Re: [ccp4bb] Phenix version 1.6.1 released
Dear Paul, this is very interesting! From the list of changes, it appears that in version 1.6.1, you use a similar idea to implement hydrogen bonds via DSSP during refinement that I used to stabilize the 4.3 A refinement of Pol II in complex with TFIIB [1]: CHANGE LOG for PHENIX distribution == Version 1.6.1 = snip - phenix.refine - much faster rotamer fixing - initial implementation of secondary structure restraints (hydrogen bonds) - automatic assignment of sec. str. using KSDSSP (from UCSF CGL) - proper treatment of charges on metal ions for scattering factors /snip Could you please give more details of how this is implemented? What are your hydrogen bond target distances and sigmas? Do you update this list during refinement? I used a simple list of additional 2.9 A target-bond-distances between N and O with a target sigma of 0.05 A. This list was determined with DSSP and a self-made Fortran95 program using a user-defined energy-threshold prior to refinement and was kept constant during refinement. Personally, I think, using secondary structure hydrogen bonds should be an option in every refinement program, especially at lower resolution!!! The BUSTER Wiki describes the procedure that I used. For REFMAC, I haven't seen anything similar, yet. Very exciting! Best regards, Dirk. [1] Nature 462, 323-330, (2009) Am 31.03.10 04:41, schrieb Paul Adams: The Phenix developers are pleased to announce that version 1.6.1 of Phenix is now available. Binary installers for Linux, and Mac OSX platforms are available at the download site: http://phenix-online.org/download/ Just some of the new features in this version (1.6.1) are: - Fast automated rotamer fixing in phenix.refine - Use of atomic charge in calculation of scattering factors - New Graphical User Interfaces for phenix.superpose_pdbs, phenix.get_cc_mtz_mtz, phenix.get_cc_mtz_pdb, and phenix.fmodel - Consolidated validation tool in GUI - New map calculation tool, phenix.maps (also used in GUI) - Rapid loading of structures in GUI selection editor - Improved geometry restraints to maintain amino acid rotameric states - Alpha version of secondary structure restraints - Automated secondary structure analysis with KSDSSP (code from UCSF CGL) For a full list of changes see: http://www.phenix-online.org/documentation/CHANGES Please note that there is a new publication that should be used to cite use of Phenix: PHENIX: a comprehensive Python-based system for macromolecular structure solution. P. D. Adams, P. V. Afonine, G. Bunkóczi, V. B. Chen, I. W. Davis, N. Echols, J. J. Headd, L.-W. Hung, G. J. Kapral, R. W. Grosse-Kunstleve, A. J. McCoy, N. W. Moriarty, R. Oeffner, R. J. Read, D. C. Richardson, J. S. Richardson, T. C. Terwilliger and P. H. Zwart. Acta Cryst. D66, 213-221 (2010). Full documentation is available here: http://www.phenix-online.org/documentation/ There is a Phenix bulletin board: http://www.phenix-online.org/mailman/listinfo/phenixbb/ Please consult the installer README file or online documentation for installation instructions. Direct questions and problem reports to the bulletin board or: h...@phenix-online.org and b...@phenix-online.org Commercial users interested in obtaining access to Phenix should visit the Phenix website for information about the Phenix Industrial Consortium. The development of Phenix is principally funded by the National Institute of General Medical Sciences (NIH) under grant P01-GM063210. We also acknowledge the generous support of the members of the Phenix Industrial Consortium. -- *** Dirk Kostrewa Gene Center, A 5.07 Ludwig-Maximilians-University Feodor-Lynen-Str. 25 81377 Munich Germany Phone: +49-89-2180-76845 Fax:+49-89-2180-76999 E-mail: kostr...@genzentrum.lmu.de WWW:www.genzentrum.lmu.de ***
[ccp4bb] Postdoctoral position at Washington University in St. Louis
