Re: [ccp4bb] about anisotrophic diffraction
Ji, probably the oil doesn't work and the glycerol gets you into problems with phase separation in 2.5 M AmS I recommend sucrose or other sugars with AmS - 20-22% (w/vol) should do and you get no phase separation At the same time you may need to increase the AmS considerably - often the protein solubility in AmS increases significantly in the presence of cosolvents. I have previously had success with crystals grown in 1.9 M AmS, that were cryoprotected by gradual transfer to 2.6 M AmS/20% sucrose. The drops were then equilibrated against 3.5M AmS for a couple of hours (dehydration) - without the final step in place (dehydration) the crystal diffraction would be anisotropic at 3.5 Å resolution rather than isotropic at 2.7 Å which was also the full potential revealed from capillary-mounted crystals. Poul On 24/06/2008, at 02.14, Ji lee wrote: Dear, I have a crystal diffracted anisotrophically. I tested with a few different cryo conditions like oil, glycerol in different concentration to get a better data but these conditions didn't help any. Using capillary method improved the diffraction (isotrophic) but the crystal couldn't survive during the data collection. Is there any methods or cryo conditions I can use to improve my diffraction data? This crystal grew in 2.5M Ammonium Sulfate. Thank you so much. Ji
Re: [ccp4bb] about anisotrophic diffraction
You could use salts such as LiSO4 for cryo-protection (also amm sulphate with small crystals has worked to a degree with us, when nothing else worked, also 1.6-1.7 M amm. sulphate was exchangable to 40% PEG 400 (very quick), although with not so great results...). which oil did you try? that would make a lot of difference.. (see also the recent discussion on LN2/propane etc and how to freeze) hth, Tommi Quoting Ji lee [EMAIL PROTECTED]: Dear, I have a crystal diffracted anisotrophically. I tested with a few different cryo conditions like oil, glycerol in different concentration to get a better data but these conditions didn't help any. Using capillary method improved the diffraction (isotrophic) but the crystal couldn't survive during the data collection. Is there any methods or cryo conditions I can use to improve my diffraction data? This crystal grew in 2.5M Ammonium Sulfate. Thank you so much. Ji -- Tommi Kajander, Ph.D. Macromolecular X-ray Crystallography Research Program in Structural Biology and Biophysics Institute of Biotechnology P.O. Box 65 (Street address: Viikinkaari 1, 4th floor) University of Helsinki FIN-00014 Helsinki, Finland Tel. +358-9-191 58903 Fax +358-9-191 59940
[ccp4bb] Michele Cianci is out of office.
I will be out of the office starting 24.06.2008 and will not return until 25.06.2008. I maybe able to reply to your message from the 03/06 till the 11/06. There will be no replies from the 12/06 till the 23rd/06. Apologies for any inconvenience. PS: Dear ccp4bber's, it seems that my auto-replies go where they shouldn't ... I will try to find out what is happening... meanwhile second round of apologies...
