Re: [ccp4bb] Sequence Alignment Question
Re sequence alignment, For those TeXnically inclined, TeXshade produces beautiful alignment figures. (Steep TeX learning curve, I have an example script if anyone wants one). http://www.ctan.org/pkg/texshade James -- Dr. James W. Murray David Phillips Research Fellow Division of Molecular Biosciences Imperial College, LONDON Tel: +44 (0)20 759 48895 From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Yuri [yuri.pom...@ufl.edu] Sent: Friday, August 19, 2011 12:31 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Sequence Alignment Question Hello Everyone, A little off topic but, what is a good way to show (publication quality) multiple sequence alignment? I am trying to show conserved regions in related proteins from different organisms. Thank you -- Yuri Pompeu
Re: [ccp4bb] Off topic_protein degradation.
Dear Bei, The first question to ask is not whether there is spontaneous disulfide bond breakage, but whether the correct disulfide bonds have been made in the first place. Extreme protease sensitivity could point to an unfolded/misfolded protein. If you know some protein NMR people, you could ask them to check. Even a 1-D NMR spectrum could give some information whether the protein is correctly folded or not. Another way to check is to see if your protein has proper enzymatic/biological activity. If this activity is ok, the folding is probably ok as well. You may have a protease contaminant, so you may want to check the purification protocol. The least you could do is to add some protease inhibitors to your crystallization setups. I once added PMSF for this purpose. Good luck! Herman From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of joybeiyang Sent: Friday, August 19, 2011 6:28 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Off topic_protein degradation. Dear all, I am trying to crystallize a protein for which the yield and solubility were both fine. However, this protein has a severe problem of degradation. When stored at RT, the protein will degrade madly into pieces, while stored at 4 degree, the degradation is much slower and a relatively stable truncate form can be get. I am going to try to crystallize the protein at 4 degree, however I still want to understand what's going on there at RT because this protein was supposed to be very stable, it is Cys rich, and the 6 Cys were predicted to form 3 disulfide bonds which hold the protein as a globule, how can a protein with 3 stabilizing disulfide bond be fully degraded like this? Is there a possibility of spontaneous disulfide bond breakage at pH 8 ? Another question is I tried limited proteolysis with this protein, however even at 1:1000(w/w, chymotrypsin), the protein is degraded into pieces in about 2 hrs (again, a relatively stable truncated form can be get between 10 min and 30 min). I am wondering how is the crystallization probability correlated with proteolysis stability? Does this phenomenon indicate that the crystallization probability of my protein is pretty low? Any comments would be greatly appreciated. Bei 2011-08-15
Re: [ccp4bb] more Computer encryption matters
With OSX 10.6 Snow Leopard, you can FileVault your sensitive home directory, but put all your non-sensitive compute || i/o-intensive files outside your home directory (eg in /Users/Stuff) I don't know whether you can do this in 10.7 Lion Phil On 18 Aug 2011, at 22:50, William G. Scott wrote: OS X 10.7 enables you to do whole-drive encryption. Here is a description from Arse Technica: http://arstechnica.com/apple/reviews/2011/07/mac-os-x-10-7.ars/13 I ain't never tried it myself. 10.7 seems to run slow enough as it is. -- Bill On Aug 18, 2011, at 5:34 AM, Andreas Förster wrote: Since we're on the subject... I've been tempted on and off to encrypt my hard drive, but after getting burned once a hundred years ago when encrypted data turned into garbled bytes all of a sudden I've been hesitant. I've gone so far as to install TrueCrypt (on a MacBook), but I haven't put it into action. Before I do, the big question: What software do people on the bb use for encryption? What can be recommended without hesitation? Thanks. Andreas On 18/08/2011 1:19, Eric Bennett wrote: John, Since so many people have said it's flawless, I'd like to point out this is not always the case. The particular version of the particular package that we have installs some system libraries that caused a program I use on a moderately frequent basis to crash every time I tried to open a file on a network drive. It took me about 9 months to figure out what the cause was, during which time I had to manually copy things to the local drive before I could open them in that particular program. The vendor of the encryption software has a newer version but our IT department is using an older version. There is another workaround but it's kind of a hack. So I'd say problems are very rare, but if you run into strange behavior, don't rule out encryption as a possible cause. -Eric -- Andreas Förster, Research Associate Paul Freemont Xiaodong Zhang Labs Department of Biochemistry, Imperial College London http://www.msf.bio.ic.ac.uk
Re: [ccp4bb] Sequence Alignment Question
Another quite powerful tool is ESPript ( http://espript.ibcp.fr/ESPript/ESPript/ ), which takes e.g. clustalw outputs and allows various manipulations (change of colors, add text, secondary structures, etc.) Matthias From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Yuri [yuri.pom...@ufl.edu] Sent: Friday, August 19, 2011 12:31 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Sequence Alignment Question Hello Everyone, A little off topic but, what is a good way to show (publication quality) multiple sequence alignment? I am trying to show conserved regions in related proteins from different organisms. Thank you -- Yuri Pompeu
Re: [ccp4bb] Sequence Alignment Question
Jalview is another option.