POSTDOCTORAL POSITION IN STRUCTURAL BIOLOGY WASHINGTON UNIVERSITY SCHOOL OF MEDICINE, ST. LOUIS MO An NIH-funded Postdoctoral position is immediately available to study the structure and function of proteins involved in obstructive pulmonary diseases (asthma and COPD). These proteins represent key therapeutic targets for the treatment of asthma and COPD. This work will be done in collaboration with individuals in the Pulmonary Division. Our lab is located in the Department of Internal Medicine (Pulmonary Division) and has state-of-the-art equipment for protein expression and purification as well as direct access to crystallization robotics in addition to instrumentation for protein characterization (CD, MALS, AUC) and probing protein-protein interactions (ITC, SPR). Instrumentation for in-house X-ray data collection is available, and we have frequent access to synchrotron sites (either directly or via remote data collection). Excellent computation facilities are available. Enthusiastic and self-motivated individuals with (or expecting) a PhD in Biochemistry or a related discipline are encouraged to apply. The ideal candidate will have extensive experience in all aspects of protein expression, purification, characterization and crystallization. Experience in X-ray crystallography and the use of biophysical methods to characterize protein-protein interactions as well as cell-binding assays would be advantageous but not required as training can be provided. Must have a strong interest in the biology of human diseases. This is an excellent position for a structural biologist wanting to learn more about chronic inflammatory diseases or (vice versa) an individual with experience in inflammatory disease research wanting to learn structural biology. Excellent oral and written communication skills are required. Interested candidates are encouraged to apply or inquire by submitting (via email) a curriculum vitae (including a summary of research experience and interests, and contact information for 2-3 references) to Tom J. Brett: tbr...@wustl.edu http://brettlab.dom.wustl.edu/ Contact Info: Tom J. Brett, PhD Assistant Professor of Medicine Division of Pulmonary and Critical Care Washington University School of Medicine Campus Box 8052, 660 S. Euclid Saint Louis, MO 63110
[ccp4bb] Staff Scientist Position in Structural Biology in Vienna
Staff scientist position is available in the research group of Kristina Djinovic-Carugo in the Department for Structural and Computational Biology, Max. F. Perutz Laboratories at the University of Vienna, Austria. Areas of the group research include structural biology of F-actin based cytoskeleton, metallo-enzymes involved in protection from oxidative damage and X-ray induced radiation damage. The Department for Structural and Computational Biology is equipped with state of the art instrumentation/facilities to carry out all steps from cloning, protein expression and purification, to X-ray diffraction, biomolecular NMR, as well as for a series of biophysical and optical spectroscopy techniques. The group has in addition access to major European synchrotron high brilliance X-ray sources. The successful candidate will help coordinate and support structural biology research in Djinovic-Carugo group, will assist in the training and supervision of undergraduate, graduate students and postdocs and will have responsibilities in lab management (equipment maintenance, safety) with help of a technical assistant. The staff scientist will be involved in (but not limited to) conducting laboratory experiments related to all aspects and steps from sample preparation and characterisation to crystal growth, X-ray diffraction, structure solution, analysis and interpretation of scientific data, collaboration with other scientists and dissemination at external events. S/he will be able to develop her/his own project in the field of structural biology of cytoskeleton. *Qualifications and experience: *a Ph.D. in biochemistry, molecular biology, physics, chemistry or a related field is required. Minimum 4-5 years post-doctoral experience in molecular biology, protein expression in prokaryotic and eukaryotic expression systems, purification, crystallisation with robotics and macromolecular crystallography is a must. Hands-on experience with