Ursula, I agree with Tim. Try RT data collection, and also as many cryo-protectants as possible. I would suggest a method we have used for many years for an easy RT crystal mounting. Use Paratone-N oil as you would for cryo, being careful to wick away as much oil from the loop as possible. At 4C the oil is so viscous as to form a solid mount. We have left protein crystals in loops for weeks without loss of diffraction. However, with our FRE we get only 10-20 minutes total exposure before the crystal is lost due to radiation damage. So data collection will require multi-crystal averaging, which is not as bad as it sounds.
The suggestion of using sucrose as a cryo-protectant is an excellent one, it has worked for us where glycerol and PEGs did not. For a good comprehensive list of useful cryo-protectants I would recommend Stura & Vera's article: "Strategies for Protein Cryocrystallography" Cryst. Growth Des. (2014) as a starting point. Last, I would suggest not moving the crystals from solution to solution as you titrate in the cryo-buffers, but to slowly add the cryo to the crystal drop to bring it up to the required concentration of cryo-protectant. Do not forget to keep any ligand you use in the cryo-buffer, otherwise it may be soaked out of the crystal. Finally, looping into Paratone-N oil will always help in the flash-freezing process, and improve its reproducibility. Best regards, Mark -- Yours sincerely, Mark A. White, Ph.D. Associate Professor of Biochemistry and Molecular Biology, Manager, Sealy Center for Structural Biology and Molecular Biophysics Macromolecular X-ray Laboratory, Basic Science Building, Room 6.630 University of Texas Medical Branch Galveston, TX 77555-0647 Tel. (409) 747-4747 Fax. (409) 747-1404 mailto://mawh...@utmb.edu http://xray.utmb.edu QQ: "Research: If we got it right the first time it would just be called search." - A. Garcia -----Original Message----- From: Tim Gruene <t...@shelx.uni-ac.gwdg.de> Reply-to: Tim Gruene <t...@shelx.uni-ac.gwdg.de> To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] cryo protection for low salt crystallization at 4 degrees Date: Mon, 2 Mar 2015 20:32:47 +0100 -----BEGIN PGP SIGNED MESSAGE----- Hash: SHA1 Dear Ursula, unless it is too long a list, maybe you could list the cryo-protectants you have used so far? Did you try sugars (e.g. glucose), or Na Malonate, or Butanediol (either 2,5 or 1,6, I don't remember exactly)? You could also increase the protein or NaCl concentration a little and decrease the temperature to 10 degrees, say, for crystallisation, then to 4 degrees to harvesting. But first I would try as Jacob suggested, collect at room temperature in a capillary. It's a very gentle treatment for crystals (except for the X-rays ;-) ). When you transfer the crystal with a pipet and the capillary, it is never exposed to air and you can work in the cold room to stabilise the temperature. Regards, Tim On 03/02/2015 07:49 PM, Ursula Schulze-Gahmen wrote: > I know there was jut recently a discussion about cryoconditions > for crystals, but I am still hoping for some new ideas for my > crystals that grow from HEPES buffer pH 7.3, 0.2 M NaCl by slowly > lowering the temperature from 20 to 4 degrees. > > These crystals are easy to grow but extremely sensitive to > temperature change, and any of the usual cryo reagents tested so > far simply dissolve the crystals. I am n ot sure how I could > counteract the solubilizing effect of glycerol or ethylen glycol, > since there is no precipitant concentration that I could increase > to stabilize the crystals. Paratone didn't work either so far. > > Any other ideas for these low salt crystals? > > Ursula > - -- - -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A -----BEGIN PGP SIGNATURE----- Version: GnuPG v1 iD8DBQFU9LrfUxlJ7aRr7hoRAg0UAJ0fZJScgnGNWNOAHS00ec4f6kJmIACffBQB Vm/WTDjFC5J+OKtGPfEYbpo= =lw4r -----END PGP SIGNATURE-----
