Re: [ccp4bb] cryo condition
Along with all the excellent suggestions so far you can also try no cryoprotectant at all: If you harvest your crystal on a mesh loop and remove all the mother liquor the crystal lattice itself will act as a cryoprotectant as long as the solvent channels are smaller than 40A in diameter (they usually are). Shameless self citation: http://scripts.iucr.org/cgi-bin/paper?S0907444911031210 Best wishes, Matt. On 05/05/2015 13:16, Clemens Grimm wrote: Hi Faisal, Did you try to simply raise the Sokalan CP7 percentage? Additives like Glycerol can increase the solubility of proteins, in that case you have to counteract by increasing also the precipitant concentration. If your crystals crack, a likely reason is osmotic shock. Particularly large crystals tend to be problematic. It is in general advisable to transfer the cryo protectant onto the crystal within the mother liquor rather than fishing the crystal out with a loop. In addition, it might be necessary to prepare intermediate concentration of the cryo protectant by mixing with reservoir solution and do a stepwise increase. I had several projects with very large crystals that definitely needed at least five gradual steps over a time span of more than half an hour to extract datasets with reasonable mosaicity. Sokalan CP7 is an acrylate copolymer. Therefore, low molecular weight sodium polycralyte as a cryo protectant (e. g. Aldrich #420344) might be worth a try, possibly also in combination with some amount of good old glycerol, PEG, Trehalose, TMAO etc. Best wishes, Clemens Zitat von Faisal Tarique faisaltari...@gmail.com: Hello everyone Can anybody suggest me a cryo condition for a crystal obtained in MIDAS screen of Molecular Dimension: G1 0.1MTris8.0G10.1Mpotassium chloride25% v/vSOKALAN®CP7 0.1MHEPES7.0 G20.3Mammonium formate20% v/vSOKALAN® CP 5 0.1MHEPES7.0 Crystals are in beautiful cuboid shaped but all sorts of PEG combinations and Glycerol formulation failed to prevent it from cracking and dissolving. Has anybody faced a similar situation as mentioned above and what precaution was taken to prevent it from cracking or dissolving. Your suggestions will be of immense help Thanks in advance -- Regards Faisal School of Life Sciences JNU -- Dr. Clemens Grimm Institut für Biochemie Biozentrum der Universität Würzburg Am Hubland D-97074 Würzburg Germany e-mail: clemens.gr...@biozentrum.uni-wuerzburg.de phone : +49 0931 31 84031 - -- Matthew Bowler Synchrotron Crystallography Group European Molecular Biology Laboratory 71 avenue des Martyrs CS 90181 F-38042 Grenoble France === Tel: +33 (0) 4.76.20.76.37 Fax: +33 (0) 4.76.20.71.99 http://www.embl.fr/ ===
Re: [ccp4bb] cryo condition
Hi Faisal, Did you try to simply raise the Sokalan CP7 percentage? Additives like Glycerol can increase the solubility of proteins, in that case you have to counteract by increasing also the precipitant concentration. If your crystals crack, a likely reason is osmotic shock. Particularly large crystals tend to be problematic. It is in general advisable to transfer the cryo protectant onto the crystal within the mother liquor rather than fishing the crystal out with a loop. In addition, it might be necessary to prepare intermediate concentration of the cryo protectant by mixing with reservoir solution and do a stepwise increase. I had several projects with very large crystals that definitely needed at least five gradual steps over a time span of more than half an hour to extract datasets with reasonable mosaicity. Sokalan CP7 is an acrylate copolymer. Therefore, low molecular weight sodium polycralyte as a cryo protectant (e. g. Aldrich #420344) might be worth a try, possibly also in combination with some amount of good old glycerol, PEG, Trehalose, TMAO etc. Best wishes, Clemens Zitat von Faisal Tarique faisaltari...@gmail.com: Hello everyone Can anybody suggest me a cryo condition for a crystal obtained in MIDAS screen of Molecular Dimension: G1 0.1MTris8.0G10.1Mpotassium chloride25% v/vSOKALAN®CP7 0.1MHEPES7.0 G20.3Mammonium formate20% v/vSOKALAN® CP 5 0.1MHEPES7.0 Crystals are in beautiful cuboid shaped but all sorts of PEG combinations and Glycerol formulation failed to prevent it from cracking and dissolving. Has anybody faced a similar situation as mentioned above and what precaution was taken to prevent it from cracking or dissolving. Your suggestions will be of immense help Thanks in advance -- Regards Faisal School of Life Sciences JNU -- Dr. Clemens Grimm Institut für Biochemie Biozentrum der Universität Würzburg Am Hubland D-97074 Würzburg Germany e-mail: clemens.gr...@biozentrum.uni-wuerzburg.de phone : +49 0931 31 84031 -
