Re: [ccp4bb] refmac5 MMA bug
Hi Ed, This is a 'compatability' option in Refmac that internally renames atoms. If you comment out 'MMA .C7 CM' in your mon_lib_list.cif file, the problem will disappear. Cheers, Robbie Date: Sun, 10 Feb 2013 23:35:25 -0500 From: epozh...@umaryland.edu Subject: [ccp4bb] refmac5 MMA bug To: CCP4BB@JISCMAIL.AC.UK I see a strange issue with a model that includes O1-methyl-mannose (three letter code MMA). Basically, refmac fails and says that C7 is missing in the model while CM is absent from the library. The problem is that there is no CM atom in the pdb file, while C7 is right there. This happens with Refmac_5.7.0029, and I see no obvious issues with the corresponding cif-file in the monomer library. -- Oh, suddenly throwing a giraffe into a volcano to make water is crazy? Julian, King of Lemurs
Re: [ccp4bb] protein crystals or salt crystals
Dear Amro, What you could try is this. Make a solution of 0.5 % (w/v) commassie brilliant blue in 10% (v/v) ethanol in water. Pipet 1 ul of this into your drop and close the cover slip. If the crystals are protein, they should turn blue after some time (typically 30 mins). Salt crystals will not turn blue as they are not stained by commassie. You could also try using Hampton's Izit crystal dye for this, but the problem I have faced with it is that the izit itself crystallizes (gives lovely blue crystals) under certain buffer conditions. cheers Ganesh Hallo my colleagues. i hope every one doing ok . i did screening since two weeks . i noticed today this crystals. i don`t know either it salt or protein crystal . my protein has zero tryptophan so i could distinguish by UV camera. the condition was conditions: 0.1M SPG buffer pH 8 and 25%PEG 1500. in addition to Nickle chlorid 1mM. best regards Amr
Re: [ccp4bb] protein crystals or salt crystals
Amro Here is an extract from our paper, describing a method that is almost infallible, and not too hard to do if you're organized. It can never give false positives and (in the 3 cases we looked at it) only gave false negatives when there was heavy precipitate in the drop. Best wishes, Patrick Ref: Patrick D. Shaw Stewart, Stefan A. Kolek, Richard A. Briggs, Naomi E. Chayen and Peter F.M. Baldock. 'Getting the most out of the random microseed matrix-screening method in protein crystallization'. Cryst. Growth Des., 2011, 11 (8), pp 3432–3441. On-line athttp://pubs.acs.org/doi/abs/10.1021/cg2001442. In the cases of crystals of the proteins concanavalin A, trypsin, and thaumatin, we used an interesting novel method of making the distinction, which is a modification of the method of Pusey et al.17 We covalently labeled 50 μL aliquots of the proteins with the fluorescent dye DyLight 350 NHS Ester (from Thermo), following the manufacturer’s instructions except that we used higher protein concentrations (30 mg/mL for trypsin and concanavalin A, 36 mg/mL for xylanase). We added 20 nL samples of labeled protein to wells containing putative protein crystals after the crystals had grown. We photographed crystals in a darkroom by illuminating with the UV Pen-280 or with an FL4BLB UV lamp (Luxina), which has a peak wavelength of 370 nm. As shown in Figure 2, crystals fluoresced brightly and were unambiguously identified as protein rather than salt. (The DyLight kits are very easy to use because all resins, columns, etc. are provided. We chose the label that is excited at 350 nm because it is not necessary to use a filter since most cameras have built-in UV filters.) The advantages of the method are (1) since it allows protein to be seen directly, it does not give false positives or negatives (except when the drop contains a lot of precipitate, see below). (2) It cannot interfere with the crystallization process. (3) Labeled protein need only be prepared if crystal identification by other methods fails; (4) even needles and small crystals can be identified. The method does not work well when the drop contains a lot of protein precipitate, which may absorb the labeled protein before it can reach the crystals. Note also that protein sometimes coats salt crystals in crystallization experiments, giving a superficially similar appearance. Such cases can, however, easily be distinguished by comparing UV images with visible light images because the protein coating is outside the salt crystal. (17) Pusey, M.; Forsythe, E.; Achari, A. Methods Mol. Biol. 2008, 426, 377–385. On 11 February 2013 09:37, Ganesh Natrajan ganesh.natra...