Re: [ccp4bb] crystal dehydration
Judith, If you can, try monitoring the diffraction during the dehydration experiment. There are plenty of ways of doing this. Perhaps the simplest is to shoot your crystals in-situ after swapping the precipitant for a more concentrated one. The is also the Free Mounting System. David Hargreaves Associate Principal Scientist _ AstraZeneca Discovery Sciences, Structure Biophysics Mereside, 50F49, Alderley Park, Cheshire, SK10 4TF Tel +44 (0)01625 518521 Fax +44 (0) 1625 232693 David.Hargreaves @astrazeneca.com Please consider the environment before printing this e-mail -- AstraZeneca UK Limited is a company incorporated in England and Wales with registered number: 03674842 and a registered office at 2 Kingdom Street, London, W2 6BD. Confidentiality Notice: This message is private and may contain confidential, proprietary and legally privileged information. If you have received this message in error, please notify us and remove it from your system and note that you must not copy, distribute or take any action in reliance on it. Any unauthorised use or disclosure of the contents of this message is not permitted and may be unlawful. Disclaimer: Email messages may be subject to delays, interception, non-delivery and unauthorised alterations. Therefore, information expressed in this message is not given or endorsed by AstraZeneca UK Limited unless otherwise notified by an authorised representative independent of this message. No contractual relationship is created by this message by any person unless specifically indicated by agreement in writing other than email. Monitoring: AstraZeneca UK Limited may monitor email traffic data and content for the purposes of the prevention and detection of crime, ensuring the security of our computer systems and checking Compliance with our Code of Conduct and Policies. -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Judith A Ronau Sent: 15 January 2013 14:47 To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] crystal dehydration Greetings! I have recently been attempting crystal dehydration experiments to improve diffraction following the procedures from the ERSF in which crystals are exposed to increased concentrations of precipitant. I would like to know if anyone knew of any alternative methods for dehydration of protein crystals. Thanks! Best, Judith
Re: [ccp4bb] crystal dehydration
Indeed! We have a short communication in Acta D that is under revision (no preprint yet, sorry!), going in the same direction than what Hagelueken et al have done (using saturated salt solutions in the reservoir and transferring your crystals in perfluoropolyether oil (see paper here that we have just seen: http://journals.iucr.org/d/issues/2012/10/00/bw5408/bw5408.pdf), but instead of transferring the crystals in the 96 wells, you grow your crystals first in 96-wells, you then pierce your tray with a thin needle and add your gradient of salt concentration in the reservoir, re-seal it, let it equilibrate and use in-situ plate screening facilities available in several European synchrotrons (@ Diamond, SLS, Bessy, ESRF-FIP beamline) or in-house facilities to get an x-ray feedback of your experiment. You would follow changes in your cell parameters (like with the HC1). The advantage of this is that hopefully you get an overall picture of whether dehydration has an effect on your samples within one experiment without handling your crystals (which could add another detrimental parameter to your samples). This is a work in progress at Diamond and we are happy to help/talk to anyone who would like to try it, just contact us: Alice Douangamath (alice.douangam...@diamond.ac.uk) Pierre Aller (pierre.al...@diamond.ac.uk) Juan Sanchez-Weatherby (juan.sanchez-weathe...@diamond.ac.uk) Jose Brandao-Neto (jose.brandao-n...@diamond.ac.uk) -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Hargreaves, David Sent: 16 January 2013 12:37 To: ccp4bb Subject: Re: [ccp4bb] crystal dehydration Judith, If you can, try monitoring the diffraction during the dehydration experiment. There are plenty of ways of doing this. Perhaps the simplest is to shoot your crystals in-situ after swapping the precipitant for a more concentrated one. The is also the Free Mounting System. David Hargreaves Associate Principal Scientist _ AstraZeneca Discovery Sciences, Structure Biophysics Mereside, 50F49, Alderley Park, Cheshire, SK10 4TF Tel +44 (0)01625 518521 Fax +44 (0) 1625 232693 David.Hargreaves @astrazeneca.com Please consider the environment before printing this e-mail -- AstraZeneca UK Limited is a company incorporated in England and Wales with registered number: 03674842 and a registered office at 2 Kingdom Street, London, W2 6BD. Confidentiality Notice: This message is private and may contain confidential, proprietary and legally privileged information. If you have received this message in error, please notify us and remove it from your system and note that you must not copy, distribute or take any action in reliance on it. Any unauthorised use or disclosure of the contents of this message is not permitted and may be unlawful. Disclaimer: Email messages may be subject to delays, interception, non-delivery and unauthorised alterations. Therefore, information expressed in this message is not given or endorsed by AstraZeneca UK Limited unless otherwise notified by an authorised representative independent of this message. No contractual relationship