Re: [ccp4bb] brute force molecular replacement
Dear Jacob, Why is it called queen of spades? As it happened, I was reading Alexander Pushkin's Queen of Spades while writing Qs. The book touches upon the subject of how close you can get to win everything and still lose it all. I thought that this was relevant for a stochastic search, and so the program got baptised. Nicholas -- Dr Nicholas M. Glykos, Department of Molecular Biology and Genetics, Democritus University of Thrace, University Campus, 68100 Alexandroupolis, Greece, Fax +302551030613 Tel ++302551030620 (77620), http://www.mbg.duth.gr/~glykos/
Re: [ccp4bb] ccp4-6.1, imosflm, phaser
Hi Andreas,
Have you a project set up in CCP4i? Make sure that your CCP4i project
directory is set before you try to start Imosflm. When run through CCP4i,
Imosflm uses this directory to write it's output files.
You can set the project directory by clicking on the
DirectoriesProjectsDir button in the interface.
Luke
Hey all,
I have a questions regarding imosflm in ccp4-6.1 on RHEL 5.2. The
installation (after automated download) went well and all works, except:
imosflm from within ccp4i doesn't start and outputs the following error:
MOSDIR is
Error in startup script: can't create directory : no such file or
directory
while executing
file mkdir $env(MOSDIR)
invoked from within
if { ! [file exists $env(MOSDIR)] } {
file mkdir $env(MOSDIR)
} {
if { ! [file isdirectory $env(MOSDIR)] } {
puts stop here; the directory f...
(file
/csb/soft/Linux/src/ccp4-6.1.0/ccp4-6.1.0/ccp4i/imosflm/imosflm.tcl
line 51)
imosflm from the command line works well. How do I get imosflm to work
from within ccp4i?
Two more issues that doesn't affect usability of ccp4.
Why is IMOSFLM_VERSION set to 0.6.1 in ccp4.setup? This should be
1.0.0, shouldn't it?
The last line of ccp4-others.setup sources
ccp4-6.1.0/src/phaser/phaser-2.1.4/build/intel-linux/setpaths.csh. This
file does not exist.
Thank you.
Andreas
--
Andreas Förster, Research Associate
Paul Freemont Xiaodong Zhang Labs
Department of Biochemistry, Imperial College London
[ccp4bb] Postdoctoral position in Protein Crystallography, Manchester UK
Posted on behalf of Lydia Tabernero (so replies to me will get ignored ;-) POSTDOCTORAL POSITION IN PROTEIN CRYSTALLOGRAPHY Applications are invited for a postdoctoral position to work on the structure determination of regulatory protein complexes critical in cell signaling pathways. This exciting project is aimed to understand the molecular basis for specific substrate recognition by protein tyrosine phosphatases that are involved in regulation of cell proliferation and growth. A better understanding of their molecular interactions with their partners, together with bioinformatics analyses will set the foundation for the rational design of novel inhibitors.This project is part of a multidisciplinary Marie Curie research training network funded by the EU-FP6 (www.ptpnet.ich.ucl.ac.uk).Postdocs will be expected to carry out novel research that contributes new knowledge to the field. They may also work for short periods in other Network laboratories, to obtain experience in other disciplines and techniques. They will have opportunities to collaborate widely and to present their work to other Network scientists at regular meetings. This post offers an excellent opportunity to join a dynamic research team in the Structural Biology group of the Faculty of Life Sciences that benefits from its superb multidisciplinary facilities and environment as well as from a highly friendly working atmosphere. The Faculty provides a stimulating and interactive environment for research staff, where a career development advisory programme is offered. Suitable candidates will have a strong background in protein crystallography and biochemistry. Good programming skills and experience with MySQL, Perl will be a plus. The position is available from 1st April 2009, and for up to 18 months. Candidates must be EU citizens (other eligibility criteria apply). Enquiries should be made to: Dr. Lydia Tabernero Michael Smith Building Faculty of Life Sciences University of Manchester Tel: 0161 275 7794 email: lydia.tabern...@manchester.ac.uk www.ls.manchester.ac.uk/research/themes/structuralbiology/ www.ls.manchester.ac.uk/research/themes/molecularcancerstudies/ -- *** * * * Dr. Martyn Winn * * * * STFC Daresbury Laboratory, Daresbury, Warrington, WA4 4AD, U.K. * * Tel: +44 1925 603455E-mail: martyn.w...@stfc.ac.uk* * Fax: +44 1925 603825Skype name: martyn.winn * * URL: http://www.ccp4.ac.uk/martyn/ * ***
Re: [ccp4bb] brute force molecular replacement
