Re: [ccp4bb] small crystals
Dear Careina, you can also apply for beamtime at PETRA-III. You get away with the same size crystals but only require a nano liter drop rather than a few ml of your sample. And you probably get beamtime much quicker because all the equipment is installed and collecting a data set takes very short time. This was demonstrated at the ECM in Warwick this year, so no need for FEL (at least for structure determination). Best, Tim On 12/10/2013 04:36 AM, Jens Kaiser wrote: Careina, If your target is interesting enough, try to reproduce the small crystals in batch and apply for FELS time. Small crystals are actually an advantage there. Cheers, Jens On Wed, 2013-12-04 at 21:41 -0800, Careina Edgooms wrote: Hi all Any advice on how to get bigger crystals from conditions that give showers of tiny crystals? I am getting small pretty looking individual crystals but they are too small and they don't seem to grow. In fact, in some instances if left for a couple of days they actually dissolve. I have fiddled around with mother liquor volume, protein concentration as well as drop volume (I am using hanging drop method) but none seem to make any difference and I always get the same tiny crystals. I think I might try microseeding but I haven't tried that yet. Any suggestions or tricks would be welcome Careina. -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A signature.asc Description: OpenPGP digital signature
Re: [ccp4bb] small crystals
Yes, Tim is right… for now… in few years, with the E-XFEL, we'll get to much less sample, and much more time available. But that's in few years. Leo On Dec 10, 2013, at 10:01 AM, Tim Gruene t...@shelx.uni-ac.gwdg.de wrote: Dear Careina, you can also apply for beamtime at PETRA-III. You get away with the same size crystals but only require a nano liter drop rather than a few ml of your sample. And you probably get beamtime much quicker because all the equipment is installed and collecting a data set takes very short time. This was demonstrated at the ECM in Warwick this year, so no need for FEL (at least for structure determination). Best, Tim On 12/10/2013 04:36 AM, Jens Kaiser wrote: Careina, If your target is interesting enough, try to reproduce the small crystals in batch and apply for FELS time. Small crystals are actually an advantage there. Cheers, Jens On Wed, 2013-12-04 at 21:41 -0800, Careina Edgooms wrote: Hi all Any advice on how to get bigger crystals from conditions that give showers of tiny crystals? I am getting small pretty looking individual crystals but they are too small and they don't seem to grow. In fact, in some instances if left for a couple of days they actually dissolve. I have fiddled around with mother liquor volume, protein concentration as well as drop volume (I am using hanging drop method) but none seem to make any difference and I always get the same tiny crystals. I think I might try microseeding but I haven't tried that yet. Any suggestions or tricks would be welcome Careina. -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A
Re: [ccp4bb] small crystals
'Will' is a fashionable word - but since even a Nobel prize was given based on promises never kept, who would be surprised... Best wishes from now, Tim On 12/10/2013 10:11 AM, CHAVAS Leonard wrote: Yes, Tim is right… for now… in few years, with the E-XFEL, we'll get to much less sample, and much more time available. But that's in few years. Leo On Dec 10, 2013, at 10:01 AM, Tim Gruene t...@shelx.uni-ac.gwdg.de wrote: Dear Careina, you can also apply for beamtime at PETRA-III. You get away with the same size crystals but only require a nano liter drop rather than a few ml of your sample. And you probably get beamtime much quicker because all the equipment is installed and collecting a data set takes very short time. This was demonstrated at the ECM in Warwick this year, so no need for FEL (at least for structure determination). Best, Tim On 12/10/2013 04:36 AM, Jens Kaiser wrote: Careina, If your target is interesting enough, try to reproduce the small crystals in batch and apply for FELS time. Small crystals are actually an advantage there. Cheers, Jens On Wed, 2013-12-04 at 21:41 -0800, Careina Edgooms wrote: Hi all Any advice on how to get bigger crystals from conditions that give showers of tiny crystals? I am getting small pretty looking individual crystals but they are too small and they don't seem to grow. In fact, in some instances if left for a couple of days they actually dissolve. I have fiddled around with mother liquor volume, protein concentration as well as drop volume (I am using hanging drop method) but none seem to make any difference and I always get the same tiny crystals. I think I might try microseeding but I haven't tried that yet. Any suggestions or tricks would be welcome Careina. -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A signature.asc Description: OpenPGP digital signature