@ibs.fr wrote: Dear Amro, What you could try is this. Make a solution of 0.5 % (w/v) commassie brilliant blue in 10% (v/v) ethanol in water. Pipet 1 ul of this into your drop and close the cover slip. If the crystals are protein, they should turn blue after some time (typically 30 mins). Salt crystals will not turn blue as they are not stained by commassie. You could also try using Hampton's Izit crystal dye for this, but the problem I have faced with it is that the izit itself crystallizes (gives lovely blue crystals) under certain buffer conditions. cheers Ganesh Hallo my colleagues. i hope every one doing ok . i did screening since two weeks . i noticed today this crystals. i don`t know either it salt or protein crystal . my protein has zero tryptophan so i could distinguish by UV camera. the condition was conditions: 0.1M SPG buffer pH 8 and 25%PEG 1500. in addition to Nickle chlorid 1mM. best regards Amr -- patr...@douglas.co.ukDouglas Instruments Ltd. Douglas House, East Garston, Hungerford, Berkshire, RG17 7HD, UK Directors: Peter Baldock, Patrick Shaw Stewart http://www.douglas.co.uk Tel: 44 (0) 148-864-9090US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36
[ccp4bb] PhD positions in crystallography - Stockholm University
The research group of Dr Martin Högbom, in the Department of Biochemistry and Biophysics at Stockholm University has two PhD student positions currently available, as part of our quarterly intake of new students. Project title: Membrane protein structural biochemistry The inner environment the living cell is controlled by protein components inserted into the membrane. Integral membrane proteins perform a remarkable number of cellular processes, including transport of small molecules and ions, energy transduction, interaction of cells with other cells or pathogens, signaling and enzymatic reactions. Membrane proteins make up more than half of all drug targets. The biomedical importance of membrane proteins is in sharp contrast to the available structural information: structures of soluble proteins outnumber those of membrane proteins by 100:1. In this project we aim to determine the structure and function of a number of medically and scientifically important membrane proteins as well as develop methodology to make membrane protein structural biology more efficient. Our main method is X-ray crystallography complemented with protein design, enzymatic assays and a variety of spectroscopic techniques. The ideal candidate has a strong background and keen interest in Biochemistry, Biophysics and/or Structural biology and is familiar with basic molecular biology and protein production/purification methods. Enthusiasm for scientific discovery and problem solving skills are key personal traits required for this position. Project title: The amazing chemistry of metalloproteins It is estimated that almost half of all enzymes utilize metal cofactors for their function, for example the respiratory complexes and the oxygen-evolving photosystem II, the most fundamental requirements for aerobic life as we know it. Before a metalloprotein can function, the correct metal has to be bound and its reactivity tuned to carry out the appropriate chemical reaction while avoiding harmful side reactions. The principles of metal affinity, specificity and tuning, as well as the interplay between these is fundamentally important, but only poorly understood. This is what we address in the proposed project. We study a number of proteins that utilize metals to perform very challenging chemical reactions such as oxygen activation, lipid oxidation and ribonucleotide reduction. The ideal candidate has a strong background and keen interest in Biochemistry, Biophysics and/or Structural biology and is familiar with basic molecular biology and protein production/purification methods. Enthusiasm for scientific discovery and problem solving skills are key personal traits required for this position. Online information about the Department, eligibility of candidates, and the application procedure can be found at the following link: http://www.dbb.su.se/en/?p=about-us#positions Informal enquiries about these positions can be made to Dr Martin Högbom ( hog...@dbb.su.se ). Applications close 28th Feb, 2013. Kind regards Matthew Bennett Högbom Group Department of Biochemistry and Biophysics Stockholm University Stockholm Sweden
