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Aurélie Martinez.
Topics of the day: 1. Bypassing phase separation for nice crystals. (3) 2. unusual sighting of a crystal structure (2) 3. Off Topic: PDB validation server 4. un-subscribe 5. Data from old tapes (2) ---------------------------------------------------------------------- Date: Mon, 18 Jul 2011 09:52:31 -0400 From: "F. Timur Senguen" Subject: Bypassing phase separation for nice crystals. Hi everyone, I have been issues with a particular protein. I have been close for a while, but yet so far. Rather than going from a clear drop to crystal, my protein first undergoes phase separation (large oily drops) in which one phase contains most, if not all, of the protein. This phase separation occurs within a day of preparing the drop. A day after phase separation the oily phase is now a large disordered crystalline mass which does not diffract very well. I have tried changing buffer concentrations, precipitant amounts, ionic strengths and pH and in all cases this phenomenon is observed. I even screened protein concentrations to see if reducing protein concentration would prevent the phase separation. Is there any way to bypass this phase separation, which I think prevents me from obtaining nice crystals. Should I try detergents, chaotropes, or other additives? Thanks in advance. Timur -- F. Timur Senguen, Ph.D. Postdoctoral Research Fellow Boston Biomedical Research Institute 64 Grove Street, Watertown, MA 02472 USA ------------------------------ Date: Mon, 18 Jul 2011 22:30:04 +0800 From: Albert Guskov Subject: Re: Bypassing phase separation for nice crystals. Hi Timur, have you tried seeding from your microstalline stuff? Might be worth to try! Cheers, Albert Albert GUSKOV (Dr) | Research Fellow | Division of Structural & Computational Biology | Nanyang Technological University Proteos 7-01, Biopolis Drive 61, Singapore 138673 Tel: (65) 6586-9690 GMT+8h | Cell: (65) 8366-2779 | Email: [log in to unmask] | Web: www.ntu.edu.sg 2011/7/18 F. Timur Senguen > Hi everyone, > > I have been issues with a particular protein. I have been close for a > while, but yet so far. > > Rather than going from a clear drop to crystal, my protein first undergoes > phase separation (large oily drops) in which one phase contains most, if not > all, of the protein. This phase separation occurs within a day of preparing > the drop. A day after phase separation the oily phase is now a large > disordered crystalline mass which does not diffract very well. I have tried > changing buffer concentrations, precipitant amounts, ionic strengths and pH > and in all cases this phenomenon is observed. I even screened protein > concentrations to see if reducing protein concentration would prevent the > phase separation. > > Is there any way to bypass this phase separation, which I think prevents me > from obtaining nice crystals. Should I try detergents, chaotropes, or other > additives? > > Thanks in advance. > > Timur > > -- > F. Timur Senguen, Ph.D. > Postdoctoral Research Fellow > Boston Biomedical Research Institute > 64 Grove Street, > Watertown, > MA 02472 USA > > ------------------------------ Date: Mon, 18 Jul 2011 17:05:07 +0200 From: Florian Sauer Subject: Re: Bypassing phase separation for nice crystals. -------- Original-Nachricht -------- Betreff: Re: [ccp4bb] Bypassing phase separation for nice crystals. Datum: Mon, 18 Jul 2011 17:01:27 +0200 Von: Florian Sauer An: F. Timur Senguen CC: [log in to unmask] Dear Timur, one possibility to handle this problem can be the change from vapor (I assume this is what you do) to the free interface diffusion method. Phase separation often occurs if the protein is immediately exposed to the full precipitant concentration while it might not "escape" into its own phase if it gets slowly equilibrated. There are commercial setups available for this method but you can also do it in a normal vapor diffusion plate. To do so, just put the protein and precipitant drops next to each other and then link them through a thin liquid bridge. Requires some practice and works best with large drops but helped me in several similar cases. Good luck! Florian Am 18.07.11 15:52, schrieb F. Timur Senguen: > Hi everyone, > > I have been issues with a particular protein. I have been close for a > while, but yet so far. > > Rather than going from a clear drop to crystal, my protein first > undergoes phase separation (large oily drops) in which one phase > contains most, if not all, of the protein. This phase separation > occurs within a day of preparing the drop. A day after phase > separation the oily phase is now a large disordered crystalline mass > which does not diffract very well. I have tried changing buffer > concentrations, precipitant amounts, ionic strengths and pH and in all > cases this phenomenon is observed. I even screened protein > concentrations to see if reducing protein concentration would prevent > the phase separation. > > Is there any way to bypass