[ccp4bb] Help with reducing crystal mosaicity
Help please! I'm looking for some new ideas. I have crystals that come out of a sitting drop with a mixture of sodium cacodylate at pH 6.5, magnesium acetate and MPD for the well solution. The MPD concentration is sufficient to act as a cryoprotectant. Currently, I directly freeze these crystals in liquid nitrogen. When I collect data, I typically have high anisotropic mosaicity; it ranges from 0.8 to 1.2. This is further complicated with a weakly diffracting crystal (4-5 A) that has a long unit cell axis of ~500 and often twinning. It has been suggested to me that the cryoprotectent is a problem. I haven't checked the diffraction at room temperature, yet. Please no suggestions of finding a different crystal form as that's not a consideration at the moment. I have my reasons. I did find one crystal that has lower mosaicity (0.5 to 0.8) but had weaker diffraction then the typical crystal. Attempts at flash cryoannealing have not helped. So, what's a good way to change the cryoprotectant if the cryoprotectant is the precipitant? I've considered trying dehydration but wasn't certain if that would help with the mosaicity. Thanks for any ideas, Mary X. Fitzgerald Postdoctoral Associate
Re: [ccp4bb] Help with reducing crystal mosaicity
What is the mosaicity of the unfrozen crystal? Jim -Original Message- From: Mary Fitzgerald <[EMAIL PROTECTED]> Date: Mon, 9 Jul 2007 18:05:10 To:CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Help with reducing crystal mosaicity Help please! I'm looking for some new ideas. I have crystals that come out of a sitting drop with a mixture of sodium cacodylate at pH 6.5, magnesium acetate and MPD for the well solution. The MPD concentration is sufficient to act as a cryoprotectant. Currently, I directly freeze these crystals in liquid nitrogen. When I collect data, I typically have high anisotropic mosaicity; it ranges from 0.8 to 1.2. This is further complicated with a weakly diffracting crystal (4-5 A) that has a long unit cell axis of ~500 and often twinning. It has been suggested to me that the cryoprotectent is a problem. I haven't checked the diffraction at room temperature, yet. Please no suggestions of finding a different crystal form as that's not a consideration at the moment. I have my reasons. I did find one crystal that has lower mosaicity (0.5 to 0.8) but had weaker diffraction then the typical crystal. Attempts at flash cryoannealing have not helped. So, what's a good way to change the cryoprotectant if the cryoprotectant is the precipitant? I've considered trying dehydration but wasn't certain if that would help with the mosaicity. Thanks for any ideas, Mary X. Fitzgerald Postdoctoral Associate
Re: [ccp4bb] Help with reducing crystal mosaicity
Here's a method that has had some success in reducing mosaicity, either with or without cryoprotectant: Chae Un Kim, Raphael Kapfer and Sol M. Gruner (2005), High Pressure Cooling of Protein Crystals without Cryoprotectants, ActaCryst. D61, 881-890 This method may be available at a synchrotron near you. Cheers, -- === With the single exception of Cornell, there is not a college in the United States where truth has ever been a welcome guest - R.G. Ingersoll === David J. Schuller modern man in a post-modern world MacCHESS, Cornell University [EMAIL PROTECTED] On Mon, 2007-07-09 at 18:05 -0400, Mary Fitzgerald wrote: > Help please! > > I'm looking for some new ideas. I have crystals that come out of a > sitting drop with a mixture of sodium cacodylate at pH 6.5, magnesium > acetate and MPD for the well solution. The MPD concentration is > sufficient to act as a cryoprotectant. Currently, I directly freeze > these crystals in liquid nitrogen. When I collect data, I typically > have high anisotropic mosaicity; it ranges from 0.8 to 1.2. This is > further complicated with a weakly diffracting crystal (4-5 A) that has > a long unit cell axis of ~500 and often twinning. > > It has been suggested to me that the cryoprotectent is a problem. I > haven't checked the diffraction at room temperature, yet. Please no > suggestions of finding a different crystal form as that's not a > consideration at the moment. I have my reasons. I did find one > crystal that has lower mosaicity (0.5 to 0.8) but had weaker > diffraction then the typical crystal. Attempts at flash cryoannealing > have not helped. > > So, what's a good way to change the cryoprotectant if the > cryoprotectant is the precipitant? I've considered trying dehydration > but wasn't certain if that would help with the mosaicity. > > Thanks for any ideas, > > Mary X. Fitzgerald > Postdoctoral Associate
Re: [ccp4bb] Help with reducing crystal mosaicity
Hi Mary, What final percent MPD do you have prior to flashcooling? Karolin Luger (and perhaps others) found that the final percent MPD had a significant effect on crystal mosaicity and final diffraction limit for nucleosome crystals. For example, if the crystals are left in mother liquor containing 15% MPD, that is suboptimal. On the other hand, if the MPD concentration was increased beyond 24%, the the crystal mosacity increased and the diffraction worsened. We stuck to 24% MPD. Did you try playing with the MPD concentrations? Also, try supplementing MPD with a small percent of sugars like sucrose, trehalose. Another note: Nucleosome crystals are prone to temperature-dependent lattice changes (always along c-axis) with a concomitant worsening of diffraction. Even if it sounds anectodal, mythological or voodoo-ish, we found that it greatly helped diffraction anisotropy and mosaicity if we dunked the crystals in liquid propane first. After the initial dunking, it was just fine to freeze the crystals in liq. N2 for longer storage and/or transport. It was the first dunk that was critical. Good luck! Raji -Included Message-- >Help please! > >I'm looking for some new ideas. I have crystals that come out of a >sitting drop with a mixture of sodium cacodylate at pH 6.5, magnesium >acetate and MPD for the well solution. The MPD concentration is >sufficient to act as a cryoprotectant. Currently, I directly freeze >these crystals in liquid nitrogen. When I collect data, I typically >have high anisotropic mosaicity; it ranges from 0.8 to 1.2. This is >further complicated with a weakly diffracting crystal (4-5 A) that has >a long unit cell axis of ~500 and often twinning. > >It has been suggested to me that the cryoprotectent is a problem. I >haven't checked the diffraction at room temperature, yet. Please no >suggestions of finding a different crystal form as that's not a >consideration at the moment. I have my reasons. I did find one >crystal that has lower mosaicity (0.5 to 0.8) but had weaker >diffraction then the typical crystal. Attempts at flash cryoannealing >have not helped. > >So, what's a good way to change the cryoprotectant if the >cryoprotectant is the precipitant? I've considered trying dehydration >but wasn't certain if that would help with the mosaicity. > >Thanks for any ideas, > >Mary X. Fitzgerald >Postdoctoral Associate > > -End of Included Message--
