[ccp4bb] Crystals grown from high ammonium sulphate
Hi everyone, We got crystals that grew in ~3.2M ammonium sulphate and some tris-buffer at 18dgC. Unfortunately the crystals take a while to grow (~4-5 weeks) and so far we only have 4-5 xtals. I tried to freeze the crystals, but as soon as I broke though the skin of the drop the ammonium sulphate started to crystallise. I got the crystals out, froze them using sort of an artifical mother liquor with sodium malonate as cryo and tested the diffraction. The freezing seems to be OK and it is definitely a protein crystal. The crystal suffered when the ammonium sulphate in the drop started to crystallise, but didn't seem to deteriorate anymore in the cryo. Well the corners had already more or less disappeared by the time I got them out of the drop... Since we only have a few xtals at the moment and I can't try out a lot of things, I was wondering if anyone has experienced and solved a similar problem? My freezing attempt so far was in an airconditioned room with 18dgC. I thought about higher humidity and temperature in the room, and/or adding the cryo directly to the drop Any ideas are very much appreciated! Sabine -- Dr. Sabine Schneider Ludwig-Maximilians-University Department of Chemistry and Pharmacy Butenandtstrasse 5-13, Building F 81377 Munich Germany Phone: +49 (0)89 2180 77846 Fax: +49 (0)89 2180 77756 http://www.carellgroup.de/
Re: [ccp4bb] Crystals grown from high ammonium sulphate
Hi Sabine, I had a similar problem years ago. Have you tried oils ? (mineral oil, paraffin oil, 50:50 mixtures of either with N-paratone) You can either 1. add a small amount (1ul or less) to the drop containing the crystals and mount from there or 2. if you are quick enough, transfer the crystal to a small drop of oil and mount it in the cryostream straight from the oil. HTH, Iain Sabine Schneider wrote: Hi everyone, We got crystals that grew in ~3.2M ammonium sulphate and some tris-buffer at 18dgC. Unfortunately the crystals take a while to grow (~4-5 weeks) and so far we only have 4-5 xtals. I tried to freeze the crystals, but as soon as I broke though the skin of the drop the ammonium sulphate started to crystallise. I got the crystals out, froze them using sort of an artifical mother liquor with sodium malonate as cryo and tested the diffraction. The freezing seems to be OK and it is definitely a protein crystal. The crystal suffered when the ammonium sulphate in the drop started to crystallise, but didn't seem to deteriorate anymore in the cryo. Well the corners had already more or less disappeared by the time I got them out of the drop... Since we only have a few xtals at the moment and I can't try out a lot of things, I was wondering if anyone has experienced and solved a similar problem? My freezing attempt so far was in an airconditioned room with 18dgC. I thought about higher humidity and temperature in the room, and/or adding the cryo directly to the drop Any ideas are very much appreciated! Sabine -- Dr. Sabine Schneider Ludwig-Maximilians-University Department of Chemistry and Pharmacy Butenandtstrasse 5-13, Building F 81377 Munich Germany Phone: +49 (0)89 2180 77846 Fax: +49 (0)89 2180 77756 http://www.carellgroup.de/
Re: [ccp4bb] Crystals grown from high ammonium sulphate
Generally crystals require a much higher precipitant concentration for nucleation than for growth and maybe even less for maintenance of their crystalline state. Therefore I would a) try micro seeding into less Ammonium Sulpahte b) reduce the ammonium sulfate concentration in the drop by adding ammonium sulphate free mother liquor to the reservoir (not the drop itself!) This would re-equilibrate the drop to lower a AS concentration. You could remove one crytal and try to find the lowest possible AS concentration with it separately. Tim -- Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A On Tue, 4 Nov 2008, Sabine Schneider wrote: Hi everyone, We got crystals that grew in ~3.2M ammonium sulphate and some tris-buffer at 18dgC. Unfortunately the crystals take a while to grow (~4-5 weeks) and so far we only have 4-5 xtals. I tried to freeze the crystals, but as soon as I broke though the skin of the drop the ammonium sulphate started to crystallise. I got the crystals out, froze them using sort of an artifical mother liquor with sodium malonate as cryo and tested the diffraction. The freezing seems to be OK and it is definitely a protein crystal. The crystal suffered when the ammonium sulphate in the drop started to crystallise, but didn't seem to deteriorate anymore in the cryo. Well the corners had already more or less disappeared by the time I got them out of the drop... Since we only have a few xtals at the moment and I can't try out a lot of things, I was wondering if anyone has experienced and solved a similar problem? My freezing attempt so far was in an airconditioned room with 18dgC. I thought about higher humidity and temperature in the room, and/or adding the cryo directly to the drop Any ideas are very much appreciated! Sabine -- Dr. Sabine Schneider Ludwig-Maximilians-University Department of Chemistry and Pharmacy Butenandtstrasse 5-13, Building F 81377 Munich Germany Phone: +49 (0)89 2180 77846 Fax: +49 (0)89 2180 77756 http://www.carellgroup.de/