[ccp4bb] cryo condition
Hello everyone Can anybody suggest me a cryo condition for a crystal obtained in MIDAS screen of Molecular Dimension: G1 0.1MTris8.0G10.1Mpotassium chloride25% v/vSOKALAN®CP7 0.1MHEPES7.0 G20.3Mammonium formate20% v/vSOKALAN® CP 5 0.1MHEPES7.0 Crystals are in beautiful cuboid shaped but all sorts of PEG combinations and Glycerol formulation failed to prevent it from cracking and dissolving. Has anybody faced a similar situation as mentioned above and what precaution was taken to prevent it from cracking or dissolving. Your suggestions will be of immense help Thanks in advance -- Regards Faisal School of Life Sciences JNU
Re: [ccp4bb] cryo condition
How long are you cryoprotecting for? You don't really need more than a few seconds. If I have sensitive crystals i tend to just swipe them slowly through the final CP solution and often that works. If I would leave them in the CP solution for more than say 30 seconds the crystals would behave like you describe. GL Bert From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Faisal Tarique [faisaltari...@gmail.com] Sent: Monday, May 04, 2015 7:43 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] cryo condition Hello everyone Can anybody suggest me a cryo condition for a crystal obtained in MIDAS screen of Molecular Dimension: G1 0.1MTris8.0G10.1Mpotassium chloride25% v/vSOKALAN®CP7 0.1MHEPES7.0 G20.3Mammonium formate20% v/vSOKALAN® CP 5 0.1MHEPES7.0 Crystals are in beautiful cuboid shaped but all sorts of PEG combinations and Glycerol formulation failed to prevent it from cracking and dissolving. Has anybody faced a similar situation as mentioned above and what precaution was taken to prevent it from cracking or dissolving. Your suggestions will be of immense help Thanks in advance -- Regards Faisal School of Life Sciences JNU
Re: [ccp4bb] cryo condition
High concentration of ammonium formate is a cryosolvent On Mon, May 4, 2015 at 5:20 PM, Tristan Croll tristan.cr...@qut.edu.au wrote: What about nature's favourite cryoprotectant, trehalose? From: CCP4 bulletin board CCP4BB@JISCMAIL.AC.UK on behalf of Roger Rowlett rrowl...@colgate.edu Sent: Tuesday, 5 May 2015 7:22 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] cryo condition We rarely use glycerol anymore, because it seems to fail so often for many of our current proteins. Try glucose, 25-30%. This is most conveniently done by weighing 125-150 mg of glucose in a microcentrifuge tube, then addding well solution to the 0.5 mL mark and mixing until completely dissolved. Then you can try dunking crystals in the cryo solution, or, you can try the no-fail method (which does fail on occasion) of cryoprotecting directly in the crystallization drop by slow addition of the cryoprotectant. See http://capsicum.colgate.edu/chwiki/tiki-index.php?page=Mounting+Protein+Crystals . We have often found the slow addition of a glucose cryoprotectant works for fragile, high solvent content crystals that are prone to cracking under osmotic stress. Other alternatives include high concentrations of sodium malonate (1-2M), or high concentrations of sodium formate (I think around 4 M?). These could also be introduced gradually if required. Good luck! ___ Roger S. Rowlett Gordon Dorothy Kline Professor 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...@colgate.edu On 5/4/2015 2:43 PM, Faisal Tarique wrote: Hello everyone Can anybody suggest me a cryo condition for a crystal obtained in MIDAS screen of Molecular Dimension: G1 0.1MTris8.0G10.1Mpotassium chloride25% v/vSOKALAN®CP7 0.1MHEPES7.0 G20.3Mammonium formate20% v/vSOKALAN® CP 5 0.1MHEPES7.0 Crystals are in beautiful cuboid shaped but all sorts of PEG combinations and Glycerol formulation failed to prevent it from cracking and dissolving. Has anybody faced a similar situation as mentioned above and what precaution was taken to prevent it from cracking or dissolving. Your suggestions will be of immense help Thanks in advance
Re: [ccp4bb] cryo condition
