Re: [ccp4bb] Cryoprotection in 3M ammonium sulfate
I third it - we used 3.2M + 5% glycerol ammonium sulfate to cryoprotect crystals grown in 1.2M ammonium suphate (Bayliss et al. JBC 2002). This really helped diffraction too. My tip when handling drops at very high salt is to make a little circle of wet tissue paper around your coverslip to make a humidity chamber. You'll have much longer to fish crystals out of the drop before salt crystals start to form. Good luck Richard Dr Richard Bayliss Royal Society University Research Fellow Career Development Team Leader Section of Structural Biology Institute of Cancer Research 237 Fulham Road London SW3 6JB UK Tel: +44 (0)20 71535557 Fax: +44 (0)20 71535457 On 19 Aug 2009, at 7:20 PM, James Holton wrote: I second that one. Ammonium sulfate is one of my favorite cryos. I recommend making up a saturated solution of ammonium sulfate, as it is actually a very good cryo all by itself, and then dilute it by adding the rest of the stuff in your condition (buffers, etc. and a little bit of water). You want to be a little below saturation so that the salt does not grow crystals of its own while you are soaking. This is especially important if your protein crystals are hexagonal rods! -James Holton MAD Scientist Savvas Savvides wrote: Hi Brenda Try 3M ammonium sulfate itself! We have tried that with great succes by cryo-cooling xtals grown at 3.2M AS straight out of their crystallization drops. You can consult the MM section in Kyndt J. et al Biochemistry 2007 Jan 9;46(1):95-105 for a more detailed description of what we did. Best of luck Savvas Savvas Savvides L-ProBE, Unit for Structural Biology Ghent University K.L. Ledeganckstraat 35 9000 Ghent, BELGIUM office: +32- (0)9-264.51.24 ; mobile: +32-(0)472-92.85.19 Email: savvas.savvi...@ugent.be http://www.lprobe.ugent.be/xray.html -Original Message- From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Schulman, Brenda Sent: Wednesday, August 19, 2009 6:19 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Cryoprotection in 3M ammonium sulfate Hello! I would be grateful for suggestions on cryoprotectants for crystals growing in 3M ammonium sulfate. Thanks! Brenda Email Disclaimer: www.stjude.org/emaildisclaimer E-mail message checked by Spyware Doctor (6.1.0.447) Database version: 6.13080 http://www.pctools.com/en/spyware-doctor-antivirus/ The Institute of Cancer Research: Royal Cancer Hospital, a charitable Company Limited by Guarantee, Registered in England under Company No. 534147 with its Registered Office at 123 Old Brompton Road, London SW7 3RP. This e-mail message is confidential and for use by the addressee only. If the message is received by anyone other than the addressee, please return the message to the sender by replying to it and then delete the message from your computer and network.
Re: [ccp4bb] Cryoprotection in 3M ammonium sulfate
Hi Brenda, Maybe you want to have a look at the cryoprotectant database for protein crystals. http://idb.exst.jaxa.jp/db_data/protein/search-e.php There you can search for your precipitant and get a list of compatible freezing conditions that worked before including the reference. It is a nice starting point and a good inspiration. Joerg On 19 Aug 2009, at 17:18, Schulman, Brenda wrote: Hello! I would be grateful for suggestions on cryoprotectants for crystals growing in 3M ammonium sulfate. Thanks! Brenda Email Disclaimer: www.stjude.org/emaildisclaimer *** Jörg Standfuss Medical Research Council Laboratory of Molecular Biology Hills Road Cambridge CB2 0QH
[ccp4bb] Cryoprotection in 3M ammonium sulfate
Hello! I would be grateful for suggestions on cryoprotectants for crystals growing in 3M ammonium sulfate. Thanks! Brenda Email Disclaimer: www.stjude.org/emaildisclaimer
Re: [ccp4bb] Cryoprotection in 3M ammonium sulfate
Hi Brenda, You can try sugars like glucose, trehalose and sucrose for high AS contents. It has been succesfully used in really hard cases such at protein RNA crystals grown in AS. see Acta Cryst (2002) D58 1664-1669 Garber et al. HTH Pascal Egea
Re: [ccp4bb] Cryoprotection in 3M ammonium sulfate
Soluble possibilities include glucose, ethylene glycol, DMSO, glycerol (15-30%). You may not need much cryoprotectant at all at these salt concentrations. PEGs are not very soluble in high ammonium sulfate solutions. (I think PEG-400 can go up to only 4% or so at 2 M ammonium sulfate.) Cheers. Schulman, Brenda wrote: Hello! I would be grateful for suggestions on cryoprotectants for crystals growing in 3M ammonium sulfate. Thanks! Brenda Email Disclaimer: www.stjude.org/emaildisclaimer -- Roger S. Rowlett Professor Colgate University Presidential Scholar 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...@mail.colgate.edu
Re: [ccp4bb] Cryoprotection in 3M ammonium sulfate
Dear Brenda Check out malonate http://www.ncbi.nlm.nih.gov/pubmed/14646118? ordinalpos=2itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubm ed_DefaultReportPanel.Pubmed_RVDocSum Malonate: a versatile cryoprotectant and stabilizing solution for salt-grown macromolecular crystals. Holyoak T, Fenn TD, Wilson MA, Moulin AG, Ringe D, Petsko GA. Regards Christine On 19/08/2009, at 9:18 AM, Schulman, Brenda wrote: Hello! I would be grateful for suggestions on cryoprotectants for crystals growing in 3M ammonium sulfate. Thanks! Brenda Email Disclaimer: www.stjude.org/emaildisclaimer
Re: [ccp4bb] Cryoprotection in 3M ammonium sulfate
