Re: [ccp4bb] Crystal rescue
Hi Zhiyi, There are two ways you can go about this (RT data collection). Either mount the crystals in capillaries (not all beam lines at synchrotrons have sufficient space on the goniometer setup to allow data collection on capillary-mounted crystals) or use a Humidifier (the latter is briefly discussed, for example, on web page http://www.esrf.eu/UsersAndScience/Experiments/MX/Cryobench/Instruments/microspec ). If nothing is done, the crystals will rapidly dry up and die. Fred. Message du 27/01/10 08:53 De : Zhiyi Wei A : CCP4BB@JISCMAIL.AC.UK Copie à : Objet : Re: [ccp4bb] Crystal rescue Thanks for so many quick responses! Actually, I have test several different cryo-protectants, including glycerol, EG, and PEG400. I did not see much differences between these cryo conditions. So, I choose glycerol. I would like to test my crystals in RT. But I don't know how to do this. Just mount crystal to the X-ray machine without cryo stream? Or I should use capillaries? Zhiyi On Wed, Jan 27, 2010 at 5:45 AM, Kelly Daughtry wrote: Tascimate can be used as the cryo as well. I have had experience with crystals in similar condition and moved the crystals to a 20% increased Tascimate solution and they froze well. I agree with Ezra, room temperature mount your crystal before freezing. It is the only way to know the true problem. Kelly *** Kelly Daughtry PhD Candidate Department of Physiology and Biophysics Boston University School of Medicine 590 Commonwealth Ave R 390 Boston MA, 02215 (P) 617-358-5548 *** On Tue, Jan 26, 2010 at 1:25 PM, Marcus Winter wrote: Dear Zhiyi, Ezra is exactly right, of course. The Oxford Diffraction PX Scanner system can assess the diffraction qualities of (putative) protein crystals in situ - in the crystallisation plate. So, directly, you would discover if your 'big and beautiful' crystals actually diffract well... in their mother liquor under ambient conditions and before the addition of any cryo-protect. Do you have a friend or neighbour with a PX Scanner ? If not, please feel most welcome to contact Oxford Diffraction: we would be pleased to assist if at all possible. Good Luck and Best Wishes, Marcus Winter. www.oxford-diffraction.com -Original Message- From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Ezra Peisach Sent: 26 January 2010 16:01 To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Crystal rescue First you need to establish if it is your cryo conditions or the crystals. Depending where you are - they might have the equipment to do a wet mount - without freezing. Yes the crystal will not last - but then you know if the problem is in the crystal. If it is - you need better crystals. If it is the cryo - you need to work on that. Tacsimate is mixture of alot of different compounds - but the smears are too close together to be a small salt crystal on top... Good luck, Ezra On 1/26/2010 10:42 AM, Zhiyi Wei wrote: Dear all, I got a problem with my crystals. I have two total different proteins that both can be crystallized in the condition with PEG3350 and Tacsimate (although the concentrations are different) with different shapes. The crystals look big and beautiful. However, when I test them in synchrotron, both of these two types of crystals showed poor diffractions. As showed in the attached diffraction image, the diffraction is up to ~4 A but smear in one direction while8 A in the other direction. The interesting thing is that the diffraction pattern is similar for all crystals (from two different proteins) that I tested without exception although they belong to different space groups. So, I wonder whether these kind of pattern is caused by Tacsimate (I don't know what it is) and how to rescue these crystals. Any suggestions or comments? Thanks a lot! Best, Zhiyi
Re: [ccp4bb] Crystal rescue
Zhiyi, You can use a thin cap over your cryo loop, just put a drop of mother liquor in the top, place over the loop and make it airtight at the base. Not sure who sells these things though, I guess you can make it from a capillary too. Then remove the cryo stream or put it at a temp above freezing, say 253K. Flip Zhiyi Wei wrote: Thanks for so many quick responses! Actually, I have test several different cryo-protectants, including glycerol, EG, and PEG400. I did not see much differences between these cryo conditions. So, I choose glycerol. I would like to test my crystals in RT. But I don't know how to do this. Just mount crystal to the X-ray machine without cryo stream? Or I should use capillaries? Zhiyi On Wed, Jan 27, 2010 at 5:45 AM, Kelly Daughtry kddau...