What about nature's favourite cryoprotectant, trehalose? From: CCP4 bulletin board CCP4BB@JISCMAIL.AC.UK on behalf of Roger Rowlett rrowl...@colgate.edu Sent: Tuesday, 5 May 2015 7:22 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] cryo condition We rarely use glycerol anymore, because it seems to fail so often for many of our current proteins. Try glucose, 25-30%. This is most conveniently done by weighing 125-150 mg of glucose in a microcentrifuge tube, then addding well solution to the 0.5 mL mark and mixing until completely dissolved. Then you can try dunking crystals in the cryo solution, or, you can try the no-fail method (which does fail on occasion) of cryoprotecting directly in the crystallization drop by slow addition of the cryoprotectant. See http://capsicum.colgate.edu/chwiki/tiki-index.php?page=Mounting+Protein+Crystals. We have often found the slow addition of a glucose cryoprotectant works for fragile, high solvent content crystals that are prone to cracking under osmotic stress. Other alternatives include high concentrations of sodium malonate (1-2M), or high concentrations of sodium formate (I think around 4 M?). These could also be introduced gradually if required. Good luck! ___ Roger S. Rowlett Gordon Dorothy Kline Professor 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...@colgate.edu On 5/4/2015 2:43 PM, Faisal Tarique wrote: Hello everyone Can anybody suggest me a cryo condition for a crystal obtained in MIDAS screen of Molecular Dimension: G1 0.1MTris8.0G10.1Mpotassium chloride25% v/vSOKALAN®CP7 0.1MHEPES7.0 G20.3Mammonium formate20% v/vSOKALAN® CP 5 0.1MHEPES7.0 Crystals are in beautiful cuboid shaped but all sorts of PEG combinations and Glycerol formulation failed to prevent it from cracking and dissolving. Has anybody faced a similar situation as mentioned above and what precaution was taken to prevent it from cracking or dissolving. Your suggestions will be of immense help Thanks in advance
Re: [ccp4bb] cryo condition
We rarely use glycerol anymore, because it seems to fail so often for many of our current proteins. Try glucose, 25-30%. This is most conveniently done by weighing 125-150 mg of glucose in a microcentrifuge tube, then addding well solution to the 0.5 mL mark and mixing until completely dissolved. Then you can try dunking crystals in the cryo solution, or, you can try the no-fail method (which does fail on occasion) of cryoprotecting directly in the crystallization drop by slow addition of the cryoprotectant. See http://capsicum.colgate.edu/chwiki/tiki-index.php?page=Mounting+Protein+Crystals. We have often found the slow addition of a glucose cryoprotectant works for fragile, high solvent content crystals that are prone to cracking under osmotic stress. Other alternatives include high concentrations of sodium malonate (1-2M), or high concentrations of sodium formate (I think around 4 M?). These could also be introduced gradually if required. Good luck! ___ Roger S. Rowlett Gordon Dorothy Kline Professor 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...@colgate.edu On 5/4/2015 2:43 PM, Faisal Tarique wrote: Hello everyone Can anybody suggest me a cryo condition for a crystal obtained in MIDAS screen of Molecular Dimension: G1 0.1MTris8.0G10.1Mpotassium chloride25% v/vSOKALAN®CP7 0.1MHEPES7.0 G20.3Mammonium formate20% v/vSOKALAN® CP 5 0.1MHEPES7.0 Crystals are in beautiful cuboid shaped but all sorts of PEG combinations and Glycerol formulation failed to prevent it from cracking and dissolving. Has anybody faced a similar situation as mentioned above and what precaution was taken to prevent it from cracking or dissolving. Your suggestions will be of immense help Thanks in advance
Re: [ccp4bb] cryo condition
If in doubt, try dragging the crystal through a 1:1 mix of Paratone-N and Mineral oil until most or all the mother liquor from surrounding the crystal has been removed. There are more tips here: http://hamptonresearch.com/tip_detail.aspx?id=99 Good luck! D On Mon, 4 May 2015 19:45 Faisal Tarique faisaltari...@gmail.com wrote: Hello everyone Can anybody suggest me a cryo condition for a crystal obtained in MIDAS screen of Molecular Dimension: G1 0.1MTris8.0G10.1Mpotassium chloride25% v/vSOKALAN®CP7 0.1MHEPES7.0 G20.3Mammonium formate20% v/vSOKALAN® CP 5 0.1MHEPES7.0 Crystals are in beautiful cuboid shaped but all sorts of PEG combinations and Glycerol formulation failed to prevent it from cracking and dissolving. Has anybody faced a similar situation as mentioned above and what precaution was taken to prevent it from cracking or dissolving. Your suggestions will be of immense help Thanks in advance -- Regards Faisal School of Life Sciences JNU
