[ccp4bb] 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
Re: [ccp4bb] 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: a.gus...@ntu.edu.sg | Web: www.ntu.edu.sg 2011/7/18 F. Timur Senguen ftseng...@gmail.com 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
Re: [ccp4bb] 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 sa...@embl-hamburg.de An: F. Timur Senguen ftseng...@gmail.com CC: CCP4BB@JISCMAIL.AC.UK 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
Re: [ccp4bb] 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 er...@pobox.com 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: j-kell...@northwestern.edu ***
Re: [ccp4bb] unusual sighting of a crystal structure
Just in case anyone want to see it IRL http://www.youtube.com/watch?v=4sYSyuuLk5ghd=1t=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: j-kell...@northwestern.edu ***
Re: [ccp4bb] 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
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[ccp4bb] 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
Re: [ccp4bb] 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
[ccp4bb] off-topic: LCD monitors
Pardon the off-topic query, but I would like to get some feedback about any personal preference for 3D LCD monitors. I am trying to decide between the following 3 monitors: Samsung 2233RZ 1680 x 1050 2D and 3d Widescreen LCD Monitor Asus VG236H 23 2ms 1920x1080 Full HD 120Hz 3D multimedia Height Swivel Adjustable WideScreen LCD Alienware OptX AW2310 23 3D Full HD Widescreen Monitor The monitor will display off of a Mac Pro Two Quad-Core Intel Xeon computer running both Apple and Linux OS. Any input will be much appreciated. Padmaja
Re: [ccp4bb] off-topic: LCD monitors
If you aren't aware of this, the use of 120 Hz LCDs to display stereoscopic 3D using Nvidia 3D Vision on Apple's Operating system(s) is/are not supported: http://www.nvidia.com/object/3d-vision-pro-requirements.html However, it will work flawlessly on Linux (or Windows if you're in the mood for that). I run the Alienware OptX AW2310 on two of our 3D Linux workstations and it looks spectacular. Make sure that you have a Quadro FX Nvidia video card with 3-pin stereo (3-pin stereo connector required for Linux, it will work without one in Windows through USB) output and not just a normal GeForce Nvidia card or you won't be able to run stereoscopic 3D in Linux. One option that supports both Mac and Linux are the Zalman brand 3D monitors. Some people like them, some people don't. Unless Zalman significantly improved the technology on their monitors (ie: the right eye can see odd numbered rows of pixels and the left eye can see even numbered rows of pixels) you lose a significant amount of resolution from displaying 3D on these. Due to half the pixels being drawn to each eye, the display on the 3D Vision system using a 120 Hz monitor will look crisper and higher quality than the equivalent Zalman monitor when displaying stereoscopic 3D. Cheers, Jim On Mon, Jul 18, 2011 at 7:12 PM, Padmaja Mehta-D'souza padmaja-mehta-dso...@omrf.org wrote: Pardon the off-topic query, but I would like to get some feedback about any personal preference for 3D LCD monitors. I am trying to decide between the following 3 monitors: *Samsung 2233RZ* 1680 x 1050 2D and 3d Widescreen LCD Monitor Asus VG236H 23 2ms 1920x1080 Full HD 120Hz 3D multimedia Height Swivel Adjustable WideScreen LCD Alienware OptX AW2310 23 3D Full HD Widescreen Monitor The monitor will display off of a Mac Pro Two Quad-Core Intel Xeon computer running both Apple and Linux OS. Any input will be much appreciated. Padmaja -- Jim Fairman, Ph D. Post-Doctoral Fellow National Institutes of Health - NIDDK The Buchanan Lab http://www-mslmb.niddk.nih.gov/buchanan/index.html Lab: 1-301-594-9229 E-mail: fairman@gmail.com james.fair...@nih.gov
[ccp4bb] Off Topic: How to delete loops from a protein
Hi all I wrote last time but got only one feedback. I know some of you guys must have this experience that how to delete loops from the protein. Please help me with suggestions. I am working with a human protein which have around 20% sequence identity with the other proteins of the same family. Structure of some of the proteins from this family have been solved. All the solved structures have around 20% identity with my protein. I am trying to crystallize the protein but it looks like very hard to get crystal. I have tried different N and C terminally truncated constructs for crystallization but no crystal. My feeling is that probably there is some flexible loops with in the protein which limiting the crystallization. So I want to delete the loops with in the protein (not to truncate in the terminal, I already have done this). I am not asking suggestion about how to delete the loop rather how to decide where the loop is. I am not sure how much it will be helpful to get a homology model of such a protein having low sequence identity. Is there any strategy to decide where the loop could be? Does anybody know any established/ rational method to do that. Waiting for your suggestions Obayed Ullah
[ccp4bb] Off Topic: How to delete loops from a protein