That may be so, but I'm still grappling with the visual of a younger Ian on all fours under the table -- or whatever it was, I deleted the email but the visual lingers, morphing disturbingly). phx. Nicholas M Glykos wrote: Dear Jacob, Why is it called queen of spades? As it happened, I was reading Alexander Pushkin's Queen of Spades while writing Qs. The book touches upon the subject of how close you can get to win everything and still lose it all. I thought that this was relevant for a stochastic search, and so the program got baptised. Nicholas
[ccp4bb] fix atomic positions in REFMAC5
Is there a possibility in refmac5 to fix atomic positions of some part of the molecule and to refine the other part? I couldn't find a proper keyword in keywords list:(
Re: [ccp4bb] fix atomic positions in REFMAC5
There is an option to do harmonic restraints: external harmonic chain [ch] residue [res] insertion [ins] atom [n] [altcode [a]] [sigma [value]] or external harmonic residues from [residue_number] [chain_name] to [residue_number] [chain_name] sigma [value] sigma 0.1 For example: external harmonic chain A residue 225 atom CA will put harmominc restraint on this atom external harmonic residues from 225 A to 250 A sigma 0.02 Have a look for description for this and other newer keywords in: http://www.ysbl.york.ac.uk/refmac/data/refmac_news.html regards Garib On 27 Jan 2009, at 13:46, Andrii Ishchenko wrote: Is there a possibility in refmac5 to fix atomic positions of some part of the molecule and to refine the other part? I couldn't find a proper keyword in keywords list:(
[ccp4bb] Job opening at MedImmune, Cambridge, UK
Job details for Research Scientist/Senior Research Scientist in Structural Biology/Bioinformatics, MedImmune, Cambridge, UK MedImmune Cambridge, with its distinctive science and culture, is central to AstraZeneca's plans to establish a major international presence in the research and development of biological therapeutics. To help us meet the many challenges that MedImmune's new horizons present we have an opportunity for a Research Scientist/Senior Research Scientist to join our Protein Sciences team, focusing on protein structure modelling and visualisation, and target cloning. You will be primarily responsible for both interpreting protein structure information derived in house and obtained from public databases, and homology modelling of target proteins. An additional role will also include the cloning of validated target genes into appropriate expression vectors, the expression of target proteins in relevant host organisms, and the purification of target proteins. Furthermore, an additional role will be the application of molecular biology skills to design and prepare expression constructs for use in the generation of structural data on antigen/Fab complexes. You should be educated to PhD level or equivalent in Structural Biology, Structural Bioinformatics or related discipline. Experience of working within an industry environment and demonstrable skills of working in a project team are also highly desirable. You should also have experience of using computational tools to analyse and interpret protein structures, with Bioinformatics expertise to prepare homology models, and be proficient with both PC and Linux operating systems. We are looking for an innovative team player with excellent communication skills and the ability to work independently and accurately to deliver goals. Cut and paste the link below to see the full information for this position: https://careers.cambridgeantibody.com/templates/cambridge/jobdetail.aspx ?raparam=4439644151735A3275467A36303642445A524A6A306274646E576B515161565 0 To apply for this position, click on 'register / apply' at the foot of this page. Please be prepared to attach your current CV (Word or PDF only). MedImmune is an Equal Opportunity Employer. We seek to empower each individual, and respect the diverse cultures, perspectives, skills and experiences within our workplace. To the extent this electronic communication or any of its attachments contain information that is not in the public domain, such information is considered by MedImmune to be confidential and proprietary. This communication is expected to be read and/or used only by the individual(s) for whom it is intended. If you have received this electronic communication in error, please reply to the sender advising of the error in transmission and delete the original message and any accompanying documents from your system immediately, without copying, reviewing or otherwise using them for any purpose. Thank you for your cooperation.
Re: [ccp4bb] Problem during refinement
Eleanor Dodson wrote: ... Many of these are unlikely, and you may well correct them when rebuilding. However the pdb file output by COOT will retain the CISPEP records.. You need to check and edit these yourself.. For the record, I believe that this is no longer the case. Coot will only write CISPEP records for real CISPEPS (|omega| 90). (Thanks to Eugene Krissinel who added support in mmdb so that Coot could do this). Paul.