http://www.jalview.org/
Sent from my HTC
- Reply message -
From: Yuri yuri.pom...@ufl.edu
Date: Fri, Aug 19, 2011 00:31
Subject: [ccp4bb] Sequence Alignment Question
To: CCP4BB@JISCMAIL.AC.UK
UID 23435 BODY[1]0 {238}
Hello Everyone,
A little off topic but, what is a good way to show (publication
quality) multiple sequence alignment?
I am trying to show conserved regions in related proteins from
different organisms.
Thank you
--
Yuri Pompeu
[ccp4bb] ccp4 at IUCr2011
Shameless advertising, but... CCP4 will have a booth, No. 47, and posters at the IUCr meeting in Madrid next week. So, I hope to see you there Charles
Re: [ccp4bb] Off topic_protein degradation.
BioSAXS can also tell you if the protein is folded or not, but in either case, you may want to purify on site since degradation is so fast. Many BioSAXS beamlines (including ours at MacCHESS) now have SEC systems on site. Richard Gillilan MacCHESS Cornell University On Aug 19, 2011, at 3:55 AM, herman.schreu...@sanofi-aventis.commailto:herman.schreu...@sanofi-aventis.com herman.schreu...@sanofi-aventis.commailto:herman.schreu...@sanofi-aventis.com wrote: Dear Bei, The first question to ask is not whether there is spontaneous disulfide bond breakage, but whether the correct disulfide bonds have been made in the first place. Extreme protease sensitivity could point to an unfolded/misfolded protein. If you know some protein NMR people, you could ask them to check. Even a 1-D NMR spectrum could give some information whether the protein is correctly folded or not. Another way to check is to see if your protein has proper enzymatic/biological activity. If this activity is ok, the folding is probably ok as well. You may have a protease contaminant, so you may want to check the purification protocol. The least you could do is to add some protease inhibitors to your crystallization setups. I once added PMSF for this purpose. Good luck! Herman From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of joybeiyang Sent: Friday, August 19, 2011 6:28 AM To: CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Off topic_protein degradation. Dear all, I am trying to crystallize a protein for which the yield and solubility were both fine. However, this protein has a severe problem of degradation. When stored at RT, the protein will degrade madly into pieces, while stored at 4 degree, the degradation is much slower and a relatively stable truncate form can be get. I am going to try to crystallize the protein at 4 degree, however I still want to understand what's going on there at RT because this protein was supposed to be very stable, it is Cys rich, and the 6 Cys were predicted to form 3 disulfide bonds which hold the protein as a globule, how can a protein with 3 stabilizing disulfide bond be fully degraded like this? Is there a possibility of spontaneous disulfide bond breakage at pH 8 ? Another question is I tried limited proteolysis with this protein, however even at 1:1000(w/w, chymotrypsin), the protein is degraded into pieces in about 2 hrs (again, a relatively stable truncated form can be get between 10 min and 30 min). I am wondering how is the crystallization probability correlated with proteolysis stability? Does this phenomenon indicate that the crystallization probability of my protein is pretty low? Any comments would be greatly appreciated. Bei 2011-08-15
[ccp4bb] Application deadline for MX-beamtime at HZB-BESSY is approaching soon at September 1, 2011 !