Re: [ccp4bb] about anisotrophic diffraction
Hi Ji, Our lab has had good luck using sodium malonate to cryoprotect salt- grown crystals. See: Acta Cryst. (2003). D59, 2356-2358 Malonate: a versatile cryoprotectant and stabilizing solution for salt- grown macromolecular crystals T. Holyoak, T. D. Fenn, M. A. Wilson, A. G. Moulin, D. Ringe and G. A. Petsko Best, Wally On Jun 24, 2008, at 2:14 AM, Ji lee wrote: Dear, I have a crystal diffracted anisotrophically. I tested with a few different cryo conditions like oil, glycerol in different concentration to get a better data but these conditions didn't help any. Using capillary method improved the diffraction (isotrophic) but the crystal couldn't survive during the data collection. Is there any methods or cryo conditions I can use to improve my diffraction data? This crystal grew in 2.5M Ammonium Sulfate. Thank you so much. Ji Walter R.P. Novak, Ph.D. Postdoctoral Fellow Rosenstiel Basic Medical Research Center Brandeis University 415 South St. MS 029 Waltham, MA 02454-9110 Phone: (781) 736-4944 Fax: (781) 736-2405
[ccp4bb] Summary sedimentation coefficient calculation programs
Dear All, After my enquiry a few days ago and some helpful responses, we are now aware of two programs to calculate solution properties from atomic structures (pdb files). HYDROPRO (available for linux and windows): http://leonardo.fcu.um.es/macromol/programs/hydropro/hydropro.htm ref: J. Garcia de la Torre, M.L. Huertas and B. Carrasco, Calculation of hydrodynamic properties of globular proteins from their atomic- level structure. Biophys. J. 78, 719-730 (2000) SOMO (linux only): http://somoauc.googlepages.com we used HYDROPRO in the end, which worked ok. Note to the authors of both programs, how about a MacOSX version? another paper: Müller JJ. Prediction of the rotational diffusion behavior of biopolymers on the basis of their solution or crystal structure. Biopolymers. 1991 Feb 5;31(2):149–160. Greetings, Mark Mark J. van Raaij Dpto de Bioquímica, Facultad de Farmacia Universidad de Santiago 15782 Santiago de Compostela Spain http://web.usc.es/~vanraaij/
[ccp4bb] program for distribution of distances
Dear all, I apologize for the off-topic question. I'm looking for some software that is able to read in (small molecule) structure files (e.g. .pdb, .cif,..) and subsequently outputs a listing of bond lengths AND 'environment' distances for each atom within a certain radius. Additionally, the listing should allow to construct a distribution diagram for each atom-atom distance. I already played a bit with Vista en Mercury (CCDC), but to my knowledge it's not possible to include such 'environment' distances... Thanks a lot Kristof -- Kristof Van Hecke, PhD Biomoleculaire Architectuur Celestijnenlaan 200 F B-3001 Heverlee (Leuven) Tel: +32(0)16327477 -- Disclaimer: http://www.kuleuven.be/cwis/email_disclaimer.htm
Re: [ccp4bb] program for distribution of distances
Hi SHELX should be able to do this if you convert your co-ordinates into the appropriate format... On 24 Jun 2008, at 12:30, Eleanor Dodson wrote: It sounds like something the CCDC software might do? Eleanor DISTANG will do it for pdb input Kristof Van Hecke wrote: Dear all, I apologize for the off-topic question. I'm looking for some software that is able to read in (small molecule) structure files (e.g. .pdb, .cif,..) and subsequently outputs a listing of bond lengths AND 'environment' distances for each atom within a certain radius. Additionally, the listing should allow to construct a distribution diagram for each atom-atom distance. I already played a bit with Vista en Mercury (CCDC), but to my knowledge it's not possible to include such 'environment' distances... Thanks a lot Kristof -- Kristof Van Hecke, PhD Biomoleculaire Architectuur Celestijnenlaan 200 F B-3001 Heverlee (Leuven) Tel: +32(0)16327477 -- Disclaimer: http://www.kuleuven.be/cwis/email_disclaimer.htm Harry -- Dr Harry Powell, MRC Laboratory of Molecular Biology, MRC Centre, Hills Road, Cambridge, CB2 2QH
Re: [ccp4bb] program for distribution of distances
