Re: [ccp4bb] Crystals Disappearing Overnight
Dear Maria have you collected the crystals before they dissolve and washed them, then either dissolved them in SDS PAGE buffer and ran them on an SDS PAGE gel (for say a western blot) or capillary mounted them, then shot them on the x-ray set to determine if they are protein or small molecule, or tried to freeze them, or washed them and sent them to mass spec to determine if they are protein? If the crystals are protein try different conditions, such as a gradient of PEG vs NaCl, seeding using the crystals crushed before they dissolve, protein concentration, ligand concentration etc and maybe a different temperature. Good luck G On May 7, 2014, at 1:52 AM, dusky dew wrote: I tried microbatch and the crystals are not stable. They dissolve overnight. I also have reproducibility issue. Can this be due to poor stability of adenosine? Best Maria On Monday, May 5, 2014, Bob Cudney b...@hrmail.commailto:b...@hrmail.com wrote: Try the microbatch first to see if the problem is related to ionic strength. When possible and practical it is good to change only one variable at a time to identify cause and effect. Kind Regards, Bob Cudney Hampton Research 34 Journey Aliso Viejo, CA 92656-3317 USA Telephone 1 949 425 1321 Extension 200 Fax 1 949 425 1611 E-mail b...@hrmail.commailto:b...@hrmail.com Web www.hamptonresearch.comhttp://www.hamptonresearch.com/ From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of dusky dew Sent: Saturday, May 03, 2014 1:29 AM To: CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Crystals Disappearing Overnight Thank you all for getting back! I will set up the drops using microbatch method. Regarding the temp, I set them up in lab and put them in incubator. The lab temp may be slightly higher. So are they not stable at lower temp? Or its the shock? So how can I take care of the temp issue? Thanks again! Maria On Saturday, May 3, 2014, Bob Cudney b...@hrmail.commailto:b...@hrmail.com wrote: Hello Maria, Check to see if there might have been a temperature change between the time the crystals were present and when the crystals disappeared. If your sample has temperature dependent solubility, in this relatively low ionic strength condition, a temperature change could mean the difference between the presence and absence of crystals. That being said, if the experiment is returned to the temperature that produced the crystals, the crystals should/might reappear. If your drop is made by mixing 1 part of protein with 1 part of reagent the initial drop concentration would be 10 mM Tris, 150 mM NaCl, 2.5% w/v PEG 8,000, 50 mM Sodium cacodylate. Is this a vapor diffusion experiment? If yes, then the reservoir would be 5% w/v PEG 8,000, 100 mM Sodium cacodylate. The ionic strength of your drop would initially be higher than the ionic strength in your reservoir. This means water vapor leaves the reservoir and vapor diffuses into the drop, lowering the protein and reagent concentration in your drop. This decrease in relative supersaturation could dissolve a crystal. Your set up would be a reserve vapor diffusion. You say the crystals appeared right after setting the experiment so your crystallization is essentially a batch experiment. Therefore you might want to change your set up from a vapor diffusion to a microbatch experiment under oil. If you need more information about how to perform a microbatch experiment, let me know and I’ll explain. Hope this helps. Kind Regards, Bob Cudney Hampton Research 34 Journey Aliso Viejo, CA 92656-3317 USA Telephone 1 949 425 1321 Extension 200 Fax 1 949 425 1611 E-mail b...@hrmail.commailto:b...@hrmail.com Web www.hamptonresearch.comhttp://www.hamptonresearch.com/ From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of dusky dew Sent: Friday, May 02, 2014 4:39 AM To: CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Crystals Disappearing Overnight Dear All, I am trying to crystallize a protein with Adenosine. My protein is in 20 mM Tris, 300 mM NaCl and the crystals appear in a condition with 5 percent PEG8K, 0.1 M Sodium Cacodylate. The protein is incubated with adenosine for 1/2 hr before setting the drop. The crystals appear right after the drop is set but unfortunately they dissolve overnight. The plate is kept at 16 degree. Could anyone elaborate on this. Is it possibly occurring because Adenosine has stability issues. Thanks for your suggestions. ~ Maria NOTICE: This email message is for the sole use of the intended recipient(s) and may contain confidential and privileged information. Any unauthorized review, use, disclosure or distribution is prohibited. If you are not the intended recipient, please contact the sender