biophysical techniques and complementary structural biology methods (e.g. ITC, DLS, SLS, differential scanning fluorimetry, small angle scattering, FRET, BiFC) will be considered an advantage. Good communication skills, organizational ability, project management experience and capacity to interact with other researchers are essential. *Contract*: A 6 year contract will be offered to the successful candidate. *Starting date: *1^st October 2010 *Closing date: *30^th April 2010 * * *Interviews planned for*: end of May - beginning of June 2010. *Further information* can be obtained from Kristina Djinovic-Carugo kristina.djino...@univie.ac.at ; tel.: + 43 1 4277 52203 *To apply* for this position, please: i) E-mail a detailed CV with concise description of research experience, letter of motivation and contact information of three professional references to: karin.pfeif...@univie.ac.at mailto:karin.pfeif...@univie.ac.at, Subject: Application for Staff Scientist Position. ***AND*** * ii) Send the application to Univ. Vienna Jobcenter by: a. Registering to Univ. Vienna Jobcenter: i. http://jobcenter.univie.ac.at/sitemap40054/?L=2 */ ii. /**Registering for the first time* https://univis.univie.ac.at/ebewerber/flow/?_flowExecutionKey=_c5FF18670-23BA-6B60-B657-2E519CCA90F9_k0F33BB5C-3340-B9E6-5E8C-A3E5AE5AD2C6*//* */b. /*Submitting the application following the link and instructions at Jobcenter of the Univ. of Vienna, referring */to Job ref number: 1068./* i. *Log in* https://univis.univie.ac.at/ebewerber/flow/bew_bewerber-flow?_flowExecutionKey=_c814E228F-81C6-4BC4-AB43-A0F10B1A5846_k85E3CFA4-A274-C834-D1C1-72868BD09589 (for external applicants - Registering has to be done) -- ** Department for Structural and Computational Biology Max F. Perutz Laboratories University of Vienna Campus Vienna Biocenter 5 A-1030 Vienna Austria e-mail: kristina.djino...@univie.ac.at Phone: +43-1-4277-52203/52201 Mobile: +43-664-602 77-522 03 Fax: +43-1-4277-9522
[ccp4bb] question about the zinc binding protein
hello everyone, recently i purify a protein conteining zinc binding domain,and i want to determine its structure.i get the crystal,but poor diffraction.so i try to adding zinc into the protein to optimize the crystal,but the protein precipitate immidiately even the znic is 1 mM.BTW,we use the protein to do zinc scan,we don't find the zinc. does anyone have some advice? 2010-03-31 dengzq1987
[ccp4bb] question about zinc binding protein
hello everyone， recently i purify a protein conteining zinc binding domain,and i want to determine its structure.i get the crystal,but poor diffraction.so i try to adding zinc into the protein to optimize the crystal,but the protein precipitate immidiately even the znic is 1 mM.BTW,we use the protein to do zinc scan,we don't find the zinc. does anyone have some advice?hello everyone， recently i purify a protein conteining zinc binding domain,and i want to determine its structure.i get the crystal,but poor diffraction.so i try to adding zinc into the protein to optimize the crystal,but the protein precipitate immidiately even the znic is 1 mM.BTW,we use the protein to do zinc scan,we don't find the zinc. does anyone have some advice?
Re: [ccp4bb] Phenix version 1.6.1 released
Hi Dirk, this is very interesting! From the list of changes, it appears that in version 1.6.1, you use a similar idea to implement hydrogen bonds via DSSP during refinement that I used to stabilize the 4.3 A refinement of Pol II in complex with TFIIB [1]: (...) Could you please give more details of how this is implemented? What are your hydrogen bond target distances and sigmas? Do you update this list during refinement? In phenix.refine documentation: http://www.phenix-online.org/documentation/refinement.htm look towards the bottom of the page for secondary_structure. All the parameters are defined there. If you still have any questions, please let Nat or me know (Nat added this option so he may provide with more details). The documentation for secondary structure restraints is still missing, sorry. Hopefully Nat adds it soon. Pavel.