There is a program called XP in the Bruker SHELXTL system that (amongst many other things) does precisely that (use the ENVI instruction) taking symmetry equivalents into account. I suggest that you find the nearest small-molecule crytallographer, maybe she/he has XP (which has no relation to the much more recent Microsoft program with the same name). George Prof. George M. Sheldrick FRS Dept. Structural Chemistry, University of Goettingen, Tammannstr. 4, D37077 Goettingen, Germany Tel. +49-551-39-3021 or -3068 Fax. +49-551-39-2582 On Tue, 24 Jun 2008, harry powell wrote: Hi SHELX should be able to do this if you convert your co-ordinates into the appropriate format... On 24 Jun 2008, at 12:30, Eleanor Dodson wrote: It sounds like something the CCDC software might do? Eleanor DISTANG will do it for pdb input Kristof Van Hecke wrote: Dear all, I apologize for the off-topic question. I'm looking for some software that is able to read in (small molecule) structure files (e.g. .pdb, .cif,..) and subsequently outputs a listing of bond lengths AND 'environment' distances for each atom within a certain radius. Additionally, the listing should allow to construct a distribution diagram for each atom-atom distance. I already played a bit with Vista en Mercury (CCDC), but to my knowledge it's not possible to include such 'environment' distances... Thanks a lot Kristof -- Kristof Van Hecke, PhD Biomoleculaire Architectuur Celestijnenlaan 200 F B-3001 Heverlee (Leuven) Tel: +32(0)16327477 -- Disclaimer: http://www.kuleuven.be/cwis/email_disclaimer.htm Harry -- Dr Harry Powell, MRC Laboratory of Molecular Biology, MRC Centre, Hills Road, Cambridge, CB2 2QH
[ccp4bb] pH gradient in Mono Q
Dear All, Sorry for off-topic question. Does anyone have any experience in purifying protein using pH gradient in Mono Q column? I have been googling for a whole day, only one paper was found to mention performing pH gradient in Mono Q, but in a mixture of amine buffering species, which is a bit too complicated (J. Chromatogr. A 1164 (2007) 181 - 188. Can Tris-Cl/Tris-base or phosphate buffer give a linear pH gradient from pH 8.0 to 4.0? Is it usual to perform pH gradient in Mono Q as I don't want to ruin my Mono Q column... Any suggestions are welcome. Thanks in advance! Kind regards, Matt Matthew LH Chu PhD Student School of Pharmacy and Pharmaceutical Sciences University of Manchester
[ccp4bb] Bug/feature in Phaser 2.1.1 over solution scoring/culling (long)
This is on OSX Tiger 10.4.11 on a G5 machine. Phaser 2.1.1, CCP4 6.0.2 Is anyone else seeing the following ? In a feature that seems new-ish in Phaser, intermediate solutions get culled after translation function and before packing tests: (begin snippet) Purge solutions according to highest LLG from TFs in other spacegroups Best Space Group so far: P 61 2 2 Percent used for purge = 0.75 Top LLG value for purge = 28.8034 Mean LLG used for purge = 6.03998 Cutoff LLG used for purge = 23.1125 Number of solutions stored before purge = 33 Number of solutions stored (deleted) after purge = 0 (33) Purging of the results of the translation function in this spacegroup using the highest LLG value so far (from searches in other spacegroups) deleted all solutions (end snippet) which is all well and good, except in this particular case the solutions for P6122 have LLGs in the 14-16 range. What Phaser appears to be doing is picking up the best case LLGs (and therefore LLG cutoffs) for translation function peaks from another space group (P622), none of which passed the packing criteria, and then inappropriately applying them to subsequent trial solutions in subsequent space groups that have lower LLGs may in fact be better candidate solutions and survive the packing test. In the normal course of events, you'd hope that the best LLG corresponds to the correct solution in the correct space group, but I'll gladly concede that I'm using a marginal model with unimpressive data, and it'll probably fail anyway. This feature in Phaser would seem to potentially speed up correct solutions with good models and data when using SGALTERNATIVE ALL but may in fact make the performance worse with bad models and poor data. Unless I've missed something here, the LLG score/cutoff test needs to be based on trial solutions that have survived the packing test, not peaks from the translation function before that test. I'm using a fairly conventional script (not the GUI) in this case. # phaser EOF MODE MR_AUTO HKLIN ptcr1680_pk_truncate.mtz LABIN F=F SIGF=SIGF TITLE Just a phaser script COMPOSITION PROTEIN MW 28000 NUMBER 3 RESOLUTION 10. 3.2 SGALTERNATIVE ALL ENSEMBLE ensemble1 PDBFILE helix_16.pdb IDENT 75.0 SEARCH ENSEMBLE ensemble1 NUMBER 1 ROOT phaser1 END EOF