Hi Brenda Try 3M ammonium sulfate itself! We have tried that with great succes by cryo-cooling xtals grown at 3.2M AS straight out of their crystallization drops. You can consult the MM section in Kyndt J. et al Biochemistry 2007 Jan 9;46(1):95-105 for a more detailed description of what we did. Best of luck Savvas Savvas Savvides L-ProBE, Unit for Structural Biology Ghent University K.L. Ledeganckstraat 35 9000 Ghent, BELGIUM office: +32-(0)9-264.51.24 ; mobile: +32-(0)472-92.85.19 Email: savvas.savvi...@ugent.be http://www.lprobe.ugent.be/xray.html -Original Message- From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Schulman, Brenda Sent: Wednesday, August 19, 2009 6:19 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Cryoprotection in 3M ammonium sulfate Hello! I would be grateful for suggestions on cryoprotectants for crystals growing in 3M ammonium sulfate. Thanks! Brenda Email Disclaimer: www.stjude.org/emaildisclaimer E-mail message checked by Spyware Doctor (6.1.0.447) Database version: 6.13080 http://www.pctools.com/en/spyware-doctor-antivirus/
Re: [ccp4bb] Cryoprotection in 3M ammonium sulfate
Dear Brenda, I'd suggest using a mixture of 75% Paratone-N oil with 25% light, white mineral oil. Mix the two thoroughly with a positive displacement pipette then add a small amount next to the mother liquor (ML) drop. Remove a crystal from the ML using a matched cryo loop (the smaller the better) and then dip it completely into the oil. Gently slide the side of the loop containing the crystal against the surface beside the ML to wick off some of the oil, which will also remove some of the aqueous layer on the crystal surface. Then dip the crystal back into the oil; it usually takes 2-3 times to completely remove the aqueous layer. This works about 80% of the time for me for crystals grown from high salt conditions. Method courtesy of A. McPherson and J. Pflugrath. HTH, Jeff -Original Message- From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Schulman, Brenda Sent: Wednesday, August 19, 2009 12:19 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Cryoprotection in 3M ammonium sulfate Hello! I would be grateful for suggestions on cryoprotectants for crystals growing in 3M ammonium sulfate. Thanks! Brenda Email Disclaimer: www.stjude.org/emaildisclaimer
Re: [ccp4bb] Cryoprotection in 3M ammonium sulfate
Or saturated LiSO4 works too. Should work in this case. -Tom Tom J. Brett, PhD Assistant Professor of Medicine Division of Pulmonary and Critical Care Washington University School of Medicine Campus Box 8052, 660 S. Euclid Saint Louis, MO 63110 From: CCP4 bulletin board [ccp...@jiscmail.ac.uk] On Behalf Of Savvas Savvides [savvas.savvi...@ugent.be] Sent: Wednesday, August 19, 2009 11:47 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Cryoprotection in 3M ammonium sulfate Hi Brenda Try 3M ammonium sulfate itself! We have tried that with great succes by cryo-cooling xtals grown at 3.2M AS straight out of their crystallization drops. You can consult the MM section in Kyndt J. et al Biochemistry 2007 Jan 9;46(1):95-105 for a more detailed description of what we did. Best of luck Savvas Savvas Savvides L-ProBE, Unit for Structural Biology Ghent University K.L. Ledeganckstraat 35 9000 Ghent, BELGIUM office: +32-(0)9-264.51.24 ; mobile: +32-(0)472-92.85.19 Email: savvas.savvi...@ugent.be http://www.lprobe.ugent.be/xray.html -Original Message- From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Schulman, Brenda Sent: Wednesday, August 19, 2009 6:19 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Cryoprotection in 3M ammonium sulfate Hello! I would be grateful for suggestions on cryoprotectants for crystals growing in 3M ammonium sulfate. Thanks! Brenda Email Disclaimer: www.stjude.org/emaildisclaimer E-mail message checked by Spyware Doctor (6.1.0.447) Database version: 6.13080 http://www.pctools.com/en/spyware-doctor-antivirus/
Re: [ccp4bb] Cryoprotection in 3M ammonium sulfate
I second that one. Ammonium sulfate is one of my favorite cryos. I recommend making up a saturated solution of ammonium sulfate, as it is actually a very good cryo all by itself, and then dilute it by adding the rest of the stuff in your condition (buffers, etc. and a little bit of water). You want to be a little below saturation so that the salt does not grow crystals of its own while you are soaking. This is especially important if your protein crystals are hexagonal rods! -James Holton MAD Scientist Savvas Savvides wrote: Hi Brenda Try 3M ammonium sulfate itself! We have tried that with great succes by cryo-cooling xtals grown at 3.2M AS straight out of their crystallization drops. You can consult the MM section in Kyndt J. et al Biochemistry 2007 Jan 9;46(1):95-105 for a more detailed description of what we did. Best of luck Savvas Savvas Savvides L-ProBE, Unit for Structural Biology Ghent University K.L. Ledeganckstraat 35 9000 Ghent, BELGIUM office: +32-(0)9-264.51.24 ; mobile: +32-(0)472-92.85.19 Email: savvas.savvi...@ugent.be http://www.lprobe.ugent.be/xray.html -Original Message- From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Schulman, Brenda Sent: Wednesday, August 19, 2009 6:19 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Cryoprotection in 3M ammonium sulfate Hello! I would be grateful for suggestions on cryoprotectants for crystals growing in 3M ammonium sulfate. Thanks! Brenda Email Disclaimer: www.stjude.org/emaildisclaimer E-mail message checked by Spyware Doctor (6.1.0.447) Database version: 6.13080 http://www.pctools.com/en/spyware-doctor-antivirus/
