Re: [ccp4bb] Enhancing Crystal Quality
Lucas, if your crystals diffract worse at room temp than cryoprotected, it looks like there may be radiation damage at room temperature, so you may need to cryoprotect. The change in osmotic pressure may be too much for your crystals when you're cryoprotecting the crystals by a sudden dunk. Also, did you just add PEG 400 to the reservoir solution already in the wells or did you make it afresh with higher precipitant concentration to compensate for removal into a solution without any soluble protein or crystalline protein to be in equilibrium with? I always made solutions afresh unless the crystal was cryoprotected and if it diffracted just fine by dragging the crystal through a reservoir+cryoprotectant solution. My crystals did not tolerate sudden changes in osmotic pressure. Harvest the crystal into 15 uL of reservoir solution with excess precipitant to compensate for loss of protein to be in equilibrium with (if I had a crystal from 8% PEG 8000, I used to use 12% PEG8000 with all of the other components having the same concentration. It was random but I found one that worked and stuck to it. Sometimes, there may be a solubility issue, so I had to cut back on a salt but just enough to keep everything soluble. So I made the solution fresh because my crystals didn't always appear at exactly the same precipitant concentration. I always set up a small range of concentrations. Add 2.5 uL of cryoprotectant (reservoir solution with excess precipitant concentration AND required concentration of precipitant present in it). Remove 2.5 uL from a different part of the drop. Wait 6 min (again, random. 5 min didn't work well, 6 min did). Repeat with 2.5 uL, 3.75 uL, 5 uL, 7.5 uL. By that time, I achieved enough cryoprotectant concentration for it to do its job. I never mixed the drop because my crystals were very fragile and obviously, I was always looking at it under the microscope to make sure I wasn't smashing or sucking up my crystal. Have you tried additive screens? I had tremendous improvement in my crystals with that. They were very mosaic and overall resolution was only about 3.7 A or so without additives. With 3% DMSO (from an additive screen), the size was great, still mosaic, but overall resolution with good outer shell completeness (I don't remember the exact numbers for data quality but it was much better than without) improved to about 2.8-ish A. Good luck! S. On Sun, Aug 12, 2012 at 3:31 PM, Yi-Liang (Lucas) Liu wrote: > Hi CCP4ers, > > I tested my crystal under room temperature. It still only has low > resolution (<7A). Are there any way to improve this? I attached the > diffraction as well. Thanks. > > Lucas > > > > On Aug 2, 2012, at 5:27 PM, Roger Rowlett wrote: > > Mitegen makes a nice little product that is a plastic tube that will slide > over one of their magnetic cap/loops. If you put some well solution in the > tube and seal the base with apiezon, you can collect quite a bit of data on > the loop mounted crystal before it dries out. > > Cheers, > > ___ > Roger S. Rowlett > Gordon & Dorothy Kline Professor > Department of Chemistry > Colgate University > 13 Oak Drive > Hamilton, NY 13346 > > tel: (315)-228-7245 > ofc: (315)-228-7395 > fax: (315)-228-7935 > email: rrowl...@colgate.edu > > > > > > > On Thu, Aug 2, 2012 at 2:14 PM, Yi-Liang Liu wrote: > >> Hi Herman and other CCP3BBers, >> >> Thanks for your suggestions. I didn't see any cracks in the crystal drops >> initially. I will certainly try to shot crystals under room temperature and >> see what happens. Does the plastic loops fit into the cryo stands Molecular >> Dimension sells? >> >> LUcas >> On Aug 2, 2012, at 2:24 AM, herman.schreu...@sanofi.com wrote: >> >> > Hi Lucas, >> > >> > The funky diffraction pattern is most likely due to a cracked crystal, >> > resulting in a mixture of slightly differently aligned diffraction >> > patterns. Were the cracks there before you added the cryprotectant? If >> > not, the cryoprotectant is definitively to blame. As has mentioned >> > before, you have to take a shot at room temperature without any >> > cryoprotectant added, to make sure the bad quality is not due to the >> > cryoprotectant. Mitegen sells plastic capillaries, which you can slide >> > over your loop to prevent the crystal from drying out. >> > >> > Good luck! >> > Herman >> > >> > -Original Message- >> > From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of >> > Yi-Liang Liu >> > Sent: Thursday, August 02, 2012 4:15 AM >> > To: CCP4BB@JISCMAIL.AC.UK >> > Subject: Re: [ccp4bb