Re: [ccp4bb] pH gradient in Mono Q
Hey Matt, it seems to me that what you're asking for is chromatofocusing. See the official GE documentation: http://www6.gelifesciences.com/aptrix/upp00919.nsf/Content/WD:Chromatofocusin(260949098-R350) The proprietary buffers are a bit expensive, but as you found out, they're a bit complicated to make. I don't know if you'd ruin your Mono Q with a pH gradient. If in doubt, buy one of the dedicated Mono P column. Hope that helps. Andreas Matthew Chu wrote: Dear All, Sorry for off-topic question. Does anyone have any experience in purifying protein using pH gradient in Mono Q column? I have been googling for a whole day, only one paper was found to mention performing pH gradient in Mono Q, but in a mixture of amine buffering species, which is a bit too complicated (J. Chromatogr. A 1164 (2007) 181 - 188. Can Tris-Cl/Tris-base or phosphate buffer give a linear pH gradient from pH 8.0 to 4.0? Is it usual to perform pH gradient in Mono Q as I don't want to ruin my Mono Q column... Any suggestions are welcome. Thanks in advance! Kind regards, Matt Matthew LH Chu PhD Student School of Pharmacy and Pharmaceutical Sciences University of Manchester
Re: [ccp4bb] pH gradient in Mono Q
Thanks Guenter and Andreas, Yea, I have taken a look for the Mono P before, I thought the material they used in Mono P is basically the same as in Mono Q and I found the bookProtein Purification Protocols: Second Edition by Paul Cutler mentioned that phosphate buffer can also generate a continuous gradientthat's why I reckon we can also perform pH gradient in Mono Q. But I am worrying if it is a good idea to go for it, as both mono Q or mono P are quite expensive. Matt 2008/6/24 Andreas Förster [EMAIL PROTECTED]: Hey Matt, it seems to me that what you're asking for is chromatofocusing. See the official GE documentation: http://www6.gelifesciences.com/aptrix/upp00919.nsf/Content/WD:Chromatofocusin(260949098-R350)http://www6.gelifesciences.com/aptrix/upp00919.nsf/Content/WD:Chromatofocusin%28260949098-R350%29 The proprietary buffers are a bit expensive, but as you found out, they're a bit complicated to make. I don't know if you'd ruin your Mono Q with a pH gradient. If in doubt, buy one of the dedicated Mono P column. Hope that helps. Andreas Matthew Chu wrote: Dear All, Sorry for off-topic question. Does anyone have any experience in purifying protein using pH gradient in Mono Q column? I have been googling for a whole day, only one paper was found to mention performing pH gradient in Mono Q, but in a mixture of amine buffering species, which is a bit too complicated (J. Chromatogr. A 1164 (2007) 181 - 188. Can Tris-Cl/Tris-base or phosphate buffer give a linear pH gradient from pH 8.0 to 4.0? Is it usual to perform pH gradient in Mono Q as I don't want to ruin my Mono Q column... Any suggestions are welcome. Thanks in advance! Kind regards, Matt Matthew LH Chu PhD Student School of Pharmacy and Pharmaceutical Sciences University of Manchester -- Matthew LH Chu PhD Student School of Pharmacy and Pharmaceutical Sciences University of Manchester
[ccp4bb] COURSE ANNOUNCEMENT - BIOCRYS2008 - REMINDER
On behalf of the organizers, Maria Armenia Carrondo and Thomas R. Schneider COURSE ANNOUNCEMENT - BIOCRYS 2008 Fundamentals of Modern Methods in Biocrystallography - 'What you always wanted to know about crystallography but never dared to ask' http://biocrys.itqb.unl.pt 4th - 11th October 2008 at the Instituto de Tecnologia Química e Biológica, Oeiras, Portugal. The topics of the second edition of this course will run from fundamentals such as symmetry, point groups and crystal systems, basic diffraction physics, reciprocal space and the Ewalds sphere, radiation damage, data processing, structure factors, Patterson function to modern methodologies including molecular replacement, SAD, MAD, MIR and maximum likelihood phasing, density modification, refinement, model building, twinning and structure validation. The course will be organized with lectures in the mornings and interactive practicals and tutorials in the afternoons. Evening lectures on current topics will be presented by the invited speakers. Instructors and speakers (confirmed): M. Archer, I. Bento, N. Berrow, G. Bunkoczi, M. Coll, K. Cowtan, Z. Dauter, D. de Sanctis, P. Donner, C. Frazão, E. Garman, C. Hermes, E. Hough, G. Leonhard, A. Leslie, P. Matias, A. Perrakis, T. Schneider, C. Vonrhein. 