[ccp4bb] Nvidia 3D
Has anyone taken the plunge and tried this setup with Coot or PyMol? Seems interesting: http://www.nvidia.com/object/GeForce_3D_Vision_Main.html Chris -- Dr. Christopher A. Waddling, Ph.D. University of California at San Francisco MC 2140 S126C 600 16th St., San Francisco, CA 94158-2517 (415) 476-8288 (office) (415) 502-7779 (lab) (415) 514-4142 (fax) (415) 810-7556 (cell) waddl...@msg.ucsf.edu AIM duckie2k1 Skype chriswaddling
Re: [ccp4bb] Nvidia 3D
https://www.jiscmail.ac.uk/cgi-bin/webadmin?A2=ind0901L=CCP4BBT=0F=S =P=192056 https://www.jiscmail.ac.uk/cgi-bin/webadmin?A2=ind0901L=CCP4BBT=0F=; S=P=192056 -Original Message- From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Chris Waddling Sent: Tuesday, January 27, 2009 12:56 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Nvidia 3D Has anyone taken the plunge and tried this setup with Coot or PyMol? Seems interesting: http://www.nvidia.com/object/GeForce_3D_Vision_Main.html Chris -- Dr. Christopher A. Waddling, Ph.D. University of California at San Francisco MC 2140 S126C 600 16th St., San Francisco, CA 94158-2517 (415) 476-8288 (office) (415) 502-7779 (lab) (415) 514-4142 (fax) (415) 810-7556 (cell) waddl...@msg.ucsf.edu AIM duckie2k1 Skype chriswaddling
[ccp4bb] [OFF TOPIC] his-tag doesn't bind
Hi ccp4 list, I am trying to purify a his-tag protein by metal affinity chromatography. The protein was expressed in inclusion bodies and its his-tag doesn't bind the Qiagen Ni resin in denatured conditions (urea 8M or GndHCl 6M). Playing with NaCl and detergents didn't help much. Any help is appreciated. Fred
Re: [ccp4bb] [OFF TOPIC] his-tag doesn't bind
Hi Fred, I used to worked with a protein which would not bind to the Qiagen NiNTA (really *NOT*) but beautifully to IDA resin (e.g. Amersham Pharmacia). This was under non-denaturing conditions, though, and I don't know whether this applies to the denatured state. It may be worth a try. Tim -- Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A On Tue, 27 Jan 2009, Fred wrote: Hi ccp4 list, I am trying to purify a his-tag protein by metal affinity chromatography. The protein was expressed in inclusion bodies and its his-tag doesn't bind the Qiagen Ni resin in denatured conditions (urea 8M or GndHCl 6M). Playing with NaCl and detergents didn't help much. Any help is appreciated. Fred
Re: [ccp4bb] [OFF TOPIC] his-tag doesn't bind
You can also try TALON cobalt affinity resin from Clontech ( http://www.clontech.com/products/detail.asp?product_id=10588tabno=2) or the high yield PrepEase resin from USB ( http://www.usbweb.com/category.asp?special=cat=234id=78806). On Tue, Jan 27, 2009 at 4:34 PM, Tim Gruene t...@shelx.uni-ac.gwdg.de wrote: Hi Fred, I used to worked with a protein which would not bind to the Qiagen NiNTA (really *NOT*) but beautifully to IDA resin (e.g. Amersham Pharmacia). This was under non-denaturing conditions, though, and I don't know whether this applies to the denatured state. It may be worth a try. Tim -- Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A On Tue, 27 Jan 2009, Fred wrote: Hi ccp4 list, I am trying to purify a his-tag protein by metal affinity chromatography. The protein was expressed in inclusion bodies and its his-tag doesn't bind the Qiagen Ni resin in denatured conditions (urea 8M or GndHCl 6M). Playing with NaCl and detergents didn't help much. Any help is appreciated. Fred -- Jim Fairman Graduate Research Assistant Department of Biochemistry, Cellular, and Molecular Biology (BCMB) University of Tennessee -- Knoxville 216-368-3337 jfair...@utk.edu james.fair...@case.edu
[ccp4bb] pseudo translation
Hi all,here is a question from a beginner. I have a home source data set that indexed and scaled in a P2 space group (a=46.704,b=59.362, c=48.783, alpha=90.0, beta=104.469, gamma=90.0) with predicted 2mol/au. After failing to get a MR solution with Phaser I ran the phenix.xtriage which showed that I have a large (89.18) Patterson peak at 0.5 0 0.5 which indicates pseudo translational symmetry. I was wondering if there is anything I could do with this data to get around this problem. Given that I don't have a lot of experience any suggestion/explanation would be fantastic. Thanks in advance K
[ccp4bb] sticky crystals
