Re: [ccp4bb] Disappearing crystals
On 11/28/11 12:04, Harman, Christine wrote: Hi All, I have just noticed a very strange thing and need some help in understanding it. I recently found two crystals in a condition from a screen (0.05M Calcium chloride dihydrate, 0.1M M Bis-Tris pH6.5 and 30% PEG MME 550). The small crystals appeared after a month and started to grow over the next 5 days after I first saw them (see pictures attached). I just check the same drop today and now the crystals are gone. So I was wondering what happened and if anyone experienced this before. Any insight or advice on what to do would be greatly appreciated. Was the heating/cooling turned down over the Turkey day holiday? -- === All Things Serve the Beam === David J. Schuller modern man in a post-modern world MacCHESS, Cornell University schul...@cornell.edu
Re: [ccp4bb] Disappearing crystals
On 11/28/11 12:04 PM, Harman, Christine wrote: Hi All, I have just noticed a very strange thing and need some help in understanding it. I recently found two crystals in a condition from a screen (0.05M Calcium chloride dihydrate, 0.1M M Bis-Tris pH6.5 and 30% PEG MME 550). The small crystals appeared after a month and started to grow over the next 5 days after I first saw them (see pictures attached). I just check the same drop today and now the crystals are gone. So I was wondering what happened and if anyone experienced this before. Any insight or advice on what to do would be greatly appreciated. Thanks Christine Small 5 days later Hi Christine, I had similar experience. In my case, another crystal showed again with different size a few days later. Sometimes, it seems like it is a common event to others as well as I heard although my case only takes about a week to be crystallized. I'd rather wait or just set up again or in a slightly different way. Wish you well. Young-Jin
Re: [ccp4bb] Disappearing crystals
When advice on crystallization is needed, it is important to give details of the protein concentration, the buffer the protein is in as well as the method used to grow the crystals. Problem: The crystallization conditions are essentially low salt: 100mM buffer and only 50mM CaCl2. So the buffer that the protein is in is very important !! Fluctuation in the reservoir/drop environment will lead to crystals dissolving. Solution: Balance the salt in the reservoir and in the protein:precipitant drop and make sure the temperature is kept constant. Since I do not have all the necessary information, the diagnosis and the solution proposed are likely to be wrong! Enrico. On Mon, 28 Nov 2011 19:19:49 +0100, YoungJin wrote: On 11/28/11 12:04 PM, Harman, Christine wrote: Hi All, I have just noticed a very strange thing and need some help in understanding it. I recently found two crystals in a condition from a screen (0.05M Calcium chloride dihydrate, 0.1M M Bis-Tris pH6.5 and 30% PEG MME 550). The small crystals appeared after a month and started to grow over the next 5 days after I first saw them (see pictures attached). I just check the same drop today and now the crystals are gone. So I was wondering what happened and if anyone experienced this before. Any insight or advice on what to do would be greatly appreciated. Thanks Christine Small 5 days later Hi Christine, I had similar experience. In my case, another crystal showed again with different size a few days later. Sometimes, it seems like it is a common event to others as well as I heard although my case only takes about a week to be crystallized. I'd rather wait or just set up again or in a slightly different way. Wish you well. Young-Jin -- Enrico A. Stura D.Phil. (Oxon) ,Tel: 33 (0)1 69 08 4302 Office Room 19, Bat.152, Tel: 33 (0)1 69 08 9449Lab LTMB, SIMOPRO, IBiTec-S, CE Saclay, 91191 Gif-sur-Yvette, FRANCE http://www-dsv.cea.fr/en/institutes/institute-of-biology-and-technology-saclay-ibitec-s/unites-de-recherche/department-of-molecular-engineering-of-proteins-simopro/molecular-toxinology-and-biotechnology-laboratory-ltmb/crystallogenesis-e.-stura http://www.chem.gla.ac.uk/protein/mirror/stura/index2.html e-mail: est...@cea.fr Fax: 33 (0)1 69 08 90 71
Re: [ccp4bb] Disappearing crystals
We have seen such behaviour connected to temperature fluctuations. Jan On Mon, Nov 28, 2011 at 6:04 PM, Harman, Christine < christine.har...@fda.hhs.gov> wrote: > Hi All, > I have just noticed a very strange thing and need some help in > understanding it. I recently found two crystals in a condition from a > screen (0.05M Calcium chloride dihydrate, 0.1M M Bis-Tris pH6.5 and 30% PEG > MME 550). The small crystals appeared after a month and started to grow > over the next 5 days after I first saw them (see pictures attached). I > just check the same drop today and now the crystals are gone. So I was > wondering what happened and if anyone experienced this before. Any insight > or advice on what to do would be greatly appreciated. > > Thanks > > Christine > > > Small 5 days later > > -- Jan Dohnalek, Ph.D Institute of Macromolecular Chemistry Academy of Sciences of the Czech Republic Heyrovskeho nam. 2 16206 Praha 6 Czech Republic Tel: +420 296 809 390 Fax: +420 296 809 410