Re: [ccp4bb] Crystals grown from high ammonium sulphate
You can try microseeding. It is really suitable for crystallization in high ammonium sulphate. I got a similar problem as well, before. Microseeding can supply nucleus. You can get more crystals and spend less time. And for the salt crystallization, I think if you transfer quickly it can be fine. Or a high humidity might be useful. Liang On Tue, 4 Nov 2008 19:34:20 +0100, Sabine Schneider wrote Hi everyone, We got crystals that grew in ~3.2M ammonium sulphate and some tris-buffer at 18dgC. Unfortunately the crystals take a while to grow (~4-5 weeks) and so far we only have 4-5 xtals. I tried to freeze the crystals, but as soon as I broke though the skin of the drop the ammonium sulphate started to crystallise. I got the crystals out, froze them using sort of an artifical mother liquor with sodium malonate as cryo and tested the diffraction. The freezing seems to be OK and it is definitely a protein crystal. The crystal suffered when the ammonium sulphate in the drop started to crystallise, but didn't seem to deteriorate anymore in the cryo. Well the corners had already more or less disappeared by the time I got them out of the drop... Since we only have a few xtals at the moment and I can't try out a lot of things, I was wondering if anyone has experienced and solved a similar problem? My freezing attempt so far was in an airconditioned room with 18dgC. I thought about higher humidity and temperature in the room, and/or adding the cryo directly to the drop Any ideas are very much appreciated! Sabine -- Dr. Sabine Schneider Ludwig-Maximilians-University Department of Chemistry and Pharmacy Butenandtstrasse 5-13, Building F 81377 Munich Germany Phone: +49 (0)89 2180 77846 Fax: +49 (0)89 2180 77756 http://www.carellgroup.de/ -- Open WebMail Project (http://openwebmail.org)
Re: [ccp4bb] Crystals grown from high ammonium sulphate
Dear Sabine, I recently dealt with a very similar situation as follows: -I ended up growing the crystals in 4+4 uL drops. Skin formation tends to be less of a problem in larger drops. This kind of experimentation is of course only possible if protein production is not a limiting factor. -For crystal manipulation, I used to add 10-20 uL of the reservoir solution directly to the drop. In this way I could easily manipulate the 5-6 crystals that grew per drop within 5 minutes without any noticeable effects on the crystals. -I found out that cryo-cooling the crystals by plunging them into liquid nitrogen straight out the drop was the only way to effectively cryo-cool such crystals. In fact the crystal condition was very similar to yours (3.2 M AS, 20 mM potassium phosphate pH 6.0). I cite the most relevant paragraph from our paper (Kyndt et al. Biochemistry. 2007 46(1):95-105.): To prepare crystals for data collection under cryogenic conditions (100 K), crystals were flash-cooled by plunging them directly from their native drops into liquid nitrogen. A series of cryocooling conditions using a variety of cryoprotecting reagents such as glycerol, sucrose, PEG 400, and paratone indicated that only crystals flash-cooled by plunging them directly from their native drops into liquid nitrogen produced diffraction of acceptable quality. Best wishes Savvas Savvas Savvides L-ProBE, Unit for Structural Biology Ghent University K.L. Ledeganckstraat 35 9000 Ghent, BELGIUM office: +32-(0)9-264.51.24 ; mobile: +32-(0)472-92.85.19 Email: [EMAIL PROTECTED] http://www.eiwitbiochemie.ugent.be/units_en/structbio_en.html -Original Message- From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Sabine Schneider Sent: Tuesday, November 04, 2008 7:34 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Crystals grown from high ammonium sulphate Hi everyone, We got crystals that grew in ~3.2M ammonium sulphate and some tris-buffer at 18dgC. Unfortunately the crystals take a while to grow (~4-5 weeks) and so far we only have 4-5 xtals. I tried to freeze the crystals, but as soon as I broke though the skin of the drop the ammonium sulphate started to crystallise. I got the crystals out, froze them using sort of an artifical mother liquor with