[ccp4bb] crystal bent once open cover slip
Hi Folks, We have some membrane protein crystals that are grown in 30%PEG400, 0.1M Na Citrate pH 4.5, 0.1M LiCl. The protein is purified in DDM. The crystals are long rods and grown under room temperature in a hanging drop set up. But once we open the cover slip, we see the rods start to break and bend in a few seconds. Since it is in high PEG400, we just directly freeze the crystals. The diffraction only goes to 10 Ang at synchrotron. Have anybody had similar problem before and any suggestions? Thanks a lot, Weikai
Re: [ccp4bb] crystal bent once open cover slip
You probably use hanging drops. It's the surface tension effect. Check if sitting drops are better. Maia Sitting drops weikai wrote: Hi Folks, We have some membrane protein crystals that are grown in 30%PEG400, 0.1M Na Citrate pH 4.5, 0.1M LiCl. The protein is purified in DDM. The crystals are long rods and grown under room temperature in a hanging drop set up. But once we open the cover slip, we see the rods start to break and bend in a few seconds. Since it is in high PEG400, we just directly freeze the crystals. The diffraction only goes to 10 Ang at synchrotron. Have anybody had similar problem before and any suggestions? Thanks a lot, Weikai
Re: [ccp4bb] crystal bent once open cover slip
you could try sitting drops. Perhaps you can fish a crystal quicker, before it degrades. In sitting drops the crystals may also be further away from the drop surface and take longer to degrade. Or quickly add mineral oil to cover the sitting drop and fish the crystals through the oil. If this is still too slow, you could try microbatch under oil and you should be able to fish the crystals without previous exposure to air. You may need to adapt the mother liquor somewhat. The mineral oil may not be compatible with your membrane protein and detergent, but without trying you will never know. Mark J van Raaij Laboratorio M-4 Dpto de Estructura de Macromoleculas Centro Nacional de Biotecnologia - CSIC c/Darwin 3, Campus Cantoblanco E-28049 Madrid, Spain tel. (+34) 91 585 4616 http://www.cnb.csic.es/content/research/macromolecular/mvraaij On 24 May 2011, at 19:19, weikai wrote: Hi Folks, We have some membrane protein crystals that are grown in 30%PEG400, 0.1M Na Citrate pH 4.5, 0.1M LiCl. The protein is purified in DDM. The crystals are long rods and grown under room temperature in a hanging drop set up. But once we open the cover slip, we see the rods start to break and bend in a few seconds. Since it is in high PEG400, we just directly freeze the crystals. The diffraction only goes to 10 Ang at synchrotron. Have anybody had similar problem before and any suggestions? Thanks a lot, Weikai
Re: [ccp4bb] crystal bent once open cover slip
Weikai, What you might be experiencing is a detergent effect, i.e., you are near a detergent-dependent crystallization boundary. We have been hit with this many times. Under vapor diffusion conditions, sitting or hanging drop, the protein-detergent complex crystallizes and the free/ bound detergent reaches an equilibrium, but when you open the well, the drop begins to dry out. Hence, the detergent concentration increases, which dissolves/cracks the crystals. We also found that this also happened when our stabilization/freezing buffers had too high a detergent concentration (lower didn't hurt nearly as badly). Anecdotally, we have experienced that too high detergent concentrations inhibited crystallization, perhaps by having too high a concentration of free detergent micelles, which may interfere with the crystallization of the protein-detergent complex (there is alway detergent exchange between the solvent and the crystal). The way we solved the problem is by setting up the crystals at lower initial detergent concentrations. Note that as the concentrations of salt and PEG increases (like during crystallization), the detergent CMC decreases, even for non-ionic detergents. Therefore, by dropping the detergent concentration, often to just below the apparent CMC, we could grow nice stable crystals. Hope this helps, Michael R. Michael Garavito, Ph.D. Professor of Biochemistry Molecular Biology 513 Biochemistry Bldg. Michigan State University East Lansing, MI 48824-1319 Office: (517) 355-9724 Lab: (517) 353-9125 FAX: (517) 353-9334Email: rmgarav...@gmail.com On May 24, 2011, at 1:19 PM, weikai wrote: Hi Folks, We have some membrane protein crystals that are grown in 30%PEG400, 0.1M Na Citrate pH 4.5, 0.1M LiCl. The protein is purified in DDM. The crystals are long rods and grown under room temperature in a hanging drop set up. But once we open the cover slip, we see the rods start to break and bend in a few seconds. Since it is in high PEG400, we just directly freeze the crystals. The diffraction only goes to 10 Ang at synchrotron. Have anybody had similar problem before and any suggestions? Thanks a lot, Weikai
