Re: [ccp4bb] Bypassing phase separation for nice crystals.
Hi Timur, I've had a crystal which sounds similar to yours. When I came to freeze it noticed that it was not exactly solid more amorphous like treacle. Laughingly I went ahead and screened it anyway and was shocked to see diffraction to around 15Ang. I was lucky enough to have other crystals in the same screen but if that had been the only hit maybe some techniques like re-annealing, dehydrating, shifting pH osmotic shock might help. Any diffraction (order!) is better than no diffraction. Dave David Hargreaves Associate Principal Scientist _ AstraZeneca DECS, CPSS Mereside, 50F49, Alderley Park, Cheshire, SK10 4TF Tel +44 (0)01625 518521 Fax +44 (0) 1625 232693 David.Hargreaves @astrazeneca.com mailto:name.surn...@astrazeneca.com Please consider the environment before printing this e-mail From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of F. Timur Senguen Sent: 18 July 2011 14:53 To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Bypassing phase separation for nice crystals. Hi everyone, I have been issues with a particular protein. I have been close for a while, but yet so far. Rather than going from a clear drop to crystal, my protein first undergoes phase separation (large oily drops) in which one phase contains most, if not all, of the protein. This phase separation occurs within a day of preparing the drop. A day after phase separation the oily phase is now a large disordered crystalline mass which does not diffract very well. I have tried changing buffer concentrations, precipitant amounts, ionic strengths and pH and in all cases this phenomenon is observed. I even screened protein concentrations to see if reducing protein concentration would prevent the phase separation. Is there any way to bypass this phase separation, which I think prevents me from obtaining nice crystals. Should I try detergents, chaotropes, or other additives? Thanks in advance. Timur -- F. Timur Senguen, Ph.D. Postdoctoral Research Fellow Boston Biomedical Research Institute 64 Grove Street, Watertown, MA 02472 USA -- 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.
Re: [ccp4bb] Bypassing phase separation for nice crystals.
Thanks for all the suggestions. I forgot to mention that I had tried microseeding and it was partially successful. I am trying this while also pursuing other venues. At Florian's suggestion I have set up a free interface diffusion method and it looks very promising. I have a few free floating crystals that seem somewhat flawed, but otherwise very nice. I will try combining microseeding techniques and free interface diffusion to make THE crystal. Timur On Mon, Jul 18, 2011 at 11:01 AM, Florian Sauer sa...@embl-hamburg.dewrote: Dear Timur, one possibility to handle this problem can be the change from vapor (I assume this is what you do) to the free interface diffusion method. Phase separation often occurs if the protein is immediately exposed to the full precipitant concentration while it might not escape into its own phase if it gets slowly equilibrated. There are commercial setups available for this method but you can also do it in a normal vapor diffusion plate. To do so, just put the protein and precipitant drops next to each other and then link them through a thin liquid bridge. Requires some practice and works best with large drops but helped me in several similar cases. Good luck! Florian Am 18.07.11 15:52, schrieb F. Timur Senguen: Hi everyone, I have been issues with a particular protein. I have been close for a while, but yet so far. Rather than going from a clear drop to crystal, my protein first undergoes phase separation (large oily drops) in which one phase contains most, if not all, of the protein. This phase separation occurs within a day of preparing the drop. A day after phase separation the oily phase is now a large disordered crystalline mass which does not diffract very well. I have tried changing buffer concentrations, precipitant amounts, ionic strengths and pH and in all cases this phenomenon is observed. I even screened protein concentrations to see if reducing protein concentration would prevent the phase separation. Is there any way to bypass this phase separation, which I think prevents me from obtaining nice crystals. Should I try detergents, chaotropes, or other additives? Thanks in advance. Timur -- F. Timur Senguen, Ph.D. Postdoctoral Research Fellow Boston Biomedical Research Institute 64 Grove Street, Watertown, MA 02472 USA -- F. Timur Senguen, Ph.D. Postdoctoral Research Fellow Boston Biomedical Research Institute 64 Grove Street, Watertown, MA 02472 USA
[ccp4bb] Bypassing phase separation for nice crystals.