36 participants will be selected with preference to scientists at the beginning of their crystallographic career and to those from Europe. A registration fee of 500 Euro for academic and of 1000 Euro for not academic applicants is requested for full board and accommodation. Selected applicants will have to pay the registration fee by bank transfer before arrival. A very limited number of grants may be available to partially cover the registration fees and will be paid upon students arrival. For application, please please fill the form on the web page http://www.embl-hamburg.de/workshops/2008/BIOCRYST08/ by the 15th of July For more information visit the course web page http://biocrys.itqb.unl.pt -- Daniele de Sanctis, PhD Homo sum humani nil alienum a me puto __ phone ++ 351 21 4469 662 fax ++ 351 21 4433 664 e-mail [EMAIL PROTECTED], [EMAIL PROTECTED] Mailing address: ITQB, Av. República, Apartado 127 2781-901 Oeiras Portugal
Re: [ccp4bb] pH gradient in Mono Q
Matthew, You're not going to ruin your column, but you won't get great performance either. Elution by pH change is a very common method, but getting a really linear pH gradient is very hard. The Mono Q matrix is a strong anion exchanger, meaning that it is insensitive to pH changes, i.e., you can't titrate it smoothly with acid or base. DEAE resins, which are weak anion exchangers, have a nice pH titration curve and lend themselves better to elution by pH change. This is the reason chromatofocusing is not a commonly used method, and its expensive. Andreas has pointed you in the general direction for chromatofocusing, but there is a poor man's way to do it. We use this method a lot, and the key is using a weak ion exchanger (like DEAE or CM) and a mix of buffers with pKas that span the titration range you want to exploit. Remember, you actually want to titrate the resin with the buffer: as the pH shifts away from the pKa of one buffer component, it moves into the buffering range of the other. If you do it correctly, you get a nice, flatter titration curve from the resin, which spreads out the release of the proteins. We have used a mixture of Tris and Bis-Tris-Propane with a HiTrap-DEAE or Sepharose- DEAE FF columns. Hope this helps, Michael R. Michael Garavito, Ph.D. Professor of Biochemistry Molecular Biology 513 Biochemistry Bldg. Michigan State University East Lansing, MI 48824-1319 Office: (517) 355-9724 Lab: (517) 353-9125 FAX: (517) 353-9334Email: [EMAIL PROTECTED] On Jun 24, 2008, at 12:53 PM, Matthew Chu wrote: Dear All, Sorry for off-topic question. Does anyone have any experience in purifying protein using pH gradient in Mono Q column? I have been googling for a whole day, only one paper was found to mention performing pH gradient in Mono Q, but in a mixture of amine buffering species, which is a bit too complicated (J. Chromatogr. A 1164 (2007) 181 - 188. Can Tris-Cl/Tris-base or phosphate buffer give a linear pH gradient from pH 8.0 to 4.0? Is it usual to perform pH gradient in Mono Q as I don't want to ruin my Mono Q column... Any suggestions are welcome. Thanks in advance! Kind regards, Matt -- -- Matthew LH Chu PhD Student School of Pharmacy and Pharmaceutical Sciences University of Manchester -- --
Re: [ccp4bb] pH gradient in Mono Q
Matt, there should be also a material called PBE94 for pH range 9 to 4 (and another one called Pharmalyte for high pH) to pack your own column. It worked pretty well for our protein and is not in the high price region of the MonoP column. Best, Guenter Matthew Chu wrote: Thanks Guenter and Andreas, Yea, I have taken a look for the Mono P before, I thought the material they used in Mono P is basically the same as in Mono Q and I found the book Protein Purification Protocols: Second Edition by Paul Cutler mentioned that phosphate buffer can also generate a continuous gradientthat's why I reckon we can also perform pH gradient in Mono Q. But I am worrying if it is a good idea to go for it, as