Dear colleagues, we have been growing crystals of a protein complex in sitting-drop geometry that stick to the bottom of the drop remarkably well. It's as if they are glued onto the plastic. This makes crystal handling next to impossible without destroying the crystals. We have tried whiskers, loops, all kinds of micro-tools, and pipetting techniques to no avail. I can say at the outset that we have been unsuccessful in growing these crystals in hanging-drops or at 4 degrees. Deglycosylating the complex also leads to nowhere. In fact, we are only able to get crystals from homogeneously glycosylated protein produced in HEK293S/I- cells. In the meantime we are playing with the idea of siliconizing the sitting-drop depressions to alter the crystal/plate interface. But then again, nucleation events on the plastic may be the reason we are getting crystals in the first place. We have also thought of trying microseeding to have more control on nucleation issues. Our protein production is quite limiting and forces us to be very selective with our experimentation. Nonetheless, while we are waiting for fresh material to explore some of these ideas we would like to make the most out of the crystals we have grown thus far. We would therefore very much appreciate any input/ideas on manipulating these crystals for data collection. 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: savvas.savvi...@ugent.be http://www.lprobe.ugent.be/xray.html From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Katarina Moravcevic Sent: Tuesday, January 27, 2009 10:52 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] pseudo translation Hi all, here is a question from a beginner. I have a home source data set that indexed and scaled in a P2 space group (a=46.704,b=59.362, c=48.783, alpha=90.0, beta=104.469, gamma=90.0) with predicted 2mol/au. After failing to get a MR solution with Phaser I ran the phenix.xtriage which showed that I have a large (89.18) Patterson peak at 0.5 0 0.5 which indicates pseudo translational symmetry. I was wondering if there is anything I could do with this data to get around this problem. Given that I don't have a lot of experience any suggestion/explanation would be fantastic. Thanks in advance K E-mail message checked by Spyware Doctor (6.0.0.386) Database version: 5.11630 http://www.pctools.com/en/spyware-doctor-antivirus/
Re: [ccp4bb] sticky crystals
Put a small piece of dry ice on the opposite side of the plastic from the crystal. Perhaps the difference in thermal expansive coefficient will let the crystal(s) break away. Don't overdo it though. This is a trick that Gary Gilliland taught me. Jim On Wed, 28 Jan 2009, Savvas Savvides wrote: Dear colleagues, we have been growing crystals of a protein complex in sitting-drop geometry that stick to the bottom of the drop remarkably well. It's as if they are glued onto the plastic. This makes crystal handling next to impossible without destroying the crystals. We have tried whiskers, loops, all kinds of micro-tools, and pipetting techniques to no avail. I can say at the outset that we have been unsuccessful in growing these crystals in hanging-drops or at 4 degrees. Deglycosylating the complex also leads to nowhere. In fact, we are only able to get crystals from homogeneously glycosylated protein produced in HEK293S/I- cells. In the meantime we are playing with the idea of siliconizing the sitting-drop depressions to alter the crystal/plate interface. But then again, nucleation events on the plastic may be the reason we are getting crystals in the first place. We have also thought of trying microseeding to have more control on nucleation issues. Our protein production is quite limiting and forces us to be very selective with our experimentation. Nonetheless, while we are waiting for fresh material to explore some of these ideas we would like to make the most out of the crystals we have grown thus far. We would therefore very much appreciate any input/ideas on manipulating these crystals for data collection. 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: savvas.savvi...@ugent.be http://www.lprobe.ugent.be/xray.html From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Katarina Moravcevic Sent: Tuesday, January 27, 2009 10:52 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] pseudo translation Hi all, here is a question from a beginner. I have a home source data set that indexed and scaled in a P2 space group (a=46.704,b=59.362, c=48.783, alpha=90.0, beta=104.469, gamma=90.0) with predicted 2mol/au. After failing to get a MR solution with Phaser I ran the phenix.xtriage which showed that I have a large (89.18) Patterson peak at 0.5 0 0.5 which indicates pseudo translational symmetry. I was wondering if there is anything I could do with this data to get around this problem. Given that I don't have a lot of experience any suggestion/explanation would be fantastic. Thanks in advance K E-mail message checked by Spyware Doctor (6.0.0.386) Database version: 5.11630 http://www.pctools.com/en/spyware-doctor-antivirus/
Re: [ccp4bb] sticky crystals