Next MX-proposal application deadline: September 1, 2011 is approaching See also: http://www.bessy.de/boat/http://www.bessy.de/boat/www/ We kindly invite new MX-proposals for beamtime applications for the next beamtime period. In order to apply for beamtime, please register at the HZB on-line access tool BOAT (http://www.bessy.de/boat/http://www.bessy.de/boat/www/) and submit a new beamtime application proposal. HZB provides beamtime at the MX-beamlines 14.1, 14.2 and 14.3. The requested beamtime is granted based on the reviewed proposal and reports from previous research activities. Reported results from previous beamtimes stated in the Experimental Reports form will affect the chances for future beamtimes significantly. Please make sure to include them if available. Experimental setup: BL14.1 setup: - Photon flux: 1.4x10¹¹ Phot/sx100mAx0.05%BW at sample position (0.1-20 sec exposure time per frame) - User defined beam shaping from 30µm-100µm diameter possible - MX-225 X-ray detector, 92mm-720mm max. distance from the sample - Microdiffractometer (MD2) with Mini-kappa goniometer MK3 (STAC controlled) - Automatic sample changer (CATS), 90 sample storage capacity (SPINE-Pin EMBL sample magazine compatibility) - 96-well crystallization plate scanning operational upon request - Dedicated eight-core XEON-CPU server, with fibre channel SAN up-link data processing environment - EDNA installed and available - Common MX-software installed including HKL2000, XDS, IMOSFLM, CCP4, Phenix, SHELXC-E, etc. - Automated data processing with ixds and xdsi available - Remotely controlled cryo-shutter for crystal annealing - Bruker AXS X-Flash XRF detector We are offering the hard- and software environment for carrying out: - UVRIP experiments at BL14.1. For further information, please visit: http://www.helmholtz-berlin.de/forschung/funkma/soft-matter/forschung/bessy-mx/ancillary-facilities/uvrip_en.html - In situ crystal screening experiments, please visit: http://www.helmholtz-berlin.de/forschung/funkma/soft-matter/forschung/bessy-mx/ancillary-facilities/insitu-screening_en.html BL14.2 setup: - Photon flux: 1.9x10¹¹ Phot/sx100mAx0.05%BW at sample position (3-20 sec exposure time per frame) - MX-225 X-ray detector, 45mm-380mm distance from the sample, 30 deg 2-Theta possible - mardtb goniometer installed - Dedicated eight-core XEON-CPU server, with fibre channel SAN up-link data processing environment - EDNA installed and available - Common MX-software installed including HKL2000, XDS, IMOSFLM, CCP4, Phenix, SHELXC-E, etc. - Automated data processing with ixds and xdsi available - Remotely controlled cryo-shutter for crystal annealing - Bruker AXS X-Flash XRF detector - Pressure chamber for noble gas derivatization (Xe, Kr available upon request) - Ultra high performance stereo microscope Leica M205A, 20-255x zoom, 8 Mpixel CCD-camera BL14.3 setup: - Photon flux: 4x10exp10 Phot/sx100mAx0.05%BW at sample position (3-20 sec exposure time per frame) - SX-165 X-ray detector, 45mm-380mm distance from the sample, 30 deg 2-Theta possible - mardtb goniometer installed - Dedicated eight-core XEON-CPU server, with fibre channel SAN up-link data processing environment - EDNA installed and available - Common MX software installed including HKL2000, XDS, IMOSFLM, CCP4, Phenix, SHELXC-E, etc. - Automated data processing with ixds and xdsi available - Remotely controlled cryo-shutter for crystal annealing - HC-1c dehydration device installed, HC1-beamtime upon request - Pressure chamber for noble gas derivatization (Xe, Kr available upon request) - Ultra high performance stereo microscope Leica M205A, 20-255x zoom, 8 Mpixel CCD-camera Please visit our web page www.helmholtz-berlin.de/bessy-mxhttp://www.helmholtz-berlin.de/bessy-mx to gain updated information about our experimental setup and other requirements. Uwe Mueller, Manfred Weiss and the HZB BESSY-MX group Dr. Uwe Mueller Soft Matter and Functional Materials Macromolecular Crystallography (BESSY-MX) | Group leader Elektronenspeicherring BESSY II Albert-Einstein-Str. 15, D-12489 Berlin, Germany Fon: +49 30 8062 14974 Fax: +49 30 8062 14975 url: www.helmholtz-berlin.de/bessy-mxhttp://www.helmholtz-berlin.de/bessy-mx email:u...