Re: [ccp4bb] Enhancing Crystal Quality
controlled dehydration with saturated salt solutions testing of various cryo conditions persistence is the key to success you have diffraction, now solve it ! Good luck Jürgen On Aug 12, 2012, at 3:31 PM, Yi-Liang (Lucas) Liu wrote: Hi CCP4ers, I tested my crystal under room temperature. It still only has low resolution (<7A). Are there any way to improve this? I attached the diffraction as well. Thanks. Lucas On Aug 2, 2012, at 5:27 PM, Roger Rowlett wrote: Mitegen makes a nice little product that is a plastic tube that will slide over one of their magnetic cap/loops. If you put some well solution in the tube and seal the base with apiezon, you can collect quite a bit of data on the loop mounted crystal before it dries out. Cheers, ___ Roger S. Rowlett Gordon & Dorothy Kline Professor Department of Chemistry Colgate University 13 Oak Drive Hamilton, NY 13346 tel: (315)-228-7245 ofc: (315)-228-7395 fax: (315)-228-7935 email: rrowl...@colgate.edu<mailto:rrowl...@colgate.edu> On Thu, Aug 2, 2012 at 2:14 PM, Yi-Liang Liu mailto:yiliang...@gmail.com>> wrote: Hi Herman and other CCP3BBers, Thanks for your suggestions. I didn't see any cracks in the crystal drops initially. I will certainly try to shot crystals under room temperature and see what happens. Does the plastic loops fit into the cryo stands Molecular Dimension sells? LUcas On Aug 2, 2012, at 2:24 AM, herman.schreu...@sanofi.com<mailto:herman.schreu...@sanofi.com> wrote: > Hi Lucas, > > The funky diffraction pattern is most likely due to a cracked crystal, > resulting in a mixture of slightly differently aligned diffraction > patterns. Were the cracks there before you added the cryprotectant? If > not, the cryoprotectant is definitively to blame. As has mentioned > before, you have to take a shot at room temperature without any > cryoprotectant added, to make sure the bad quality is not due to the > cryoprotectant. Mitegen sells plastic capillaries, which you can slide > over your loop to prevent the crystal from drying out. > > Good luck! > Herman > > -Original Message- > From: CCP4 bulletin board > [mailto:CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>] On Behalf Of > Yi-Liang Liu > Sent: Thursday, August 02, 2012 4:15 AM > To: CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK> > Subject: Re: [ccp4bb] Enhancing Crystal Quality > > Hi, > > Thanks for the kindly answers from everyone. I actually haven't tried > different cryoprotectants. I might will give a try next time. I usually > only use mother liquor+30% PEG400. It is noticeable that it has some > "patterns (cracks (?))" on the crystal. However, it didn't form icy > rings or etc. The diffraction pattern looks funky too. It looks like it > is twin and the diffraction spot has tails. Does this indicate the > cryoprotectant problem? > > Lucas > On Aug 1, 2012, at 2:11 PM, Antony Oliver wrote: > >> Have you tried different cryoprotectants? Can make a huge difference. > Also, have you shot an xtal at room temp - to see what the intrinsic > diffraction limit is? Additive screens? If all else fails you may well > need to explore a different expression construct. >> >> Tony. >> >> Sent from my iPhone >> >> On 1 Aug 2012, at 19:52, "Yi-Liang Liu" >> mailto:yiliang...@gmail.com>> wrote: >> >>> Hi CCP4BBers, >>> >>> I have a protein crystallized with 0.2mM Mg acetate and PEG 8000 or > 0.1mM cacodylate, pH 6.5, 0.2 Mg acetate, PEG 3350. Both of the > conditions gave triangle pyramid like crystals. I brought the crystals > to synchrotron using 30% PEG 400 as cryoprotactant, the resolution was > only be able to reach 4A or worse. I have tried changing pH and > concentrations of PEG, PEG types. I found out this crystal only grew > between pH 6.5~7.5 and PEG types did not change the result of > diffraction dramatically. I have also tried the seeding (break it down > and reseed in the same condition. Maybe I did it wrong?). It gave me the > similar results, not improving. Is there any simple way of improving it > before jumping into reengineering the protein. >>> >>> Thanks, >>> >>> Lucas .. Jürgen Bosch Johns Hopkins University Bloomberg School of Public Health Department of Biochemistry & Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Office: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-2926 http://lupo.jhsph.edu
Re: [ccp4bb] Enhancing Crystal Quality