Hi, I had this exact problem before. The method below worked for me: Take a sturdy needle (like one of the microneedles from a Hampton kit or a very thin syringe needle) and (while observing the whole thing under a scope) stick the needle into the plastic a bit away from the crystal. Push hard. If you're using polarizers, you may b abe to visualize the stress forces in the plastic by the shifting of the colors. The basic idea here is to stress the plastic under the crystal w/o touching the crystal in any way. In my case the bloody things just popped off. Sometimes you have to push quite hard, and to wiggle the needle a bit - and beware, they can slip and ruin your drop. But this really worked quite well! Artem _ From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Savvas Savvides Sent: Tuesday, January 27, 2009 6:06 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] sticky crystals Dear colleagues, we have been growing crystals of a protein complex in sitting-drop geometry that stick to the bottom of the drop remarkably well. It's as if they are glued onto the plastic. This makes crystal handling next to impossible without destroying the crystals. We have tried whiskers, loops, all kinds of micro-tools, and pipetting techniques to no avail. I can say at the outset that we have been unsuccessful in growing these crystals in hanging-drops or at 4 degrees. Deglycosylating the complex also leads to nowhere. In fact, we are only able to get crystals from homogeneously glycosylated protein produced in HEK293S/I- cells. In the meantime we are playing with the idea of siliconizing the sitting-drop depressions to alter the crystal/plate interface. But then again, nucleation events on the plastic may be the reason we are getting crystals in the first place. We have also thought of trying microseeding to have more control on nucleation issues. Our protein production is quite limiting and forces us to be very selective with our experimentation. Nonetheless, while we are waiting for fresh material to explore some of these ideas we would like to make the most out of the crystals we have grown thus far. We would therefore very much appreciate any input/ideas on manipulating these crystals for data collection. 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: savvas.savvi...@ugent.be http://www.lprobe.ugent.be/xray.html From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Katarina Moravcevic Sent: Tuesday, January 27, 2009 10:52 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] pseudo translation Hi all, here is a question from a beginner. I have a home source data set that indexed and scaled in a P2 space group (a=46.704,b=59.362, c=48.783, alpha=90.0, beta=104.469, gamma=90.0) with predicted 2mol/au. After failing to get a MR solution with Phaser I ran the phenix.xtriage which showed that I have a large (89.18) Patterson peak at 0.5 0 0.5 which indicates pseudo translational symmetry. I was wondering if there is anything I could do with this data to get around this problem. Given that I don't have a lot of experience any suggestion/explanation would be fantastic. Thanks in advance K E-mail message checked by Spyware Doctor (6.0.0.386) Database version: 5.11630 http://www.pctools.com/spyware-doctor-antivirus/ http://www.pctools.com/en/spyware-doctor-antivirus/
Re: [ccp4bb] sticky crystals
If you have good and bad crystals in the same drop, I've had success pushing a crummy crystal into a good crystal and having it release that way. Additionally, once I realized this was going to be a long term problem, I started coating the sitting drop depressions with a thin layer of vacuum grease. The crystals just slid right off the grease and I never saw any changes in the diffraction data to suggest the grease was giving me issues. Chris On Wed, 28 Jan 2009, Savvas Savvides wrote: Dear colleagues, we have been growing crystals of a protein complex in sitting-drop geometry that stick to the bottom of the drop remarkably well. It's as if they are glued onto the plastic. This makes crystal handling next to impossible without destroying the crystals. We have tried whiskers, loops, all kinds of micro-tools, and pipetting techniques to no avail. I can say at the outset that we have been unsuccessful in growing these crystals in hanging-drops or at 4 degrees. Deglycosylating the complex also leads to nowhere. In fact, we are only able to get crystals from homogeneously glycosylated protein produced in HEK293S/I- cells. In the meantime we are playing with the idea of siliconizing the sitting-drop depressions to alter the crystal/plate interface. But then again, nucleation events on the plastic may be the reason we are getting crystals in the first place. We have also thought of trying microseeding to have more control on nucleation issues. Our protein production is quite limiting and forces us to be very selective with our experimentation. Nonetheless, while we are waiting for fresh material to explore some of these ideas we would like to make the most out of the crystals we have grown thus far. We would therefore very much appreciate any input/ideas on manipulating these crystals for data collection. 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: savvas.savvi...