Re: [ccp4bb] Help with reducing crystal mosaicity
Wow, thanks. I'm going to try to answer most of the questions I've received in one message as I'm overwhelmed by the quick multitude of responses. As I stated earlier, I haven't collected any room temperature data, yet. So, I don't know the unfrozen mosaicity. It is very possible that the crystals are simply mosaic at RT, too. I have tried flash-cooling in liquid nitrogen and "gas cooling" in the cryo-stream, I didn't notice a difference in the diffraction pattern. My solvent content is on the high side about 67%. Additional crystal details, I set the trays up at 20 degrees. The crystals take about 2 weeks to appear and another week to grow to full size. The crystals are bipyrimidal and often 150x150x300 although sometimes smaller. Typically, there's about 34-39% MPD in the well solutions. The well solution freezes clear and I don't get any ice rings in my diffraction patterns. I've tried looping the crystals directly out of the drop or adding a few microletters of well solution and then looping, I didn't notice a difference. I've been hesitant to start adding extras fearing crystal cracking. I did however switch to freezing directly in the automounter puck which was my one low mosacity crystal but I had plenty of other crystals in the puck that weren't better then before. I was wondering if the MPD concentration could cause problems and sounds like it might be. So, with the crystals I have I'll try higher MPD to dehydrate and try freezing in lower MPD concentration or add some other cryoprotectants to my current well solution. Although, I might have to go find some propane. If that doesn't work maybe, I'll try seeding at lower MPD concentrations or pressure freezing. Thanks again, Mary
Re: [ccp4bb] Help with reducing crystal mosaicity
On Tuesday 10 July 2007 09:16, Mary Fitzgerald wrote: > If that doesn't work maybe, I'll try seeding at lower MPD > concentrations or pressure freezing. > > Thanks again, > Mary With such a high MPD condition I would probably first try micro-seeding at 25-35% MPD before doing anything else. It sounds like a very good candidate. -- Tim Grune Australian Synchrotron 800 Blackburn Road Clayton, VIC 3168 Australia pgpEJuDKsMqme.pgp Description: PGP signature
Re: [ccp4bb] Help with reducing crystal mosaicity
Hi Mary, not sure if you received this suggestion already. Despite the low resolution diffraction 4-5 A as you report, try mounting smaller crystals and see how well they perform. Very often our crystals had a better freezing experience when they were small, even if sufficient cryo protectant was present. Another thing to try is once you have mounted a crystal, how does it behave after flash annealing / wet annealing / multiple rounds of annealing ? Juergen -- Jürgen Bosch University of Washington Dept. of Biochemistry, K-426 1705 NE Pacific Street Seattle, WA 98195 Box 357742 Phone: +1-206-616-4510 FAX: +1-206-685-7002
Re: [ccp4bb] Help with reducing crystal mosaicity
Mary, freezing habitually increases mosaicity. In your case, the high water content adds to the problem. Try not to freeze the crystal but collect at sub-zero temperature (in short glass capillaries or use oil plugs instead). You have to optimize the "close to freezing" data-collection temperature. I collected complete synchrotron datasets (of GCPII in buffer with PEG1500 and PEG400) at 260-263 Kelvin which resulted in mosaicity values of as small as 0.07 degrees! At 277 K, the crystals only last for a few images and freezing did not work (for the buffer mentioned before). - J. - -Original Message- From: Mary Fitzgerald <[EMAIL PROTECTED]> Date: Mon, 9 Jul 2007 18:05:10 To:CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Help with reducing crystal mosaicity Help please! I'm looking for some new ideas. I have crystals that come out of a sitting drop with a mixture of sodium cacodylate at pH 6.5, magnesium acetate and MPD for the well solution. The MPD concentration is sufficient to act as a cryoprotectant. Currently, I directly freeze these crystals in liquid nitrogen. When I collect data, I typically have high anisotropic mosaicity; it ranges from 0.8 to 1.2. This is further complicated with a weakly diffracting crystal (4-5 A) that has a long unit cell axis of ~500 and often twinning. It has been suggested to me that the cryoprotectent is a problem. I haven't checked the diffraction at room temperature, yet. Please no suggestions of finding a different crystal form as that's not a consideration at the moment. I have my reasons. I did find one crystal that has lower mosaicity (0.5 to 0.8) but had weaker diffraction then the typical crystal. Attempts at flash cryoannealing have not helped. So, what's a good way to change the cryoprotectant if the cryoprotectant is the precipitant? I've considered trying dehydration but wasn't certain if that would help with the mosaicity. Thanks for any ideas, Mary X. Fitzgerald Postdoctoral Associate -- Jeroen Raymundus Mesters, Ph.D. Institut fuer Biochemie, Universitaet zu Luebeck Zentrum fuer Medizinische Struktur und Zellbiologie Ratzeburger Allee 160, D-23538 Luebeck Tel: +49-451-5004070, Fax: +49-451-5004068 E-mail: [EMAIL PROTECTED] Http://www.biochem.uni-luebeck.de Http://www.iobcr.org Http://www.opticryst.org -- If you can look into the seeds of time and say which grain will grow and which will not - speak then to me (Macbeth) --
Re: [ccp4bb] Help with reducing crystal mosaicity