@bu.edu wrote: Tascimate can be used as the cryo as well. I have had experience with crystals in similar condition and moved the crystals to a 20% increased Tascimate solution and they froze well. I agree with Ezra, room temperature mount your crystal before freezing. It is the only way to know the true problem. Kelly *** Kelly Daughtry PhD Candidate Department of Physiology and Biophysics Boston University School of Medicine 590 Commonwealth Ave R 390 Boston MA, 02215 (P) 617-358-5548 *** On Tue, Jan 26, 2010 at 1:25 PM, Marcus Winter marcus.win...@oxford-diffraction.com wrote: Dear Zhiyi, Ezra is exactly right, of course. The Oxford Diffraction PX Scanner system can assess the diffraction qualities of (putative) protein crystals in situ - in the crystallisation plate. So, directly, you would discover if your 'big and beautiful' crystals actually diffract well... in their mother liquor under ambient conditions and before the addition of any cryo-protect. Do you have a friend or neighbour with a PX Scanner ? If not, please feel most welcome to contact Oxford Diffraction: we would be pleased to assist if at all possible. Good Luck and Best Wishes, Marcus Winter. www.oxford-diffraction.com -Original Message- From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Ezra Peisach Sent: 26 January 2010 16:01 To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Crystal rescue First you need to establish if it is your cryo conditions or the crystals. Depending where you are - they might have the equipment to do a wet mount - without freezing. Yes the crystal will not last - but then you know if the problem is in the crystal. If it is - you need better crystals. If it is the cryo - you need to work on that. Tacsimate is mixture of alot of different compounds - but the smears are too close together to be a small salt crystal on top... Good luck, Ezra On 1/26/2010 10:42 AM, Zhiyi Wei wrote: Dear all, I got a problem with my crystals. I have two total different proteins that both can be crystallized in the condition with PEG3350 and Tacsimate (although the concentrations are different) with different shapes. The crystals look big and beautiful. However, when I test them in synchrotron, both of these two types of crystals showed poor diffractions. As showed in the attached diffraction image, the diffraction is up to ~4 A but smear in one direction while8 A in the other direction. The interesting thing is that the diffraction pattern is similar for all crystals (from two different proteins) that I tested without exception although they belong to different space groups. So, I wonder whether these kind of pattern is caused by Tacsimate (I don't know what it is) and how to rescue these crystals. Any suggestions or comments? Thanks a lot! Best, Zhiyi
Re: [ccp4bb] Crystal rescue
Hi Fred and Zhiyi, the EMBL and ESRF have also developed a dehydration device that can also be used for room temperature data collection, see the website here: http://www.esrf.fr/UsersAndScience/Experiments/MX/About_our_beamlines/ID14-2/HC1b http://scripts.iucr.org/cgi-bin/paper?S0907444909037822 Cheers, Matt. Frederic VELLIEUX wrote: Hi Zhiyi, There are two ways you can go about this (RT data collection). Either mount the crystals in capillaries (not all beam lines at synchrotrons have sufficient space on the goniometer setup to allow data collection on capillary-mounted crystals) or use a Humidifier (the latter is briefly discussed, for example, on web page http://www.esrf.eu/UsersAndScience/Experiments/MX/Cryobench/Instruments/microspec ). If nothing is done, the crystals will rapidly dry up and die. Fred. Message du 27/01/10 08:53 De : Zhiyi Wei A : CCP4BB@JISCMAIL.AC.UK Copie à : Objet : Re: [ccp4bb] Crystal rescue Thanks for so many quick responses! Actually, I have test several different cryo-protectants, including glycerol, EG, and PEG400. I did not see much differences between these cryo conditions. So, I choose glycerol. I would like to test my crystals in RT. But I don't know how to do this. Just mount crystal to the X-ray machine without cryo stream? Or I should use capillaries? Zhiyi On Wed, Jan 27, 2010 at 5:45 AM, Kelly Daughtry wrote: Tascimate can be used as the cryo as well. I have had experience with crystals in similar condition and moved the crystals to a 20% increased Tascimate solution and they froze well. I agree with Ezra, room temperature mount your crystal before freezing. It is the only way to know the true problem. Kelly *** Kelly Daughtry PhD Candidate Department of Physiology and Biophysics Boston University School of Medicine 590 Commonwealth Ave R 390 Boston MA, 02215 (P) 617-358-5548 *** On Tue, Jan 26, 2010 at 1:25 PM, Marcus Winter wrote: Dear Zhiyi, Ezra is exactly right, of course. The Oxford Diffraction PX Scanner system can assess the diffraction qualities of (putative) protein crystals in situ - in the crystallisation plate. So, directly, you would discover if your 'big and beautiful' crystals actually diffract well... in their mother liquor under ambient conditions