Re: [ccp4bb] Tough 'shell' on disturbed drop
Hi, You're working with a very 'rich' crystallization condition. It probably was supersaturated or close to super-saturated with respect to something, and that something crashed out on the surface (where liquid contacted air) forming a crust. Your loop (while perfectly clean) can also be the source of nucleation. Just a mere act of opening a drop can cause phenomena like this one. How old was your drop (older partially evaporated drops tend to do this more often)? Sometimes it's possible to slow this crust formation down by coating the drop with a film of oil, as soon as you open the reservoir. I would also recommend avoiding complex buffers like that one unless there's literally no other way to grow your crystals - there's a lot of inherent trouble (especially with phosphate). Artem On Thu, Nov 25, 2010 at 8:45 AM, Rick mbp08...@sheffield.ac.uk wrote: Dear CCP4 I looped a v.thin rod emerging from a cluster of v.thin rods that grew in 29%PEG1500 and 0.1M SPG buffer at pH7.5 (succinic acid, sodium dihydrogen orthophospate and glycine). The loop i used had been washed more than 10 times with deionised water (so assumed as 'clean'). The crystals had grown at 17degreesC, and looped out probably just below room temperature (~20-23 degreesC). When transferred to 5% glycerol cryo-buffer the crystal disintegrated (maybe due to glycerol being an unfavourable addition to the mother-liquor). When i looked back at the original cluster-containing drop, a very tough shell had formed over the surface of the drop, from which chunks could be dug out...the nearest analogy is maybe like when you blow-torch sugar on top of creme brulee, and have to crack it with your spoon. The crystals within had also disintegrated. Any clues to what might have caused this very tough shell to form, and maybe how to deal with it? Much appreciated Rick Salmon
Re: [ccp4bb] Tough 'shell' on disturbed drop
The shell may be denatured protein. Remove the protein from the experiment and the problem will likely go away. On 11/25/10 09:45, Rick wrote: Dear CCP4 I looped a v.thin rod emerging from a cluster of v.thin rods that grew in 29%PEG1500 and 0.1M SPG buffer at pH7.5 (succinic acid, sodium dihydrogen orthophospate and glycine). The loop i used had been washed more than 10 times with deionised water (so assumed as 'clean'). The crystals had grown at 17degreesC, and looped out probably just below room temperature (~20-23 degreesC). When transferred to 5% glycerol cryo-buffer the crystal disintegrated (maybe due to glycerol being an unfavourable addition to the mother-liquor). When i looked back at the original cluster-containing drop, a very tough shell had formed over the surface of the drop, from which chunks could be dug out...the nearest analogy is maybe like when you blow-torch sugar on top of creme brulee, and have to crack it with your spoon. The crystals within had also disintegrated. Any clues to what might have caused this very tough shell to form, and maybe how to deal with it? Much appreciated Rick Salmon -- === All Things Serve the Beam === David J. Schuller modern man in a post-modern world MacCHESS, Cornell University schul...@cornell.edu
Re: [ccp4bb] Tough 'shell' on disturbed drop
Hi, In our hands, the crystallisation droplets of glycosomal pyruvate phosphate dikinase had a 'skin' of what I thought was denatured protein at the surface of every crystallisation droplet. We had to learn to use the crystal microtools (such as a microknife, or a micro-needle can't remember what we have - sold by Hampton Research and I do not own shares in this company) to be able to cut this skin and drag it to the side of the droplet before being able to suck out the crystals. A bit like dissection under the binoculars. Fred. Message du 25/11/10 15:56 De : Rick A : CCP4BB@JISCMAIL.AC.UK Copie à : Objet : [ccp4bb] Tough 'shell' on disturbed drop Dear CCP4 I looped a v.thin rod emerging from a cluster of v.thin rods that grew in 29%PEG1500 and 0.1M SPG buffer at pH7.5 (succinic acid, sodium dihydrogen orthophospate and glycine). The loop i used had been washed more than 10 times with