Re: [ccp4bb] cryo condition
Thank you everybody for their nice suggestions.. On Thu, May 23, 2013 at 7:39 PM, Matthew BOWLER mbow...@embl.fr wrote: I keep sending mails by accident today - apologies for the spam. The last sentence of my should read: This could of course be due to too high a concentration of mother liquor but quite often it occurs at relative humidity values where the concentration of the mother liquor components will not have increased by very much. Cheers, Matt. On 2013-05-23 15:32, Ed Pozharski wrote: Matt, with this technique, how do you prevent crystal from drying up (other than doing it fast)? I know Thorne's group does this trick under oil. If you take no extra precautions, do you have an estimate of how often diffraction is destroyed by this? On the other hand, it's quite possible that what destroys resolution when crystals dry up is increase in concentration of non-volatile mother liquor components, which shouldn't be happening here to the same degree. Cheers, Ed. On Thu, 2013-05-23 at 14:38 +0200, Matthew BOWLER wrote: Hi Faisal, if your solvent channels are smaller than 40A in the largest dimension (most are) you can use a mesh loop to pick up the crystal and then wick away all of the mother liquor. You can then flash cool your crystal without having to transfer the crystal to another solution. Good luck, Matt -- 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/ ==**= -- Regards Faisal School of Life Sciences JNU
[ccp4bb] cryo condition
Dear all Can anybody tell me the appropriate cryo condition for the crystals obtained in 2M Ammonium sulphate and tris pH8.5..I tried to add 10% glycerol to it but still the ice ring is forming.. Thanx in advance -- Regards Faisal School of Life Sciences JNU
Re: [ccp4bb] cryo condition
80% saturated Li2SO4 On Thu, 23 May 2013 11:42:09 +0200, Faisal Tarique faisaltari...@gmail.com wrote: Dear all Can anybody tell me the appropriate cryo condition for the crystals obtained in 2M Ammonium sulphate and tris pH8.5..I tried to add 10% glycerol to it but still the ice ring is forming.. Thanx in advance -- Enrico A. Stura D.Phil. (Oxon) ,Tel: 33 (0)1 69 08 4302 Office Room 19, Bat.152, Tel: 33 (0)1 69 08 9449Lab http://www-dsv.cea.fr/ibitecs/simopro/ltmb/cristallogenese LTMB, SIMOPRO, IBiTec-S, CE Saclay, 91191 Gif-sur-Yvette, FRANCE http://scholar.google.com/citations?hl=enuser=Kvm06WIoPAsCpagesize=100sortby=pubdate http://www.chem.gla.ac.uk/protein/mirror/stura/index2.html e-mail: est...@cea.fr Fax: 33 (0)1 69 08 90 71
Re: [ccp4bb] cryo condition
Hi, Paraton oil worked nicely for me for these conditions. Boaz Boaz Shaanan, Ph.D. Dept. of Life Sciences Ben-Gurion University of the Negev Beer-Sheva 84105 Israel E-mail: bshaa...@bgu.ac.il Phone: 972-8-647-2220Skype: boaz.shaanan Fax: 972-8-647-2992 or 972-8-646-1710 From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Faisal Tarique [faisaltari...@gmail.com] Sent: Thursday, May 23, 2013 12:42 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] cryo condition Dear all Can anybody tell me the appropriate cryo condition for the crystals obtained in 2M Ammonium sulphate and tris pH8.5..I tried to add 10% glycerol to it but still the ice ring is forming.. Thanx in advance -- Regards Faisal School of Life Sciences JNU
Re: [ccp4bb] cryo condition
30% glycerol or 25% glucose should be sufficient for 1-2 M ammonium sulfate. Roger Rowlett On May 23, 2013 5:42 AM, Faisal Tarique faisaltari...@gmail.com wrote: Dear all Can anybody tell me the appropriate cryo condition for the crystals obtained in 2M Ammonium sulphate and tris pH8.5..I tried to add 10% glycerol to it but still the ice ring is forming.. Thanx in advance -- Regards Faisal School of Life Sciences JNU
Re: [ccp4bb] cryo condition