Re: [ccp4bb] secondary structure restraints [was: Phenix version 1.6.1 released]
On Wed, Mar 31, 2010 at 8:47 AM, Dirk Kostrewa kostr...@genzentrum.lmu.dewrote: this is very interesting! From the list of changes, it appears that in version 1.6.1, you use a similar idea to implement hydrogen bonds via DSSP during refinement that I used to stabilize the 4.3 A refinement of Pol II in complex with TFIIB [1]: . . . Could you please give more details of how this is implemented? What are your hydrogen bond target distances and sigmas? Do you update this list during refinement? We're still testing this method (thus alpha version in Paul's email); my initial experiments indicate that it may improve R-free by up to 0.5% at moderate resolution, and generally keeps R-work and R-free closer than would otherwise be the case. It can also make R-free worse, although I think this happens less frequently. I had expected this to be resolution-dependent, but I tried it on partially built structures at either 2.25A and 3.1A and the results looked similar. Part of the problem is deciding how to filter outliers; the default behavior is to throw out any bonds greater than a specified threshold (e.g. 2.5A for H-O). KSDSSP (UCSF's free clone) has some quirks with respect to helix assignment which result in incorrect bonding if you take the results as-is without filtering. However, for poor models at low resolution, leaving the outliers in may be essential. All of the restraint parameters are adjustable, but I found that 1.975A for H-O was appropriate, and I left the sigma at 0.05 but 0.02 is sometimes better. We don't update the bonds during refinement, but I suppose we could - haven't done enough testing yet to know whether this is helpful. I used a simple list of additional 2.9 A target-bond-distances between N and O with a target sigma of 0.05 A. This list was determined with DSSP and a self-made Fortran95 program using a user-defined energy-threshold prior to refinement and was kept constant during refinement. Currently PHENIX uses a 3.0A distance for N-O (sigma also 0.05), but that wasn't rigorously derived. My gut feeling is that N-O bonds are problematic, partly because the distribution of distances (in the Richardson lab's Top500 database) appears to be bimodal - I think this results from the difference between parallel and anti-parallel sheets. We probably need to account for this when calculating the bonds. I also found that the restraints usually improved R-free more when hydrogens were used, but I haven't tried anything close to a 4.3A structure yet. At that resolution, anything that keeps the helices wound probably helps. I hadn't realized that you used similar restraints in the PolII/TFIIB paper - I'd be interested in knowing more about this, since I haven't found many useful references so far. (I couldn't find the page on the BUSTER wiki, by the way.) Personally, I think, using secondary structure hydrogen bonds should be an option in every refinement program, especially at lower resolution!!! The BUSTER Wiki describes the procedure that I used. For REFMAC, I haven't seen anything similar, yet. If you don't mind using PHENIX for this: phenix.secondary_structure_restraints model.pdb format=refmac I haven't made it as far as testing the output with REFMAC, but it's there if anyone wants to try. FYI, the program is still clumsy about switching to N-O bonds when hydrogens are absent, so the argument h_bond_restraints.substitute_n_for_h=True may be necessary. -Nat
Re: [ccp4bb] question about zinc binding protein
Not strange at all: Reading from: http://www.chem.gla.ac.uk/research/groups/protein/mirror/stura/cryst/add.html Zinc acetate or sulfate 0.2-5mM It inevitably reduces protein solubility. It can act as an inhibitor. Essential for activity of various enzymes Zinc reduces protein solubility and often aggregates proteins. In a case of a zinc binding protein you may need to add 100-200 mM imidazole to control even 0.2mM concentrations of this ion. Without good control, you will end up by depleating the zinc within the precipitate and end up without zinc in your binding site. This is what you got. Enrico. On Wed, 31 Mar 2010 18:11:46 +0200, zq deng dengzq1...@gmail.com wrote: hello everyone, recently i purify a protein conteining zinc binding domain,and i want to determine its structure.i get the crystal,but poor diffraction.so i try to adding zinc into the protein to optimize the crystal,but the protein precipitate immidiately even the znic is 1 mM.BTW,we use the protein to do zinc scan,we don't find the zinc. does anyone have some advice?hello everyone, recently i purify a protein conteining zinc binding domain,and i want to determine its structure.i get the crystal,but poor diffraction.so i try to adding zinc into the protein to optimize the crystal,but the protein precipitate immidiately even the znic is 1 mM.BTW,we use the protein to do zinc scan,we don't find the zinc. does anyone have some advice? -- Enrico A. Stura D.Phil. (Oxon) ,Tel: 33 (0)1 69 08 4302 Office Room 19, Bat.152, Tel: 33 (0)1 69 08 9449 Lab LTMB, SIMOPRO, IBiTec-S, CEA Saclay, 91191 Gif-sur-Yvette Cedex FRANCE http://www-dsv.cea.fr/en/ibitecs/82 http://www.chem.gla.ac.uk/protein/mirror/stura/index2.html e-mail: est...@cea.fr Fax: 33 (0)1 69 08 90 71