@ugent.be http://www.lprobe.ugent.be/xray.html From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Katarina Moravcevic Sent: Tuesday, January 27, 2009 10:52 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] pseudo translation Hi all, here is a question from a beginner. I have a home source data set that indexed and scaled in a P2 space group (a=46.704,b=59.362, c=48.783, alpha=90.0, beta=104.469, gamma=90.0) with predicted 2mol/au. After failing to get a MR solution with Phaser I ran the phenix.xtriage which showed that I have a large (89.18) Patterson peak at 0.5 0 0.5 which indicates pseudo translational symmetry. I was wondering if there is anything I could do with this data to get around this problem. Given that I don't have a lot of experience any suggestion/explanation would be fantastic. Thanks in advance K E-mail message checked by Spyware Doctor (6.0.0.386) Database version: 5.11630 http://www.pctools.com/en/spyware-doctor-antivirus/ Christopher L. Colbert, Ph.D. InstructorPhone: (214) 645 5944 University of Texas Southwestern Medical Center FAX: (214) 645 5945 6001 Forest Park Lane Dallas, TX 75390
Re: [ccp4bb] [OFF TOPIC] his-tag doesn't bind
You don't have EDTA left over from your protease inhibitor, do you? Some commercial cocktails include it. You may have to read the fine print. James On Jan 27, 2009, at 1:00 PM, Fred wrote: Hi ccp4 list, I am trying to purify a his-tag protein by metal affinity chromatography. The protein was expressed in inclusion bodies and its his-tag doesn't bind the Qiagen Ni resin in denatured conditions (urea 8M or GndHCl 6M). Playing with NaCl and detergents didn't help much. Any help is appreciated. Fred
Re: [ccp4bb] sticky crystals
Suggestions so far have been good ones. However, the MiTeGen microtools kit: http://mitegen.com/products/microtools/microtools_kit1.shtml comes with a MicroSaw, which is a 10-micron thick kapton saw that is intended for this purpose. That is, you don't pry the crystal off the surface, but rather rest this saw against the surface, bring it over to the edge of the stuck crystal and then work it back and forth until you have cut underneath the crystal. Did you try this tool? -James Holton MAD Scientist Savvas Savvides wrote: Dear colleagues, we have been growing crystals of a protein complex in sitting-drop geometry that stick to the bottom of the drop remarkably well. It’s as if they are glued onto the plastic. This makes crystal handling next to impossible without destroying the crystals. We have tried whiskers, loops, all kinds of micro-tools, and pipetting techniques to no avail. I can say at the outset that we have been unsuccessful in growing these crystals in hanging-drops or at 4 degrees. Deglycosylating the complex also leads to nowhere. In fact, we are only able to get crystals from homogeneously glycosylated protein produced in HEK293S/I- cells. In the meantime we are playing with the idea of siliconizing the sitting-drop depressions to alter the crystal/plate interface. But then again, nucleation events on the plastic may be the reason we are getting crystals in the first place. We have also thought of trying microseeding to have more control on nucleation issues. Our protein production is quite limiting and forces us to be very selective with our experimentation. Nonetheless, while we are waiting for fresh material to explore some of these ideas we would like to make the most out of the crystals we have grown thus far. We would therefore very much appreciate any input/ideas on manipulating these crystals for data collection. 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: savvas.savvi...@ugent.be http://www.lprobe.ugent.be/xray.html *From:* CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] *On Behalf Of *Katarina Moravcevic *Sent:* Tuesday, January 27, 2009 10:52 PM *To:* CCP4BB@JISCMAIL.AC.UK *Subject:* [ccp4bb] pseudo translation Hi all, here is a question from a beginner. I have a home source data set that indexed and scaled in a P2 space group (a=46.704,b=59.362, c=48.783, alpha=90.0, beta=104.469, gamma=90.0) with predicted 2mol/au. After failing to get a MR solution with Phaser I ran the phenix.xtriage which showed that I have a large (89.18) Patterson peak at 0.5 0 0.5 which indicates pseudo translational symmetry. I was wondering if there is anything I could do with this data to get around this problem. Given that I don't have a lot of experience any suggestion/explanation would be fantastic. Thanks in advance K * E-mail message checked by Spyware Doctor (6.0.0.386) Database version: 5.11630 http://www.pctools.com/spyware-doctor-antivirus/ http://www.pctools.com/en/spyware-doctor-antivirus/ *
Re: [ccp4bb] sticky crystals