Re: [ccp4bb] Crystals Disappearing Overnight
Hi Maria, Adenosine is quite stable but not very soluble in aqueous solution. A labmate of mine would routinely observe adenosine precipitating in his crystal trays, which later dissolved, presumably as his protein slowly took up ligand. To follow the previous comments, I'd make sure what you see is really protein and not simply crystals of adenosine. Note that crystals of adenosine may still give you a signal under UV fluorescence, so best to trust other tests for protein, or of course the one test that really matters, diffraction. Shane Caldwell McGill University On Fri, May 9, 2014 at 12:39 PM, Clayton, Gina clayt...@njhealth.orgwrote: Dear Maria have you collected the crystals before they dissolve and washed them, then either dissolved them in SDS PAGE buffer and ran them on an SDS PAGE gel (for say a western blot) or capillary mounted them, then shot them on the x-ray set to determine if they are protein or small molecule, or tried to freeze them, or washed them and sent them to mass spec to determine if they are protein? If the crystals are protein try different conditions, such as a gradient of PEG vs NaCl, seeding using the crystals crushed before they dissolve, protein concentration, ligand concentration etc and maybe a different temperature. Good luck G On May 7, 2014, at 1:52 AM, dusky dew wrote: I tried microbatch and the crystals are not stable. They dissolve overnight. I also have reproducibility issue. Can this be due to poor stability of adenosine? Best Maria On Monday, May 5, 2014, Bob Cudney b...@hrmail.com wrote: Try the microbatch first to see if the problem is related to ionic strength. When possible and practical it is good to change only one variable at a time to identify cause and effect. Kind Regards, Bob Cudney Hampton Research 34 Journey Aliso Viejo, CA 92656-3317 USA Telephone 1 949 425 1321 Extension 200 Fax 1 949 425 1611 E-mail b...@hrmail.com Web www.hamptonresearch.com From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of dusky dew Sent: Saturday, May 03, 2014 1:29 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Crystals Disappearing Overnight Thank you all for getting back! I will set up the drops using microbatch method. Regarding the temp, I set them up in lab and put them in incubator. The lab temp may be slightly higher. So are they not stable at lower temp? Or its the shock? So how can I take care of the temp issue? Thanks again! Maria On Saturday, May 3, 2014, Bob Cudney b...@hrmail.com wrote: Hello Maria, Check to see if there might have been a temperature change between the time the crystals were present and when the crystals disappeared. If your sample has temperature dependent solubility, in this relatively low ionic strength condition, a temperature change could mean the difference between the presence and absence of crystals. That being said, if the experiment is returned to the temperature that produced the crystals, the crystals should/might reappear. If your drop is made by mixing 1 part of protein with 1 part of reagent the initial drop concentration would be 10 mM Tris, 150 mM NaCl, 2.5% w/v PEG 8,000, 50 mM Sodium cacodylate. Is this a vapor diffusion experiment? If yes, then the reservoir would be 5% w/v PEG 8,000, 100 mM Sodium cacodylate. The ionic strength of your drop would initially be higher than the ionic strength in your reservoir. This means water vapor leaves the reservoir and vapor diffuses into the drop, lowering the protein and reagent concentration in your drop. This decrease in relative supersaturation could dissolve a crystal. Your set up would be a reserve vapor diffusion. You say the crystals appeared right after setting the experiment so your crystallization is essentially a batch experiment. Therefore you might want to change your set up from a vapor diffusion to a microbatch experiment under oil. If you need more information about how to perform a microbatch experiment, let me know and I’ll explain. Hope this helps. Kind Regards, Bob Cudney Hampton Research 34 Journey Aliso Viejo, CA 92656-3317 USA Telephone 1 949 425 1321 Extension 200 Fax 1 949 425 1611 E-mail b...@hrmail.com Web www.hamptonresearch.com From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of dusky dew Sent: Friday, May 02, 2014 4:39 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Crystals Disappearing Overnight Dear All, I am trying to crystallize a protein with Adenosine. My protein is in 20 mM Tris, 300 mM NaCl and the crystals appear in a condition with 5 percent PEG8K, 0.1 M Sodium Cacodylate. The protein is incubated with adenosine for 1/2 hr before setting the drop. The crystals appear right after the drop is set but unfortunately they dissolve