@helmholtz-berlin.demailto:u...@helmholtz-berlin.de Helmholtz-Zentrum Berlin für Materialien und Energie GmbH Hahn-Meitner-Platz 1, 14109 Berlin Vorsitzender des Aufsichtsrats: Prof. Dr. Dr. h.c. mult. Joachim Treusch Stellvertretende Vorsitzende: Dr. Beatrix Vierkorn-Rudolph Geschäftsführer: Prof. Dr. Anke Rita Kaysser-Pyzalla, Dr. Ulrich Breuer Sitz der Gesellschaft: Berlin Handelsregister: AG Charlottenburg, 89 HRB 5583 Helmholtz-Zentrum Berlin für Materialien und Energie GmbH Mitglied der Hermann von Helmholtz-Gemeinschaft Deutscher Forschungszentren e.V. Aufsichtsrat: Vorsitzender Prof. Dr. Dr. h.c. mult. Joachim Treusch, stv. Vorsitzende Dr. Beatrix Vierkorn-Rudolph Geschäftsführer:
[ccp4bb] Neat and tidy Re: [ccp4bb] more Computer encryption matters
That is neat and tidy! I don't suppose you know if Windows 7 might have such a facility? Anyway it's a good tip and I will start looking in that direction, Thanks, John Prof John R Helliwell DSc On 19 Aug 2011, at 09:05, Phil Evans p...@mrc-lmb.cam.ac.uk wrote: With OSX 10.6 Snow Leopard, you can FileVault your sensitive home directory, but put all your non-sensitive compute || i/o-intensive files outside your home directory (eg in /Users/Stuff) I don't know whether you can do this in 10.7 Lion Phil On 18 Aug 2011, at 22:50, William G. Scott wrote: OS X 10.7 enables you to do whole-drive encryption. Here is a description from Arse Technica: http://arstechnica.com/apple/reviews/2011/07/mac-os-x-10-7.ars/13 I ain't never tried it myself. 10.7 seems to run slow enough as it is. -- Bill On Aug 18, 2011, at 5:34 AM, Andreas Förster wrote: Since we're on the subject... I've been tempted on and off to encrypt my hard drive, but after getting burned once a hundred years ago when encrypted data turned into garbled bytes all of a sudden I've been hesitant. I've gone so far as to install TrueCrypt (on a MacBook), but I haven't put it into action. Before I do, the big question: What software do people on the bb use for encryption? What can be recommended without hesitation? Thanks. Andreas On 18/08/2011 1:19, Eric Bennett wrote: John, Since so many people have said it's flawless, I'd like to point out this is not always the case. The particular version of the particular package that we have installs some system libraries that caused a program I use on a moderately frequent basis to crash every time I tried to open a file on a network drive. It took me about 9 months to figure out what the cause was, during which time I had to manually copy things to the local drive before I could open them in that particular program. The vendor of the encryption software has a newer version but our IT department is using an older version. There is another workaround but it's kind of a hack. So I'd say problems are very rare, but if you run into strange behavior, don't rule out encryption as a possible cause. -Eric -- Andreas Förster, Research Associate Paul Freemont Xiaodong Zhang Labs Department of Biochemistry, Imperial College London http://www.msf.bio.ic.ac.uk
[ccp4bb] Diamond MX BAG Training - November 2011
To all Diamond MX BAG Users, Diamond Light Source will be holding the next training day for MX BAG Users on Wednesday 2nd November 2011. The aim of the day is to provide BAG users with sufficient training to be able to operate any of the Diamond MX beamlines efficiently and get the most benefit from their beamtime. Sessions will include: -Automated sample handling (ACTOR CATS) -Data Acquisition Analysis Software -Remote Access -Sample Humidity Control (HC1) -Microbeam Crystallography Registration is free-of-charge with lunch provided on the 2nd and accommodation and dinner for the night of the 1st. Travelling expenses within the UK will also be provided. The training is targeted at any BAG members who require it and is not limited to students and post docs. Individuals wishing to register should reply to this e-mail with their name, BAG, contact details, accommodation and any other requirements. Early registration is recommended as places are limited to twenty. Best wishes, James. Dr James Nicholson, Senior Beamline Scientist MX, Diamond Light Source Ltd. Harwell Science and Innovation Campus, Didcot, Oxfordshire, OX11 0DE, UK.