Re: [ccp4bb] Disappearing crystals
Yes, and if they don't reappear it may be worth moving the trail to an incubator set at a slightly different temperature than "room temperature". You may have to try both lower or higher temperatures if you have no idea how the temperature may have changed (it's the temperature of the room when the crystals showed up that matters, obviously). Alternatively you could set up "fresh" trails with same conditions but for a deliberate and controlled screening around what the room temperature typically is. Good luck Thierry From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Jan Dohnalek Sent: Monday, November 28, 2011 1:47 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Disappearing crystals We have seen such behaviour connected to temperature fluctuations. Jan On Mon, Nov 28, 2011 at 6:04 PM, Harman, Christine mailto:christine.har...@fda.hhs.gov>> wrote: Hi All, I have just noticed a very strange thing and need some help in understanding it. I recently found two crystals in a condition from a screen (0.05M Calcium chloride dihydrate, 0.1M M Bis-Tris pH6.5 and 30% PEG MME 550). The small crystals appeared after a month and started to grow over the next 5 days after I first saw them (see pictures attached). I just check the same drop today and now the crystals are gone. So I was wondering what happened and if anyone experienced this before. Any insight or advice on what to do would be greatly appreciated. Thanks Christine Small 5 days later -- Jan Dohnalek, Ph.D Institute of Macromolecular Chemistry Academy of Sciences of the Czech Republic Heyrovskeho nam. 2 16206 Praha 6 Czech Republic Tel: +420 296 809 390 Fax: +420 296 809 410 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 privileged. It is intended solely for the use of the individual or entity named on this message. If you are not the intended recipient, and have received this message in error, please notify us immediately by reply e-mail and then delete it from your system.
Re: [ccp4bb] Disappearing crystals
Hi, Thank you so much for your replies. A lot of you have mentioned fluctuations in temp as the major contributor. And a few of you have asked for more details of my protein/buffer and set up. To my knowledge, the tray has been kept at constant 20 C (in an incubator) with exception of course to when I remove the tray to view the drops. It could be possible that my inspection of the tray might have contributed to an increase in temp, but only temporarily. I am very careful about the time the drop sees intense light, but it is possible the temp could have changed enough to cause this problem. Just to give a few more details. My protein (a Fab fragment/peptide complex (hopefully) is in buffer containing 100mM Sodium Acetate pH 5.5 with 150mM NaCl at a protein concentration of ~4.3mg/mL. After setting up my drop with reservoir solution I add NaCl to well to give ~75mM NaCl to match ionic strength of protein in drop which is diluted 1:1 with well solution. I do hope this problem is temperature. Although I am a little sad to not be able to freeze those crystals I did see, I still consider myself lucky to get such good result from a condition right from the screen so there will be some definite optimization set ups with this condition. Could I safely say though that the crystals I observed are not salt..:) I guess that is one good thing to take from this. Any more suggestions on optimization would be very welcomed. Thanks again to all you, Christine -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Enrico Stura Sent: Monday, November 28, 2011 1:45 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Disappearing crystals When advice on crystallization is needed, it is important to give details of the protein concentration, the buffer the protein is in as well as the method used to grow the crystals. Problem: The crystallization conditions are essentially low salt: 100mM buffer and only 50mM CaCl2. So the buffer that the protein is in is very important !! Fluctuation in the reservoir/drop environment will lead to crystals dissolving. Solution: Balance the salt in the reservoir and in the protein:precipitant drop and make sure the temperature is kept constant. Since I do not have all the necessary information, the diagnosis and the solution proposed are likely to be wrong! Enrico. On Mon, 28 Nov 2011 19:19:49 +0100, YoungJin wrote: > On 11/28/11 12:04 PM, Harman, Christine wrote: >> Hi All, >> I have just noticed a very strange thing and need some help in >> understanding it. I recently found two crystals in a condition from a >> screen (0.05M Calcium chloride dihydrate, 0.1M M Bis-Tris pH6.5 and >> 30% PEG MME 550). The small crystals appeared after a month and >> started to grow over the next 5 days after I first saw them (see >> pictures attached). I just check the same drop today and now the >> crystals are gone. So I was wondering what happened and if anyone >> experienced this before. Any insight or advice on what to do would be >> greatly appreciated. >> Thanks >> Christine >> Small 5 days later > Hi Christine, > I had similar experience. In my case, another crystal showed again with > different size a few days later. Sometimes, it seems like it is a common > event to others as well as I heard although my case only takes about a > week to be crystallized. I'd rather wait or just set up again or in a > slightly different way. > > Wish you well. > > Young-Jin > -- Enrico A. Stura D.Phil. (Oxon) ,Tel: 33 (0)1 69 08 4302 Office Room 19, Bat.152, Tel: 33 (0)1 69 08 9449Lab LTMB, SIMOPRO, IBiTec-S, CE Saclay, 91191 Gif-sur-Yvette, FRANCE http://www-dsv.cea.fr/en/institutes/institute-of-biology-and-technology-saclay-ibitec-s/unites-de-recherche/department-of-molecular-engineering-of-proteins-simopro/molecular-toxinology-and-biotechnology-laboratory-ltmb/crystallogenesis-e.-stura http://www.chem.gla.ac.uk/protein/mirror/stura/index2.html e-mail: est...@cea.fr Fax: 33 (0)1 69 08 90 71
Re: [ccp4bb] Disappearing crystals
Hi Christine, Another minor point (mainly for hanging drops) not mentioned in the messages. When you bring the crystallization tray from lower temperature to room temp. there is a possibility for condensation and slowly droplets near the protein drops gets absorbed into the protein drop. If this happens, depending on the condition and character of the protein, the crystals could crack or dissolve. In your case, condensation might have happened during your last check and the crystal may have affected by this issue. To avoid this problem: while taking the tray from the incubator place another tray on the top of your protein tray and keep them outside for few mts before checking with the microscope. best wishes, Mathews From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] On Behalf Of Harman, Christine [christine.har...@fda.hhs.gov] Sent: Monday, November 28, 2011 12:43 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Disappearing crystals Hi, Thank you so much for your replies. A lot of you have mentioned fluctuations in temp as the major contributor. And a few of you have asked for more details of my protein/buffer and set up. To my knowledge, the tray has been kept at constant 20 C (in an incubator) with exception of course to when I remove the tray to view the drops. It could be possible that my inspection of the tray might have contributed to an increase in temp, but only temporarily. I am very careful about the time the drop sees intense light, but it is possible the temp could have changed enough to cause this problem. Just to give a few more details. My protein (a Fab fragment/peptide complex (hopefully) is in buffer containing 100mM Sodium Acetate pH 5.5 with 150mM NaCl at a protein concentration of ~4.3mg/mL. After setting up my drop with reservoir solution I add NaCl to well to give ~75mM NaCl to match ionic strength of protein in drop which is diluted 1:1 with well solution. I do hope this problem is temperature. Although I am a little sad to not be able to freeze those crystals I did see, I still consider myself lucky to get such good result from a condition right from the screen so there will be some definite optimization set ups with this condition. Could I safely say though that the crystals I observed are not salt..:) I guess that is one good thing to take from this. Any more suggestions on optimization would be very welcomed. Thanks again to all you, Christine -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Enrico Stura Sent: Monday, November 28, 2011 1:45 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Disappearing crystals When advice on crystallization is needed, it is important to give details of the protein concentration, the buffer the protein is in as well as the method used to grow the crystals. Problem: The crystallization conditions are essentially low salt: 100mM buffer and only 50mM CaCl2. So the buffer that the protein is in is very important !! Fluctuation in the reservoir/drop environment will lead to crystals dissolving. Solution: Balance the salt in the reservoir and in the protein:precipitant drop and make sure the temperature is kept constant. Since I do not have all the necessary information, the diagnosis and the solution proposed are likely to be wrong! Enrico. On Mon, 28 Nov 2011 19:19:49 +0100, YoungJin wrote: > On 11/28/11 12:04 PM, Harman, Christine wrote: >> Hi All, >> I have just noticed a very strange thing and need some help in >> understanding it. I recently found two crystals in a condition from a >> screen (0.05M Calcium chloride dihydrate, 0.1M M Bis-Tris pH6.5 and >> 30% PEG MME 550). The small crystals appeared after a month and >> started to grow over the next 5 days after I first saw them (see >> pictures attached). I just check the same drop