and before the addition of any cryo-protect. Do you have a friend or neighbour with a PX Scanner ? If not, please feel most welcome to contact Oxford Diffraction: we would be pleased to assist if at all possible. Good Luck and Best Wishes, Marcus Winter. www.oxford-diffraction.com -Original Message- From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Ezra Peisach Sent: 26 January 2010 16:01 To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Crystal rescue First you need to establish if it is your cryo conditions or the crystals. Depending where you are - they might have the equipment to do a wet mount - without freezing. Yes the crystal will not last - but then you know if the problem is in the crystal. If it is - you need better crystals. If it is the cryo - you need to work on that. Tacsimate is mixture of alot of different compounds - but the smears are too close together to be a small salt crystal on top... Good luck, Ezra On 1/26/2010 10:42 AM, Zhiyi Wei wrote: Dear all, I got a problem with my crystals. I have two total different proteins that both can be crystallized in the condition with PEG3350 and Tacsimate (although the concentrations are different) with different shapes. The crystals look big and beautiful. However, when I test them in synchrotron, both of these two types of crystals showed poor diffractions. As showed in the attached diffraction image, the diffraction is up to ~4 A but smear in one direction while8 A in the other direction. The interesting thing is that the diffraction pattern is similar for all crystals (from two different proteins) that I tested without exception although they belong to different space groups. So, I wonder whether these kind of pattern is caused by Tacsimate (I don't know what it is) and how to rescue these crystals. Any suggestions or comments? Thanks a lot! Best, Zhiyi -- Matthew Bowler Structural Biology Group European Synchrotron Radiation Facility B.P. 220, 6 rue Jules Horowitz F-38043 GRENOBLE CEDEX FRANCE === Tel: +33 (0) 4.76.88.29.28 Fax: +33 (0) 4.76.88.29.04 http://www.esrf.fr/UsersAndScience/Experiments/MX/ ===
Re: [ccp4bb] Crystal rescue
MiTeGen (www.mitegen.com) sells a RT device - plastic capillary to put over loop... I do not know how well it would work for a long data collection - but people here have used it to evaluate their crystals Ezra On 1/27/2010 3:58 AM, Flip Hoedemaeker wrote: Zhiyi, You can use a thin cap over your cryo loop, just put a drop of mother liquor in the top, place over the loop and make it airtight at the base. Not sure who sells these things though, I guess you can make it from a capillary too. Then remove the cryo stream or put it at a temp above freezing, say 253K. Flip Zhiyi Wei wrote: Thanks for so many quick responses! Actually, I have test several different cryo-protectants, including glycerol, EG, and PEG400. I did not see much differences between these cryo conditions. So, I choose glycerol. I would like to test my crystals in RT. But I don't know how to do this. Just mount crystal to the X-ray machine without cryo stream? Or I should use capillaries? Zhiyi On Wed, Jan 27, 2010 at 5:45 AM, Kelly Daughtry kddau...@bu.edu wrote: Tascimate can be used as the cryo as well. I have had experience with crystals in similar condition and moved the crystals to a 20% increased Tascimate solution and they froze well. I agree with Ezra, room temperature mount your crystal before freezing. It is the only way to know the true problem. Kelly *** Kelly Daughtry PhD Candidate Department of Physiology and Biophysics Boston University School of Medicine 590 Commonwealth Ave R 390 Boston MA, 02215 (P) 617-358-5548 *** On Tue, Jan 26, 2010 at 1:25 PM, Marcus Winter marcus.win...@oxford-diffraction.com wrote: Dear Zhiyi, Ezra is exactly right, of course. The Oxford Diffraction PX Scanner system can assess the diffraction qualities of (putative) protein crystals in situ - in the crystallisation plate. So, directly, you would discover if your 'big and beautiful' crystals actually diffract well... in their mother liquor under ambient conditions and before the addition of any cryo-protect. Do you have a friend or neighbour with a PX Scanner ? If not, please feel most welcome to contact Oxford Diffraction: we would be pleased to assist if at all possible. Good Luck and Best Wishes, Marcus Winter. www.oxford-diffraction.com -Original Message- From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Ezra Peisach Sent: 26 January 2010 16:01 To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Crystal rescue First you need to establish if it is your cryo conditions or the crystals. Depending where you are - they might have the equipment to do a wet mount - without freezing. Yes the crystal will not last - but then you know if the problem is in the crystal. If it is - you need better crystals. If it is the cryo - you need to work on that. Tacsimate is mixture of alot of different compounds - but the smears are too close together to be a small salt crystal on top... Good luck, Ezra On 1/26/2010 10:42 AM, Zhiyi Wei wrote: Dear all, I got a problem with my crystals. I have two total different proteins that both can be crystallized in the condition with PEG3350 and Tacsimate (although the concentrations are different) with different shapes. The crystals look big and beautiful. However, when I test them in synchrotron, both of these two types of crystals showed poor diffractions. As showed in the attached diffraction image, the diffraction is up to ~4 A but smear in one direction while8 A in the other direction. The interesting thing is that the diffraction pattern is similar for all crystals (from two different proteins) that I tested without exception although they belong to different space groups. So, I wonder whether these kind of pattern is caused by Tacsimate (I don't know what it is) and how to rescue these crystals. Any suggestions or comments? Thanks a lot! Best, Zhiyi
Re: [ccp4bb] Crystal rescue
MiTeGen have instructions on their website for using their system. In practice I usually put a little silicon grease at the base of the capillary as sometimes the capillaries fall off plus you need reasonable steady hand eye coordination to put the capillary over the loop...I have found MiTeGen RT system very useful. Good luck! -Original Message- From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Ezra Peisach Sent: Wednesday, January 27, 2010 7:41 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Crystal rescue MiTeGen (www.mitegen.com) sells a RT device - plastic capillary to put over loop... I do not know how well it would work for a long data collection - but people here have used it to evaluate their crystals Ezra On 1/27/2010 3:58 AM, Flip Hoedemaeker wrote: Zhiyi, You can use a thin cap over your cryo loop, just put a drop of mother liquor in the top, place over the loop and make it airtight at the base. Not sure who sells these things though, I guess you can make it from a capillary too. Then remove the cryo stream or put it at a temp above freezing, say 253K. Flip Zhiyi Wei wrote: Thanks for so many quick responses! Actually, I have test several different cryo-protectants, including glycerol, EG, and PEG400. I did not see much differences between these cryo conditions. So, I choose glycerol. I would like to test my crystals in RT. But I don't know how to do this. Just mount crystal to the X-ray machine without cryo stream? Or I should use capillaries? Zhiyi On Wed, Jan 27, 2010 at 5:45 AM, Kelly Daughtry kddau...@bu.edu wrote: Tascimate can be used as the cryo as well. I have had experience with crystals in similar condition and moved the crystals to a 20% increased Tascimate solution and they froze well. I agree with Ezra, room temperature mount your crystal before freezing. It is the only way to know the true problem. Kelly *** Kelly Daughtry PhD Candidate Department of Physiology and Biophysics Boston University School of Medicine 590 Commonwealth Ave R 390 Boston MA, 02215 (P) 617-358-5548 *** On Tue, Jan 26, 2010 at 1:25 PM, Marcus Winter marcus.win...@oxford-diffraction.com wrote: Dear Zhiyi, Ezra is exactly right, of course. The Oxford Diffraction PX Scanner system can assess the diffraction qualities of (putative) protein crystals in situ - in the crystallisation plate. So, directly, you would discover if your 'big and beautiful' crystals actually diffract well... in their mother liquor under ambient conditions and before the addition of any cryo-protect. Do you have a friend or neighbour with a PX Scanner ? If not, please feel most welcome to contact Oxford Diffraction: we would be pleased to assist if at all possible. Good Luck and Best Wishes, Marcus Winter. www.oxford-diffraction.com -Original Message- From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Ezra Peisach Sent: 26 January 2010 16:01 To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Crystal rescue First you need to establish if it is your cryo conditions or the crystals. Depending where you are - they might have the equipment to do a wet mount - without freezing. Yes the crystal will not last - but then you know if the problem is in the crystal. If it is - you need better crystals. If it is the cryo - you need to work on that. Tacsimate is mixture of alot of different compounds - but the smears are too close together to be a small salt crystal on top... Good luck, Ezra On 1/26/2010 10:42 AM, Zhiyi Wei wrote: Dear all, I got a problem with my crystals. I have two total different proteins that both can be crystallized in the condition with PEG3350 and Tacsimate (although the concentrations are different) with