You might also want to loook into using parathone for freezing... Or collection at 260K indeed! Flip -Original Message- From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of mesters Sent: Tuesday, July 10, 2007 11:01 To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Help with reducing crystal mosaicity Mary, freezing habitually increases mosaicity. In your case, the high water content adds to the problem. Try not to freeze the crystal but collect at sub-zero temperature (in short glass capillaries or use oil plugs instead). You have to optimize the "close to freezing" data-collection temperature. I collected complete synchrotron datasets (of GCPII in buffer with PEG1500 and PEG400) at 260-263 Kelvin which resulted in mosaicity values of as small as 0.07 degrees! At 277 K, the crystals only last for a few images and freezing did not work (for the buffer mentioned before). - J. - > -Original Message- > From: Mary Fitzgerald <[EMAIL PROTECTED]> > Date: Mon, 9 Jul 2007 18:05:10 > To:CCP4BB@JISCMAIL.AC.UK > Subject: [ccp4bb] Help with reducing crystal mosaicity > > Help please! > > I'm looking for some new ideas. I have crystals that come out of a > sitting drop with a mixture of sodium cacodylate at pH 6.5, magnesium > acetate and MPD for the well solution. The MPD concentration is > sufficient to act as a cryoprotectant. Currently, I directly freeze > these crystals in liquid nitrogen. When I collect data, I typically > have high anisotropic mosaicity; it ranges from 0.8 to 1.2. This is > further complicated with a weakly diffracting crystal (4-5 A) that has > a long unit cell axis of ~500 and often twinning. > > It has been suggested to me that the cryoprotectent is a problem. I > haven't checked the diffraction at room temperature, yet. Please no > suggestions of finding a different crystal form as that's not a > consideration at the moment. I have my reasons. I did find one > crystal that has lower mosaicity (0.5 to 0.8) but had weaker > diffraction then the typical crystal. Attempts at flash cryoannealing > have not helped. > > So, what's a good way to change the cryoprotectant if the > cryoprotectant is the precipitant? I've considered trying dehydration > but wasn't certain if that would help with the mosaicity. > > Thanks for any ideas, > > Mary X. Fitzgerald > Postdoctoral Associate > -- Jeroen Raymundus Mesters, Ph.D. Institut fuer Biochemie, Universitaet zu Luebeck Zentrum fuer Medizinische Struktur und Zellbiologie Ratzeburger Allee 160, D-23538 Luebeck Tel: +49-451-5004070, Fax: +49-451-5004068 E-mail: [EMAIL PROTECTED] Http://www.biochem.uni-luebeck.de Http://www.iobcr.org Http://www.opticryst.org -- If you can look into the seeds of time and say which grain will grow and which will not - speak then to me (Macbeth) --
Re: [ccp4bb] Help with reducing crystal mosaicity
Hi Mary, There are many excellent suggestions already but you may want to try tweaking your cryo a bit. This is described very nicely by Ed Mitchell and Elspeth Garman in a paper on mosaic spread as a function of cryoprotectants concentration - J. Appl. Cryst. (1994) 27, 1070-1074. You already have a cryoprotectant, the MPD, and could up the concentration a little by successive transfer or adding a little to the drop. I've also found that (2R,3R)-(-)-2,3-butanediol works as a 'magic' elixir in very small concentrations when added directly to the drop. I'd say try room temperature but at that resolution you are going to need all the X-rays you can get onto it. Cheers, Eddie. Edward Snell Ph.D. Assistant Prof. Department of Structural Biology, SUNY Buffalo, Hauptman-Woodward Medical Research Institute 700 Ellicott Street, Buffalo, NY 14203-1102 Phone: (716) 898 8631 Fax: (716) 898 8660 Email: [EMAIL PROTECTED] Telepathy: 42.2 GHz Heisenberg was probably here! Mary, freezing habitually increases mosaicity. In your case, the high water content adds to the problem. Try not to freeze the crystal but collect at sub-zero temperature (in short glass capillaries or use oil plugs instead). You have to optimize the "close to freezing" data-collection temperature. I collected complete synchrotron datasets (of GCPII in buffer with PEG1500 and PEG400) at 260-263 Kelvin which resulted in mosaicity values of as small as 0.07 degrees! At 277 K, the crystals only last for a few images and freezing did not work (for the buffer mentioned before). - J. - > -Original Message- > From: Mary Fitzgerald <[EMAIL PROTECTED]> > Date: Mon, 9 Jul 2007 18:05:10 > To:CCP4BB@JISCMAIL.AC.UK > Subject: [ccp4bb] Help with reducing crystal mosaicity > > Help please! > > I'm looking for some new ideas. I have crystals that come out of a > sitting drop with a mixture of sodium cacodylate at pH 6.5, magnesium > acetate and MPD for the well solution. The MPD concentration is > sufficient to act as a cryoprotectant. Currently, I directly freeze > these crystals in liquid nitrogen. When I collect data, I typically > have high anisotropic mosaicity; it ranges from 0.8 to 1.2. This is > further complicated with a weakly diffracting crystal (4-5 A) that has > a long unit cell axis of ~500 and often twinning. > > It has been suggested to me that the cryoprotectent is a problem. I > haven't checked the diffraction at room temperature, yet. Please no > suggestions of finding a different crystal form as that's not a > consideration at the moment. I have my reasons. I did find one > crystal that has lower mosaicity (0.5 to 0.8) but had weaker > diffraction then the typical crystal. Attempts at flash cryoannealing > have not helped. > > So, what's a good way to change the cryoprotectant if the > cryoprotectant is the precipitant? I've considered trying dehydration > but wasn't certain if that would help with the mosaicity. > > Thanks for any ideas, > > Mary X. Fitzgerald > Postdoctoral Associate > -- Jeroen Raymundus Mesters, Ph.D. Institut fuer Biochemie, Universitaet zu Luebeck Zentrum fuer Medizinische Struktur und Zellbiologie Ratzeburger Allee 160, D-23538 Luebeck Tel: +49-451-5004070, Fax: +49-451-5004068 E-mail: [EMAIL PROTECTED] Http://www.biochem.uni-luebeck.de Http://www.iobcr.org Http://www.opticryst.org -- If you can look into the seeds of time and say which grain will grow and which will not - speak then to me (Macbeth) --
Re: [ccp4bb] Help with reducing crystal mosaicity