this phase separation, which I think > prevents me from obtaining nice crystals. Should I try detergents, > chaotropes, or other additives? > > Thanks in advance. > > Timur > > -- > F. Timur Senguen, Ph.D. > Postdoctoral Research Fellow > Boston Biomedical Research Institute > 64 Grove Street, > Watertown, > MA 02472 USA > ------------------------------ Date: Mon, 18 Jul 2011 11:59:18 -0500 From: Jacob Keller Subject: Re: unusual sighting of a crystal structure Why don't they ask us first what would be scariest? We could really come up with some good stuff.... JPK On Sun, Jul 17, 2011 at 2:57 PM, Eric Bennett wrote: > > It would be even scarier if they used an NMR structure. > -Eric > > > On Jul 16, 2011, at 1:20 PM, Robbie Joosten wrote: > > Hi Artem, > > Thank for that nice example of a protein structure used to pimp a movie. > Ribbon representations are always the scariest. > > Cheers, > Robbie > > -- ******************************************* Jacob Pearson Keller Northwestern University Medical Scientist Training Program cel: 773.608.9185 email: [log in to unmask] ******************************************* ------------------------------ Date: Mon, 18 Jul 2011 12:03:54 -0500 From: Jacob Keller Subject: Re: unusual sighting of a crystal structure > Just in case anyone want to see it IRL > http://www.youtube.com/watch?v=4sYSyuuLk5g&hd=1&t=38s Wow! This movie is the perfect propaganda for immunology grants! JPK ******************************************* Jacob Pearson Keller Northwestern University Medical Scientist Training Program cel: 773.608.9185 email: [log in to unmask] ******************************************* ------------------------------ Date: Mon, 18 Jul 2011 18:06:09 +0100 From: Thomas Womack Subject: Re: Off Topic: PDB validation server On 8 Jul 2011, at 19:13, Katherine Sippel wrote: > I know that the PDB updated its validation server in May as described in their news link but it seemed to indicate an increase in output options rather than a change in criteria. Is anyone aware of what changes were made to the validation server in regards to the preferred geometrical and stereochemical features? As far as I can tell empirically, if I run the validation server today it complains about a) waters which make a perfectly good contact with a residue in a different ASU b) waters which make a perfectly good contact with metal ions or with other waters which themselves make a perfectly good contact with the protein. and this means it's really not much use for validation of large complicated proteins with hundreds of waters. Tom ------------------------------ Date: Mon, 18 Jul 2011 14:11:04 -0400 From: Szilvia Szep Subject: un-subscribe Please un-subscribe me from the list! Thank you! Szilvia Szep ------------------------------ Date: Mon, 18 Jul 2011 11:54:21 -0700 From: "Min, Xiaoshan" Subject: Data from old tapes Dear CCP4 community, We have a few datasets that reside in tapes (Sony QG-112M 8 mm tape and Maxell DDS-2 4 mm tape). I have been searching the internet for tape drives ( and cable) but haven't found anything. Does anyone know where we can purchase compatible tape drives for these lovely tapes? Or if you have a spare working set sitting in your graphic room and would like to sell them, that will be wonderful. Thanks. Xiaoshan Min. Ph.D. Molecular Structure Amgen San Francisco 1120 Veterans Blvd. South San Francisco, CA 94080 ------------------------------ Date: Mon, 18 Jul 2011 16:01:33 -0400 From: "Edward A. Berry" Subject: Re: Data from old tapes My exabyte eliant 8 mm tape just went on the blink, so I've sent it to Pacific Data (http://www.pacificdata.com/tape_drive.html) for repair. They also sell tape drives, but looks like mainly newer ones. Probably have some old ones from the repair business. Search ebay for exabyte or "dat tape" and buy a junker, pacific data can probably fix it for $200-300. If the tape happens to be vax backup format, there is a utility available for reading them on unix/linux. I also have a procedure for using unix with a tape drive to make tape images for the Simh vax emulator running on linux or windows to read, you can then FTP files from the emulator to linux. This preserves a little more of the metadata than unix vmsbackup utility eab Min, Xiaoshan wrote: > Dear CCP4 community, > > We have a few datasets that reside in tapes (Sony QG-112M 8 mm tape and > Maxell DDS-2 4 mm tape). I have been searching the internet for tape > drives ( and cable) but haven’t found anything. Does anyone know where > we can purchase compatible tape drives for these lovely tapes? Or if you > have a spare working set sitting in your graphic room and would like to > sell them, that will be wonderful. Thanks. > > Xiaoshan Min. Ph.D. > > Molecular Structure > > Amgen San Francisco > > 1120 Veterans Blvd. > > South San Francisco, CA 94080 > ------------------------------ End of CCP4BB Digest - 17 Jul 2011 to 18 Jul 2011 (#2011-199) ************************************************************* |