different shapes. The crystals look big and beautiful. However, when I test them in synchrotron, both of these two types of crystals showed poor diffractions. As showed in the attached diffraction image, the diffraction is up to ~4 A but smear in one direction while8 A in the other direction. The interesting thing is that the diffraction pattern is similar for all crystals (from two different proteins) that I tested without exception although they belong to different space groups. So, I wonder whether these kind of pattern is caused by Tacsimate (I don't know what it is) and how to rescue these crystals. Any suggestions or comments? Thanks a lot! Best, Zhiyi Notice: This e-mail message, together with any attachments, contains information of Merck Co., Inc. (One Merck Drive, Whitehouse Station, New Jersey, USA 08889), and/or its affiliates Direct contact information for affiliates is available at http://www.merck.com/contact/contacts.html) that may be confidential, proprietary copyrighted and/or legally
Re: [ccp4bb] Crystal rescue
in my opinion one should always first check RT diffraction and we use the Mitegen plastic cap over the loop. We find the solutions slowly dry out (6-18 hours), which can be enough to get a useful RT dataset on the rotating anode. If you like, the crystal can also be recovered after RT diffraction checking for addition of cryosolution and freezing. Mark Quoting Clayton, Gina Martyn gina_clay...@merck.com: MiTeGen have instructions on their website for using their system. In practice I usually put a little silicon grease at the base of the capillary as sometimes the capillaries fall off plus you need reasonable steady hand eye coordination to put the capillary over the loop...I have found MiTeGen RT system very useful. Good luck! -Original Message- From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Ezra Peisach Sent: Wednesday, January 27, 2010 7:41 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Crystal rescue MiTeGen (www.mitegen.com) sells a RT device - plastic capillary to put over loop... I do not know how well it would work for a long data collection - but people here have used it to evaluate their crystals Ezra On 1/27/2010 3:58 AM, Flip Hoedemaeker wrote: Zhiyi, You can use a thin cap over your cryo loop, just put a drop of mother liquor in the top, place over the loop and make it airtight at the base. Not sure who sells these things though, I guess you can make it from a capillary too. Then remove the cryo stream or put it at a temp above freezing, say 253K. Flip Zhiyi Wei wrote: Thanks for so many quick responses! Actually, I have test several different cryo-protectants, including glycerol, EG, and PEG400. I did not see much differences between these cryo conditions. So, I choose glycerol. I would like to test my crystals in RT. But I don't know how to do this. Just mount crystal to the X-ray machine without cryo stream? Or I should use capillaries? Zhiyi On Wed, Jan 27, 2010 at 5:45 AM, Kelly Daughtry kddau...@bu.edu wrote: Tascimate can be used as the cryo as well. I have had experience with crystals in similar condition and moved the crystals to a 20% increased Tascimate solution and they froze well. I agree with Ezra, room temperature mount your crystal before freezing. It is the only way to know the true problem. Kelly *** Kelly Daughtry PhD Candidate Department of Physiology and Biophysics Boston University School of Medicine 590 Commonwealth Ave R 390 Boston MA, 02215 (P) 617-358-5548 *** On Tue, Jan 26, 2010 at 1:25 PM, Marcus Winter marcus.win...@oxford-diffraction.com wrote: Dear Zhiyi, Ezra is exactly right, of course. The Oxford Diffraction PX Scanner system can assess the diffraction qualities of (putative) protein crystals in situ - in the crystallisation plate. So, directly, you would discover if your 'big and beautiful' crystals actually diffract well... in their mother liquor under ambient conditions and before the addition of any cryo-protect. Do you have a friend or neighbour with a PX Scanner ? If not, please feel most welcome to contact Oxford Diffraction: we would be pleased to assist if at all possible. Good Luck and Best Wishes, Marcus Winter. www.oxford-diffraction.com -Original Message- From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Ezra Peisach Sent: 26 January 2010 16:01 To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Crystal rescue First you need to establish if it is your cryo conditions or the crystals. Depending where you are - they might have the equipment to do a wet mount - without freezing. Yes the crystal will not last - but then you know if the problem is in the crystal. If it is - you need better crystals. If it is the cryo - you need to work on that. Tacsimate is mixture of alot of different compounds - but the smears are too close