ca 200 residues, with 2 free cysteines, 5 histidines, 2 methionines. We have nice diffraction for the native crystals, that grow in 150 mM KSCN, 17% PEG 3350, bis tris propane pH 8.8. We are crystallising the SeMet derivative, but I'm not completely sure I will be able to have nice crystals by Saturday, when we have tunable time at the ESRF. I was thinking of trying with some heavy atom soaks, but only have like 30 crystals, so limited trials allowed! Which compound would you advice as more likely to work, and thus worth testing? Thanks in advance for the suggestions, ciao s -- Sebastiano Pasqualato, PhD IFOM-IEO Campus Dipartimento di Oncologia Sperimentale Istituto Europeo di Oncologia via Adamello, 16 20139 - Milano Italy tel +39 02 9437 5094 -- Xiaoli Xiong PhD Candidate Department of Cellular and Molecular Medicine School of Medical Sciences University of Bristol University Walk Bristol BS8 1TD, UK x.l.xi...@bristol.ac.uk
Re: [ccp4bb] heavy atom derivative choice
I would also include a NaI and NaBr quick soak in 250 mM, SCN- is considered a pseudohalide cheers Preben Sebastiano Pasqualato wrote: Hi all, I've got crystals of a protein of ca 200 residues, with 2 free cysteines, 5 histidines, 2 methionines. We have nice diffraction for the native crystals, that grow in 150 mM KSCN, 17% PEG 3350, bis tris propane pH 8.8. We are crystallising the SeMet derivative, but I'm not completely sure I will be able to have nice crystals by saturday, when we have tunable time at the ESRF. I was thinking of trying with some heavy atom soaks, but only have like 30 crystals, so limited trials allowed! Which compound would you advice as more likely to work, and thus worth testing? Thanks in advance for the suggestions, ciao s --Sebastiano Pasqualato, PhD IFOM-IEO Campus Dipartimento di Oncologia Sperimentale Istituto Europeo di Oncologia via Adamello, 16 20139 - Milano Italy tel +39 02 9437 5094 -- Jens Preben Morth, Ph.D Aarhus University Department of Molecular Biology Gustav Wieds Vej 10 C DK - 8000 Aarhus C Tel. +45 8942 5257, Fax. +45 8612 3178 j...@mb.au.dk website: http://person.au.dk/da/j...@mb
Re: [ccp4bb] heavy atom derivative choice
Hi Sebastiano, In addition to the heavy atom database server and the suggestions made here, you could try the strategy described in this paper: Lott JS et al. (2003) Acta Cryst D Biol Crystallogr, Dec (Pt 12): 2242-6. Making the most of two crystals: structural analysis of a conserved hypothetical protein using native gel screening and SAD phasing. I second the sulfur SAD suggestion made earlier. 2 Met and 2Cys for a 200 residue protein would be doable. You can collect at 1.9 Å wavelength to enhance the signal. Best, Sarma On Jul 15, 2009, at 6:37 AM Jul 15, 2009, Savvas Savvides wrote: Hi Sebastiano, Given that your crystallization condition contains KSCN, I would certainly give Hg(SCN)2 a go among other Hg options for a protein containing free cysteines. Hg(SCN)2 might end up being more compatible with your crystal form, and might thus minimize non-isomorphism issues. Best of luck Savvas -Original Message- From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of David Briggs Sent: Wednesday, July 15, 2009 3:27 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] heavy atom derivative choice Hi Sebastiano, Free Cys' are crying out for Mercury derivatives. Try 2-3 with varying sizes of additional groups - HgCl2, K2HgI4, PCMB, from the "Magic seven" would be a good place to start. Watch out for problems with your low pH, though. Some salts will form insoluble hydroxide salts at pH 8.8. (HgCl2 & K2HgI4 should be okay) Bart Hazes used to have an excellent set of notes online about heavy atom derivatisation - I can't seem to find the URL right now... HTH, David 2009/7/15 Sebastiano Pasqualato >: Hi all, I've got crystals of a protein of ca 200 residues, with 2 free