both mono Q or mono P are quite expensive. Matt 2008/6/24 Andreas Förster [EMAIL PROTECTED] mailto:[EMAIL PROTECTED]: Hey Matt, it seems to me that what you're asking for is chromatofocusing. See the official GE documentation: http://www6.gelifesciences.com/aptrix/upp00919.nsf/Content/WD:Chromatofocusin(260949098-R350) http://www6.gelifesciences.com/aptrix/upp00919.nsf/Content/WD:Chromatofocusin%28260949098-R350%29 The proprietary buffers are a bit expensive, but as you found out, they're a bit complicated to make. I don't know if you'd ruin your Mono Q with a pH gradient. If in doubt, buy one of the dedicated Mono P column. Hope that helps. Andreas Matthew Chu wrote: Dear All, Sorry for off-topic question. Does anyone have any experience in purifying protein using pH gradient in Mono Q column? I have been googling for a whole day, only one paper was found to mention performing pH gradient in Mono Q, but in a mixture of amine buffering species, which is a bit too complicated (J. Chromatogr. A 1164 (2007) 181 - 188. Can Tris-Cl/Tris-base or phosphate buffer give a linear pH gradient from pH 8.0 to 4.0? Is it usual to perform pH gradient in Mono Q as I don't want to ruin my Mono Q column... Any suggestions are welcome. Thanks in advance! Kind regards, Matt Matthew LH Chu PhD Student School of Pharmacy and Pharmaceutical Sciences University of Manchester -- Matthew LH Chu PhD Student School of Pharmacy and Pharmaceutical Sciences University of Manchester -- *** Priv.Doz.Dr. Guenter Fritz Fachbereich Biologie Sektion Naturwissenschaften Universitaet Konstanz http://www.biologie.uni-konstanz.de/fritz Universitaetsstrasse 10 Postfach M665 D-78457 Konstanz e-mail: [EMAIL PROTECTED] Tel. Office: +49-(0)7531 88 3205 Tel. Lab : +49-(0)7531 88 3687 Fax: +49-(0)7531 88 2966
Re: [ccp4bb] pH gradient in Mono Q
Michael, Well, why do you need to titrate the exchanger rather then the proteins themselves? MonoQ is a lot simpler to adjust pHwise, as with DEAE you actually titrate both the matrix and the proteins. Recommended buffers to use are Goods' (pKa from 8 to 6.15 at 20 °C) + acetic acid (pKa 4.76). An equimolar (50mM) mixture of these with Buffer A titrated to 8.0 and Buffer titrated to 4.0 has been shown (in my hands) to yield a very linear gradient (must not be too steep, though). Matthew's question does not seem to concern chromatofocusing. Hth, Nadir -- Pr. Nadir T. Mrabet Cellular Molecular Biochemistry INSERM U-724 Nancy University, School of Medicine 9, Avenue de la Foret de Haye, BP 184 54505 Vandoeuvre-les-Nancy Cedex France Phone: +33 (0)3.83.68.32.73 Fax: +33 (0)3.83.68.32.79 E-mail: [EMAIL PROTECTED] R.M. Garavito wrote: Matthew, You're not going to ruin your column, but you won't get great performance either. Elution by pH change is a very common method, but getting a really linear pH gradient is very hard. The Mono Q matrix is a strong anion exchanger, meaning that it is insensitive to pH changes, i.e., you can't titrate it smoothly with acid or base. DEAE resins, which are weak anion exchangers, have a nice pH titration curve and lend themselves better to elution by pH change. This is the reason chromatofocusing is not a commonly used method, and its expensive. Andreas has pointed you in the general direction for chromatofocusing, but there is a poor man's way to do it. We use this method a lot, and the key is using a weak ion exchanger (like DEAE or CM) and a mix of buffers with pKas that span the titration range you want to exploit. Remember, you actually want to titrate the resin with the buffer: as the pH shifts away from the pKa of one buffer component, it moves into the buffering range of the other. If you do it correctly, you get a nice, flatter titration curve from the resin, which spreads out the release of the proteins. We have used a mixture of Tris and Bis-Tris-Propane with a HiTrap-DEAE or Sepharose-DEAE FF columns. Hope this helps, Michael // /R. Michael Garavito, Ph.D./ /Professor of Biochemistry Molecular Biology/ /513 Biochemistry Bldg. / /Michigan State University / /East Lansing, MI 48824-1319/ /Office:// //(517) 355-9724 Lab: (517) 353-9125/ /FAX: (517) 353-9334Email: [EMAIL PROTECTED] mailto:[EMAIL PROTECTED]/ // On Jun 24, 2008, at 12:53 PM, Matthew Chu wrote: Dear All, Sorry for off-topic