Re: [ccp4bb] refmac5 MMA bug
On Mon, 2013-02-11 at 09:56 +0100, Robbie Joosten wrote: This is a 'compatability' option in Refmac that internally renames atoms. If you comment out 'MMA .C7 CM' in your mon_lib_list.cif file, the problem will disappear. Robbie, thanks a lot - this fixes it. Is this still considered a bug? From what I understand, the data_comp_synonym_atom_list entry indicates that whenever MMA C7 atom is encountered, it will be internally renamed to CM. However, the $CCP4_LIB/data/monomers/m/MMA.cif should then refer to CM as well. But that cif-file still uses C7. Maybe this gets fixed in ccp4 updates, which reminds me to get that set up at last. Cheers, Ed. -- After much deep and profound brain things inside my head, I have decided to thank you for bringing peace to our home. Julian, King of Lemurs
Re: [ccp4bb] refmac5 MMA bug
Hi Ed, C7 is the correct name for the atom. Instead of commenting out the line you could swap the C7 and the CM and then Refmac would correct the atom name if it is wrong. This is of course very user friendly, but it also keeps users from using the correct atom names (similar to the nucleic acid naming problem). So I prefer causing an error message. Cheers, Robbie -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Ed Pozharski Sent: Monday, February 11, 2013 15:07 To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] refmac5 MMA bug On Mon, 2013-02-11 at 09:56 +0100, Robbie Joosten wrote: This is a 'compatability' option in Refmac that internally renames atoms. If you comment out 'MMA .C7 CM' in your mon_lib_list.cif file, the problem will disappear. Robbie, thanks a lot - this fixes it. Is this still considered a bug? From what I understand, the data_comp_synonym_atom_list entry indicates that whenever MMA C7 atom is encountered, it will be internally renamed to CM. However, the $CCP4_LIB/data/monomers/m/MMA.cif should then refer to CM as well. But that cif-file still uses C7. Maybe this gets fixed in ccp4 updates, which reminds me to get that set up at last. Cheers, Ed. -- After much deep and profound brain things inside my head, I have decided to thank you for bringing peace to our home. Julian, King of Lemurs
Re: [ccp4bb] protein crystals or salt crystals
If one tries to use a dye to determine if crystals are protein or salt, then I recommend that they use both a positive and a negative control. So have some handy salt or sugar crystals ready along with some known protein crystals to use as controls. Jim From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Ganesh Natrajan [ganesh.natra...@ibs.fr] Sent: Monday, February 11, 2013 3:37 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] protein crystals or salt crystals Dear Amro, What you could try is this. Make a solution of 0.5 % (w/v) commassie brilliant blue in 10% (v/v) ethanol in water. Pipet 1 ul of this into your drop and close the cover slip. If the crystals are protein, they should turn blue after some time (typically 30 mins). Salt crystals will not turn blue as they are not stained by commassie. You could also try using Hampton's Izit crystal dye for this, but the problem I have faced with it is that the izit itself crystallizes (gives lovely blue crystals) under certain buffer conditions. cheers Ganesh Hallo my colleagues. i hope every one doing ok . i did screening since two weeks . i noticed today this crystals. i don`t know either it salt or protein crystal . my protein has zero tryptophan so i could distinguish by UV camera. the condition was conditions: 0.1M SPG buffer pH 8 and 25%PEG 1500. in addition to Nickle chlorid 1mM. best regards Amr