I would try 2.5M or 3M sodium malonate pH 7 Janet Newman Principal Scientist / Director, Collaborative Crystallisation Centre CSIRO Material Science and Engineering 343 Royal Parade Parkville. VIC. 3052 Australia Tel +613 9662 7326 Email janet.new...@csiro.au From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Roger Rowlett [rrowl...@colgate.edu] Sent: 23 May 2013 21:21 To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] cryo condition 30% glycerol or 25% glucose should be sufficient for 1-2 M ammonium sulfate. Roger Rowlett On May 23, 2013 5:42 AM, Faisal Tarique faisaltari...@gmail.commailto:faisaltari...@gmail.com wrote: Dear all Can anybody tell me the appropriate cryo condition for the crystals obtained in 2M Ammonium sulphate and tris pH8.5..I tried to add 10% glycerol to it but still the ice ring is forming.. Thanx in advance -- Regards Faisal School of Life Sciences JNU
Re: [ccp4bb] cryo condition
Hi Faisal, 3 - 3.5M Ammonium sulphate with your buffer should work too. Take a small loop. Jan -- Jan Abendroth Emerald BioStructures Seattle / Bainbridge Island WA, USA home: Jan.Abendroth_at_gmail.com work: JAbendroth_at_embios.com http://www.emeraldbiostructures.com On May 23, 2013, at 2:42 AM, Faisal Tarique faisaltari...@gmail.com wrote: Dear all Can anybody tell me the appropriate cryo condition for the crystals obtained in 2M Ammonium sulphate and tris pH8.5..I tried to add 10% glycerol to it but still the ice ring is forming.. Thanx in advance -- Regards Faisal School of Life Sciences JNU
Re: [ccp4bb] cryo condition
Hi Faisal, On 2013-05-23 11:42, Faisal Tarique wrote: Dear all Can anybody tell me the appropriate cryo condition for the crystals obtained in 2M Ammonium sulphate and tris pH8.5..I tried to add 10% glycerol to it but still the ice ring is forming.. Thanx in advance-- Regards Faisal School of Life Sciences JNU -- 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] cryo condition
Hi Faisal, if your solvent channels are smaller than 40A in the largest dimension (most are) you can use a mesh loop to pick up the crystal and then wick away all of the mother liquor. You can then flash cool your crystal without having to transfer the crystal to another solution. Good luck, Matt On 2013-05-23 11:42, Faisal Tarique wrote: Dear all Can anybody tell me the appropriate cryo condition for the crystals obtained in 2M Ammonium sulphate and tris pH8.5..I tried to add 10% glycerol to it but still the ice ring is forming.. Thanx in advance-- Regards Faisal School of Life Sciences JNU -- 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] cryo condition
Just to follow up, the paper that was attached is based on the far more original work by Garman and Mitchell Elspeth Garman and Edward Mitchell. (1996) Glycerol concentrations required for cryoprotection of 50 typical protein crystallisation solutions. J.Appl. Cryst. 29, 584-587. And there is also a further paper Glycerol concentrations required for the successful vitrification of cocktail conditions in a high-throughput crystallization screen.Kempkes R, Stofko E, Lam K, Snell EH.(2003) Acta Cryst D 64, 287-301. There are a few other papers on high salt concentrations as a cryoprotectant including: Todd Holyoak, Timothy D. Fenn, Mark A. Wilson, Aaron G. Moulin, Dagmar Ringe and Gregory A. Petsko. (2003) Malonate: a versatile cryoprotectant and stabilizing solution for salt-grown macromolecular crystals. Acta D. 59, 2356-2358. Cheers, Eddie. Edward Snell Ph.D. Assistant Prof. Department of Structural Biology, SUNY Buffalo, Senior Scientist, Hauptman-Woodward Medical Research Institute 700 Ellicott Street, Buffalo, NY 14203-1102 Phone: (716) 898 8631 Fax: (716) 898 8660 Skype: eddie.snell Email: esn...@hwi.buffalo.edu Telepathy: 42.2 GHz Heisenberg was probably here! -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Thibaut Crepin Sent: Thursday, May 23, 2013 6:24 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] cryo condition Dear Faisal, this paper can be really useful. Regards Thibaut On 23/05/2013 11:42, Faisal Tarique wrote: Dear all Can anybody tell me the appropriate cryo condition for the crystals obtained in 2M Ammonium sulphate and tris pH8.5..I tried to add 10% glycerol to it but still the ice ring is forming.. Thanx in advance -- Regards Faisal School of Life Sciences JNU
Re: [ccp4bb] cryo condition
L-proline works well with ammonium sulfate: http://www.ncbi.nlm.nih.gov/pubmed/22868767 Sent from Jack's iPad On May 23, 2013, at 4:42 AM, Faisal Tarique faisaltari...@gmail.com wrote: Dear all Can anybody tell me the appropriate cryo condition for the crystals obtained in 2M Ammonium sulphate and tris pH8.5..I tried to add 10% glycerol to it but still the ice ring is forming.. Thanx in advance -- Regards Faisal School of Life Sciences JNU