Hi, I had a similar story like yours.Then I added a drop of 10ul simulated mother liquot which contains much higher concentrations of all components in the normal mother liquot. Sometimes, the crystals attached to the plastic would float to the surface. If not, take another 10ul, but blew it to the bottom plastic with a pippetman back and forth, and some crystals would also leave the plastic(But you have to be very careful to do this.) My friend Jill solved her similar problem by hanging drop setup instead of sitting drop. Good luck. Deliang - Original Message - From: Savvas Savvides To: CCP4BB@JISCMAIL.AC.UK Sent: Tuesday, January 27, 2009 3:05 PM Subject: [ccp4bb] sticky crystals Dear colleagues, we have been growing crystals of a protein complex in sitting-drop geometry that stick to the bottom of the drop remarkably well. It's as if they are glued onto the plastic. This makes crystal handling next to impossible without destroying the crystals. We have tried whiskers, loops, all kinds of micro-tools, and pipetting techniques to no avail. I can say at the outset that we have been unsuccessful in growing these crystals in hanging-drops or at 4 degrees. Deglycosylating the complex also leads to nowhere. In fact, we are only able to get crystals from homogeneously glycosylated protein produced in HEK293S/I- cells. In the meantime we are playing with the idea of siliconizing the sitting-drop depressions to alter the crystal/plate interface. But then again, nucleation events on the plastic may be the reason we are getting crystals in the first place. We have also thought of trying microseeding to have more control on nucleation issues. Our protein production is quite limiting and forces us to be very selective with our experimentation. Nonetheless, while we are waiting for fresh material to explore some of these ideas we would like to make the most out of the crystals we have grown thus far. We would therefore very much appreciate any input/ideas on manipulating these crystals for data collection. 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: savvas.savvi...@ugent.be http://www.lprobe.ugent.be/xray.html From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Katarina Moravcevic Sent: Tuesday, January 27, 2009 10:52 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] pseudo translation Hi all, here is a question from a beginner. I have a home source data set that indexed and scaled in a P2 space group (a=46.704,b=59.362, c=48.783, alpha=90.0, beta=104.469, gamma=90.0) with predicted 2mol/au. After failing to get a MR solution with Phaser I ran the phenix.xtriage which showed that I have a large (89.18) Patterson peak at 0.5 0 0.5 which indicates pseudo translational symmetry. I was wondering if there is anything I could do with this data to get around this problem. Given that I don't have a lot of experience any suggestion/explanation would be fantastic. Thanks in advance K E-mail message checked by Spyware Doctor (6.0.0.386) Database version: 5.11630 http://www.pctools.com/spyware-doctor-antivirus/
Re: [ccp4bb] [OFF TOPIC] his-tag doesn't bind
To go along with the EDTA comment, did you check the pH of your binding solution? Sometimes lysing E. coli causes the pH to drop below the pKa of the histidines. On Tue, Jan 27, 2009 at 8:10 PM, James Stroud xtald...@gmail.com wrote: You don't have EDTA left over from your protease inhibitor, do you? Some commercial cocktails include it. You may have to read the fine print. James On Jan 27, 2009, at 1:00 PM, Fred wrote: Hi ccp4 list, I am trying to purify a his-tag protein by metal affinity chromatography. The protein was expressed in inclusion bodies and its his-tag doesn't bind the Qiagen Ni resin in denatured conditions (urea 8M or GndHCl 6M). Playing with NaCl and detergents didn't help much. Any help is appreciated. Fred -- Jim Fairman Graduate Research Assistant Department of Biochemistry, Cellular, and Molecular Biology (BCMB) University of Tennessee -- Knoxville 216-368-3337 jfair...@utk.edu james.fair...@case.edu
Re: [ccp4bb] sticky crystals
Hi Savvas, You can collect data on your crystal still in the drop, on our beamline (FIP-BM30A at the ESRF) if you are interested. Provided space group is not P1 We do that routinely. Let me know if you are interested. JL Savvas Savvides wrote: Dear colleagues, we have been growing crystals of a protein complex in sitting-drop geometry that stick to the bottom of the drop remarkably well. Its as if they are glued onto the plastic. This makes crystal handling next to impossible without destroying the crystals. We have tried whiskers, loops, all kinds of micro-tools, and pipetting techniques to no avail. I can say at the outset that we have been unsuccessful in growing these crystals in hanging-drops or at 4 degrees. Deglycosylating the complex also leads to nowhere. In fact, we are only able to get crystals from homogeneously glycosylated protein produced in HEK293S/I- cells. In the meantime we are playing with the idea of siliconizing the sitting-drop depressions to alter the crystal/plate interface. But then again, nucleation events on the plastic may be the reason we are getting crystals in the first place. We have also thought of trying microseeding to have more control on nucleation issues. Our protein production is quite limiting and forces us to be very selective with our experimentation. Nonetheless, while we are waiting for fresh material to explore some of these ideas we would like to make the most out of the crystals we have grown thus far. We would therefore very much appreciate any input/ideas on manipulating these crystals for data collection. 