Jeroen brings up a good point. Back in the old days, around 5 B. C. (Before Cryo), we would use a chilled air generator to blow a stream of cold air along the capillary axis to keep the crystals just above their freezing point--it made a huge difference in crystal lifetime. I recall a colleague devising an apparatus from a 50 ml conical tube. The bottom was cut off and cold air was blown in from the other end. Windows were cut in either side to allow the beam to pass & covered in mylar. This way the entire capillary was contained within the cold tube, so no temperature gradients formed along the length of the capillary (temp gradient => distillation => dead crystal). Later, we purchased a very clever goniometer head from Nonius that had a plastic cylinder attached to goniometer head, with a swivel, so the hose supplying cold air didn't get tangled during data collection... I've often thought duplicating this apparatus when we encounter cryo problems, but I'm always stymied when trying to find a cheap and simple source of cold air. Any bright ideas? On Jul 10, 2007, at 5:00 AM, mesters wrote: Mary, freezing habitually increases mosaicity. In your case, the high water content adds to the problem. Try not to freeze the crystal but collect at sub-zero temperature (in short glass capillaries or use oil plugs instead). You have to optimize the "close to freezing" data-collection temperature. I collected complete synchrotron datasets (of GCPII in buffer with PEG1500 and PEG400) at 260-263 Kelvin which resulted in mosaicity values of as small as 0.07 degrees! At 277 K, the crystals only last for a few images and freezing did not work (for the buffer mentioned before). - J. - --- Patrick J. Loll, Ph. D. (215) 762-7706 Associate Professor FAX: (215) 762-4452 Department of Biochemistry & Molecular Biology Director, Biochemistry Graduate Program Drexel University College of Medicine Room 10-102 New College Building 245 N. 15th St., Mailstop 497 Philadelphia, PA 19102-1192 USA [EMAIL PROTECTED]
Re: [ccp4bb] Help with reducing crystal mosaicity
CCP4 bulletin board wrote on 07/10/2007 02:07:37 PM: > Jeroen brings up a good point. Back in the old days, around 5 B. C. > (Before Cryo), we would use a chilled air generator to blow a stream > of cold air along the capillary axis to keep the crystals just above > their freezing point--it made a huge difference in crystal lifetime. > I recall a colleague devising an apparatus from a 50 ml conical > tube. The bottom was cut off and cold air was blown in from the > other end. Windows were cut in either side to allow the beam to pass > & covered in mylar. This way the entire capillary was contained > within the cold tube, so no temperature gradients formed along the > length of the capillary (temp gradient => distillation => dead > crystal). Later, we purchased a very clever goniometer head from > Nonius that had a plastic cylinder attached to goniometer head, with > a swivel, so the hose supplying cold air didn't get tangled during > data collection... > > I've often thought duplicating this apparatus when we encounter cryo > problems, but I'm always stymied when trying to find a cheap and > simple source of cold air. Any bright ideas? > Hi Patrick - Many cryosystems (definitely the Cryojet, and I believe the Cryostream) can be set to run at any temperature between room temp and liquid nitrogen temp. I'm not sure of the temperature stability at temps > 0 C, and you might burn out the heaters prematurely if you do this all the time, but it should work. Then you just need to move the nozzle so it's coaxial with your goniostat's rotation axis, and aim the capillary down the nozzle. You could probably even move the capillary *inside* the cryo nozzle a bit, so only the bit with the crystal is in the free stream. It's not a cheap solution, but you've almost certainly got a cryosystem already... - Matt -- Matthew Franklin , Ph.D. Senior Scientist, ImClone Systems 180 Varick Street, 6th floor New York, NY 10014 phone:(917)606-4116 fax:(212)645-2054 Confidentiality Note: This e-mail, and any attachment to it, contains privileged and confidential information intended only for the use of the individual(s) or entity named on the e-mail. If the reader of this e-mail is not the intended recipient, or the employee or agent responsible for delivering it to the intended recipient, you are hereby notified that reading it is strictly prohibited. If you have received this e-mail in error, please immediately return it to the sender and delete it from your system. Thank you.
Re: [ccp4bb] Help with reducing crystal mosaicity
Matthew, I have not had any problem with our Cryojet XL (90 - 300 Kelvin), it is very stable at sub-zero temperatures, I checked it over a longer period using a highly sensitive electronic device. Also, you can buy the Cryojet HT that can be operated from 90 – 490 Kelvin! The heaters will be okay for both devices. But more important, coaxial mounting does not help unless the capillary is really short! If the capillary is to long, water will again collect at the point closest to the nozzle, especially if the data collection takes more than one or two hours! I am not aware of any cheaper devices that deliver a stable temperature over a prolonged time-span. - J. - [EMAIL PROTECTED] wrote: Hi Patrick - Many cryosystems (definitely the Cryojet, and I believe the Cryostream) can be set to run at any temperature between room temp and liquid nitrogen temp. I'm not sure of the temperature stability at temps > 0 C, and you might burn out the heaters prematurely if you do this all the time, but it should work. Then you just need to move the nozzle so it's coaxial with your goniostat's rotation axis, and aim the capillary down the nozzle. You could probably even move the capillary *inside* the cryo nozzle a bit, so only the bit with the crystal is in the free stream. It's not a cheap solution, but you've almost certainly got a cryosystem already... - Matt -- Matthew Franklin , Ph.D. Senior Scientist, ImClone Systems 180 Varick Street, 6th floor New York, NY 10014 phone:(917)606-4116 fax:(212)645-2054 Confidentiality Note: This e-mail, and any attachment to it, contains privileged and confidential information intended only for the use of the individual(s) or entity named on the e-mail. If the reader of this e-mail is not the intended recipient, or the employee or agent responsible for delivering it to the intended recipient, you are hereby notified that reading it is strictly prohibited. If you have received this e-mail in error, please immediately return it to the sender and delete it from your system. Thank you. CCP4 bulletin board wrote on 07/10/2007 02:07:37 PM: Jeroen brings up a good point. Back in the old days, around 5 B. C. (Before Cryo), we would use a chilled air generator to blow a stream of cold air along the capillary axis to keep the crystals just above their freezing point--it made a huge difference in crystal lifetime. I recall a colleague devising an apparatus from a 50 ml conical tube. The bottom was cut off and cold air was blown in from the other end. Windows were cut in either side to allow the beam to pass & covered in mylar. This way the entire capillary was contained within the cold tube, so no temperature gradients formed along the length of the capillary (temp gradient => distillation => dead crystal). Later, we purchased a very clever goniometer head from Nonius that had a plastic cylinder attached to goniometer head, with a swivel, so the hose supplying cold air didn't get tangled during data collection... I've often thought duplicating this apparatus when we encounter cryo problems, but I'm always stymied when trying to find a cheap and simple source of cold air. Any bright ideas? -- Jeroen Raymundus Mesters, Ph.D. Institut fuer Biochemie, Universitaet zu Luebeck Zentrum fuer Medizinische Struktur und Zellbiologie Ratzeburger Allee 160, D-23538 Luebeck Tel: +49-451-5004070, Fax: +49-451-5004068 E-mail: [EMAIL PROTECTED] Http://www.biochem.uni-luebeck.de Http://www.iobcr.org Http://www.opticryst.org -- If you can look into the seeds of time and say which grain will grow and which will not - speak then to me (Macbeth) --