Re: [ccp4bb] Off topic_protein degradation.
If you are unsure about whether the disulfides have formed treat a small amount of protein with N-ethylmaleimide. If the disulfides have not formed, when you perform mass spec on the protein you should see an increase of mass of 125Da for each exposed cysteine.
Re: [ccp4bb] Sequence Alignment Question- Thank you everyone
Thanks everyone, for the help. -- Yuri Pompeu
Re: [ccp4bb] crystal Packing
Dear Eswar, You could try to slow down crystallization somehow. There are several ways to accomplish this: 1) increase drop size 2) add small amounts of glycerol (1-5% v/v) 3) cover the reservoir solution with paraffin or silicon oil or a mixture of both in order to reduce the vapor-diffusion rate 4) find another additive that would change the crystallization regime (e.g. kosmotropic/chaotropic agents) Good luck! christian On 08/18/2011 04:24 AM, eswar reddy wrote: Dear All I was working two domain protein and i have an hexagonal look like crystals from additive screen, now i am facing problem these crystal are growing in 2 minutes even in cold room even at lower protein concentration @2mg/ml . and i have an smeary data of 7 Å, and we thinking one domain might be flexible and other is stable is there anyway to increase the crystal packing Any suggestions are welcome. Eswar Reddy -- __ Dr. Christian Biertümpfel Research Fellow Vaccine Research Center/NIAID/NIH phone: +1 301 594 8726 40 Convent Dr, Rm. 4613B fax:+1 301 480 2658 Bethesda, MD 20892-3027 USA __
Re: [ccp4bb] crystal Packing
A really simple trick is to dilute your drops. This has worked to suppress excessive nucleation for us many times, and nearly always increases crystal size. For example, instead of setting drops with 1 uL of protein and 1 uL of well solution, try setting the drop with 1 uL of protein, 1 uL of water and 1 uL of well solution. You can increase the water volume even more if desired, up to 6-10x the protein/well volumes. The extra evaporation time will postpone concentration into the supersaturation zone. Cheers, On 8/19/2011 10:32 AM, Christian Biertuempfel wrote: Dear Eswar, You could try to slow down crystallization somehow. There are several ways to accomplish this: 1) increase drop size 2) add small amounts of glycerol (1-5% v/v) 3) cover the reservoir solution with paraffin or silicon oil or a mixture of both in order to reduce the vapor-diffusion rate 4) find another additive that would change the crystallization regime (e.g. kosmotropic/chaotropic agents) Good luck! christian On 08/18/2011 04:24 AM, eswar reddy wrote: Dear All I was working two domain protein and i have an hexagonal look like crystals from additive screen, now i am facing problem these crystal are growing in 2 minutes even in cold room even at lower protein concentration @2mg/ml . and i have an smeary data of 7 , and we thinking one domain might be flexible and other is stable is there anyway to increase the crystal packing Any suggestions are welcome. Eswar Reddy -- Roger S. Rowlett Gordon Dorothy Kline Professor Department of Chemistry Colgate University 13 Oak Drive Hamilton, NY 13346 tel: (315)-228-7245 ofc: (315)-228-7395 fax: (315)-228-7935 email: rrowl...@colgate.edu
[ccp4bb] phaser openmp
Dear all, it would be really great if someone could point out to me how to enable the openmp option for phaser during the compilation of the latest ccp4 release. The system will be a SUSE 11.3 64bit using gcc. Cheers, Jochen - Dr. Jochen Kuper Rudolf Virchow Center for Biomedical Research Josef Schneider Strasse 2, Haus D15 97080 Wuerzburg +49 (0) 9313180391 jochen.ku...@virchow.uni-wuerzburg.de
Re: [ccp4bb] Off topic_protein degradation.