cysteines, 5 histidines, 2 methionines. We have nice diffraction for the native crystals, that grow in 150 mM KSCN, 17% PEG 3350, bis tris propane pH 8.8. We are crystallising the SeMet derivative, but I'm not completely sure I will be able to have nice crystals by saturday, when we have tunable time at the ESRF. I was thinking of trying with some heavy atom soaks, but only have like 30 crystals, so limited trials allowed! Which compound would you advice as more likely to work, and thus worth testing? Thanks in advance for the suggestions, ciao s -- Sebastiano Pasqualato, PhD IFOM-IEO Campus Dipartimento di Oncologia Sperimentale Istituto Europeo di Oncologia via Adamello, 16 20139 - Milano Italy tel +39 02 9437 5094 -- David C. Briggs PhD Father & Crystallographer http://drdavidcbriggs.googlepages.com/home Skype: DocDCB E-mail message checked by Spyware Doctor (6.0.1.441) Database version: 6.12830 http://www.pctools.com/en/spyware-doctor-antivirus/
Re: [ccp4bb] heavy atom derivative choice
Sebastiano-- http://www.sbg.bio.ic.ac.uk/had/ good table of HA and pH ranges http://www.shapirolab.org/lab-links/had.html links to a variety of HA databases HTH! annie Annie Hassell Glaxo Smithkline 5 Moore Drive RTP, NC 27709 919/483-3228 919/483-0368 (FAX) annie.m.hass...@gsk.com "Miguel Ortiz Lombardia" Sent by: "CCP4 bulletin board" 15-Jul-2009 09:21 Please respond to "Miguel Ortiz Lombardia" To CCP4BB@JISCMAIL.AC.UK cc Subject Re: [ccp4bb] heavy atom derivative choice Hi Sebastiano, Have a look at HATODAS: http://hatodas.harima.riken.go.jp/ Good luck, Miguel Le 15 juil. 09 à 14:33, Sebastiano Pasqualato a écrit : > Hi all, > I've got crystals of a protein of ca 200 residues, with 2 free > cysteines, 5 histidines, 2 methionines. > We have nice diffraction for the native crystals, that grow in 150 > mM KSCN, 17% PEG 3350, bis tris propane pH 8.8. > We are crystallising the SeMet derivative, but I'm not completely > sure I will be able to have nice crystals by saturday, when we have > tunable time at the ESRF. > I was thinking of trying with some heavy atom soaks, but only have > like 30 crystals, so limited trials allowed! > Which compound would you advice as more likely to work, and thus > worth testing? > Thanks in advance for the suggestions, > ciao > s > > > > -- > Sebastiano Pasqualato, PhD > IFOM-IEO Campus > Dipartimento di Oncologia Sperimentale > Istituto Europeo di Oncologia > via Adamello, 16 > 20139 - Milano > Italy > > tel +39 02 9437 5094 > > -- > This message has been scanned for viruses and > dangerous content by MailScanner, and is > believed to be clean. > -- Miguel Ortiz Lombardía Architecture et Fonction des Macromolécules Biologiques (UMR6098) CNRS, Universités d'Aix-Marseille I & II Case 932 163 Avenue de Luminy 13288 Marseille cedex 9 France Tel : +33(0) 491 82 55 93 Fax: +33(0) 491 26 67 20 e-mail: miguel.ortiz-lombar...@afmb.univ-mrs.fr Web: http://www.pangea.org/mol/spip.php?rubrique2 [attachment "emfalert.txt" deleted by Annie M Hassell/PharmRD/GSK]
Re: [ccp4bb] heavy atom derivative choice
Dear Sebastiano, why not have a look at http://sis.niaid.nih.gov/cgi-bin/heavyatom_reactivity.cgi. A reprint of the original paper can be found at http://sis.niaid.nih.gov/pub_pdf/Agniswamy-et-al-2008.pdf We did not specifically look at Bis-Tris Propane but did look at Tris pH 8.5 and Hepes pH 8. We certainly found greater reactivity in Hepes buffer with a number of mercury compounds and perhaps if your crystals can survive, switching to this buffer for the heavy atom soaking experiments should improve reactivity. I would definitely recommend short soaks (2-10 min) with high compound concentration (1-10 mM). Due to your limited crystal supply, the most reactive compounds we have found are in order. Ranking of the most reactive compounds % Derivitization Ethyl mercury (II) phosphate 69.4 Methyl