today and now the >> crystals are gone. So I was wondering what happened and if anyone >> experienced this before. Any insight or advice on what to do would be >> greatly appreciated. >> Thanks >> Christine >> Small 5 days later > Hi Christine, > I had similar experience. In my case, another crystal showed again with > different size a few days later. Sometimes, it seems like it is a common > event to others as well as I heard although my case only takes about a > week to be crystallized. I'd rather wait or just set up again or in a > slightly different way. > > Wish you well. > > Young-Jin > -- Enrico A. Stura D.Phil. (Oxon) ,Tel: 33 (0)1 69 08 4302 Office Room 19, Bat.152, Tel: 33 (0)1 69 08 9449Lab LTMB, SIMOPRO, IBiTec-S, CE Saclay, 91191 Gif-sur-Yvette, FRANCE http://www-dsv.cea.fr/en/institutes/institute-of-biology-and-technology-saclay-ibitec-s/unites-de-recherche/department
Re: [ccp4bb] Disappearing crystals
In addition to others' excellent suggestions, I'd like to offer more option to consider: Your crystallization set-up has some form of chemical non-equilibrium with respect to protein. It may just be my imagination but something is happening to the yellow precipitate in your drops, namely it's going away. Is it possible that you have a slow proteolysis (or something) happening to your protein, where one of the proteolytic products is crystallizing, however it's not the *final* proteolytic product so crystals are transient for as long as a 'steady state pool' of the appropriate form is above the solubility threshold (this implies equilibrium between soluble protein and crystals, which is not a huge stretch). If this is the case, I'd check your drops with MS after a few days to see if anything interesting is happening. Options include but are not limited to: proteolysis, oxidation, chemical reaction of protein, chemical reaction of buffers and/or additives, and so on :) Artem P.S. It could simply be that your protein is initially precipitated but then driven back into solution? It happens. A. On Mon, Nov 28, 2011 at 4:56 PM, Mathews, Irimpan I. < iimat...@slac.stanford.edu> wrote: > Hi Christine, > > Another minor point (mainly for hanging drops) not mentioned in the > messages. > > When you bring the crystallization tray from lower temperature to room > temp. there is a possibility for condensation and slowly droplets near the > protein drops gets absorbed into the protein drop. If this happens, > depending on the condition and character of the protein, the crystals > could crack or dissolve. > > In your case, condensation might have happened during your last check and > the crystal may have affected by this issue. To avoid this problem: while > taking the tray from the incubator place another tray on the top of your > protein tray and keep them outside for few mts before checking with the > microscope. > > best wishes, > Mathews > > > From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] On Behalf Of Harman, > Christine [christine.har...@fda.hhs.gov] > Sent: Monday, November 28, 2011 12:43 PM > To: CCP4BB@JISCMAIL.AC.UK > Subject: Re: [ccp4bb] Disappearing crystals > > Hi, > Thank you so much for your replies. A lot of you have mentioned > fluctuations in temp as the major contributor. And a few of you have asked > for more details of my protein/buffer and set up. To my knowledge, the > tray has been kept at constant 20 C (in an incubator) with exception of > course to when I remove the tray to view the drops. It could be possible > that my inspection of the tray might have contributed to an increase in > temp, but only temporarily. I am very careful about the time the drop sees > intense light, but it is possible the temp could have changed enough to > cause this problem. Just to give a few more details. My protein (a Fab > fragment/peptide complex (hopefully) is in buffer containing 100mM Sodium > Acetate pH 5.5 with 150mM NaCl at a protein concentration of ~4.3mg/mL. > After setting up my drop with reservoir solution I add NaCl to well to > give ~75mM NaCl to match ionic strength of protein in drop which is diluted > 1:1 with well solution. I do hope this problem is temperature. Although I > am a little sad to not be able to freeze those crystals I did see, I still > consider myself lucky to get such good result from a condition right from > the screen so there will be some definite optimization set ups with this > condition. Could I safely say though that the crystals I observed are not > salt..:) I guess that is one good thing to take from this. Any more > suggestions on optimization would be very welcomed. > > Thanks again to all you, > > Christine > > > > -Original Message- > From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of > Enrico Stura > Sent: Monday, November 28, 2011 1:45 PM > To: CCP4BB@JISCMAIL.AC.UK > Subject: Re: [ccp4bb] Disappearing crystals > > When advice on crystallization is needed, it is important to give details > of > the protein concentration, the buffer the protein is in as well as the > method > used to grow the crystals. > > Problem: The crystallization conditions are essentially low salt: 100mM > buffer > and only 50mM CaCl2. So the buffer that the protein is in is very > important !! > Fluctuation in the reservoir/drop environment will lead to crystals > dissolving. > > Solution: Balance the salt in the reservoir and in the protein:precipitant > drop and make sure > the temperature is kept constant. > > Since I do not have all the necessary information, the diagnosis and the > solution proposed > are like