Re: [ccp4bb] small crystals
Indeed nothing is predictable. We can only assume things on what is *planed* and *promised*, and you can take my words on the fact that I will do my best to have it done properly and as close to the expectations as possible. But again, nobody can really predict the future. Finger crossed. Cheers, Leo On Dec 10, 2013, at 10:13 AM, Tim Gruene t...@shelx.uni-ac.gwdg.de wrote: 'Will' is a fashionable word - but since even a Nobel prize was given based on promises never kept, who would be surprised... Best wishes from now, Tim On 12/10/2013 10:11 AM, CHAVAS Leonard wrote: Yes, Tim is right… for now… in few years, with the E-XFEL, we'll get to much less sample, and much more time available. But that's in few years. Leo On Dec 10, 2013, at 10:01 AM, Tim Gruene t...@shelx.uni-ac.gwdg.de wrote: Dear Careina, you can also apply for beamtime at PETRA-III. You get away with the same size crystals but only require a nano liter drop rather than a few ml of your sample. And you probably get beamtime much quicker because all the equipment is installed and collecting a data set takes very short time. This was demonstrated at the ECM in Warwick this year, so no need for FEL (at least for structure determination). Best, Tim On 12/10/2013 04:36 AM, Jens Kaiser wrote: Careina, If your target is interesting enough, try to reproduce the small crystals in batch and apply for FELS time. Small crystals are actually an advantage there. Cheers, Jens On Wed, 2013-12-04 at 21:41 -0800, Careina Edgooms wrote: Hi all Any advice on how to get bigger crystals from conditions that give showers of tiny crystals? I am getting small pretty looking individual crystals but they are too small and they don't seem to grow. In fact, in some instances if left for a couple of days they actually dissolve. I have fiddled around with mother liquor volume, protein concentration as well as drop volume (I am using hanging drop method) but none seem to make any difference and I always get the same tiny crystals. I think I might try microseeding but I haven't tried that yet. Any suggestions or tricks would be welcome Careina. -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A
Re: [ccp4bb] small crystals
Careina, If your target is interesting enough, try to reproduce the small crystals in batch and apply for FELS time. Small crystals are actually an advantage there. Cheers, Jens On Wed, 2013-12-04 at 21:41 -0800, Careina Edgooms wrote: Hi all Any advice on how to get bigger crystals from conditions that give showers of tiny crystals? I am getting small pretty looking individual crystals but they are too small and they don't seem to grow. In fact, in some instances if left for a couple of days they actually dissolve. I have fiddled around with mother liquor volume, protein concentration as well as drop volume (I am using hanging drop method) but none seem to make any difference and I always get the same tiny crystals. I think I might try microseeding but I haven't tried that yet. Any suggestions or tricks would be welcome Careina.
Re: [ccp4bb] small crystals
Dear Jens, Careina this is not really an *advantage*, but rather a *convenience*. You can still use big crystals if you'd like to, but as they usually never survive more than one shot (few femtosec), you'd need a lot of these bigger crystals to collect a full data. And yes, I would also highly recommend XFELs! Cheers, Leo On Dec 10, 2013, at 4:36 AM, Jens Kaiser kai...@caltech.edu wrote: Careina, If your target is interesting enough, try to reproduce the small crystals in batch and apply for FELS time. Small crystals are actually an advantage there. Cheers, Jens On Wed, 2013-12-04 at 21:41 -0800, Careina Edgooms wrote: Hi all Any advice on how to get bigger crystals from conditions that give showers of tiny crystals? I am getting small pretty looking individual crystals but they are too small and they don't seem to grow. In fact, in some instances if left for a couple of days they actually dissolve. I have fiddled around with mother liquor volume, protein concentration as well as drop volume (I am using hanging drop method) but none seem to make any difference and I always get the same tiny crystals. I think I might try microseeding but I haven't tried that yet. Any suggestions or tricks would be welcome Careina.
