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 <photo.JPG> 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<tel:%28315%29-228-7245> ofc: (315)-228-7395<tel:%28315%29-228-7395> fax: (315)-228-7935<tel:%28315%29-228-7935> email: rrowl...@colgate.edu<mailto:rrowl...@colgate.edu> On Thu, Aug 2, 2012 at 2:14 PM, Yi-Liang Liu <yiliang...@gmail.com<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" >> <yiliang...@gmail.com<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