question. Does anyone have any experience in purifying protein using pH gradient in Mono Q column? I have been googling for a whole day, only one paper was found to mention performing pH gradient in Mono Q, but in a mixture of amine buffering species, which is a bit too complicated (J. Chromatogr. A 1164 (2007) 181 - 188. Can Tris-Cl/Tris-base or phosphate buffer give a linear pH gradient from pH 8.0 to 4.0? Is it usual to perform pH gradient in Mono Q as I don't want to ruin my Mono Q column... Any suggestions are welcome. Thanks in advance! Kind regards, Matt Matthew LH Chu PhD Student School of Pharmacy and Pharmaceutical Sciences University of Manchester
[ccp4bb] Phaser nucleic acid nomenclature
To the CCP4 community, I have been trying to run PHASER to phase a nucleic acid structure by MR using a partial template model. The problem is that symmetry-related molecules generated from the output .pdb file coordinates spatially overlap with extensive clashes. I understand from the PHASER FAQ page that this is commonly due to a misalignment of the nucleic acid residue name in the .pdb file. I have tried the suggested solutions by changing to either single-letter nucleic acid nomenclature alinged to column 20 in the .pdb file or 3-letter nomenclature. Unfortunately, these suggestions have failed to correct the problem. The FAQ also indicated that this issue can be caused by an incorrect nomenclature for the trace atoms. I assume this is my problem since the aforementioned solutions did not work. I would like to confirm that my atom names are correct in terms of what PHASER expects, but I have not been able to find relevant documentation for atom naming in any of the PHASER documentation websites (i.e. linked from PHENIX, CCP4 or the PHASER homepage) or the CCP4bb archives. Could anyone indicate where I could find information regarding the nomenclature that PHASER expects for nucleic acid trace atoms (i.e. the phosphate and ribose carbon atoms)? I'm sure it's a standard nomenclature, but nothing I've tried has corrected the clashing problem, which makes me think I may have some incorrect nomenclature (e.g. 'O1P' vs. 'OP1' if the non-bridging phosphate oxygens are included). Alternatively, are there any other suggestions of possible causes or solutions to this clashing problem? Thank you very much for your time, -Andy Torelli
[ccp4bb] BIOMEDICAL CONFERENCE IN AFRICA
*The first Ghana Biomedical Convention* will take place in Accra- Ghana from 13th - 15 August 2008. Please if you are interested visit: http://www.ghanabiomedicalconvention.org/ Note that everyone is invited to attend. Sam SANBI/NBN South Africa
Re: [ccp4bb] Packing broke for 2nd molecule in phaser?
Thanks to those of you that replied. The packing bug turns out to have been observed in Phaser 1.3 with ensembles. We upgraded to Phaser 2.1 and the packing problem has disappeared. Thanks Randy. Shane Atwell Director, Crystallization SGX Pharmaceuticals 10505 Roselle Street San Diego, CA 92121 phone: (858) 228-1622 fax: (858) 457-5362 [EMAIL PROTECTED] -Original Message- From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Shane Atwell Sent: Friday, June 20, 2008 1:21 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Packing broke for 2nd molecule in phaser? I'm attempting a difficult molecular replacement using phaser and low resolution data (3.5A). There's almost certainly only one molecule in the asymmetric unit. Since the entire structure of a homologous multidomain protein doesn't work, I've broken it down in domains and have been attempting to find them sequentially. The problem is the following. The solutions with the first domain work fine, pack, have ok Z-scores etc. They look fine visually. Then when I fix the first solution(s) and attempt to find the next domain, the packing function doesn't seem to work. When I look at the solution and generate symmetry mates, the 2nd domain invariably clashes with itself (but not with the 1st, nor does the 1st with itself). I don't remember seeing a similar problem when finding a 2nd copy of the same molecule. My suspicion is that phaser is using the wrong symmetry information for the 2nd molecule. Although why would the packing for the 1st molecule relative to itself remain the same (i.e. have no clashes)? Is this a known problem? Am I doing something wrong? The script file looks like this: MODE MR_AUTO HKLIn truncated.mtz RESOLUTION 3.5 LABIN F=F SIGF=SIGF COMPOSITION PROTEIN MW 75000 NUM 1 ENSEmble mol1 PDBFile helical.pdb IDENtity 0.20 ENSEmble mol2 PDBFile Cterm.pdb IDENtity 0.27 SOLU SET RFZ=2.7 TFZ=4.9 PAK=0 LLG=32 LLG=32 SOLU 6DIM ENSE mol1 EULER 13.345 119.350 196.414 FRAC -0.14439 1.86117 0.01283 SEARCH ENSEmble Cterm NUM 1