Re: [ccp4bb] small crystals
Dear Careina – Orthogonal crystallization methods can be of great utility when optimizing crystallization conditions, since they offer a different sampling of the crystallization space. Orthogonal methods include: liquid-liquid diffusion, microbatch and dialysis. Liquid-liquid diffusion is known to be a productive crystallization method and a recent paper from the Sundberg lab demonstrated that crystallization by liquid-liquid diffusion could improve both the size and diffraction quality of crystals of an endo-b-N-acetylglucosaminidase, EndoS, from Streptococcus pyogenes. The paper can be found here: http://scripts.iucr.org/cgi-bin/paper?nj5178 Description of the crystallization device can be found here: http://scripts.iucr.org/cgi-bin/paper?S1744309111024456 and http://www.microlytic.com/crystal-former Using microseed matrix screening could also improve your crystal quality. You can find information on the approach here: http://pubs.acs.org/doi/abs/10.1021/cg2001442?journalCode=cgdefu Best regards, Morten Original message Subject:[SURESPAM] [ccp4bb] small crystals From:Careina Edgooms careinaedgo...@yahoo.commailto:careinaedgo...@yahoo.com To:CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK Cc: Hi all Any advice on how to get bigger crystals from conditions that give showers of tiny crystals? I am getting small pretty looking individual crystals but they are too small and they don't seem to grow. In fact, in some instances if left for a couple of days they actually dissolve. I have fiddled around with mother liquor volume, protein concentration as well as drop volume (I am using hanging drop method) but none seem to make any difference and I always get the same tiny crystals. I think I might try microseeding but I haven't tried that yet. Any suggestions or tricks would be welcome Careina. -- TotalCare Message Security: Click below to verify authenticity http://www.exchangedefender.com/verify.asp?id=rB5EYXWs004083from=m...@microlytic.com
Re: [ccp4bb] small crystals
Hi, can you give a bit more information... Can you concentrate the protein easily to a higher concentration, let's say 2-3 times from what you have now, without precipitation? What is the buffer of your protein stock solution at the moment? At what temperature and what precipitant are you using? - Jeroen - showers of crystals Original message Subject:[SURESPAM] [ccp4bb] small crystals From:Careina Edgooms careinaedgo...@yahoo.com To:CCP4BB@JISCMAIL.AC.UK Cc: Hi all Any advice on how to get bigger crystals from conditions that give showers of tiny crystals? I am getting small pretty looking individual crystals but they are too small and they don't seem to grow. In fact, in some instances if left for a couple of days they actually dissolve. I have fiddled around with mother liquor volume, protein concentration as well as drop volume (I am using hanging drop method) but none seem to make any difference and I always get the same tiny crystals. I think I might try microseeding but I haven't tried that yet. Any suggestions or tricks would be welcome Careina. TotalCare Message Security: Check Authenticity -- Dr. Jeroen R. Mesters Deputy and Group Leader Institute of Biochemistry, University of Lübeck Ratzeburger Allee 160, 23538 Lübeck, Germany phone: +49-451-5004065 (secretariate 5004061) fax: +49-451-5004068 http://www.biochem.uni-luebeck.de http://www.iobcr.org -- If you can look into the seeds of time and tell which grain will grow and which will not, speak then to me who neither beg nor fear (Shakespeare's Macbeth, Act I, Scene 3) -- Disclaimer * This message contains confidential information and is intended only for the individual named. If you are not the named addressee you should not disseminate, distribute or copy this e-mail. Please notify the sender immediately by e-mail if you have received this e-mail by mistake and delete this e-mail from your system. * E-mail transmission cannot be guaranteed to be secure or error-free as information could be intercepted, corrupted, lost, destroyed, arrive late or incomplete, or contain viruses. The sender therefore does not accept liability for any errors or omissions in the contents of this message, which arise as a result of e-mail transmission. If verification is required please request a hard-copy version. Please send us by fax any message containing deadlines as incoming e-mails are not screened for response deadlines. * Employees of the Institute are expressly required not to make defamatory statements and not to infringe or authorize any infringement of copyright or any other legal right by email communications. Any such communication is contrary to Institute policy and outside the scope of the employment of the individual concerned. The Institute will not accept any liability in respect of such communication, and the employee responsible will be personally liable for any damages or other liability arising. Employees who receive such an email must notify their supervisor immediately. --
Re: [ccp4bb] small crystals