Mitegen makes a nice little product that is a plastic tube that will slide over one of their magnetic cap/loops. If you put some well solution in the tube and seal the base with apiezon, you can collect quite a bit of data on the loop mounted crystal before it dries out. Cheers, ___ Roger S. Rowlett Gordon & Dorothy Kline Professor Department of Chemistry Colgate University 13 Oak Drive Hamilton, NY 13346 tel: (315)-228-7245 ofc: (315)-228-7395 fax: (315)-228-7935 email: rrowl...@colgate.edu On Thu, Aug 2, 2012 at 2:14 PM, Yi-Liang Liu wrote: > Hi Herman and other CCP3BBers, > > Thanks for your suggestions. I didn't see any cracks in the crystal drops > initially. I will certainly try to shot crystals under room temperature and > see what happens. Does the plastic loops fit into the cryo stands Molecular > Dimension sells? > > LUcas > On Aug 2, 2012, at 2:24 AM, herman.schreu...@sanofi.com wrote: > > > Hi Lucas, > > > > The funky diffraction pattern is most likely due to a cracked crystal, > > resulting in a mixture of slightly differently aligned diffraction > > patterns. Were the cracks there before you added the cryprotectant? If > > not, the cryoprotectant is definitively to blame. As has mentioned > > before, you have to take a shot at room temperature without any > > cryoprotectant added, to make sure the bad quality is not due to the > > cryoprotectant. Mitegen sells plastic capillaries, which you can slide > > over your loop to prevent the crystal from drying out. > > > > Good luck! > > Herman > > > > -Original Message- > > From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of > > Yi-Liang Liu > > Sent: Thursday, August 02, 2012 4:15 AM > > To: CCP4BB@JISCMAIL.AC.UK > > Subject: Re: [ccp4bb] Enhancing Crystal Quality > > > > Hi, > > > > Thanks for the kindly answers from everyone. I actually haven't tried > > different cryoprotectants. I might will give a try next time. I usually > > only use mother liquor+30% PEG400. It is noticeable that it has some > > "patterns (cracks (?))" on the crystal. However, it didn't form icy > > rings or etc. The diffraction pattern looks funky too. It looks like it > > is twin and the diffraction spot has tails. Does this indicate the > > cryoprotectant problem? > > > > Lucas > > On Aug 1, 2012, at 2:11 PM, Antony Oliver wrote: > > > >> Have you tried different cryoprotectants? Can make a huge difference. > > Also, have you shot an xtal at room temp - to see what the intrinsic > > diffraction limit is? Additive screens? If all else fails you may well > > need to explore a different expression construct. > >> > >> Tony. > >> > >> Sent from my iPhone > >> > >> On 1 Aug 2012, at 19:52, "Yi-Liang Liu" wrote: > >> > >>> Hi CCP4BBers, > >>> > >>> I have a protein crystallized with 0.2mM Mg acetate and PEG 8000 or > > 0.1mM cacodylate, pH 6.5, 0.2 Mg acetate, PEG 3350. Both of the > > conditions gave triangle pyramid like crystals. I brought the crystals > > to synchrotron using 30% PEG 400 as cryoprotactant, the resolution was > > only be able to reach 4A or worse. I have tried changing pH and > > concentrations of PEG, PEG types. I found out this crystal only grew > > between pH 6.5~7.5 and PEG types did not change the result of > > diffraction dramatically. I have also tried the seeding (break it down > > and reseed in the same condition. Maybe I did it wrong?). It gave me the > > similar results, not improving. Is there any simple way of improving it > > before jumping into reengineering the protein. > >>> > >>> Thanks, > >>> > >>> Lucas >
Re: [ccp4bb] Enhancing Crystal Quality
Hi Herman and other CCP3BBers, Thanks for your suggestions. I didn't see any cracks in the crystal drops initially. I will certainly try to shot crystals under room temperature and see what happens. Does the plastic loops fit into the cryo stands Molecular Dimension sells? LUcas On Aug 2, 2012, at 2:24 AM, herman.schreu...@sanofi.com wrote: > Hi Lucas, > > The funky diffraction pattern is most likely due to a cracked crystal, > resulting in a mixture of slightly differently aligned diffraction > patterns. Were the cracks there before you added the cryprotectant? If > not, the cryoprotectant is definitively to blame. As has mentioned > before, you have to take a shot at room temperature without any > cryoprotectant added, to make sure the bad quality is not due to the > cryoprotectant. Mitegen sells plastic capillaries, which you can slide > over your loop to prevent the crystal from drying out. > > Good luck! > Herman > > -Original Message- > From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of > Yi-Liang Liu > Sent: Thursday, August 02, 2012 4:15 AM > To: CCP4BB@JISCMAIL.AC.UK > Subject: Re: [ccp4bb] Enhancing Crystal Quality > > Hi, > > Thanks for the kindly answers from everyone. I actually haven't tried > different cryoprotectants. I might will give a try next time. I usually > only use mother liquor+30% PEG400. It is noticeable that it has some > "patterns (cracks (?))" on the crystal. However, it didn't form icy > rings or etc. The diffraction pattern looks funky too. It looks like it > is twin and the diffraction spot has tails. Does this indicate the > cryoprotectant problem? > > Lucas > On Aug 1, 2012, at 2:11 PM, Antony Oliver wrote: > >> Have you tried different cryoprotectants? Can make a huge difference. > Also, have you shot an xtal at room temp - to see what the intrinsic > diffraction limit is? Additive screens? If all else fails you may well > need to explore a different expression construct. >> >> Tony. >> >> Sent from my iPhone >> >> On 1 Aug 2012, at 19:52, "Yi-Liang Liu" wrote: >> >>> Hi CCP4BBers, >>> >>> I have a protein crystallized with 0.2mM Mg acetate and PEG 8000 or > 0.1mM cacodylate, pH 6.5, 0.2 Mg acetate, PEG 3350. Both of the > conditions gave triangle pyramid like crystals. I brought the crystals > to synchrotron using 30% PEG 400 as cryoprotactant, the resolution was > only be able to reach 4A or worse. I have tried changing pH and > concentrations of PEG, PEG types. I found out this crystal only grew > between pH 6.5~7.5 and PEG types did not change the result of > diffraction dramatically. I have also tried the seeding (break it down > and reseed in the same condition. Maybe I did it wrong?). It gave me the > similar results, not improving. Is there any simple way of improving it > before jumping into reengineering the protein. >>> >>> Thanks, >>> >>> Lucas
Re: [ccp4bb] Enhancing Crystal Quality
Hi Lucas, The funky diffraction pattern is most likely due to a cracked crystal, resulting in a mixture of slightly differently aligned diffraction patterns. Were the cracks there before you added the cryprotectant? If not, the cryoprotectant is definitively to blame. As has mentioned before, you have to take a shot at room temperature without any cryoprotectant added, to make sure the bad quality is not due to the cryoprotectant. Mitegen sells plastic capillaries, which you can slide over your loop to prevent the crystal from drying out. Good luck! Herman -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Yi-Liang Liu Sent: Thursday, August 02, 2012 4:15 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Enhancing Crystal Quality Hi, Thanks for the kindly answers from everyone. I actually haven't tried different cryoprotectants. I might will give a try next time. I usually only use mother liquor+30% PEG400. It is noticeable that it has some "patterns (cracks (?))" on the crystal. However, it didn't form icy rings or etc. The diffraction pattern looks funky too. It looks like it is twin and the diffraction spot has tails. Does this indicate the cryoprotectant problem? Lucas On Aug 1, 2012, at 2:11 PM, Antony Oliver wrote: > Have you tried different cryoprotectants? Can make a huge difference. Also, have you shot an xtal at room temp - to see what the intrinsic diffraction limit is? Additive screens? If all else fails you may well need to explore a different expression construct. > > Tony. > > Sent from my iPhone > > On 1 Aug 2012, at 19:52, "Yi-Liang Liu" wrote: > >> Hi CCP4BBers, >> >> I have a protein crystallized with 0.2mM Mg acetate and PEG 8000 or 0.1mM cacodylate, pH 6.5, 0.2 Mg acetate, PEG 3350. Both of the conditions gave triangle pyramid like crystals. I brought the crystals to synchrotron using 30% PEG 400 as cryoprotactant, the resolution was only be able to reach 4A or worse. I have tried changing pH and concentrations of PEG, PEG types. I found out this crystal only grew between pH 6.5~7.5 and PEG types did not change the result of diffraction dramatically. I have also tried the seeding (break it down and reseed in the same condition. Maybe I did it wrong?). It gave me the similar results, not improving. Is there any simple way of improving it before jumping into reengineering the protein. >> >> Thanks, >> >> Lucas
Re: [ccp4bb] Enhancing Crystal Quality