together to be a small salt crystal on top... Good luck, Ezra On 1/26/2010 10:42 AM, Zhiyi Wei wrote: Dear all, I got a problem with my crystals. I have two total different proteins that both can be crystallized in the condition with PEG3350 and Tacsimate (although the concentrations are different) with different shapes. The crystals look big and beautiful. However, when I test them in synchrotron, both of these two types of crystals showed poor diffractions. As showed in the attached diffraction image, the diffraction is up to ~4 A but smear in one direction while8 A in the other direction. The interesting thing is that the diffraction pattern is similar for all crystals (from two different proteins) that I tested without exception although they belong to different space groups. So, I wonder whether these kind of pattern is caused by Tacsimate (I don't know what it is) and how to rescue these crystals. Any suggestions or comments? Thanks a lot
Re: [ccp4bb] Crystal rescue
Hi Zhiyi, I think the easiest way is to mount your crystal in an air-impermeable, viscous oil like Paratone in a loop. Of course, your crystals may not tolerate oil. ho
Re: [ccp4bb] Crystal rescue
First you need to establish if it is your cryo conditions or the crystals. Depending where you are - they might have the equipment to do a wet mount - without freezing. Yes the crystal will not last - but then you know if the problem is in the crystal. If it is - you need better crystals. If it is the cryo - you need to work on that. Tacsimate is mixture of alot of different compounds - but the smears are too close together to be a small salt crystal on top... Good luck, Ezra On 1/26/2010 10:42 AM, Zhiyi Wei wrote: Dear all, I got a problem with my crystals. I have two total different proteins that both can be crystallized in the condition with PEG3350 and Tacsimate (although the concentrations are different) with different shapes. The crystals look big and beautiful. However, when I test them in synchrotron, both of these two types of crystals showed poor diffractions. As showed in the attached diffraction image, the diffraction is up to ~4 A but smear in one direction while8 A in the other direction. The interesting thing is that the diffraction pattern is similar for all crystals (from two different proteins) that I tested without exception although they belong to different space groups. So, I wonder whether these kind of pattern is caused by Tacsimate (I don't know what it is) and how to rescue these crystals. Any suggestions or comments? Thanks a lot! Best, Zhiyi
Re: [ccp4bb] Crystal rescue
http://hamptonresearch.com/documents/product/hr000175_what_is_tacsimate_new.pdf turns up once you use google's I'm feeling lucky button. On Tue, 2010-01-26 at 15:42 +, Zhiyi Wei wrote: Dear all, I got a problem with my crystals. I have two total different proteins that both can be crystallized in the condition with PEG3350 and Tacsimate (although the concentrations are different) with different shapes. The crystals look big and beautiful. However, when I test them in synchrotron, both of these two types of crystals showed poor diffractions. As showed in the attached diffraction image, the diffraction is up to ~4 A but smear in one direction while 8 A in the other direction. The interesting thing is that the diffraction pattern is similar for all crystals (from two different proteins) that I tested without exception although they belong to different space groups. So, I wonder whether these kind of pattern is caused by Tacsimate (I don't know what it is) and how to rescue these crystals. Any suggestions or comments? Thanks a lot! Best, Zhiyi -- Edwin Pozharski, PhD, Assistant Professor University of Maryland, Baltimore -- When the Way is forgotten duty and justice appear; Then knowledge and wisdom are born along with hypocrisy. When harmonious relationships dissolve then respect and devotion arise; When a nation falls to chaos then loyalty and patriotism are born. -- / Lao Tse /
Re: [ccp4bb] Crystal rescue
I forgot to mention a phenomenon when I did crystal mounting. I found that if open a coverslip, phase separation will appear in drops in minutes and damage to the crystals. And I have tried additive screen. The optimized crystals are really big (a few hundred microns) with sharp edge but diffraction pattern is similar. On Tue, Jan 26, 2010 at 10:42 AM, Zhiyi Wei ustcwebri...@gmail.com wrote: Dear all, I got a problem with my crystals. I have two total different proteins that both can be crystallized in the condition with PEG3350 and Tacsimate (although the concentrations are different) with different shapes. The crystals look big and beautiful. However, when I test them in synchrotron, both of these two types of crystals showed poor diffractions. As showed in the attached diffraction image, the diffraction is up to ~4 A but smear in one direction while 8 A in the other direction. The interesting thing is that the diffraction pattern is similar for all crystals (from two different proteins) that I tested without exception although they belong to different space groups. So, I wonder whether these kind of pattern is caused by Tacsimate (I don't know what it is) and how to rescue these crystals. Any suggestions or comments? Thanks a lot! Best, Zhiyi