Re: [ccp4bb] cryo condition
Matt, with this technique, how do you prevent crystal from drying up (other than doing it fast)? I know Thorne's group does this trick under oil. If you take no extra precautions, do you have an estimate of how often diffraction is destroyed by this? On the other hand, it's quite possible that what destroys resolution when crystals dry up is increase in concentration of non-volatile mother liquor components, which shouldn't be happening here to the same degree. Cheers, Ed. On Thu, 2013-05-23 at 14:38 +0200, Matthew BOWLER wrote: Hi Faisal, if your solvent channels are smaller than 40A in the largest dimension (most are) you can use a mesh loop to pick up the crystal and then wick away all of the mother liquor. You can then flash cool your crystal without having to transfer the crystal to another solution. Good luck, Matt -- After much deep and profound brain things inside my head, I have decided to thank you for bringing peace to our home. Julian, King of Lemurs
Re: [ccp4bb] cryo condition
Hi Ed, good question. I have found that you have a good 30 seconds to remove the surrounding liquid - so while you have to do it fast you have enough time that it doesn't need a robot and even a malcoordinate such as myself can do it. I'm afraid that I have no estimate for how often diffraction is lost this way more info here http://scripts.iucr.org/cgi-bin/paper?S0907444911031210 I started looking at this when doing dehydration experiments where it has long been observed that you can directly cool the crystals after dehydration - I have a feeling that 'over drying' a crystal leads to a complete loss of lattice order - I have no evidence for this but all systems that we have looked at that benefit from dehydration, or not, come to quite a sharp cutoff where suddenly there is no more diffraction, one can often recover diffraction by rehydration. This could of course be due to too high a concentration of mother liquor but quite often it occurs at relative humidity values Cheers, Matt. On 2013-05-23 15:32, Ed Pozharski wrote: Matt, with this technique, how do you prevent crystal from drying up (other than doing it fast)? I know Thorne's group does this trick under oil. If you take no extra precautions, do you have an estimate of how often diffraction is destroyed by this? On the other hand, it's quite possible that what destroys resolution when crystals dry up is increase in concentration of non-volatile mother liquor components, which shouldn't be happening here to the same degree. Cheers, Ed. On Thu, 2013-05-23 at 14:38 +0200, Matthew BOWLER wrote: Hi Faisal, if your solvent channels are smaller than 40A in the largest dimension (most are) you can use a mesh loop to pick up the crystal and then wick away all of the mother liquor. You can then flash cool your crystal without having to transfer the crystal to another solution. Good luck, Matt -- 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] cryo condition
I keep sending mails by accident today - apologies for the spam. The last sentence of my should read: This could of course be due to too high a concentration of mother liquor but quite often it occurs at relative humidity values where the concentration of the mother liquor components will not have increased by very much. Cheers, Matt. On 2013-05-23 15:32, Ed Pozharski wrote: Matt, with this technique, how do you prevent crystal from drying up (other than doing it fast)? I know Thorne's group does this trick under oil. If you take no extra precautions, do you have an estimate of how often diffraction is destroyed by this? On the other hand, it's quite possible that what destroys resolution when crystals dry up is increase in concentration of non-volatile mother liquor components, which shouldn't be happening here to the same degree. Cheers, Ed. On Thu, 2013-05-23 at 14:38 +0200, Matthew BOWLER wrote: Hi Faisal, if your solvent channels are smaller than 40A in the largest dimension (most are) you can use a mesh loop to pick up the crystal and then wick away all of the mother liquor. You can then flash cool your crystal without having to transfer the crystal to another solution. Good luck, Matt -- 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/ ===