deionised water (so assumed as 'clean'). The crystals had grown at 17degreesC, and looped out probably just below room temperature (~20-23 degreesC). When transferred to 5% glycerol cryo-buffer the crystal disintegrated (maybe due to glycerol being an unfavourable addition to the mother-liquor). When i looked back at the original cluster-containing drop, a very tough shell had formed over the surface of the drop, from which chunks could be dug out...the nearest analogy is maybe like when you blow-torch sugar on top of creme brulee, and have to crack it with your spoon. The crystals within had also disintegrated. Any clues to what might have caused this very tough shell to form, and maybe how to deal with it? Much appreciated Rick Salmon
Re: [ccp4bb] Tough 'shell' on disturbed drop
If it's on a glass coverslip, another good trick is to (carefully) cut through the skin around the crystal with a razor blade. With some practice, one manages not to get the crystal entangled in the skin. On Thu, 2010-11-25 at 16:03 +, Frederic VELLIEUX wrote: Hi, In our hands, the crystallisation droplets of glycosomal pyruvate phosphate dikinase had a 'skin' of what I thought was denatured protein at the surface of every crystallisation droplet. We had to learn to use the crystal microtools (such as a microknife, or a micro-needle can't remember what we have - sold by Hampton Research and I do not own shares in this company) to be able to cut this skin and drag it to the side of the droplet before being able to suck out the crystals. A bit like dissection under the binoculars. Fred. Message du 25/11/10 15:56 De : Rick A : CCP4BB@JISCMAIL.AC.UK Copie à : Objet : [ccp4bb] Tough 'shell' on disturbed drop Dear CCP4 I looped a v.thin rod emerging from a cluster of v.thin rods that grew in 29%PEG1500 and 0.1M SPG buffer at pH7.5 (succinic acid, sodium dihydrogen orthophospate and glycine). The loop i used had been washed more than 10 times with deionised water (so assumed as 'clean'). The crystals had grown at 17degreesC, and looped out probably just below room temperature (~20-23 degreesC). When transferred to 5% glycerol cryo-buffer the crystal disintegrated (maybe due to glycerol being an unfavourable addition to the mother-liquor). When i looked back at the original cluster-containing drop, a very tough shell had formed over the surface of the drop, from which chunks could be dug out...the nearest analogy is maybe like when you blow-torch sugar on top of creme brulee, and have to crack it with your spoon. The crystals within had also disintegrated. Any clues to what might have caused this very tough shell to form, and maybe how to deal with it? Much appreciated Rick Salmon
Re: [ccp4bb] Tough 'shell' on disturbed drop
Often you can also avoid this skin formation by adding a bit of your reservoir solution first to make it a larger droplet. Then the microtools as mentioned earlier or just two loops Jürgen .. Jürgen Bosch Johns Hopkins Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Phone: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-3655 http://web.mac.com/bosch_lab/ On Nov 25, 2010, at 6:45, Rick mbp08...@sheffield.ac.uk wrote: Dear CCP4 I looped a v.thin rod emerging from a cluster of v.thin rods that grew in 29%PEG1500 and 0.1M SPG buffer at pH7.5 (succinic acid, sodium dihydrogen orthophospate and glycine). The loop i used had been washed more than 10 times with deionised water (so assumed as 'clean'). The crystals had grown at 17degreesC, and looped out probably just below room temperature (~20-23 degreesC). When transferred to 5% glycerol cryo-buffer the crystal disintegrated (maybe due to glycerol being an unfavourable addition to the mother-liquor). When i looked back at the original cluster-containing drop, a very tough shell had formed over the surface of the drop, from which chunks could be dug out...the nearest analogy is maybe like when you blow-torch sugar on top of creme brulee, and have to crack it with your spoon. The crystals within had also disintegrated. Any clues to what might have caused this very tough shell to form, and maybe how to deal with it? Much appreciated Rick Salmon