[ccp4bb] B factor Coloring in Pymol
Dear Everyone Slightly off topic... I am trying to colour a structure by B factor in Pymol. More specifically I am colouring conserved residues (value in b factor). I want to use 4 colours - say white, yellow, orange, red. However it seems that Pymol now only allows 3 colours to be used - I tried the script from the Wiki site, but Pymol will not accept the 4 rgb colours. Does anyone have an idea how I can colour with 4 colours (but not rainbow too confusing). Thanks in advance for any help Best Gina Notice: This e-mail message, together with any attachments, contains information of Merck Co., Inc. (One Merck Drive, Whitehouse Station, New Jersey, USA 08889), and/or its affiliates Direct contact information for affiliates is available at http://www.merck.com/contact/contacts.html) that may be confidential, proprietary copyrighted and/or legally privileged. It is intended solely for the use of the individual or entity named on this message. If you are not the intended recipient, and have received this message in error, please notify us immediately by reply e-mail and then delete it from your system.
Re: [ccp4bb] B factor Coloring in Pymol
Hi Ron Mmmm... not really but thanks for the info:) I have my Bfactor column assigned in percent conservation so the B value ranges from say 30% to 100%. I want to colour my molecule accordingly. Pymol allows B factor colouring but seems only to allow 3 colour choices. It used to allow 4 colour choices say WYOR I was wondering if any one had an idea how I could trick Pymol into accepting say 4 colours. Sorry if I was not clearer in the previous. Thanks again advance. Gina -Original Message- From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Rob Nicholls Sent: Wednesday, March 31, 2010 3:14 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] B factor Coloring in Pymol Hi, I'm aware of two ways to do this: by writing the colour as a word, e.g: color white, (mypdbfile//A/*/*) which will colour a whole chain. Or by using rgb colours, e.g: set_color newcolor0 = [1,1,1]; color newcolor0, (mypdbfile//A/2/*) which will colour individual residues (residue 2 in this case) Pymol should accept a script containing such lines. Hope that helps, Rob On 31 Mar 2010, at 15:00, Clayton, Gina Martyn wrote: Dear Everyone Slightly off topic... I am trying to colour a structure by B factor in Pymol. More specifically I am colouring conserved residues (value in b factor). I want to use 4 colours - say white, yellow, orange, red. However it seems that Pymol now only allows 3 colours to be used - I tried the script from the Wiki site, but Pymol will not accept the 4 rgb colours. Does anyone have an idea how I can colour with 4 colours (but not rainbow too confusing). Thanks in advance for any help Best Gina Notice: This e-mail message, together with any attachments, contains information of Merck Co., Inc. (One Merck Drive, Whitehouse Station, New Jersey, USA 08889), and/or its affiliates Direct contact information for affiliates is available at http://www.merck.com/contact/contacts.html) that may be confidential, proprietary copyrighted and/or legally privileged. It is intended solely for the use of the individual or entity named on this message. If you are not the intended recipient, and have received this message in error, please notify us immediately by reply e-mail and then delete it from your system. Notice: This e-mail message, together with any attachments, contains information of Merck Co., Inc. (One Merck Drive, Whitehouse Station, New Jersey, USA 08889), and/or its affiliates Direct contact information for affiliates is available at http://www.merck.com/contact/contacts.html) that may be confidential, proprietary copyrighted and/or legally privileged. It is intended solely for the use of the individual or entity named on this message. If you are not the intended recipient, and have received this message in error, please notify us immediately by reply e-mail and then delete it from your system.