Re: [ccp4bb] Crystals Disappearing Overnight
*** This message has been scanned by the InterScan for CSC SSM by IICT security policy and found to be free of known security risks. *** Maria, From my experience with co-crystallization experiments, if crystals appear immediately after setting up the drop in presence of small molecules, they most likely are crystals of small molecules and not of protein. Since you have 8% 8K PEG, initially the solution is heterogeneous with pockets of high viscosity that will precipitate the small molecules. Over the period of time, solution becomes homogenous that may dissolve the crystals. To rule out this, set up two crystallization experiments under similar conditions. 1) only the protein 2) only the adenosine Regards Anthony - Dr. Anthony Addlagatta Center for Chemical Biology Indian Institute of Chemical Technology [IICT] Tarnaka, Hyderabad AP-500 607, INDIA Tel:91-40-27191812 Web: https://sites.google.com/site/chembioliict/home/dr-anthony-addlagatta-1 -- Original Message --- From: dusky dew duskyde...@gmail.com To: CCP4BB@JISCMAIL.AC.UK Sent: Wed, 7 May 2014 00:52:22 -0700 Subject: Re: [ccp4bb] Crystals Disappearing Overnight *** This message has been scanned by the InterScan for CSC SSM at IICT and found to be free of known security risks. *** I tried microbatch and the crystals are not stable. They dissolve overnight. I also have reproducibility issue. Can this be due to poor stability of adenosine? Best Maria On Monday, May 5, 2014, Bob Cudney b...@hrmail.com wrote: Try the microbatch first to see if the problem is related to ionic strength. When possible and practical it is good to change only one variable at a time to identify cause and effect. Kind Regards, Bob Cudney Hampton Research 34 Journey Aliso Viejo, CA 92656-3317 USA Telephone 1 949 425 1321 Extension 200 Fax 1 949 425 1611 E-mail b...@hrmail.com Web www.hamptonresearch.com From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of dusky dew Sent: Saturday, May 03, 2014 1:29 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Crystals Disappearing Overnight Thank you all for getting back! I will set up the drops using microbatch method. Regarding the temp, I set them up in lab and put them in incubator. The lab temp may be slightly higher. So are they not stable at lower temp? Or its the shock? So how can I take care of the temp issue? Thanks again! Maria On Saturday, May 3, 2014, Bob Cudney b...@hrmail.com wrote: Hello Maria, Check to see if there might have been a temperature change between the time the crystals were present and when the crystals disappeared. If your sample has temperature dependent solubility, in this relatively low ionic strength condition, a temperature change could mean the difference between the presence and absence of crystals. That being said, if the experiment is returned to the temperature that produced the crystals, the crystals should/might reappear. If your drop is made by mixing 1 part of protein with 1 part of reagent the initial drop concentration would be 10 mM Tris, 150 mM NaCl, 2.5% w/v PEG 8,000, 50 mM Sodium cacodylate. Is this a vapor diffusion experiment? If yes, then the reservoir would be 5% w/v PEG 8,000, 100 mM Sodium cacodylate. The ionic strength of your drop would initially be higher than the ionic strength in your reservoir. This means water vapor leaves the reservoir and vapor diffuses into the drop, lowering the protein and reagent concentration in your drop. This decrease in relative supersaturation could dissolve a crystal. Your set up would be a reserve vapor diffusion. You say the crystals appeared right after setting the experiment so your crystallization is essentially a batch experiment. Therefore you might want to change your set up from a vapor diffusion to a microbatch experiment under oil. If you need more information about how to perform a microbatch experiment, let me know and I’ll explain. Hope this helps. Kind Regards, Bob Cudney Hampton Research 34 Journey Aliso Viejo, CA 92656-3317 USA Telephone 1 949 425 1321 Extension 200 Fax 1 949 425 1611 E-mail b...@hrmail.com Web www.hamptonresearch.com From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of dusky dew Sent: Friday, May 02, 2014 4:39 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Crystals Disappearing Overnight Dear All, I am trying to crystallize a protein with Adenosine. My protein is in 20 mM Tris, 300 mM NaCl and the crystals appear in a condition with 5 percent PEG8K, 0.1 M Sodium Cacodylate. The protein is incubated with adenosine for 1/2 hr before setting the drop. The crystals appear
Re: [ccp4bb] Crystals Disappearing Overnight