Re: [ccp4bb] Crystal rescue
how much %PEG3350 ? Try different additives such as glycerol, EG, MPD or smaller PEGs 200 or 400. Try flash annealing Try freezing directly in liquid nitrogen if you have previously frozen your crystals in the stream Try liquid annealing by dipping the frozen crystal into a new cryo solution and re-freeze it. The list can be continued quite a bit. If you should find out that one of the cryo additives helps then try to grow your crystals in the presence of those additives. Good luck, Jürgen On Jan 26, 2010, at 10:42 AM, Zhiyi Wei wrote: Dear all, I got a problem with my crystals. I have two total different proteins that both can be crystallized in the condition with PEG3350 and Tacsimate (although the concentrations are different) with different shapes. The crystals look big and beautiful. However, when I test them in synchrotron, both of these two types of crystals showed poor diffractions. As showed in the attached diffraction image, the diffraction is up to ~4 A but smear in one direction while 8 A in the other direction. The interesting thing is that the diffraction pattern is similar for all crystals (from two different proteins) that I tested without exception although they belong to different space groups. So, I wonder whether these kind of pattern is caused by Tacsimate (I don't know what it is) and how to rescue these crystals. Any suggestions or comments? Thanks a lot! Best, Zhiyi bad.png - Jürgen Bosch Johns Hopkins Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Phone: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-3655 http://web.mac.com/bosch_lab/
Re: [ccp4bb] Crystal rescue
Tascimate can be used as the cryo as well. I have had experience with crystals in similar condition and moved the crystals to a 20% increased Tascimate solution and they froze well. I agree with Ezra, room temperature mount your crystal before freezing. It is the only way to know the true problem. Kelly *** Kelly Daughtry PhD Candidate Department of Physiology and Biophysics Boston University School of Medicine 590 Commonwealth Ave R 390 Boston MA, 02215 (P) 617-358-5548 *** On Tue, Jan 26, 2010 at 1:25 PM, Marcus Winter marcus.win...@oxford-diffraction.com wrote: Dear Zhiyi, Ezra is exactly right, of course. The Oxford Diffraction PX Scanner system can assess the diffraction qualities of (putative) protein crystals in situ - in the crystallisation plate. So, directly, you would discover if your 'big and beautiful' crystals actually diffract well... in their mother liquor under ambient conditions and before the addition of any cryo-protect. Do you have a friend or neighbour with a PX Scanner ? If not, please feel most welcome to contact Oxford Diffraction: we would be pleased to assist if at all possible. Good Luck and Best Wishes, Marcus Winter. www.oxford-diffraction.com -Original Message- From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Ezra Peisach Sent: 26 January 2010 16:01 To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Crystal rescue First you need to establish if it is your cryo conditions or the crystals. Depending where you are - they might have the equipment to do a wet mount - without freezing. Yes the crystal will not last - but then you know if the problem is in the crystal. If it is - you need better crystals. If it is the cryo - you need to work on that. Tacsimate is mixture of alot of different compounds - but the smears are too close together to be a small salt crystal on top... Good luck, Ezra On 1/26/2010 10:42 AM, Zhiyi Wei wrote: Dear all, I got a problem with my crystals. I have two total different proteins that both can be crystallized in the condition with PEG3350 and Tacsimate (although the concentrations are different) with different shapes. The crystals look big and beautiful. However, when I test them in synchrotron, both of these two types of crystals showed poor diffractions. As showed in the attached diffraction image, the diffraction is up to ~4 A but smear in one direction while8 A in the other direction. The interesting thing is that the diffraction pattern is similar for all crystals (from two different proteins) that I tested without exception although they belong to different space groups. So, I wonder whether these kind of pattern is caused by Tacsimate (I don't know what it is) and how to rescue these crystals. Any suggestions or comments? Thanks a lot! Best, Zhiyi
