Hi Enrico, I was just looking at non-optimal cryo-conditions and the original posters starting point. Of course if you have a good cryo bigger is better for the reasons you write but if you have no clue how your crystals will perform then I'd rather go for small to be cautious and also have those around and not only the big ones which everybody mounts because they looks so nice. To be disappointed by big crystals is often not a surprise to me and if you have not tried small crystals from the same batch well then you missed 50% of your chances to solve s structure with the first light the crystals saw.
Jürgen On Feb 7, 2012, at 11:51 AM, Enrico Stura wrote: BIGGER is not always BETTER? Theoretically it should be better because you have more scattering matter. If it is not something has gone wrong in prior steps: Purification: You were less selective and picked up more heterogeneous protein. Crystallization: The bigger crystals grew under conditions that were less controlled because of changes in the state of protein supersaturation, precipitant or temperature. These inhomogeneities contributed to give you bigger, but not better crystals. Cryo-soak: Bigger crystals are more prone to be shocked when transfered to a less than optimal cryo-solution. This is a critical step, and crystals do not like too much the cryo-chemicals. To test this I tried lower concentrations of various cryo-compounds instead of a huge quantity of a single component and in most cases the mixture was better tollerated than the single components. Flash-freezing: Bigger crystals will cool more unevenly than small ones. A change from liquid nitrogen to liquid ethane could achieve faster cooling because of the greater heat capacity of the latter liquid. Large crystals are more difficult to handle than small ones but after experimenting with small ones we can build up a good experimental protocol so that the big crystals will give exceptionally good results. I compared small crystals on high intensity beamlines at the ESRF against large crystals on BM30 and the big crystals were statistically better. Unfortunately, it takes a lot of time and a certain amount of expertize to optimize the conditions. So ... although I strongly disagree with Jürgen, I will also advise to start with small ones. Enrico. On Tue, 07 Feb 2012 16:58:04 +0100, Bosch, Juergen <jubo...@jhsph.edu<mailto:jubo...@jhsph.edu>> wrote: Something to add into this discussion is also go fro the tiny crystals versus the big ones. BIGGER is not always BETTER - in particular if you try to freeze directly out of your conditions without an additional cryo-protectant. And with small or tiny I mean 10 micron, whatever you are capable of mounting. It is also important to keep the amount of liquid volume around the crystal low, so rather use a loop in which you scoop the crystal up instead of having a large loop with lots of liquid. Then one last remark, LN2 versus cryo-stream freeze. Dipping in LN2 leads to a quicker freeze of your material. If you have the option to anneal your crystal after testing it in the beam try it out and assess the success or damage, this will be very different depending on what cryo-additives you have around. Good luck, Jürgen On Feb 7, 2012, at 9:28 AM, Jacob Keller wrote: One last thing--sometimes crystals can be frozen as is, particularly if you use mitegen mounts and get nearly all of the mother liquor off the crystals by dabbing the loop on the dry surface next to the drop several times. So simple it is always worth a try.... JPK On Tue, Feb 7, 2012 at 2:37 AM, Mark J van Raaij <mjvanra...@cnb.csic.es<mailto:mjvanra...@cnb.csic.es><mailto:mjvanra...@cnb.csic.es>> wrote: Rationalising it completely may only be possible once you know the nature of the crystal contacts, i.e. when you have solved the structure. Until then it is mainly a matter of experimenting. -----Original Message----- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Theresa H. Hsu Sent: Monday, February 06, 2012 11:00 PM To: CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK><mailto:CCP4BB@JISCMAIL.AC.UK> Subject: Re: [ccp4bb] Freezing crystal Hi all Thanks for all the suggestions which I will try soon. How do the crystallization condition (PEG vs. salts like ammonium sulfate) affect the croyprotectant condition? Do factors like presence of low concentration of high molecular weight PEG (> 2000) mean PEG is better? Do buffers and salts in protein also important? Trying to rationalize it :) Theresa -- ******************************************* Jacob Pearson Keller Northwestern University Medical Scientist Training Program email: j-kell...@northwestern.edu<mailto:j-kell...@northwestern.edu><mailto:j-kell...@northwestern.edu> ******************************************* ...................... 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://web.mac.com/bosch_lab/ -- Enrico A. Stura D.Phil. (Oxon) , Tel: 33 (0)1 69 08 4302 Office Room 19, Bat.152, Tel: 33 (0)1 69 08 9449 Lab 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 ...................... 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://web.mac.com/bosch_lab/