mercury (II) Acetate 66.6 Sodium tetrachloro aurate 61.1 Potassium tetrabromo palatinate 55.5 Potassium tetrachloro aurate 52.7 Ammonium tetrachloro palatinate 50.0 Gold (III) chloride 47.2 Diamino platinum dinitrate 47.2 Thiomersal 47.2 Mercury (II) acetate 47.2 PCMBS 47.2 Potassium tetrachloro palatinate 44.4 Potassium tetranitro palatinate 44.4 Lead acetate 43.3 Potassium hexabromo palatinate 41.7 Methyl mercury (II) chloride 38.8 Mersalyl 38.8 Mercury(II)bromide 36.1 Mercury(II)cyanide 33.3 Gold chloride 33.3 Platinum potassium thiocyanate 33.3 Lead nitrate 33.3 Yours, Gordon M. Gordon Joyce, Visiting Fellow, Structural Immunology Section, Laboratory of Immunogenetics, NIH/NIAID, 12441 Parklawn Drive, Rockville, MD 20851 Phone: 301 594 0242 Office 301 496 3792 Lab
Re: [ccp4bb] heavy atom derivative choice
Dear All, I never tried it myself but could it be a sulphur SAD/MAD a possible alternative? In my experience with heavy atoms (luckily just 2 proteins!) you may waste all of your crystals without finding any heavy metal bound! Or may be the first one you try works Ciao Stefano -Original Message- From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Sebastiano Pasqualato Sent: Wednesday, 15 July, 2009 14:34 To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] heavy atom derivative choice Hi all, I've got crystals of a protein of ca 200 residues, with 2 free cysteines, 5 histidines, 2 methionines. We have nice diffraction for the native crystals, that grow in 150 mM KSCN, 17% PEG 3350, bis tris propane pH 8.8. We are crystallising the SeMet derivative, but I'm not completely sure I will be able to have nice crystals by saturday, when we have tunable time at the ESRF. I was thinking of trying with some heavy atom soaks, but only have like 30 crystals, so limited trials allowed! Which compound would you advice as more likely to work, and thus worth testing? Thanks in advance for the suggestions, ciao s -- Sebastiano Pasqualato, PhD IFOM-IEO Campus Dipartimento di Oncologia Sperimentale Istituto Europeo di Oncologia via Adamello, 16 20139 - Milano Italy tel +39 02 9437 5094
Re: [ccp4bb] heavy atom derivative choice
Hi, Mercury will likely form a precipitate with you SCN- anions... solubility in water is 0.6 g/L (about 3 mM) at 25°C... Since you are not in water, at less than 25°C, and with you high concentration of SCN- ions, don't expect to have high concentrations of *soluble* Hg++ in you medium. Prolongated soaking migh well allow some Hg++ to dissolve from Hg(SCN)2 and go into your crystal... but will this be okay for your synchrotron trip on saturday ?? I would therefore not rely only on HgII salts for this trip. With about 40 crystals, you might want to try other compounds. good luck ! Laurent David Briggs a écrit : Hi Sebastiano, Free Cys' are crying out for Mercury derivatives. Try 2-3 with varying sizes of additional groups - HgCl2, K2HgI4, PCMB, from the "Magic seven" would be a good place to start. Watch out for problems with your low pH, though. Some salts will form insoluble hydroxide salts at pH 8.8. (HgCl2 & K2HgI4 should be okay) Bart Hazes used to have an excellent set of notes online about heavy atom derivatisation - I can't seem to find the URL right now... HTH, David 2009/7/15 Sebastiano Pasqualato : Hi all, I've got crystals of a protein of ca 200 residues, with 2 free cysteines, 5 histidines, 2 methionines. We have nice diffraction for the native crystals, that grow in 150 mM KSCN, 17% PEG 3350, bis tris propane pH 8.8. We are crystallising the SeMet derivative, but I'm not completely sure I will be able to have nice crystals by saturday, when we have tunable time at the ESRF. I was thinking of trying with some heavy atom soaks, but only have like 30 crystals, so limited trials allowed! Which compound would you advice as more likely to work, and thus worth testing? Thanks in advance for the suggestions, ciao s -- Sebastiano Pasqualato, PhD IFOM-IEO Campus Dipartimento di Oncologia Sperimentale Istituto Europeo di Oncologia via Adamello, 16 20139 - Milano Italy tel +39 02 9437 5094 -- --- Laurent Maveyraud laurent.maveyraud AT ipbs DOT fr Université Paul Sabatier Toulouse III I.P.B.S. UMR 5089 Groupe de Biophysique Structurale Département Mécanismes Moléculaires des Infections Mycobactériennes 205 route de Narbonne 31077 TOULOUSE Cedex FRANCE Tél: +33 (0)561 175 435Fax : +33 (0)561 175 994 ---