[ccp4bb] Recent IUPAC definition of the hydrogen bond
Hi, I wonder how much of an impact should there be on structure refinement, validation and, last but not least, molecular modeling, if we take into account the recent (2011) re-definition of the hydrogen bond made by the IUPAC. Ref: Elangannan Arunan, Gautam R. Desiraju, Roger A. Klein, Joanna Sadlej, Steve Scheiner, Ibon Alkorta, David C. Clary, Robert H. Crabtree, Joseph J. Dannenberg, Pavel Hobza, Henrik G. Kjaergaard, Anthony C. Legon, Benedetta Mennucci, and David J. Nesbitt Pure Appl. Chem., Vol. 83, No. 8, pp. 1619–1636, 2011. doi:10.1351/PAC-REP-10-01-01 © 2011 IUPAC, Publication date (Web): 8 July 2011 Defining the hydrogen bond: An account (IUPAC Technical Report)* Thanks for your input and interest. Greetings, Nadir Mrabet -- Pr. Nadir T. Mrabet Structural Molecular Biochemistry N-gere - INSERM U-954 University of Lorraine, Nancy School of Sciences and Technologies 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: Nadir.Mrabet at univ-lorraine.fr
Re: [ccp4bb] how to update phenix
-BEGIN PGP SIGNED MESSAGE- Hash: SHA1 Dear Bill, I disagree to your criticism. From http://www.ccp4.ac.uk/ccp4bb.php: CCP4bb is an electronic mailing list intended to host discussions about topics of general interest to macromolecular crystallographers.[...] Personally I am only subscribed to three mailing lists and I refrain from subscribing to more which is one of the reasons why I welcome the liberal topic description of the ccp4bb. Cheers, Tim On 02/10/2013 06:20 PM, William G. Scott wrote: On Feb 10, 2013, at 8:23 AM, LISA science...@gmail.com wrote: Hi all, My mac has the old version of phenix. How can i update to the new verison? Should I delete the old version and download the new version to install as the fist time ? Thanks lisa You can delete it and download a new version, or simply keep both. phenix has version labels on their binaries, for the enjoyment of those who use shell auto-completion. e.g.: fennario-% phenix.refine external command phenix.refine phenix.refine_1.8.1-1168 BTW, there is also a phenix bb. Asking about this here is kind of like asking my wife what I should get my (purely hypothetical) mistress for valentine's day. - -- - -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A -BEGIN PGP SIGNATURE- Version: GnuPG v1.4.12 (GNU/Linux) Comment: Using GnuPG with Mozilla - http://enigmail.mozdev.org/ iD8DBQFRGSZyUxlJ7aRr7hoRAokcAJ9u49SQJlPGpwn1oUDq5a1NuXkKIQCffCqH nEahBP42PZl763fdhR0NG2U= =ORTo -END PGP SIGNATURE-
Re: [ccp4bb] how to update phenix
On Mon, Feb 11, 2013 at 12:12 PM, Tim Gruene t...@shelx.uni-ac.gwdg.de wrote: -BEGIN PGP SIGNED MESSAGE-- Hash: SHA1 Dear Bill, I disagree to your criticism. From http://www.ccp4.ac.uk/ccp4bb.php: CCP4bb is an electronic mailing list intended to host discussions about topics of general interest to macromolecular crystallographers.[...] Personally I am only subscribed to three mailing lists and I refrain from subscribing to more which is one of the reasons why I welcome the liberal topic description of the ccp4bb. So the more appropriate analogy would be asking my mistress what to get my wife for V-day. Cheers, Tim On 02/10/2013 06:20 PM, William G. Scott wrote: On Feb 10, 2013, at 8:23 AM, LISA science...@gmail.com wrote: Hi all, My mac has the old version of phenix. How can i update to the new verison? Should I delete the old version and download the new version to install as the fist time ? Thanks lisa You can delete it and download a new version, or simply keep both. phenix has version labels on their binaries, for the enjoyment of those who use shell auto-completion. e.g.: fennario-% phenix.refine external command phenix.refine phenix.refine_1.8.1-1168 BTW, there is also a phenix bb. Asking about this here is kind of like asking my wife what I should get my (purely hypothetical) mistress for valentine's day. - -- - -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A -BEGIN PGP SIGNATURE- Version: GnuPG v1.4.12 (GNU/Linux) Comment: Using GnuPG with Mozilla - http://enigmail.mozdev.org/ iD8DBQFRGSZyUxlJ7aRr7hoRAokcAJ9u49SQJlPGpwn1oUDq5a1NuXkKIQCffCqH nEahBP42PZl763fdhR0NG2U= =ORTo -END PGP SIGNATURE-