Hi All On similar lines, I have been trying to optimize crystallization conditions for my protein. Initially, I had showers of needles in a PEG screen which did not really improve after screening around the condition. So, I seeded these needles into all the screens that I have available and I have plate like crystals which do not diffract at home in MPD (~15%), 0.1 M sodium acetate, 0.2 Mgcl2/cacl2 at 294 K. I tried incubating at 287 K, but that did not yield any useful results.The protein itself is in 50mM MOPS, 10% glycerol pH 7.5. I could try to take of glycerol but i cannot concentrate the protein more than ~ 5mg/ml which clearly was not sufficient to achieve crystallization. Any advice is deeply appreciated. Thank you cheers Mahesh On Thu, Dec 5, 2013 at 10:03 AM, mesters mest...@biochem.uni-luebeck.dewrote: Hi, can you give a bit more information... Can you concentrate the protein easily to a higher concentration, let's say 2-3 times from what you have now, without precipitation? What is the buffer of your protein stock solution at the moment? At what temperature and what precipitant are you using? - Jeroen - showers of crystals Original message Subject:[SURESPAM] [ccp4bb] small crystals From:Careina Edgooms careinaedgo...@yahoo.com To:CCP4BB@JISCMAIL.AC.UK Cc: Hi all Any advice on how to get bigger crystals from conditions that give showers of tiny crystals? I am getting small pretty looking individual crystals but they are too small and they don't seem to grow. In fact, in some instances if left for a couple of days they actually dissolve. I have fiddled around with mother liquor volume, protein concentration as well as drop volume (I am using hanging drop method) but none seem to make any difference and I always get the same tiny crystals. I think I might try microseeding but I haven't tried that yet. Any suggestions or tricks would be welcome Careina. *TotalCare* Message Security: Check Authenticityhttp://www.exchangedefender.com/verify.asp?id=rB5EYXWs004083from=m...@microlytic.com -- Dr. Jeroen R. Mesters Deputy and Group Leader Institute of Biochemistry, University of Lübeck Ratzeburger Allee 160, 23538 Lübeck, Germany phone: +49-451-5004065 (secretariate 5004061) fax: +49-451-5004068 http://www.biochem.uni-luebeck.de Http://www.biochem.uni-luebeck.de http://www.iobcr.org Http://www.iobcr.org -- If you can look into the seeds of time and tell which grain will grow and which will not, speak then to me who neither beg nor fear (Shakespeare's Macbeth, Act I, Scene 3) -- Disclaimer * This message contains confidential information and is intended only for the individual named. If you are not the named addressee you should not disseminate, distribute or copy this e-mail. Please notify the sender immediately by e-mail if you have received this e-mail by mistake and delete this e-mail from your system. * E-mail transmission cannot be guaranteed to be secure or error-free as information could be intercepted, corrupted, lost, destroyed, arrive late or incomplete, or contain viruses. The sender therefore does not accept liability for any errors or omissions in the contents of this message, which arise as a result of e-mail transmission. If verification is required please request a hard-copy version. Please send us by fax any message containing deadlines as incoming e-mails are not screened for response deadlines. * Employees of the Institute are expressly required not to make defamatory statements and not to infringe or authorize any infringement of copyright or any other legal right by email communications. Any such communication is contrary to Institute policy and outside the scope of the employment of the individual concerned. The Institute will not accept any liability in respect of such communication, and the employee responsible will be personally liable for any damages or other liability arising. Employees who receive such an email must notify their supervisor immediately. --
Re: [ccp4bb] small crystals
depending on how extensively you have screened so far, the most efficient thing to do may be to change the protein: different orthologs, truncations, mutagenesis of entropy rich clusters, change of tag location or tag cleavage etc. From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Mahesh Lingaraju [mxl1...@psu.edu] Sent: Thursday, December 05, 2013 5:38 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] small crystals Hi All On similar lines, I have been trying to optimize crystallization conditions for my protein. Initially, I had showers of needles in a PEG screen which did not really improve after screening around the condition. So, I seeded these needles into all the screens that I have available and I have plate like crystals which do not diffract at home in MPD (~15%), 0.1 M sodium acetate, 0.2 Mgcl2/cacl2 at 294 K. I tried incubating at 287 K, but that did not yield any useful results.The protein itself is in 50mM MOPS, 10% glycerol pH 7.5. I could try to take of glycerol but i cannot concentrate the protein more than ~ 5mg/ml which clearly was not sufficient to achieve crystallization. Any advice is deeply appreciated. Thank you cheers Mahesh On Thu, Dec 5, 2013 at 10:03 AM, mesters mest...@biochem.uni-luebeck.demailto:mest...@biochem.uni-luebeck.de wrote: Hi, can you give a bit more information... Can you concentrate the protein easily to a higher concentration, let's say 2-3 times from what you have now, without precipitation? What is the buffer of your protein stock solution at the moment? At what temperature and what precipitant are you using? - Jeroen - showers of crystals Original message Subject:[SURESPAM] [ccp4bb] small crystals From:Careina Edgooms careinaedgo...@yahoo.commailto:careinaedgo...