Re: [ccp4bb] Help with reducing crystal mosaicity
We used to use FTS-cooler for stable temperatures in Laue-experiments on protein crystals in capillaries. Check out BioCARS at the APS for this device. http://cars9.uchicago.edu/biocars/pages/timeresolved.html It has a big opening for constant air flow and temperature along the capillary and it can go to sub 0 deg C. Marius > Matthew, > > I have not had any problem with our Cryojet XL (90 - 300 Kelvin), it > is > very stable at sub-zero temperatures, I checked it over a longer > period > using a highly sensitive electronic device. Also, you can buy the > Cryojet HT that can be operated from 90 – 490 Kelvin! > The heaters will be okay for both devices. > > But more important, coaxial mounting does not help unless the > capillary > is really short! If the capillary is to long, water will again > collect > at the point closest to the nozzle, especially if the data collection > takes more than one or two hours! > > I am not aware of any cheaper devices that deliver a stable > temperature > over a prolonged time-span. > > - J. - > > > [EMAIL PROTECTED] wrote: > > Hi Patrick - > > Many cryosystems (definitely the Cryojet, and I believe the > Cryostream) can > be set to run at any temperature between room temp and liquid > nitrogen > temp. I'm not sure of the temperature stability at temps > 0 C, and > you > might burn out the heaters prematurely if you do this all the time, > but it > should work. Then you just need to move the nozzle so it's coaxial > with > your goniostat's rotation axis, and aim the capillary down the > nozzle. You > could probably even move the capillary *inside* the cryo nozzle a > bit, so > only the bit with the crystal is in the free stream. > > It's not a cheap solution, but you've almost certainly got a > cryosystem > already... > > - Matt > > -- > Matthew Franklin , Ph.D. > Senior Scientist, ImClone Systems > 180 Varick Street, 6th floor > New York, NY 10014 > phone:(917)606-4116 fax:(212)645-2054 > > Confidentiality Note: This e-mail, and any attachment to it, > contains > privileged and confidential information intended only for the use of > the > individual(s) or entity named on the e-mail. If the reader of this > e-mail > is not the intended recipient, or the employee or agent responsible > for > delivering it to the intended recipient, you are hereby notified that > reading it is strictly prohibited. If you have received this e-mail > in > error, please immediately return it to the sender and delete it from > your > system. Thank you. > >> CCP4 bulletin board wrote on 07/10/2007 02:07:37 >> PM: >> >> >>> Jeroen brings up a good point. Back in the old days, around 5 B. C. >>> (Before Cryo), we would use a chilled air generator to blow a stream >>> of cold air along the capillary axis to keep the crystals just above >>> their freezing point--it made a huge difference in crystal lifetime. >>> I recall a colleague devising an apparatus from a 50 ml conical >>> tube. The bottom was cut off and cold air was blown in from the >>> other end. Windows were cut in either side to allow the beam to pass >>> & covered in mylar. This way the entire capillary was contained >>> within the cold tube, so no temperature gradients formed along the >>> length of the capillary (temp gradient => distillation => dead >>> crystal). Later, we purchased a very clever goniometer head from >>> Nonius that had a plastic cylinder attached to goniometer head, with >>> a swivel, so the hose supplying cold air didn't get tangled during >>> data collection... >>> >>> I've often thought duplicating this apparatus when we encounter cryo >>> problems, but I'm always stymied when trying to find a cheap and >>> simple source of cold air. Any bright ideas? >>> >>> >> >> > > -- > Jeroen Raymundus Mesters, Ph.D. > Institut fuer Biochemie, Universitaet zu Luebeck > Zentrum fuer Medizinische Struktur und Zellbiologie > Ratzeburger Allee 160, D-23538 Luebeck > Tel: +49-451-5004070, Fax: +49-451-5004068 > E-mail: [EMAIL PROTECTED] > Http://www.biochem.uni-luebeck.de > Http://www.iobcr.org > Http://www.opticryst.org > -- > If you can look into the seeds of time and say > which grain will grow and which will not - speak then to me (Macbeth) > -- PD Dr. habil. Marius Schmidt Physikdepartment E17 Technische Universitaet Muenchen James Franck Strasse 85747 Garching/Germany email: [EMAIL PROTECTED] http://users.physik.tu-muenchen.de/marius/ phone: +49-(0)89-2891-2550 fax: +49-(0)89-2891-2548
Re: [ccp4bb] Help with reducing crystal mosaicity
a couple of points from long BC about approx. 0 C cooling...for what its worth! i) don't forget that high concentrations of salts can have a significant effect on the freezing point ( remember freezing point depression?). We used to collect data at -15C from crystals grown in 2.4M Ammonium Sulphate ii) cool slowly and gently over a couple of hours iii) put the mother liquor at only the colder end of the capillary to prevent condensation iv) we found a transition at about -5 for above crystals, so be careful. Peter Moody Henry Wellcome Laboratories of Structural Biology Department of Biochemistry Henry Wellcome Building University of Leicester LE1 9HN 0116 229 7097 From: CCP4 bulletin board on behalf of mesters Sent: Wed 11/07/2007 08:46 To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Help with reducing crystal mosaicity snipped
Re: [ccp4bb] Help with reducing crystal mosaicity