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: savvas.savvi...@ugent.be http://www.lprobe.ugent.be/xray.html From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Katarina Moravcevic Sent: Tuesday, January 27, 2009 10:52 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] pseudo translation Hi all, here is a question from abeginner. I have a home source data set that indexed and scaled in a P2 space group (a=46.704,b=59.362, c=48.783, alpha=90.0, beta=104.469,gamma=90.0) with predicted 2mol/au. After failing to get a MR solution with Phaser I ran the phenix.xtriage which showed that I have a large (89.18) Patterson peak at 0.5 0 0.5 which indicates pseudo translational symmetry. I was wondering if there is anything I could do with this data to get around this problem. Given that I don't have a lot of experience any suggestion/explanation would be fantastic. Thanks in advance K E-mail message checked by Spyware Doctor (6.0.0.386) Database version: 5.11630 http://www.pctools.com/spyware-doctor-antivirus/ -- Jean-Luc Ferrer |---| |Institut de Biologie Structurale J.P. Ebel CEA/CNRS/UJF| | 41 rue Jules Horowitz, 38027 Grenoble cedex 1, FRANCE | |tel.: +33 (0)4-38-78-59-10, fax : +33 (0)4-38-78-51-22| |---|
Re: [ccp4bb] sticky crystals
Hi All One more method that I heard about but never tried is to put the plate on a sonication bath and to let for a short sonication pulse. That should vibrate some liquid under your crystal. Raz -- Raz Zarivach, Ph.D. Department of Life Sciences and the National Institute of Biotechnology in the Negev Ben-Gurion University of the Negev POB 653 Zip code 84105 Beer-Sheva Israel Home page: http://raz189.tripod.com/ tel: +972-8-646-1999 cell: +972-50-5754808 fax: +972-8-6472970 skype: zarivach ra...@yahoo.com
Re: [ccp4bb] sticky crystals
Most obvious (maybe you did it already but that is not clear from your email), why not try seeding? - J. - Savvas Savvides wrote: Dear colleagues, we have been growing crystals of a protein complex in sitting-drop geometry that stick to the bottom of the drop remarkably well. It’s as if they are glued onto the plastic. This makes crystal handling next to impossible without destroying the crystals. We have tried whiskers, loops, all kinds of micro-tools, and pipetting techniques to no avail. I can say at the outset that we have been unsuccessful in growing these crystals in hanging-drops or at 4 degrees. Deglycosylating the complex also leads to nowhere. In fact, we are only able to get crystals from homogeneously glycosylated protein produced in HEK293S/I- cells. In the meantime we are playing with the idea of siliconizing the sitting-drop depressions to alter the crystal/plate interface. But then again, nucleation events on the plastic may be the reason we are getting crystals in the first place. We have also thought of trying microseeding to have more control on nucleation issues. Our protein production is quite limiting and forces us to be very selective with our experimentation. Nonetheless, while we are waiting for fresh material to explore some of these ideas we would like to make the most out of the crystals we have grown thus far. We would therefore very much appreciate any input/ideas on manipulating these crystals for data collection. 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: savvas.savvi...@ugent.be http://www.lprobe.ugent.be/xray.html *From:* CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] *On Behalf Of *Katarina Moravcevic *Sent:* Tuesday, January 27, 2009 10:52 PM *To:* CCP4BB@JISCMAIL.AC.UK *Subject:* [ccp4bb] pseudo translation Hi all, here is a question from a beginner. I have a home source data set that indexed and scaled in a P2 space group (a=46.704,b=59.362, c=48.783, alpha=90.0, beta=104.469, gamma=90.0) with predicted 2mol/au. After failing to get a MR solution with Phaser I ran the phenix.xtriage which showed that I have a large (89.18) Patterson peak at 0.5 0 0.5 which indicates pseudo translational symmetry. I was wondering if there is anything I could do with this data to get around this problem. Given that I don't have a lot of experience any suggestion/explanation would be fantastic. Thanks in advance K * E-mail message checked by Spyware Doctor (6.0.0.386) Database version: 5.11630 http://www.pctools.com/spyware-doctor-antivirus/ http://www.pctools.com/en/spyware-doctor-antivirus/ * -- Dr. Jeroen R. Mesters Gruppenleiter Strukturelle Neurobiologie und Kristallogenese Institut für Biochemie, Universität zu Lübeck Zentrum für Medizinische Struktur- und Zellbiologie Ratzeburger Allee 160, D-23538 Lübeck Tel: +49-451-5004065, Fax: +49-451-5004068 Http://www.biochem.uni-luebeck.de Http://www.iobcr.org Http://www.selfish-brain.org Http://www.opticryst.org -- If you can look into the seeds of time and say which grain will grow and which will not - speak then to me (Macbeth) --