Hi, Thanks for the kindly answers from everyone. I actually haven't tried different cryoprotectants. I might will give a try next time. I usually only use mother liquor+30% PEG400. It is noticeable that it has some "patterns (cracks (?))" on the crystal. However, it didn't form icy rings or etc. The diffraction pattern looks funky too. It looks like it is twin and the diffraction spot has tails. Does this indicate the cryoprotectant problem? Lucas On Aug 1, 2012, at 2:11 PM, Antony Oliver wrote: > Have you tried different cryoprotectants? Can make a huge difference. Also, > have you shot an xtal at room temp - to see what the intrinsic diffraction > limit is? Additive screens? If all else fails you may well need to explore a > different expression construct. > > Tony. > > Sent from my iPhone > > On 1 Aug 2012, at 19:52, "Yi-Liang Liu" wrote: > >> Hi CCP4BBers, >> >> I have a protein crystallized with 0.2mM Mg acetate and PEG 8000 or 0.1mM >> cacodylate, pH 6.5, 0.2 Mg acetate, PEG 3350. Both of the conditions gave >> triangle pyramid like crystals. I brought the crystals to synchrotron using >> 30% PEG 400 as cryoprotactant, the resolution was only be able to reach 4A >> or worse. I have tried changing pH and concentrations of PEG, PEG types. I >> found out this crystal only grew between pH 6.5~7.5 and PEG types did not >> change the result of diffraction dramatically. I have also tried the seeding >> (break it down and reseed in the same condition. Maybe I did it wrong?). It >> gave me the similar results, not improving. Is there any simple way of >> improving it before jumping into reengineering the protein. >> >> Thanks, >> >> Lucas
Re: [ccp4bb] Enhancing Crystal Quality
You might be killing crystals with the cryosolvent. Try soaking the crystals briefly in well solution + 25-30% glucose or sucrose prior to flash cooling. You can also try other cryoprotectants: glycerol, EG, etc. You can also try sequential brief soaks in gradually increasing cryoprotectant concentration. This sometimes works when a direct dunk does not. Or paratone oil. Roger Rowlett On Aug 1, 2012 2:52 PM, "Yi-Liang Liu" wrote: > Hi CCP4BBers, > > I have a protein crystallized with 0.2mM Mg acetate and PEG 8000 or 0.1mM > cacodylate, pH 6.5, 0.2 Mg acetate, PEG 3350. Both of the conditions gave > triangle pyramid like crystals. I brought the crystals to synchrotron using > 30% PEG 400 as cryoprotactant, the resolution was only be able to reach 4A > or worse. I have tried changing pH and concentrations of PEG, PEG types. I > found out this crystal only grew between pH 6.5~7.5 and PEG types did not > change the result of diffraction dramatically. I have also tried the > seeding (break it down and reseed in the same condition. Maybe I did it > wrong?). It gave me the similar results, not improving. Is there any simple > way of improving it before jumping into reengineering the protein. > > Thanks, > > Lucas
Re: [ccp4bb] Enhancing Crystal Quality
Have you tried different cryoprotectants? Can make a huge difference. Also, have you shot an xtal at room temp - to see what the intrinsic diffraction limit is? Additive screens? If all else fails you may well need to explore a different expression construct. Tony. Sent from my iPhone On 1 Aug 2012, at 19:52, "Yi-Liang Liu" wrote: > Hi CCP4BBers, > > I have a protein crystallized with 0.2mM Mg acetate and PEG 8000 or 0.1mM > cacodylate, pH 6.5, 0.2 Mg acetate, PEG 3350. Both of the conditions gave > triangle pyramid like crystals. I brought the crystals to synchrotron using > 30% PEG 400 as cryoprotactant, the resolution was only be able to reach 4A or > worse. I have tried changing pH and concentrations of PEG, PEG types. I found > out this crystal only grew between pH 6.5~7.5 and PEG types did not change > the result of diffraction dramatically. I have also tried the seeding (break > it down and reseed in the same condition. Maybe I did it wrong?). It gave me > the similar results, not improving. Is there any simple way of improving it > before jumping into reengineering the protein. > > Thanks, > > Lucas
[ccp4bb] Enhancing Crystal Quality
Hi CCP4BBers, I have a protein crystallized with 0.2mM Mg acetate and PEG 8000 or 0.1mM cacodylate, pH 6.5, 0.2 Mg acetate, PEG 3350. Both of the conditions gave triangle pyramid like crystals. I brought the crystals to synchrotron using 30% PEG 400 as cryoprotactant, the resolution was only be able to reach 4A or worse. I have tried changing pH and concentrations of PEG, PEG types. I found out this crystal only grew between pH 6.5~7.5 and PEG types did not change the result of diffraction dramatically. I have also tried the seeding (break it down and reseed in the same condition. Maybe I did it wrong?). It gave me the similar results, not improving. Is there any simple way of improving it before jumping into reengineering the protein. Thanks, Lucas