Re: [ccp4bb] Disappearing crystals
Dear Christine, I had guessed that you had more salt in the protein solution than in the reservoir with a relatively low protein concentration. The protein is in: 100mM Sodium Acetate pH 5.5 with 150mM NaCl at a protein concentration of ~4.3mg/mL. and the reservoir is: (0.05M Calcium chloride dihydrate, 0.1M M Bis-Tris pH6.5 and 30% PEG MME 550). We can suspect that no matter what temperature the equilibrium will move towards dilution in the drop. Basic suggestion: Set up a range from 20-30% PEG MME 550 with at least 150mM NaCl in the reservoir. Fine tuning: Add high MW PEG (10K or 20K 1-5%) it should have a stabilizing effect Try a range of salts apart from NaCl: The clasics are: Li2SO4, KSCN, MgCl2. Try additive amounts od MPD 0.5-3%. Try a small tight range of various pH. Enrico. On Mon, 28 Nov 2011 21:43:51 +0100, Harman, Christine wrote: Hi, Thank you so much for your replies. A lot of you have mentioned fluctuations in temp as the major contributor. And a few of you have asked for more details of my protein/buffer and set up. To my knowledge, the tray has been kept at constant 20 C (in an incubator) with exception of course to when I remove the tray to view the drops. It could be possible that my inspection of the tray might have contributed to an increase in temp, but only temporarily. I am very careful about the time the drop sees intense light, but it is possible the temp could have changed enough to cause this problem. Just to give a few more details. My protein (a Fab fragment/peptide complex (hopefully) is in buffer containing 100mM Sodium Acetate pH 5.5 with 150mM NaCl at a protein concentration of ~4.3mg/mL. After setting up my drop with reservoir solution I add NaCl to well to give ~75mM NaCl to match ionic strength of protein in drop which is diluted 1:1 with well solution. I do hope this problem is temperature. Although I am a little sad to not be able to freeze those crystals I did see, I still consider myself lucky to get such good result from a condition right from the screen so there will be some definite optimization set ups with this condition. Could I safely say though that the crystals I observed are not salt..:) I guess that is one good thing to take from this. Any more suggestions on optimization would be very welcomed. Thanks again to all you, Christine -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Enrico Stura Sent: Monday, November 28, 2011 1:45 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Disappearing crystals When advice on crystallization is needed, it is important to give details of the protein concentration, the buffer the protein is in as well as the method used to grow the crystals. Problem: The crystallization conditions are essentially low salt: 100mM buffer and only 50mM CaCl2. So the buffer that the protein is in is very important !! Fluctuation in the reservoir/drop environment will lead to crystals dissolving. Solution: Balance the salt in the reservoir and in the protein:precipitant drop and make sure the temperature is kept constant. Since I do not have all the necessary information, the diagnosis and the solution proposed are likely to be wrong! Enrico. On Mon, 28 Nov 2011 19:19:49 +0100, YoungJin wrote: On 11/28/11 12:04 PM, Harman, Christine wrote: Hi All, I have just noticed a very strange thing and need some help in understanding it. I recently found two crystals in a condition from a screen (0.05M Calcium chloride dihydrate, 0.1M M Bis-Tris pH6.5 and 30% PEG MME 550). The small crystals appeared after a month and started to grow over the next 5 days after I first saw them (see pictures attached). I just check the same drop today and now the crystals are gone. So I was wondering what happened and if anyone experienced this before. Any insight or advice on what to do would be greatly appreciated. Thanks Christine Small 5 days later Hi Christine, I had similar experience. In my case, another crystal showed again with different size a few days later. Sometimes, it seems like it is a common event to others as well as I heard although my case only takes about a week to be crystallized. I'd rather wait or just set up again or in a slightly different way. Wish you well. Young-Jin -- Enrico A. Stura D.Phil. (Oxon) ,Tel: 33 (0)1 69 08 4302 Office Room 19, Bat.152, Tel: 33 (0)1 69 08 9449Lab LTMB, SIMOPRO, IBiTec-S, CE Saclay, 91191 Gif-sur-Yvette, FRANCE http://www-dsv.cea.fr/en/institutes/institute-of-biology-and-technology-saclay-ibitec-s/unites-de-recherche/department-of-molecular-engineering-of-proteins-simopro/molecular-toxinology-and-biotechnology-laboratory-ltmb/crystallogenesis-e.-stura http://www.chem.gla.ac.uk/protein/mirror/stura/index2.html e-mail: est...@cea.fr Fax: 33 (0)1 69 08 90 71