Re: [ccp4bb] heavy atom derivative choice
Whps! Watch out for problems with your *HIGH* pH, though. Some salts will form insoluble hydroxide salts at pH 8.8. (HgCl2 & K2HgI4 should be okay). 2009/7/15 Sebastiano Pasqualato : > Hi all, > I've got crystals of a protein of ca 200 residues, with 2 free cysteines, 5 > histidines, 2 methionines. > We have nice diffraction for the native crystals, that grow in 150 mM KSCN, > 17% PEG 3350, bis tris propane pH 8.8. > We are crystallising the SeMet derivative, but I'm not completely sure I > will be able to have nice crystals by saturday, when we have tunable time at > the ESRF. > I was thinking of trying with some heavy atom soaks, but only have like 30 > crystals, so limited trials allowed! > Which compound would you advice as more likely to work, and thus worth > testing? > Thanks in advance for the suggestions, > ciao > s > > > > -- > Sebastiano Pasqualato, PhD > IFOM-IEO Campus > Dipartimento di Oncologia Sperimentale > Istituto Europeo di Oncologia > via Adamello, 16 > 20139 - Milano > Italy > > tel +39 02 9437 5094 > -- David C. Briggs PhD Father & Crystallographer http://drdavidcbriggs.googlepages.com/home Skype: DocDCB
Re: [ccp4bb] heavy atom derivative choice
Hi Sebastiano, Given that your crystallization condition contains KSCN, I would certainly give Hg(SCN)2 a go among other Hg options for a protein containing free cysteines. Hg(SCN)2 might end up being more compatible with your crystal form, and might thus minimize non-isomorphism issues. Best of luck Savvas -Original Message- From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of David Briggs Sent: Wednesday, July 15, 2009 3:27 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] heavy atom derivative choice Hi Sebastiano, Free Cys' are crying out for Mercury derivatives. Try 2-3 with varying sizes of additional groups - HgCl2, K2HgI4, PCMB, from the "Magic seven" would be a good place to start. Watch out for problems with your low pH, though. Some salts will form insoluble hydroxide salts at pH 8.8. (HgCl2 & K2HgI4 should be okay) Bart Hazes used to have an excellent set of notes online about heavy atom derivatisation - I can't seem to find the URL right now... HTH, David 2009/7/15 Sebastiano Pasqualato : > Hi all, > I've got crystals of a protein of ca 200 residues, with 2 free cysteines, 5 > histidines, 2 methionines. > We have nice diffraction for the native crystals, that grow in 150 mM KSCN, > 17% PEG 3350, bis tris propane pH 8.8. > We are crystallising the SeMet derivative, but I'm not completely sure I > will be able to have nice crystals by saturday, when we have tunable time at > the ESRF. > I was thinking of trying with some heavy atom soaks, but only have like 30 > crystals, so limited trials allowed! > Which compound would you advice as more likely to work, and thus worth > testing? > Thanks in advance for the suggestions, > ciao > s > > > > -- > Sebastiano Pasqualato, PhD > IFOM-IEO Campus > Dipartimento di Oncologia Sperimentale > Istituto Europeo di Oncologia > via Adamello, 16 > 20139 - Milano > Italy > > tel +39 02 9437 5094 > -- David C. Briggs PhD Father & Crystallographer http://drdavidcbriggs.googlepages.com/home Skype: DocDCB E-mail message checked by Spyware Doctor (6.0.1.441) Database version: 6.12830 http://www.pctools.com/en/spyware-doctor-antivirus/