Re: [ccp4bb] Crystal rescue
Hi, Zhiyi: You can always put a layer of oil on the top of your drop when you open the coverslip. It will get you more time to mount the crystals. Good luck Meng-Chiao Ho On Tue, 2010-01-26 at 11:48 -0500, Zhiyi Wei wrote: I forgot to mention a phenomenon when I did crystal mounting. I found that if open a coverslip, phase separation will appear in drops in minutes and damage to the crystals. And I have tried additive screen. The optimized crystals are really big (a few hundred microns) with sharp edge but diffraction pattern is similar. On Tue, Jan 26, 2010 at 10:42 AM, Zhiyi Wei ustcwebri...@gmail.com wrote: Dear all, I got a problem with my crystals. I have two total different proteins that both can be crystallized in the condition with PEG3350 and Tacsimate (although the concentrations are different) with different shapes. The crystals look big and beautiful. However, when I test them in synchrotron, both of these two types of crystals showed poor diffractions. As showed in the attached diffraction image, the diffraction is up to ~4 A but smear in one direction while 8 A in the other direction. The interesting thing is that the diffraction pattern is similar for all crystals (from two different proteins) that I tested without exception although they belong to different space groups. So, I wonder whether these kind of pattern is caused by Tacsimate (I don't know what it is) and how to rescue these crystals. Any suggestions or comments? Thanks a lot! Best, Zhiyi
Re: [ccp4bb] Crystal rescue
Thanks for so many quick responses! Actually, I have test several different cryo-protectants, including glycerol, EG, and PEG400. I did not see much differences between these cryo conditions. So, I choose glycerol. I would like to test my crystals in RT. But I don't know how to do this. Just mount crystal to the X-ray machine without cryo stream? Or I should use capillaries? Zhiyi On Wed, Jan 27, 2010 at 5:45 AM, Kelly Daughtry kddau...@bu.edu wrote: Tascimate can be used as the cryo as well. I have had experience with crystals in similar condition and moved the crystals to a 20% increased Tascimate solution and they froze well. I agree with Ezra, room temperature mount your crystal before freezing. It is the only way to know the true problem. Kelly *** Kelly Daughtry PhD Candidate Department of Physiology and Biophysics Boston University School of Medicine 590 Commonwealth Ave R 390 Boston MA, 02215 (P) 617-358-5548 *** On Tue, Jan 26, 2010 at 1:25 PM, Marcus Winter marcus.win...@oxford-diffraction.com wrote: Dear Zhiyi, Ezra is exactly right, of course. The Oxford Diffraction PX Scanner system can assess the diffraction qualities of (putative) protein crystals in situ - in the crystallisation plate. So, directly, you would discover if your 'big and beautiful' crystals actually diffract well... in their mother liquor under ambient conditions and before the addition of any cryo-protect. Do you have a friend or neighbour with a PX Scanner ? If not, please feel most welcome to contact Oxford Diffraction: we would be pleased to assist if at all possible. Good Luck and Best Wishes, Marcus Winter. www.oxford-diffraction.com -Original Message- From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Ezra Peisach Sent: 26 January 2010 16:01 To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Crystal rescue First you need to establish if it is your cryo conditions or the crystals. Depending where you are - they might have the equipment to do a wet mount - without freezing. Yes the crystal will not last - but then you know if the problem is in the crystal. If it is - you need better crystals. If it is the cryo - you need to work on that. Tacsimate is mixture of alot of different compounds - but the smears are too close together to be a small salt crystal on top... Good luck, Ezra On 1/26/2010 10:42 AM, Zhiyi Wei wrote: Dear all, I got a problem with my crystals. I have two total different proteins that both can be crystallized in the condition with PEG3350 and Tacsimate (although the concentrations are different) with different shapes. The crystals look big and beautiful. However, when I test them in synchrotron, both of these two types of crystals showed poor diffractions. As showed in the attached diffraction image, the diffraction is up to ~4 A but smear in one direction while8 A in the other direction. The interesting thing is that the diffraction pattern is similar for all crystals (from two different proteins) that I tested without exception although they belong to different space groups. So, I wonder whether these kind of pattern is caused by Tacsimate (I don't know what it is) and how to rescue these crystals. Any suggestions or comments? Thanks a lot! Best, Zhiyi