Another option is DTNB (aka Ellman's Reagent). This compound reacts with free thiols and produces a yellow color. The reaction is stoichiometric, so if you have access to a very basic spectrophotometer and know your protein concentration you can quantify the free thiols quite easily. Basic pH doesn't break disulfides, if anything it stabilizes them. I would be more concerned that they are incorrectly formed as described by Herman. Mike - Original Message - From: D Bonsor dbon...@ihv.umaryland.edu To: CCP4BB@JISCMAIL.AC.UK Sent: Friday, August 19, 2011 6:21:41 AM GMT -08:00 US/Canada Pacific Subject: Re: [ccp4bb] Off topic_protein degradation. If you are unsure about whether the disulfides have formed treat a small amount of protein with N-ethylmaleimide. If the disulfides have not formed, when you perform mass spec on the protein you should see an increase of mass of 125Da for each exposed cysteine. -- Michael C. Thompson Graduate Student Biochemistry Molecular Biology Division Department of Chemistry Biochemistry University of California, Los Angeles mi...@chem.ucla.edu
[ccp4bb] Electostatic surface at various pH
Hi All, Apologies for this slight off topic, however this could very well be the best bulletin board to seek help. I need to calculate the electrostatic surface and make surface figures for a protein at various pHs, say 4.0, 7.0 9.0. I'm doing this kind of wok for the first time. Could you suggest me a user-friendly software, tutorials and tips. Sincere thanks and appreciation. John
Re: [ccp4bb] Electostatic surface at various pH
John, You can use APBS in conjunction with PDB2PQR and PROPKA (all available from the ABPS website) for the calculations and Pymol for visualization. Good luck Carsten -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of john peter Sent: Friday, August 19, 2011 2:35 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Electostatic surface at various pH Hi All, Apologies for this slight off topic, however this could very well be the best bulletin board to seek help. I need to calculate the electrostatic surface and make surface figures for a protein at various pHs, say 4.0, 7.0 9.0. I'm doing this kind of wok for the first time. Could you suggest me a user-friendly software, tutorials and tips. Sincere thanks and appreciation. John
Re: [ccp4bb] Electostatic surface at various pH
Hi John, I would probably use Pdb2pqr, to assign charges and radii for the atoms at different pHs, and then APBS integrated in Pymol software for visualization: http://kryptonite.nbcr.net/pdb2pqr/ http://www.poissonboltzmann.org/apbs/ Hope this helps, Florian On Aug 19, 2011, at 2:35 PM, john peter wrote: Hi All, Apologies for this slight off topic, however this could very well be the best bulletin board to seek help. I need to calculate the electrostatic surface and make surface figures for a protein at various pHs, say 4.0, 7.0 9.0. I'm doing this kind of wok for the first time. Could you suggest me a user-friendly software, tutorials and tips. Sincere thanks and appreciation. John