sodium malonate as cryo and tested the diffraction. The freezing seems to be OK and it is definitely a protein crystal. The crystal suffered when the ammonium sulphate in the drop started to crystallise, but didn't seem to deteriorate anymore in the cryo. Well the corners had already more or less disappeared by the time I got them out of the drop... Since we only have a few xtals at the moment and I can't try out a lot of things, I was wondering if anyone has experienced and solved a similar problem? My freezing attempt so far was in an airconditioned room with 18dgC. I thought about higher humidity and temperature in the room, and/or adding the cryo directly to the drop Any ideas are very much appreciated! Sabine -- Dr. Sabine Schneider Ludwig-Maximilians-University Department of Chemistry and Pharmacy Butenandtstrasse 5-13, Building F 81377 Munich Germany Phone: +49 (0)89 2180 77846 Fax: +49 (0)89 2180 77756 http://www.carellgroup.de/ E-mail message checked by Spyware Doctor (6.0.0.386) Database version: 5.11050 http://www.pctools.com/en/spyware-doctor-antivirus/
Re: [ccp4bb] Crystals grown from high ammonium sulphate
also, don't forget to measure at least one crystal at RT, probably most easily done in a Mitegen loop-and-sleeve - to have an idea of the intrinsic diffraction quality of the crystals without freezing. Mark Quoting Savvas Savvides [EMAIL PROTECTED]: Dear Sabine, I recently dealt with a very similar situation as follows: -I ended up growing the crystals in 4+4 uL drops. Skin formation tends to be less of a problem in larger drops. This kind of experimentation is of course only possible if protein production is not a limiting factor. -For crystal manipulation, I used to add 10-20 uL of the reservoir solution directly to the drop. In this way I could easily manipulate the 5-6 crystals that grew per drop within 5 minutes without any noticeable effects on the crystals. -I found out that cryo-cooling the crystals by plunging them into liquid nitrogen straight out the drop was the only way to effectively cryo-cool such crystals. In fact the crystal condition was very similar to yours (3.2 M AS, 20 mM potassium phosphate pH 6.0). I cite the most relevant paragraph from our paper (Kyndt et al. Biochemistry. 2007 46(1):95-105.): To prepare crystals for data collection under cryogenic conditions (100 K), crystals were flash-cooled by plunging them directly from their native drops into liquid nitrogen. A series of cryocooling conditions using a variety of cryoprotecting reagents such as glycerol, sucrose, PEG 400, and paratone indicated that only crystals flash-cooled by plunging them directly from their native drops into liquid nitrogen produced diffraction of acceptable quality. Best wishes Savvas Savvas Savvides L-ProBE, Unit for Structural Biology Ghent University K.L. Ledeganckstraat 35 9000 Ghent, BELGIUM office: +32-(0)9-264.51.24 ; mobile: +32-(0)472-92.85.19 Email: [EMAIL PROTECTED] http://www.eiwitbiochemie.ugent.be/units_en/structbio_en.html -Original Message- From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Sabine Schneider Sent: Tuesday, November 04, 2008 7:34 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Crystals grown from high ammonium sulphate Hi everyone, We got crystals that grew in ~3.2M ammonium sulphate and some tris-buffer at 18dgC. Unfortunately the crystals take a while to grow (~4-5 weeks) and so far we only have 4-5 xtals. I tried to freeze the crystals, but as soon as I broke though the skin of the drop the ammonium sulphate started to crystallise. I got the crystals out, froze them using sort of an artifical mother liquor with sodium malonate as cryo and tested the diffraction. The freezing seems to be OK and it is definitely a protein crystal. The crystal suffered when the ammonium sulphate in the drop started to crystallise, but didn't seem to deteriorate anymore in the cryo. Well the corners had already more or less disappeared by the time I got them out of the drop... Since we only have a few xtals at the moment and I can't try out a lot of things, I was wondering if anyone has experienced and solved a similar problem? My freezing attempt so far was in an airconditioned room with 18dgC. I thought about higher humidity and temperature in the room, and/or adding the cryo directly to the drop Any ideas are very much appreciated! Sabine -- Dr. Sabine Schneider Ludwig-Maximilians-University Department of Chemistry and Pharmacy Butenandtstrasse 5-13, Building F 81377 Munich Germany Phone: +49 (0)89 2180 77846 Fax: +49 (0)89 2180 77756 http://www.carellgroup.de/ E-mail message checked by Spyware Doctor (6.0.0.386) Database version: 5.11050 http://www.pctools.com/en/spyware-doctor-antivirus/