Hi all I wrote last time but got only one feedback. I know some of you guys must have this experience that how to delete loops from the protein. Please help me with suggestions. I am working with a human protein which have around 20% sequence identity with the other proteins of the same family. Structure of some of the proteins from this family have been solved. All the solved structures have around 20% identity with my protein. I am trying to crystallize the protein but it looks like very hard to get crystal. I have tried different N and C terminally truncated constructs for crystallization but no crystal. My feeling is that probably there is some flexible loops with in the protein which limiting the crystallization. So I want to delete the loops with in the protein (not to truncate in the terminal, I already have done this). I am not asking suggestion about how to delete the loop rather how to decide where the loop is. I am not sure how much it will be helpful to get a homology model of such a protein having low sequence identity. Is there any strategy to decide where the loop could be? Does anybody know any established/ rational method to do that. Waiting for your suggestions Obayed Ullah
[ccp4bb] Off Topic: How to delete loops from a protein
Hi all I wrote last time but got only one feedback. I know some of you guys must have this experience that how to delete loops from the protein. Please help me with suggestions. I am working with a human protein which have around 20% sequence identity with the other proteins of the same family. Structure of some of the proteins from this family have been solved. All the solved structures have around 20% identity with my protein. I am trying to crystallize the protein but it looks like very hard to get crystal. I have tried different N and C terminally truncated constructs for crystallization but no crystal. My feeling is that probably there is some flexible loops with in the protein which limiting the crystallization. So I want to delete the loops with in the protein (not to truncate in the terminal, I already have done this). I am not asking suggestion about how to delete the loop rather how to decide where the loop is. I am not sure how much it will be helpful to get a homology model of such a protein having low sequence identity. Is there any strategy to decide where the loop could be? Does anybody know any established/ rational method to do that. Waiting for your suggestions Obayed Ullah
[ccp4bb] Off Topic: How to delete loops from a protein
Hi all I wrote last time but got only one feedback. I know some of you guys must have this experience that how to delete loops from the protein. Please help me with suggestions. I am working with a human protein which have around 20% sequence identity with the other proteins of the same family. Structure of some of the proteins from this family have been solved. All the solved structures have around 20% identity with my protein. I am trying to crystallize the protein but it looks like very hard to get crystal. I have tried different N and C terminally truncated constructs for crystallization but no crystal. My feeling is that probably there is some flexible loops with in the protein which limiting the crystallization. So I want to delete the loops with in the protein (not to truncate in the terminal, I already have done this). I am not asking suggestion about how to delete the loop rather how to decide where the loop is. I am not sure how much it will be helpful to get a homology model of such a protein having low sequence identity. Is there any strategy to decide where the loop could be? Does anybody know any established/ rational method to do that. Waiting for your suggestions Obayed Ullah
Re: [ccp4bb] Off Topic: How to delete loops from a protein
You could run your sequence through this web server first: http://ffas.burnham.org/XtalPred-cgi/xtal.pl Then regarding your loops you could use MUSTANG (or any other 3D alignment tool which generates a superimposed ensemble of structures) http://www.ncbi.nlm.nih.gov/pubmed/16736488 to identify with your other homologs which regions should be trimmed off. Then you turn to your favorite site directed mutagenesis kit and chop off the loops. How big is your protein ? Have you tried different expression systems ? Good luck ! Jürgen On Jul 18, 2011, at 9:01 PM, Obayed Ullah wrote: Hi all I wrote last time but got only one feedback. I know some of you guys must have this experience that how to delete loops from the protein. Please help me with suggestions. I am working with a human protein which have around 20% sequence identity with the other proteins of the same family. Structure of some of the proteins from this family have been solved. All the solved structures have around 20% identity with my protein. I am trying to crystallize the protein but it looks like very hard to get crystal. I have tried different N and C terminally truncated constructs for crystallization but no crystal. My feeling is that probably there is some flexible loops with in the protein which limiting the crystallization. So I want to delete the loops with in the protein (not to truncate in the terminal, I already have done this). I am not asking suggestion about how to delete the loop rather how to decide where the loop is. I am not sure how much it will be helpful to get a homology model of such a protein having low sequence identity. Is there any strategy to decide where the loop could be? Does anybody know any established/ rational method to do that. Waiting for your suggestions Obayed Ullah .. 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/