Re: [ccp4bb] B factor Coloring in Pymol
Hi Robert That seems like a great suggestion! I will give it a shot and let CCP4BB know if it works Thanks Gina -Original Message- From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Robert Campbell Sent: Wednesday, March 31, 2010 4:29 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] B factor Coloring in Pymol Hi Gina, On Wed, 31 Mar 2010 15:00:34 -0400 Clayton, Gina Martyn gina_clay...@merck.com wrote: I am trying to colour a structure by B factor in Pymol. More specifically I am colouring conserved residues (value in b factor). I want to use 4 colours - say white, yellow, orange, red. However it seems that Pymol now only allows 3 colours to be used - I tried the script from the Wiki site, but Pymol will not accept the 4 rgb colours. Does anyone have an idea how I can colour with 4 colours (but not rainbow too confusing). Say, your b-factor values range from 0-10, and your object is called protein, then you could do: color white, protein color yellow, protein b 2.5 color orange, protein b 5.0 color red, protein b 7.5 If you want to automate that process, then you need to write a script that determines, the b-factor limits for the 4 colours based on the b-factors present in your structure. Cheers, Rob -- Robert L. Campbell, Ph.D. Senior Research Associate/Adjunct Assistant Professor Botterell Hall Rm 644 Department of Biochemistry, Queen's University, Kingston, ON K7L 3N6 Canada Tel: 613-533-6821Fax: 613-533-2497 robert.campb...@queensu.ca http://pldserver1.biochem.queensu.ca/~rlc Notice: This e-mail message, together with any attachments, contains information of Merck Co., Inc. (One Merck Drive, Whitehouse Station, New Jersey, USA 08889), and/or its affiliates Direct contact information for affiliates is available at http://www.merck.com/contact/contacts.html) that may be confidential, proprietary copyrighted and/or legally privileged. It is intended solely for the use of the individual or entity named on this message. If you are not the intended recipient, and have received this message in error, please notify us immediately by reply e-mail and then delete it from your system.
[ccp4bb] Crystals Soaking in Divalent metals salts.
Dear all; I am working with protein which are supposed to be bound with Magnesium and Cobalt metals.I have now its crystals which are diffracting quite nicely.I want to know the Magnesium and Cobalt binding residues in the target protein. When I add magnesium or Cobalt salts to protein solution even 1mM about 50 percent of the protein precipitated out. When I soaked the crystals in about 10mM of the metals salt it lose diffraction. I varied the soaking time and the metal salt concentration,and even the types of metals salts as well but its seems that it does n,t work. Any idea would be highly appreciated. Bashir -- Muhammad Bashir Khan Department for Biomolecular Structural Chemistry Max F. Perutz Laboratories University of Vienna Campus Vienna Biocenter 5 A-1030 Vienna Austria Phone: +43(1)427752224 Fax: +43(1)42779522
Re: [ccp4bb] Crystals Soaking in Divalent metals salts.
On Wed, Mar 31, 2010 at 5:57 PM, Muhammed bashir Khan muhammad.bashir.k...@univie.ac.at wrote: I am working with protein which are supposed to be bound with Magnesium and Cobalt metals.I have now its crystals which are diffracting quite nicely.I want to know the Magnesium and Cobalt binding residues in the target protein. When I add magnesium or Cobalt salts to protein solution even 1mM about 50 percent of the protein precipitated out. Sometimes this doesn't matter. I used to work with a protein that partially precipitated when I added 2mM manganese (the physiological metal ion), but I just spun down the solution at top speed in a microcentrifuge and continued. The purification yield was always high enough that I still had plenty left for trays (and if you have a Mosquito robot or the equivalent, just 8 microliters is enough), and whatever stayed in solution was still easy to crystallize. You might need to re-concentrate, however. An alternate solution would be to co-purify with 1mM magnesium in all buffers (may or may not work, but it can't hurt to try). I don't know whether this is feasible using cobalt - are the metals interchangeable, or does your protein really bind two distinct sets of ions? Cobalt has the advantage of being much larger than water and has an easily measurable anomalous signal, but magnesium is usually obvious from the coordination geometry (and in my experience it tends to have a much lower B-factor too), and usually gentler towards proteins. -Nat