is created by this message by any person unless specifically indicated by agreement in writing other than email. Monitoring: AstraZeneca UK Limited may monitor email traffic data and content for the purposes of the prevention and detection of crime, ensuring the security of our computer systems and checking Compliance with our Code of Conduct and Policies. -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Judith A Ronau Sent: 15 January 2013 14:47 To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] crystal dehydration Greetings! I have recently been attempting crystal dehydration experiments to improve diffraction following the procedures from the ERSF in which crystals are exposed to increased concentrations of precipitant. I would like to know if anyone knew of any alternative methods for dehydration of protein crystals. Thanks! Best, Judith
[ccp4bb] crystal dehydration
Greetings! I have recently been attempting crystal dehydration experiments to improve diffraction following the procedures from the ERSF in which crystals are exposed to increased concentrations of precipitant. I would like to know if anyone knew of any alternative methods for dehydration of protein crystals. Thanks! Best, Judith
Re: [ccp4bb] crystal dehydration
Dear Judith, in Acta Cryst D61 (2005), 1173-1180 different handling methods for dehydration are described besides other post-crystallization treatments to improve diffraction quality. You can also try Dehydration Salts. A screen with ready made salt solutions is available from Jena Bioscience. Useful might also be the MicroRT System from Mitegen. Ulrike
Re: [ccp4bb] crystal dehydration
Dear Judith, Indeed we do. Please see our website for further details. http://www.diamond.ac.uk/Home/Beamlines/MX/I02/Humidifier.html Let me know if you have any questions or need any further information. Hope it helps Juan Juan Sanchez-Weatherby, PhD Beamline Scientist - I02 Tel: +44 (0)1235 778661 Mob:+44 (0)7795 641259 -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Judith A Ronau Sent: 15 January 2013 14:47 To: ccp4bb Subject: [ccp4bb] crystal dehydration Greetings! I have recently been attempting crystal dehydration experiments to improve diffraction following the procedures from the ERSF in which crystals are exposed to increased concentrations of precipitant. I would like to know if anyone knew of any alternative methods for dehydration of protein crystals. Thanks! Best, Judith
Re: [ccp4bb] crystal dehydration
Hi Judith, a very effective method is the use of a humidity control device - this was actually the reason we developed the equations that predict the RH in equilibrium with precipitant solutions. It has the great advantage that you can characterize changes that occur and also move straight to data collection. There are several HC1 devices (developed here at the EMBL) in Europe and at least 1 in the USA - there is also the FMS. Below are sole links that might help, best wishes, Matt. Website for experiments: http://www.esrf.eu/UsersAndScience/Experiments/MX/About_our_beamlines/ID14-2/HC1b Calculation server for mother liquor RH equilibria: http://go.esrf.eu/RH Paper describing above: http://scripts.iucr.org/cgi-bin/paper?S1744309111054029 Papers describing device and methods: http://journals.iucr.org/d/issues/2009/12/00/gm5010/index.html and http://www.sciencedirect.com/science/article/pii/S1047847711000499 Interface in MXCuBE that allows the design of dehydration gradients, data collection and analysis of the images http://www.esrf.eu/UsersAndScience/Experiments/MX/How_to_use_our_beamlines/workflows/dehydration-workflow. On 2013-01-15 15:46, Judith A Ronau wrote: Greetings! I have recently been attempting crystal dehydration experiments to improve diffraction following the procedures from the ERSF in which crystals are exposed to increased concentrations of precipitant. I would like to know if anyone knew of any alternative methods for dehydration of protein crystals. Thanks! Best, Judith -- Matthew Bowler Synchrotron Science Group European Molecular Biology Laboratory BP 181, 6 rue Jules Horowitz 38042 Grenoble Cedex 9 France === Tel: +33 (0) 4.76.20.76.37 Fax: +33 (0) 4.76.88.29.04 http://www.embl.fr/ ===
Re: [ccp4bb] crystal dehydration
In case if dehydration needs to be done slowly and under tight control of all parameters, one possibility is to use micro-dialysis buttons. We used it for a large membrane protein complex and diffraction improved from ~7 to 2.7 A. The crystal is fished out and put into mother liquor solution in the button, sealed with dialysis membrane and the button is then placed into about 5 mls of mother liquor with slightly higher PEG concentration. Then you just exchange outside buffer every day or so for solutions containing higher concentrations of PEG. We went from ~9 to 30 % PEG4000 in about a week. You can easily observe crystal under microscope and if it cracks - you went too far/too quickly with PEG and need to use a bit less next time. Also, this method allows you to control all other components of the dehydrating solution - we needed to decrease salt concentration at the same time as increasing PEG. You can also introduce/increase cryo-protectant concentration at the same time. With these crystals, otherwise excellent dehydration machines already mentioned did not work, possibly because the process had to be really slow. The reference is here: http://www.ncbi.nlm.nih.gov/pubmed/21822288 Best wishes.