Thank you all for getting back! I will set up the drops using microbatch method. Regarding the temp, I set them up in lab and put them in incubator. The lab temp may be slightly higher. So are they not stable at lower temp? Or its the shock? So how can I take care of the temp issue? Thanks again! Maria On Saturday, May 3, 2014, Bob Cudney b...@hrmail.com wrote: Hello Maria, Check to see if there might have been a temperature change between the time the crystals were present and when the crystals disappeared. If your sample has temperature dependent solubility, in this relatively low ionic strength condition, a temperature change could mean the difference between the presence and absence of crystals. That being said, if the experiment is returned to the temperature that produced the crystals, the crystals should/might reappear. If your drop is made by mixing 1 part of protein with 1 part of reagent the initial drop concentration would be 10 mM Tris, 150 mM NaCl, 2.5% w/v PEG 8,000, 50 mM Sodium cacodylate. Is this a vapor diffusion experiment? If yes, then the reservoir would be 5% w/v PEG 8,000, 100 mM Sodium cacodylate. The ionic strength of your drop would initially be higher than the ionic strength in your reservoir. This means water vapor leaves the reservoir and vapor diffuses into the drop, lowering the protein and reagent concentration in your drop. This decrease in relative supersaturation could dissolve a crystal. Your set up would be a reserve vapor diffusion. You say the crystals appeared right after setting the experiment so your crystallization is essentially a batch experiment. Therefore you might want to change your set up from a vapor diffusion to a microbatch experiment under oil. If you need more information about how to perform a microbatch experiment, let me know and I’ll explain. Hope this helps. Kind Regards, Bob Cudney Hampton Research 34 Journey Aliso Viejo, CA 92656-3317 USA Telephone 1 949 425 1321 Extension 200 Fax 1 949 425 1611 E-mail b...@hrmail.com Web www.hamptonresearch.com From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of dusky dew Sent: Friday, May 02, 2014 4:39 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Crystals Disappearing Overnight Dear All, I am trying to crystallize a protein with Adenosine. My protein is in 20 mM Tris, 300 mM NaCl and the crystals appear in a condition with 5 percent PEG8K, 0.1 M Sodium Cacodylate. The protein is incubated with adenosine for 1/2 hr before setting the drop. The crystals appear right after the drop is set but unfortunately they dissolve overnight. The plate is kept at 16 degree. Could anyone elaborate on this. Is it possibly occurring because Adenosine has stability issues. Thanks for your suggestions. ~ Maria
[ccp4bb] Crystals Disappearing Overnight
Dear All, I am trying to crystallize a protein with Adenosine. My protein is in 20 mM Tris, 300 mM NaCl and the crystals appear in a condition with 5 percent PEG8K, 0.1 M Sodium Cacodylate. The protein is incubated with adenosine for 1/2 hr before setting the drop. The crystals appear right after the drop is set but unfortunately they dissolve overnight. The plate is kept at 16 degree. Could anyone elaborate on this. Is it possibly occurring because Adenosine has stability issues. Thanks for your suggestions. ~ Maria
Re: [ccp4bb] Crystals Disappearing Overnight
-BEGIN PGP SIGNED MESSAGE- Hash: SHA1 Dear Maria, this is similar to the condition I used for my PhD thesis. I suppose the following happens: when you mix the drop the protein crystallises by inverse salting-in because you dilute the high-salt concentration. This happens instantaneously and you are lucky enough to observe crystals. As you leave the drop overnight you are observing the opposite from what normally happens: your drop gets larger instead of smaller because the hygroscopy of the 150mM NaCl is stronger than that of 2.5% PEG8K and you further dilute the drop so that the crystal disappears. Check if your drops get bigger overnight to check if my assumption is correct. During my PhD I worked with 0.5M NaCl and maybe I am wrong that 150mM NaCl is stronger than 2.5% PEG8k. Best, Tim On 05/02/2014 01:39 PM, dusky