[ccp4bb] problem about Chainsaw
Dear all, I have used chainsaw for several times and no problem happened before yesterday. When I used it last evening, the chainsaw couldn't run as usual. I checked the log file and saw the error message as follow CHAINSAW: Chainsaw is having trouble reconciling input pdb with sequence . In fact, the pdb file I used for model was just being downloaded for PDB database. So I wonder what happened or what I should do to manage this problem. ps: To do the work by chainsaw manually is terrible . Thank you for your attention. Xiang Liu the third year Ph.D candidate lab of structural biology, college of life science Peking University, Beijing, P.R.China 100871 Tel:86-10-62755714 - 雅虎邮箱，您的终生邮箱！
[ccp4bb] cell line
Hi everyone, can anyone tell me the protocol to revive human corneal cell lineor any cell line which requires serum free media i usually used serum based media initially.later switched it to serum free media for subculturingonly in case of human corneal cell line with the help of flask coated with fibronectin, BSA, and collagen..but recently i m finding difficult to revive itit deattaches after 1 or 2 days..what might be the reason? also we are unable to revive pRSV-T cell line using flask coating method. can anyone help on the same. thanks
Re: [ccp4bb] cell line
Highly recommended: Animal Cell Culture: A Practical Approach by R. Ian Freshney (Editor) Artem -Original Message- From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Exec Sent: Tuesday, June 24, 2008 10:59 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] cell line Hi everyone, can anyone tell me the protocol to revive human corneal cell lineor any cell line which requires serum free media i usually used serum based media initially.later switched it to serum free media for subculturingonly in case of human corneal cell line with the help of flask coated with fibronectin, BSA, and collagen..but recently i m finding difficult to revive itit deattaches after 1 or 2 days..what might be the reason? also we are unable to revive pRSV-T cell line using flask coating method. can anyone help on the same. thanks
[ccp4bb] Off-topic: Headhunters
Hi folks: Sorry for the off-topic nature of the following question, but I thought this question (or more specifically, answers to it) might be of general interest, given the current economic situation. Has anyone had any experience using a headhunter to find either consulting positions or other academic positions? I've got to do something to better my situation (and my wife's, who does HIV research) or I'm going to be pushing around a shopping cart with all three of my belongings on my way home from divorce court. Many thanks in advance. Bill William G. Scott Contact info: http://chemistry.ucsc.edu/~wgscott/
Re: [ccp4bb] Off-topic: Headhunters
Lets see. Bill Scott Publications in the last 5 years include (but definitely not limited to): Nature: I Plos Bio: II Chemistry Biology: I Nature: II Science: I Cell: I JMB: II And you are considering getting out of academia? What about this makes me worry? James On Jun 24, 2008, at 8:09 PM, William G. Scott wrote: Hi folks: Sorry for the off-topic nature of the following question, but I thought this question (or more specifically, answers to it) might be of general interest, given the current economic situation. Has anyone had any experience using a headhunter to find either consulting positions or other academic positions? I've got to do something to better my situation (and my wife's, who does HIV research) or I'm going to be pushing around a shopping cart with all three of my belongings on my way home from divorce court. Many thanks in advance. Bill William G. Scott Contact info: http://chemistry.ucsc.edu/~wgscott/ -- James Stroud UCLA-DOE Institute for Genomics and Proteomics Box 951570 Los Angeles, CA 90095 http://www.jamesstroud.com