Re: [ccp4bb] crystal dehydration
Hi Partha, as far as I know the HC1 is available at the CLS and soon at the APS and ALS, best wishes, Matthew. On 2013-01-15 22:27, Parthasarathy Sampathkumar wrote: Dear Dr. Bowler, Which beamline in USA has this Humidity Control Device? Best Wishes, Partha On Tue, Jan 15, 2013 at 11:02 AM, Matthew BOWLER mbow...@embl.fr [10] wrote: Hi Judith, a very effective method is the use of a humidity control device - this was actually the reason we developed the equations that predict the RH in equilibrium with precipitant solutions. It has the great advantage that you can characterize changes that occur and also move straight to data collection. There are several HC1 devices (developed here at the EMBL) in Europe and at least 1 in the USA - there is also the FMS. Below are sole links that might help, best wishes, Matt. Website for experiments: http://www.esrf.eu/UsersAndScience/Experiments/MX/About_our_beamlines/ID14-2/HC1b [1] Calculation server for mother liquor RH equilibria: http://go.esrf.eu/RH [2] Paper describing above: http://scripts.iucr.org/cgi-bin/paper?S1744309111054029 [3] Papers describing device and methods: http://journals.iucr.org/d/issues/2009/12/00/gm5010/index.html [4] and http://www.sciencedirect.com/science/article/pii/S1047847711000499 [5] Interface in MXCuBE that allows the design of dehydration gradients, data collection and analysis of the images http://www.esrf.eu/UsersAndScience/Experiments/MX/How_to_use_our_beamlines/workflows/dehydration-workflow [6]. On 2013-01-15 15:46, Judith A Ronau wrote: Greetings! I have recently been attempting crystal dehydration experiments to improve diffraction following the procedures from the ERSF in which crystals are exposed to increased concentrations of precipitant. I would like to know if anyone knew of any alternative methods for dehydration of protein crystals. Thanks! Best, Judith -- Matthew Bowler Synchrotron Science Group European Molecular Biology Laboratory BP 181, 6 rue Jules Horowitz 38042 Grenoble Cedex 9 France === Tel: +33 (0) 4.76.20.76.37 [7] Fax: +33 (0) 4.76.88.29.04 [8] http://www.embl.fr/ [9] === Links: -- [1] http://www.esrf.eu/UsersAndScience/Experiments/MX/About_our_beamlines/ID14-2/HC1b [2] http://go.esrf.eu/RH [3] http://scripts.iucr.org/cgi-bin/paper?S1744309111054029 [4] http://journals.iucr.org/d/issues/2009/12/00/gm5010/index.html [5] http://www.sciencedirect.com/science/article/pii/S1047847711000499 [6] http://www.esrf.eu/UsersAndScience/Experiments/MX/How_to_use_our_beamlines/workflows/dehydration-workflow [7] http://embl.fr/tel:%2B33%20%280%29%204.76.20.76.37 [8] http://embl.fr/tel:%2B33%20%280%29%204.76.88.29.04 [9] http://www.embl.fr/ [10] mailto:mbow...@embl.fr -- Matthew Bowler Synchrotron Science Group European Molecular Biology Laboratory BP 181, 6 rue Jules Horowitz 38042 Grenoble Cedex 9 France === Tel: +33 (0) 4.76.20.76.37 Fax: +33 (0) 4.76.88.29.04 http://www.embl.fr/ ===