dew wrote: Dear All, I am trying to crystallize a protein with Adenosine. My protein is in 20 mM Tris, 300 mM NaCl and the crystals appear in a condition with 5 percent PEG8K, 0.1 M Sodium Cacodylate. The protein is incubated with adenosine for 1/2 hr before setting the drop. The crystals appear right after the drop is set but unfortunately they dissolve overnight. The plate is kept at 16 degree. Could anyone elaborate on this. Is it possibly occurring because Adenosine has stability issues. Thanks for your suggestions. ~ Maria - -- - -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A -BEGIN PGP SIGNATURE- Version: GnuPG v1.4.12 (GNU/Linux) Comment: Using GnuPG with Icedove - http://www.enigmail.net/ iD8DBQFTY4gDUxlJ7aRr7hoRAvZcAJ9fnqKmsjWvsvQifgB2oG3EH4/h9wCghJVA 9Ng4dZzXQG5+LQiPwZVdXXQ= =LbR6 -END PGP SIGNATURE-
Re: [ccp4bb] Crystals Disappearing Overnight
Dear Tim, you are right that 150mM NaCl is 'stronger' than 2.5% PEG. If you calculate the relative humidity difference between 150 and 300mM Nacl you get 99.5 vs 98.9% whereas the difference between 5 and 2.5% P8K is 99.96 vs 99.99% ie virtually nothing. You can calculate these values here: http://go.esrf.eu/RH. Cheers, Matt. On 2014-05-02 13:56, Tim Gruene wrote: -BEGIN PGP SIGNED MESSAGE- Hash: SHA1 Dear Maria, this is similar to the condition I used for my PhD thesis. I suppose the following happens: when you mix the drop the protein crystallises by inverse salting-in because you dilute the high-salt concentration. This happens instantaneously and you are lucky enough to observe crystals. As you leave the drop overnight you are observing the opposite from what normally happens: your drop gets larger instead of smaller because the hygroscopy of the 150mM NaCl is stronger than that of 2.5% PEG8K and you further dilute the drop so that the crystal disappears. Check if your drops get bigger overnight to check if my assumption is correct. During my PhD I worked with 0.5M NaCl and maybe I am wrong that 150mM NaCl is stronger than 2.5% PEG8k. Best, Tim On 05/02/2014 01:39 PM, dusky dew wrote: Dear All, I am trying to crystallize a protein with Adenosine. My protein is in 20 mM Tris, 300 mM NaCl and the crystals appear in a condition with 5 percent PEG8K, 0.1 M Sodium Cacodylate. The protein is incubated with adenosine for 1/2 hr before setting the drop. The crystals appear right after the drop is set but unfortunately they dissolve overnight. The plate is kept at 16 degree. Could anyone elaborate on this. Is it possibly occurring because Adenosine has stability issues. Thanks for your suggestions. ~ Maria - -- - -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A -BEGIN PGP SIGNATURE- Version: GnuPG v1.4.12 (GNU/Linux) Comment: Using GnuPG with Icedove - http://www.enigmail.net/ iD8DBQFTY4gDUxlJ7aRr7hoRAvZcAJ9fnqKmsjWvsvQifgB2oG3EH4/h9wCghJVA 9Ng4dZzXQG5+LQiPwZVdXXQ= =LbR6 -END PGP SIGNATURE- -- 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] Crystals Disappearing Overnight
Hi Maria - I think you already have a likely answer, but here's another one which I have observed many times: Temperature differences. If your crystallization plate is moved from one temperature to another (lab vs. crystal incubator), or even if it's left on the bench but the lab temperature varies overnight, you can get a temperature difference between your reservoir solution and the rest of the crystallization chamber. This can cause water condensation on the cover slip, which will dilute a hanging drop (and even a sitting drop if it's high enough up) and cause things to dissolve. The solution to this one is just careful control of the crystal plate environment. The same is true after the crystals are done growing - you can lose an entire drop of nice crystals from taking the plate out of the incubator to look at it under the microscope! Hope that helps, Matt On 5/2/14 7:39 AM, dusky dew wrote: Dear All, I am trying to crystallize a protein with Adenosine. My protein is in 20 mM Tris, 300 mM NaCl and the crystals appear in a condition with 5 percent PEG8K, 0.1 M Sodium Cacodylate. The protein is incubated with adenosine for 1/2 hr before setting the drop. The crystals appear right after the drop is set but unfortunately they dissolve overnight. The plate is kept at 16 degree. Could anyone elaborate on this. Is it possibly occurring because Adenosine has stability issues. Thanks for your suggestions. ~ Maria -- Matthew Franklin, Ph. D. Senior Scientist New York Structural Biology Center 89 Convent Avenue, New York, NY 10027 (212) 939-0660 ext. 9374