@yahoo.com To:CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK Cc: Hi all Any advice on how to get bigger crystals from conditions that give showers of tiny crystals? I am getting small pretty looking individual crystals but they are too small and they don't seem to grow. In fact, in some instances if left for a couple of days they actually dissolve. I have fiddled around with mother liquor volume, protein concentration as well as drop volume (I am using hanging drop method) but none seem to make any difference and I always get the same tiny crystals. I think I might try microseeding but I haven't tried that yet. Any suggestions or tricks would be welcome Careina. TotalCare Message Security: Check Authenticityhttp://www.exchangedefender.com/verify.asp?id=rB5EYXWs004083from=m...@microlytic.com -- Dr. Jeroen R. Mesters Deputy and Group Leader Institute of Biochemistry, University of Lübeck Ratzeburger Allee 160, 23538 Lübeck, Germany phone: +49-451-5004065tel:%2B49-451-5004065 (secretariate 5004061) fax: +49-451-5004068tel:%2B49-451-5004068 http://www.biochem.uni-luebeck.deHttp://www.biochem.uni-luebeck.de http://www.iobcr.orgHttp://www.iobcr.org [X] -- If you can look into the seeds of time and tell which grain will grow and which will not, speak then to me who neither beg nor fear (Shakespeare's Macbeth, Act I, Scene 3) -- Disclaimer * This message contains confidential information and is intended only for the individual named. If you are not the named addressee you should not disseminate, distribute or copy this e-mail. Please notify the sender immediately by e-mail if you have received this e-mail by mistake and delete this e-mail from your system. * E-mail transmission cannot be guaranteed to be secure or error-free as information could be intercepted, corrupted, lost, destroyed, arrive late or incomplete, or contain viruses. The sender therefore does not accept liability for any errors or omissions in the contents of this message, which arise as a result of e-mail transmission. If verification is required please request a hard-copy version. Please send us by fax any message containing deadlines as incoming e-mails are not screened for response deadlines. * Employees of the Institute are expressly required not to make defamatory statements and not to infringe or authorize any infringement of copyright or any other legal right by email communications. Any such communication is contrary to Institute policy and outside the scope of the employment of the individual concerned. The Institute will not accept any liability in respect of such communication, and the employee responsible will be personally liable for any damages or other liability arising. Employees who receive such an email must notify their supervisor immediately. --
Re: [ccp4bb] small crystals
Hi, sorry, it should read salt in not inverse salt in ... - J. - Am 05.12.13 19:55, schrieb mesters: Hi, Showers of crystals often occur if the protein is not that soluble/happy/stable in the solute. The solubility of a protein depends on its concentration, its pI, pH of solute, temperature, compounds in the solute (e.g. salts and small organic molecules), construct used, to name a few. So, to increase the solubility, you need to change one these parameters. Salts in general are said to inverse salt in the protein thus making it more soluble. Hofmeister published an interesting paper on this in 1888! NaCl for example is placed in the middle of the Hofmeister series, neutral so to speak, and practice shows it is often a good choice to start with at a concentration of 150-200 mM. However, occasionally it is not a good choice and will have negative effects. The choice of salt basically depends on the pI of the protein and the pH of the solute/crystallization condition. Have a serious look at the chapter by Madeleine Riess-Kautt about the Hofmeister series in A. Ducruix und R. Giegé book with title Crystallization of Nucleic Acids Proteins. A practical Approach (Oxford IRL-University Press (1992)). For a normal hen egg white protein (pI/IEP below the pH, protein overal negatively charged) he was working on, ammoniumsulfate will precipitate (stabilize/crystallize) the protein while a perchlorate salt (destabilize) will dissolve the protein completely at high concentrations. As you generally need high protein concentrations for starters in crystal growth, you thus need to add a salt that dissolves the protein slightly without denaturing it (low concentration 50 -200 mM in the solute). Then, you can add a second salt (on the opposite site of the series) or precipitant like PEG to crystallize it. However, for several proteins the pI is located above the pH of the solute. In these cases the protein is positively charged and the Hofmeister series turns around! We recently had a case like this producing lost of tiny plate-like crystals. Changing the protein concentration or the precipitant concentration, or hanging to sitting etc. did not help at all. We finally found out we had to add some ammoniumsulfate to solubilize/dissolve the protein first and got very nice single and large crystals in the end. In your case, try to replace the glycerol by the proper choice of salt! You can also try to exchange MOPS (zwitterion) for another buffer compound. All this will also change the crystallizaiton behaviour. You will most probably need to redo the screening... - J. -