Hi everyone, I have been issues with a particular protein. I have been close for a while, but yet so far. Rather than going from a clear drop to crystal, my protein first undergoes phase separation (large oily drops) in which one phase contains most, if not all, of the protein. This phase separation occurs within a day of preparing the drop. A day after phase separation the oily phase is now a large disordered crystalline mass which does not diffract very well. I have tried changing buffer concentrations, precipitant amounts, ionic strengths and pH and in all cases this phenomenon is observed. I even screened protein concentrations to see if reducing protein concentration would prevent the phase separation. Is there any way to bypass this phase separation, which I think prevents me from obtaining nice crystals. Should I try detergents, chaotropes, or other additives? Thanks in advance. Timur -- F. Timur Senguen, Ph.D. Postdoctoral Research Fellow Boston Biomedical Research Institute 64 Grove Street, Watertown, MA 02472 USA
Re: [ccp4bb] Bypassing phase separation for nice crystals.
Hi Timur, have you tried seeding from your microstalline stuff? Might be worth to try! Cheers, Albert Albert GUSKOV (Dr) | Research Fellow | Division of Structural Computational Biology | Nanyang Technological University Proteos 7-01, Biopolis Drive 61, Singapore 138673 Tel: (65) 6586-9690 GMT+8h | Cell: (65) 8366-2779 | Email: a.gus...@ntu.edu.sg | Web: www.ntu.edu.sg 2011/7/18 F. Timur Senguen ftseng...@gmail.com Hi everyone, I have been issues with a particular protein. I have been close for a while, but yet so far. Rather than going from a clear drop to crystal, my protein first undergoes phase separation (large oily drops) in which one phase contains most, if not all, of the protein. This phase separation occurs within a day of preparing the drop. A day after phase separation the oily phase is now a large disordered crystalline mass which does not diffract very well. I have tried changing buffer concentrations, precipitant amounts, ionic strengths and pH and in all cases this phenomenon is observed. I even screened protein concentrations to see if reducing protein concentration would prevent the phase separation. Is there any way to bypass this phase separation, which I think prevents me from obtaining nice crystals. Should I try detergents, chaotropes, or other additives? Thanks in advance. Timur -- F. Timur Senguen, Ph.D. Postdoctoral Research Fellow Boston Biomedical Research Institute 64 Grove Street, Watertown, MA 02472 USA
Re: [ccp4bb] Bypassing phase separation for nice crystals.
Original-Nachricht Betreff:Re: [ccp4bb] Bypassing phase separation for nice crystals. Datum: Mon, 18 Jul 2011 17:01:27 +0200 Von:Florian Sauer sa...@embl-hamburg.de An: F. Timur Senguen ftseng...@gmail.com CC: CCP4BB@JISCMAIL.AC.UK Dear Timur, one possibility to handle this problem can be the change from vapor (I assume this is what you do) to the free interface diffusion method. Phase separation often occurs if the protein is immediately exposed to the full precipitant concentration while it might not escape into its own phase if it gets slowly equilibrated. There are commercial setups available for this method but you can also do it in a normal vapor diffusion plate. To do so, just put the protein and precipitant drops next to each other and then link them through a thin liquid bridge. Requires some practice and works best with large drops but helped me in several similar cases. Good luck! Florian Am 18.07.11 15:52, schrieb F. Timur Senguen: Hi everyone, I have been issues with a particular protein. I have been close for a while, but yet so far. Rather than going from a clear drop to crystal, my protein first undergoes phase separation (large oily drops) in which one phase contains most, if not all, of the protein. This phase separation occurs within a day of preparing the drop. A day after phase separation the oily phase is now a large disordered crystalline mass which does not diffract very well. I have tried changing buffer concentrations, precipitant amounts, ionic strengths and pH and in all cases this phenomenon is observed. I even screened protein concentrations to see if reducing protein concentration would prevent the phase separation. Is there any way to bypass this phase separation, which I think prevents me from obtaining nice crystals. Should I try detergents, chaotropes, or other additives? Thanks in advance. Timur -- F. Timur Senguen, Ph.D. Postdoctoral Research Fellow Boston Biomedical Research Institute 64 Grove Street, Watertown, MA 02472 USA