Just a thought, Mary - going back to your original question about MPD. I extracted the crystallization conditions from REMARK 280 of 3939 PDB entries a couple of years ago. The average concentration of the MPD used was high - 38.6%, while PEGs tended to be used at lower concs, e.g. PEG400 25.7%. You can see the data at www.douglas.co.uk/top14.htm I thought this information could be useful if you want to replace some of the MPD with another precipitant (or cryoprotectant). Best wishes Patrick Shaw Stewart -- [EMAIL PROTECTED] Douglas Instruments Ltd. DouglasHouse, EastGarston, Hungerford, Berkshire, RG177HD, UK Directors: Peter Baldock, Patrick Shaw Stewart, James Smith http://douglas.co.uk or http://www.douglasinstruments.com Tel: 44 (0) 148-864-9090 US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36 > -Original Message- > From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Mary > Fitzgerald > Sent: 09 July 2007 23:05 > To: CCP4BB@JISCMAIL.AC.UK > Subject: [ccp4bb] Help with reducing crystal mosaicity > > Help please! > > I'm looking for some new ideas. I have crystals that come out of a > sitting drop with a mixture of sodium cacodylate at pH 6.5, magnesium > acetate and MPD for the well solution. The MPD concentration is > sufficient to act as a cryoprotectant. Currently, I directly freeze > these crystals in liquid nitrogen. When I collect data, I typically > have high anisotropic mosaicity; it ranges from 0.8 to 1.2. This is > further complicated with a weakly diffracting crystal (4-5 A) that has > a long unit cell axis of ~500 and often twinning. > > It has been suggested to me that the cryoprotectent is a problem. I > haven't checked the diffraction at room temperature, yet. Please no > suggestions of finding a different crystal form as that's not a > consideration at the moment. I have my reasons. I did find one > crystal that has lower mosaicity (0.5 to 0.8) but had weaker > diffraction then the typical crystal. Attempts at flash cryoannealing > have not helped. > > So, what's a good way to change the cryoprotectant if the > cryoprotectant is the precipitant? I've considered trying dehydration > but wasn't certain if that would help with the mosaicity. > > Thanks for any ideas, > > Mary X. Fitzgerald > Postdoctoral Associate
Re: [ccp4bb] Help with reducing crystal mosaicity
Dear Mary, the problem encountered with cryoprotectans is the change in the solution surrounding your crystal as they may not be present in your crystallisation conditions or you need to increase their concentration to make them act as cryoprotectant. So I don't thik is the cryoprotectant itself as it is you mother liquor.. test it in a capillary so you could rule out that freezing is the problem of course you don't want to find new crystallisation conditions when you tried everything and I know what it means!! I would try to improve crystal quality using additives or ligand etc, your long axis is very worrying as well!!! may be some magic additive could change the packing and givew better diffraction too I also had crystals growing from MPD, unfortunately there was a non-cleavable his-tag so I could not hope that metals would help. and other additives did not make any difference then my contract finished and so did the project! I hope you still have a lot of time available to try different things but I would not waste time trying to change your cryo ciao Stefano Stefano Benini PhD AstraZeneca UK Structural Biology Mereside 50S38 Alderley Park -Original Message- From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] Behalf Of Patrick Shaw Stewart Sent: 11 July 2007 12:10 To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Help with reducing crystal mosaicity Just a thought, Mary - going back to your original question about MPD. I extracted the crystallization conditions from REMARK 280 of 3939 PDB entries a couple of years ago. The average concentration of the MPD used was high - 38.6%, while PEGs tended to be used at lower concs, e.g. PEG400 25.7%. You can see the data at www.douglas.co.uk/top14.htm I thought this information could be useful if you want to replace some of the MPD with another precipitant (or cryoprotectant). Best wishes Patrick Shaw Stewart -- [EMAIL PROTECTED] Douglas Instruments Ltd. DouglasHouse, EastGarston, Hungerford, Berkshire, RG177HD, UK Directors: Peter Baldock, Patrick Shaw Stewart, James Smith http://douglas.co.uk or http://www.douglasinstruments.com Tel: 44 (0) 148-864-9090 US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36 > -Original Message- > From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Mary > Fitzgerald > Sent: 09 July 2007 23:05 > To: CCP4BB@JISCMAIL.AC.UK > Subject: [ccp4bb] Help with reducing crystal mosaicity > > Help please! > > I'm looking for some new ideas. I have crystals that come out of a > sitting drop with a mixture of sodium cacodylate at pH 6.5, magnesium > acetate and MPD for the well solution. The MPD concentration is > sufficient to act as a cryoprotectant. Currently, I directly freeze > these crystals in liquid nitrogen. When I collect data, I typically > have high anisotropic mosaicity; it ranges from 0.8 to 1.2. This is > further complicated with a weakly diffracting crystal (4-5 A) that has > a long unit cell axis of ~500 and often twinning. > > It has been suggested to me that the cryoprotectent is a problem. I > haven't checked the diffraction at room temperature, yet. Please no > suggestions of finding a different crystal form as that's not a > consideration at the moment. I have my reasons. I did find one > crystal that has lower mosaicity (0.5 to 0.8) but had weaker > diffraction then the typical crystal. Attempts at flash cryoannealing > have not helped. > > So, what's a good way to change the cryoprotectant if the > cryoprotectant is the precipitant? I've considered trying dehydration > but wasn't certain if that would help with the mosaicity. > > Thanks for any ideas, > > Mary X. Fitzgerald > Postdoctoral Associate
Re: [ccp4bb] Help with reducing crystal mosaicity
At the risk of sounding too commercial, here are some suggestions: 1. See if you can either place your crystallization tray in a cold room or crystallize it there. Mount your crystal in a capillary here so that at whatever lower temperature you use, the distillation of solvent will be reduced. 2. If capillaries are too hard to work with, even a quick diffraction image out of a loop, while the crystal is drying out, may give you some information. Alternately, I have saturated my stock solution with sugar from the coffee room and swished my crystal through this for getting a quick diffraction image. In a rare case, the crystal actually diffracted for several hours at room temperature. 3. Someone mentioned using Paratone oil. This is good, but a little thick and sticky. So, I use PerFluoroPolyEther (PFPE) as it is much less viscous and is colorless. I buy FOMBLIN Y VAC 14/6 from SigmaAldrich (Cat # 317934-100G). PFPE is mildly hydroscopic, so it is best to open the bottle infrequently (I withdraw ten 0.5 ml aliquots whenever I open the bottle). 