Re: [ccp4bb] Disappearing crystals
Christine, I have a couple more comments First, if those crystals come back, I would certainly try microseeding into *random *screens. You should be able to pick up new conditions that have a little more salt in them, and are therefore stable regarding the movement of water. (If you can't get the crystals back, I would try microseeding with the precipitate that you have.) Second, it's helpful to understand that the movement of water in and out of the drop is connected to heat *flows*, not temperature. The heat flow drives the movement of moisture (in addition to the salt etc. of course). Once you understand that you can easily get rid of e.g. condensation on the tape by putting e.g. a warm book on top of the plate in the cold room (or a book from the cold room under the plate at room temp). Similarly, you can prevent dilution of your drops by thinking about heat flows. Some say that it's essential to use Blundell and Johnson. Others say only Bergfors on crystallization, Lord of the Rings or even Harry Potter can provide the necessary inspiration. Good luck Patrick Ref for microseeding: Allan D’Arcy, Frederic Villarda, May Marsh. An automated microseed matrix-screening method for protein crystallization. Acta Crystallographica section D 63 (2007), 550–554. http://www.douglas.co.uk/mms.htm On Mon, Nov 28, 2011 at 8:43 PM, Harman, Christine < christine.har...@fda.hhs.gov> wrote: > Hi, > Thank you so much for your replies. A lot of you have mentioned > fluctuations in temp as the major contributor. And a few of you have asked > for more details of my protein/buffer and set up. To my knowledge, the > tray has been kept at constant 20 C (in an incubator) with exception of > course to when I remove the tray to view the drops. It could be possible > that my inspection of the tray might have contributed to an increase in > temp, but only temporarily. I am very careful about the time the drop sees > intense light, but it is possible the temp could have changed enough to > cause this problem. Just to give a few more details. My protein (a Fab > fragment/peptide complex (hopefully) is in buffer containing 100mM Sodium > Acetate pH 5.5 with 150mM NaCl at a protein concentration of ~4.3mg/mL. > After setting up my drop with reservoir solution I add NaCl to well to > give ~75mM NaCl to match ionic strength of protein in drop which is diluted > 1:1 with well solution. I do hope this problem is temperature. Although I > am a little sad to not be able to freeze those crystals I did see, I still > consider myself lucky to get such good result from a condition right from > the screen so there will be some definite optimization set ups with this > condition. Could I safely say though that the crystals I observed are not > salt..:) I guess that is one good thing to take from this. Any more > suggestions on optimization would be very welcomed. > > Thanks again to all you, > > Christine > > > > -Original Message- > From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of > Enrico Stura > Sent: Monday, November 28, 2011 1:45 PM > To: CCP4BB@JISCMAIL.AC.UK > Subject: Re: [ccp4bb] Disappearing crystals > > When advice on crystallization is needed, it is important to give details > of > the protein concentration, the buffer the protein is in as well as the > method > used to grow the crystals. > > Problem: The crystallization conditions are essentially low salt: 100mM > buffer > and only 50mM CaCl2. So the buffer that the protein is in is very > important !! > Fluctuation in the reservoir/drop environment will lead to crystals > dissolving. > > Solution: Balance the salt in the reservoir and in the protein:precipitant > drop and make sure > the temperature is kept constant. > > Since I do not have all the necessary information, the diagnosis and the > solution proposed > are likely to be wrong! > > Enrico. > > On Mon, 28 Nov 2011 19:19:49 +0100, YoungJin wrote: > > > On 11/28/11 12:04 PM, Harman, Christine wrote: > >> Hi All, > >> I have just noticed a very strange thing and need some help in > >> understanding it. I recently found two crystals in a condition from a > >> screen (0.05M Calcium chloride dihydrate, 0.1M M Bis-Tris pH6.5 and > >> 30% PEG MME 550). The small crystals appeared after a month and > >> started to grow over the next 5 days after I first saw them (see > >> pictures attached). I just check the same drop today and now the > >> crystals are gone. So I was wondering what happened and if anyone > >> experienced this before. Any insight or advice on what to do would be > >> greatly appreciated. > >> Thanks > >> Christine > >> Small