Re: [ccp4bb] Off Topic: How to delete loops from a protein
Hi Obayed, you could give in situ protolysis a try. This is where you add a bit of protease along with you target protein to the crystallization drop. It has been quite successful for the folks at the SGC. Here are the relevant references: Dong A, et al. In situ proteolysis for protein crystallization and structure determination. Nat Methods. 2007 Dec;4(12):1019-21.PMID: 17982461. (http://www.ncbi.nlm.nih.gov/pubmed/17982461) Wernimont A, Edwards A. In situ proteolysis to generate crystals for structure determination: an update. PLoS One. 2009;4(4):e5094. PMID: 19352432. (http://www.ncbi.nlm.nih.gov/pubmed/19352432) good luck, Eric Eric T. Larson, PhD Biomolecular Structure Center Department of Biochemistry Box 357742 University of Washington Seattle, WA 98195 email: larso...@u.washington.edu On Mon, 18 Jul 2011, Obayed Ullah wrote: Hi all I wrote last time but got only one feedback. I know some of you guys must have this experience that how to delete loops from the protein. Please help me with suggestions. I am working with a human protein which have around 20% sequence identity with the other proteins of the same family. Structure of some of the proteins from this family have been solved. All the solved structures have around 20% identity with my protein. I am trying to crystallize the protein but it looks like very hard to get crystal. I have tried different N and C terminally truncated constructs for crystallization but no crystal. My feeling is that probably there is some flexible loops with in the protein which limiting the crystallization. So I want to delete the loops with in the protein (not to truncate in the terminal, I already have done this). I am not asking suggestion about how to delete the loop rather how to decide where the loop is. I am not sure how much it will be helpful to get a homology model of such a protein having low sequence identity. Is there any strategy to decide where the loop could be? Does anybody know any established/ rational method to do that. Waiting for your suggestions Obayed Ullah
Re: [ccp4bb] Off Topic: How to delete loops from a protein
Hi Obayed, If I understood your question well, you are looking for something called secondary structure prediction. I googled these keywords and found this server: http://bioinf.cs.ucl.ac.uk/psipred/ You may find other interesting servers on the web and some literature comparing them. I think such methods need only the sequence of your protein to predict its secondary structures. Hope this helps, Francois. On 07/19/2011 02:14 PM, Eric Larson wrote: Hi Obayed, you could give in situ protolysis a try. This is where you add a bit of protease along with you target protein to the crystallization drop. It has been quite successful for the folks at the SGC. Here are the relevant references: Dong A, et al. In situ proteolysis for protein crystallization and structure determination. Nat Methods. 2007 Dec;4(12):1019-21.PMID: 17982461. (http://www.ncbi.nlm.nih.gov/pubmed/17982461) Wernimont A, Edwards A. In situ proteolysis to generate crystals for structure determination: an update. PLoS One. 2009;4(4):e5094. PMID: 19352432. (http://www.ncbi.nlm.nih.gov/pubmed/19352432) good luck, Eric Eric T. Larson, PhD Biomolecular Structure Center Department of Biochemistry Box 357742 University of Washington Seattle, WA 98195 email: larso...@u.washington.edu On Mon, 18 Jul 2011, Obayed Ullah wrote: Hi all I wrote last time but got only one feedback. I know some of you guys must have this experience that how to delete loops from the protein. Please help me with suggestions. I am working with a human protein which have around 20% sequence identity with the other proteins of the same family. Structure of some of the proteins from this family have been solved. All the solved structures have around 20% identity with my protein. I am trying to crystallize the protein but it looks like very hard to get crystal. I have tried different N and C terminally truncated constructs for crystallization but no crystal. My feeling is that probably there is some flexible loops with in the protein which limiting the crystallization. So I want to delete the loops with in the protein (not to truncate in the terminal, I already have done this). I am not asking suggestion about how to delete the loop rather how to decide where the loop is. I am not sure how much it will be helpful to get a homology model of such a protein having low sequence identity. Is there any strategy to decide where the loop could be? Does anybody know any established/ rational method to do that. Waiting for your suggestions Obayed Ullah