4. The Free Mounting System (FMS) is ideal for RT screening. So long as you do not have a highly volatile component in solution, then you can use the standard FMS at temperatures in the range of 15 to 25 C. If you do have a volatile component, then Proteros bistructures GmbH (www.proteros.com) has the only FMS with the capability of adding solvents to the gas stream...and with some compensation, they will gladly help you with this. Kris Kris F. Tesh, Ph D Director, Macromolecular Products Rigaku Americas Corporation 9009 New Trails Drive The Woodlands, TX 77381 USA 001 281 362 2300 x 144 -Original Message- From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Benini, Stefano Sent: Wednesday, July 11, 2007 7:17 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Help with reducing crystal mosaicity Dear Mary, the problem encountered with cryoprotectans is the change in the solution surrounding your crystal as they may not be present in your crystallisation conditions or you need to increase their concentration to make them act as cryoprotectant. So I don't thik is the cryoprotectant itself as it is you mother liquor.. test it in a capillary so you could rule out that freezing is the problem of course you don't want to find new crystallisation conditions when you tried everything and I know what it means!! I would try to improve crystal quality using additives or ligand etc, your long axis is very worrying as well!!! may be some magic additive could change the packing and givew better diffraction too I also had crystals growing from MPD, unfortunately there was a non-cleavable his-tag so I could not hope that metals would help. and other additives did not make any difference then my contract finished and so did the project! I hope you still have a lot of time available to try different things but I would not waste time trying to change your cryo ciao Stefano Stefano Benini PhD AstraZeneca UK Structural Biology Mereside 50S38 Alderley Park -Original Message- From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] Behalf Of Patrick Shaw Stewart Sent: 11 July 2007 12:10 To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Help with reducing crystal mosaicity Just a thought, Mary - going back to your original question about MPD. I extracted the crystallization conditions from REMARK 280 of 3939 PDB entries a couple of years ago. The average concentration of the MPD used was high - 38.6%, while PEGs tended to be used at lower concs, e.g. PEG400 25.7%. You can see the data at www.douglas.co.uk/top14.htm I thought this information could be useful if you want to replace some of the MPD with another precipitant (or cryoprotectant). Best wishes Patrick Shaw Stewart -- [EMAIL PROTECTED] Douglas Instruments Ltd. DouglasHouse, EastGarston, Hungerford, Berkshire, RG177HD, UK Directors: Peter Baldock, Patrick Shaw Stewart, James Smith http://douglas.co.uk or http://www.douglasinstruments.com Tel: 44 (0) 148-864-9090 US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36 > -Original Message- > From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Mary > Fitzgerald > Sent: 09 July 2007 23:05 > To: CCP4BB@JISCMAIL.AC.UK > Subject: [ccp4bb] Help with reducing crystal mosaicity > > Help please! > > I'm looking for some new ideas. I have crystals that come out of a > sitting drop with a mixture of sodium cacodylate at pH 6.5, magnesium > acetate and MPD for the well solution. The MPD concentration is > sufficient to act as a cryoprotectant. Currently, I directly freeze > these crystals in liquid nitrogen. When I collect data, I typically > have high anisotropic mosaicity; it ranges from 0.8 to 1.2. This is > further complicated with a weakly diffracting crystal (4-5 A) that
Re: [ccp4bb] Help with reducing crystal mosaicity
Hi Mary, There is another expt. you might look into after trying out the different cryoprotectants, small vs. large crystals, capillary mounts, etc. as suggested. Dr. Robert Thorne's group (from Mitegen & Cornell U.)) has been doing some studies on flash freezing procedures and effect on mosaicity/diffraction quality. Some of the conclusions and recommendations from these experiments deals with the effect of cooling rate during flash freezing (by dunking in liquid nitrogen) and the effect on mosaicity. If I remember correctly, they found that crystals they tested had much lower mosaicity during diffraction data collection if instead of dunking the looped-out crystals directly in the container with liquid nitrogen, they dunk it in the container which has dry liquid nitrogen blowing laterally along the surface. This changes the environment at the liquid/air interface near the container allowing for much faster cooling rates than otherwise, resulting in an improved diffraction quality and reduced mosaicity. I haven't tried it myself but the results seemed convincing. I know that sector 8 beamlines at the ALS have a modified apparatus to do this (look for Corie Ralston). You can get more information from them or contact Robert Thorne directly. BTW, ALS beamline 12.3.1 has the FMS system if you want to try that out. Regards, Debanu. --- Debanu Das, Stanford Synchrotron Research Laboratory, SLAC, Menlo Park, California. From: CCP4 bulletin board on behalf of Kris Tesh Sent: Wed 7/11/2007 8:35 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Help with reducing crystal mosaicity At the risk of sounding too commercial, here are some suggestions: 1. See if you can either place your crystallization tray in a cold room or crystallize it there. Mount your crystal in a capillary here so that at whatever lower temperature you use, the distillation of solvent will be reduced. 2. If capillaries are too hard to work with, even a quick diffraction image out of a loop, while the crystal is drying out, may give you some information. Alternately, I have saturated my stock solution with sugar from the coffee room and swished my crystal through this for getting a quick diffraction image. In a rare case, the crystal actually diffracted for several hours at room temperature. 3. Someone mentioned using Paratone oil. This is good, but a little thick and sticky. So, I use PerFluoroPolyEther (PFPE) as it is much less viscous and is colorless. I buy FOMBLIN Y VAC 14/6 from SigmaAldrich (Cat # 317934-100G). PFPE is mildly hydroscopic, so it is best to open the bottle infrequently (I withdraw ten 0.5 ml aliquots whenever I open the bottle). 