Re: [ccp4bb] heavy atom derivative choice
Hi Sebastiano, Free Cys' are crying out for Mercury derivatives. Try 2-3 with varying sizes of additional groups - HgCl2, K2HgI4, PCMB, from the "Magic seven" would be a good place to start. Watch out for problems with your low pH, though. Some salts will form insoluble hydroxide salts at pH 8.8. (HgCl2 & K2HgI4 should be okay) Bart Hazes used to have an excellent set of notes online about heavy atom derivatisation - I can't seem to find the URL right now... HTH, David 2009/7/15 Sebastiano Pasqualato : > Hi all, > I've got crystals of a protein of ca 200 residues, with 2 free cysteines, 5 > histidines, 2 methionines. > We have nice diffraction for the native crystals, that grow in 150 mM KSCN, > 17% PEG 3350, bis tris propane pH 8.8. > We are crystallising the SeMet derivative, but I'm not completely sure I > will be able to have nice crystals by saturday, when we have tunable time at > the ESRF. > I was thinking of trying with some heavy atom soaks, but only have like 30 > crystals, so limited trials allowed! > Which compound would you advice as more likely to work, and thus worth > testing? > Thanks in advance for the suggestions, > ciao > s > > > > -- > Sebastiano Pasqualato, PhD > IFOM-IEO Campus > Dipartimento di Oncologia Sperimentale > Istituto Europeo di Oncologia > via Adamello, 16 > 20139 - Milano > Italy > > tel +39 02 9437 5094 > -- David C. Briggs PhD Father & Crystallographer http://drdavidcbriggs.googlepages.com/home Skype: DocDCB
Re: [ccp4bb] heavy atom derivative choice
Hi Sebastiano, Have a look at HATODAS: http://hatodas.harima.riken.go.jp/ Good luck, Miguel Le 15 juil. 09 à 14:33, Sebastiano Pasqualato a écrit : Hi all, I've got crystals of a protein of ca 200 residues, with 2 free cysteines, 5 histidines, 2 methionines. We have nice diffraction for the native crystals, that grow in 150 mM KSCN, 17% PEG 3350, bis tris propane pH 8.8. We are crystallising the SeMet derivative, but I'm not completely sure I will be able to have nice crystals by saturday, when we have tunable time at the ESRF. I was thinking of trying with some heavy atom soaks, but only have like 30 crystals, so limited trials allowed! Which compound would you advice as more likely to work, and thus worth testing? Thanks in advance for the suggestions, ciao s -- Sebastiano Pasqualato, PhD IFOM-IEO Campus Dipartimento di Oncologia Sperimentale Istituto Europeo di Oncologia via Adamello, 16 20139 - Milano Italy tel +39 02 9437 5094 -- This message has been scanned for viruses and dangerous content by MailScanner, and is believed to be clean. -- Miguel Ortiz Lombardía Architecture et Fonction des Macromolécules Biologiques (UMR6098) CNRS, Universités d'Aix-Marseille I & II Case 932 163 Avenue de Luminy 13288 Marseille cedex 9 France Tel : +33(0) 491 82 55 93 Fax: +33(0) 491 26 67 20 e-mail: miguel.ortiz-lombar...@afmb.univ-mrs.fr Web: http://www.pangea.org/mol/spip.php?rubrique2 smime.p7s Description: S/MIME cryptographic signature
[ccp4bb] heavy atom derivative choice
Hi all, I've got crystals of a protein of ca 200 residues, with 2 free cysteines, 5 histidines, 2 methionines. We have nice diffraction for the native crystals, that grow in 150 mM KSCN, 17% PEG 3350, bis tris propane pH 8.8. We are crystallising the SeMet derivative, but I'm not completely sure I will be able to have nice crystals by saturday, when we have tunable time at the ESRF. I was thinking of trying with some heavy atom soaks, but only have like 30 crystals, so limited trials allowed! Which compound would you advice as more likely to work, and thus worth testing? Thanks in advance for the suggestions, ciao s -- Sebastiano Pasqualato, PhD IFOM-IEO Campus Dipartimento di Oncologia Sperimentale Istituto Europeo di Oncologia via Adamello, 16 20139 - Milano Italy tel +39 02 9437 5094