4. The Free Mounting System (FMS) is ideal for RT screening. So long as you do not have a highly volatile component in solution, then you can use the standard FMS at temperatures in the range of 15 to 25 C. If you do have a volatile component, then Proteros bistructures GmbH (www.proteros.com) has the only FMS with the capability of adding solvents to the gas stream...and with some compensation, they will gladly help you with this. Kris Kris F. Tesh, Ph D Director, Macromolecular Products Rigaku Americas Corporation 9009 New Trails Drive The Woodlands, TX 77381 USA 001 281 362 2300 x 144 -Original Message- From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Benini, Stefano Sent: Wednesday, July 11, 2007 7:17 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Help with reducing crystal mosaicity Dear Mary, the problem encountered with cryoprotectans is the change in the solution surrounding your crystal as they may not be present in your crystallisation conditions or you need to increase their concentration to make them act as cryoprotectant. So I don't thik is the cryoprotectant itself as it is you mother liquor.. test it in a capillary so you could rule out that freezing is the problem of course you don't want to find new crystallisation conditions when you tried everything and I know what it means!! I would try to improve crystal quality using additives or ligand etc, your long axis is very worrying as well!!! may be some magic additive could change the packing and givew better diffraction too I also had crystals growing from MPD, unfortunately there was a non-cleavable his-tag so I could not hope that metals would help. and other additives did not make any difference then my contract finished and so did the project! I hope you still have a lot of time available to try different things but I would not waste time trying to change your cryo ciao Stefano Stefano Benini PhD AstraZeneca UK Structural Biology Mereside 50S38 Alderley Park -Original Message- From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] Behalf Of Patrick Shaw Stewart Sent: 11 July 2007 12:10 To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [cc
Re: [ccp4bb] Help with reducing crystal mosaicity
You can also try to use the micromount loops (look like ink-pen nibs) sold by Mitegen. They are a much easier alternative to capillary mounts; they are easy to handle and work well for room temperature data collection. Good luck. Raji -Included Message-- >At the risk of sounding too commercial, here are some suggestions: > >1. See if you can either place your crystallization tray in a cold room or >crystallize it there. Mount your crystal in a capillary here so that at >whatever lower temperature you use, the distillation of solvent will be >reduced. > >2. If capillaries are too hard to work with, even a quick diffraction image >out of a loop, while the crystal is drying out, may give you some >information. Alternately, I have saturated my stock solution with sugar >from the coffee room and swished my crystal through this for getting a quick >diffraction image. In a rare case, the crystal actually diffracted for >several hours at room temperature. > >3. Someone mentioned using Paratone oil. This is good, but a little thick >and sticky. So, I use PerFluoroPolyEther (PFPE) as it is much less viscous >and is colorless. I buy FOMBLIN Y VAC 14/6 from SigmaAldrich (Cat # >317934-100G). PFPE is mildly hydroscopic, so it is best to open the bottle >infrequently (I withdraw ten 0.5 ml aliquots whenever I open the bottle). > >4. The Free Mounting System (FMS) is ideal for RT screening. So long as >you do not have a highly volatile component in solution, then you can use >the standard FMS at temperatures in the range of 15 to 25 C. If you do have >a volatile component, then Proteros bistructures GmbH (www.proteros.com) has >the only FMS with the capability of adding solvents to the gas stream...and >with some compensation, they will gladly help you with this. > >Kris > > >Kris F. Tesh, Ph D >Director, Macromolecular Products >Rigaku Americas Corporation >9009 New Trails Drive >The Woodlands, TX 77381 USA >001 281 362 2300 x 144 >-Original Message- >From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of >Benini, Stefano >Sent: Wednesday, July 11, 2007 7:17 AM >To: CCP4BB@JISCMAIL.AC.UK >Subject: Re: [ccp4bb] Help with reducing crystal mosaicity > >Dear Mary, > >the problem encountered with cryoprotectans is the change in the solution >surrounding your crystal as they may not be present in your crystallisation >conditions or you need to increase their concentration to make them act as >cryoprotectant. >So I don't thik is the cryoprotectant itself as it is you mother liquor.. > >test it in a capillary so you could rule out that freezing is the problem > >of course you don't want to find new crystallisation conditions when you >tried everything and I know what it means!! > >I would try to improve crystal quality using additives or ligand etc, > >your long axis is very worrying as well!!! may be some magic additive could >change the packing and givew better diffraction too > >I also had crystals growing from MPD, unfortunately there was a >non-cleavable his-tag >so I could not hope that metals would help. and other additives did not >make any difference >then my contract finished and so did the project! > >I hope you still have a lot of time available to try different things but I >would not waste time trying to change your cryo > >ciao > >Stefano > >Stefano Benini PhD >AstraZeneca UK >Structural Biology >Mereside 50S38 >Alderley Park > > > > > > > > >-Original Message- >From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] Behalf Of >Patrick Shaw Stewart >Sent: 11 July 2007 12:10 >To: CCP4BB@JISCMAIL.AC.UK >Subject: Re: [ccp4bb] Help with reducing crystal mosaicity > > >Just a thought, Mary - going back to your original question about MPD. I >extracted the crystallization conditions from REMARK 280 of 3939 PDB entries >a couple of years ago. The average concentration of the MPD used was high - >38.6%, while PEGs tended to be used at lower concs, e.g. PEG400 25.7%. You >can see the data at www.douglas.co.uk/top14.htm > >I thought this information could be useful if you want to replace some of >the MPD with another precipitant (or cryoprotectant). > >Best wishes > >Patrick Shaw Stewart > > >-- >[EMAIL PROTECTED]Douglas Instruments Ltd. >DouglasHouse, EastGarston, Hungerford, Berkshire, RG177HD, UK >Directors: Peter Baldock, Patrick Shaw Stewart, James Smith >http://douglas.co.uk or http://www.douglasinstruments.com >Tel: 44 (0) 148-864-9090US toll-free 1-877-225-2034 >Regd. England 2177994, VAT Reg. GB 480 7371 36 > > >> -Original Message-