Re: [ccp4bb] how to update phenix
Dear Bill, whatever analogy you want to draw, you are getting yourself into serious trouble here However, regarding cheating, did anybody read the latest publication of Bernhard Rupp and co-workers: Pozharski, E. et al., Acta Cryst. (2013). D69, 150-167 http://journals.iucr.org/d/issues/2013/02/00/wd5191/index.html There seems to be a serious issue here. Regards, Yves -- Prof. Yves Muller Phone: +49-(0)-9131-8523082, 8523081 Lehrstuhl fuer BiotechnikFAX: +49-(0)-9131-8523080 www.biologie.uni-erlangen.de/biotechnik Department Biologie Friedrich-Alexander-Universität, Erlangen-Nuernberg Im IZMP, Henkestrasse 91, D-91052 Erlangen
Re: [ccp4bb] Self rotation function interpretation
Emma, You need to provide more information. Luckily, I222 is orthorhombic, so a* is along a, b* is along b, and c* is along c. This makes things so much easier to interpret. I hope the plotting conventions you used are typical: b along +y (North pole), a along +x (to the right), and c perpendicular to the page. Let us also assume that you have contours at every 10% of the origin peak height. If so, my quick assessment is that there is not much. At each of the crystallographic axes in the chi=180 map, you see an origin peak arising from the crystallographic 2-fold symmetry; the mirror symmetry is expected with orthorhombic space groups. At ~45 degrees in the ac-plane there is a small peak that is ~40% of the origin peak. An interpretation is that there may be 2-fold NCS, with the axis ~45 away from the crystallographic a- and c-axes, but the noise across the map is kind of high. However, without more details about how you set the SRF up (data completeness, resolution, etc.), matthew's coefficient, and what you expect to see, little more can be done. Good luck, Michael R. Michael Garavito, Ph.D. Professor of Biochemistry Molecular Biology 603 Wilson Rd., Rm. 513 Michigan State University East Lansing, MI 48824-1319 Office: (517) 355-9724 Lab: (517) 353-9125 FAX: (517) 353-9334Email: rmgarav...@gmail.com On Feb 9, 2013, at 11:00 PM, Emma Littlejohn wrote: Hi all, I have generated a self rotation function using MOLREP for a low res data set which indexed in space group I222 (attached). I am hoping it may provide some information on the oligomeric state of the protein. I am new to analysing SF and was wondering if anyone can help with the interpretation or has any tips that might be able to help me shed some light on what the SF is revealing? Thanks in advance. Emma SF.jpg
[ccp4bb] Which program sequence to use in transforming from P1 to orthorhombic?
Hi all, I've downloaded a structure factor file from the PDB that presents itself as being triclinic. It contains F, sig(F), and Rfree only. The P1-ness of this structure is dubious, however. Pointless is 99.6% sure it's orthorhombic and puts out an mtz file in P212121 containing I SIGI BATCH M/ISYM where the batch numbers are all 1 and ISYM runs from 1 to 8. So far so good, but now I'm stuck. I can't persuade Scala or Aimless to merge the symmetry mates and report a merging R factor.Is there a trick to this? Some other program sequence? Ethan -- Ethan A Merritt Biomolecular Structure Center, K-428 Health Sciences Bldg University of Washington, Seattle 98195-7742
Re: [ccp4bb] Which program sequence to use in transforming from P1 to orthorhombic?
Ethan, The last time I attempted similar things, I had to run rotaprep to convince scala of using most things that did not come directly out of mosflm, but that was before the pointless days. As the reflections are already scaled in P1, I would consider it safe to rely on the Pointless Rmerge -- but that's just a guess (and you can't do much with the data downstream). I would assume sftools might be able to merge the reindexed file output by pointless. Nevertheless, if I were faced with the same problem nowadays, I would convert to a shelx hkl file and use xprep for the merging and statistics -- that's painless. Cheers, Jens On Mon, 2013-02-11 at 13:56 -0800, Ethan Merritt wrote: Hi all, I've downloaded a structure factor file from the PDB that presents itself as being triclinic. It contains F, sig(F), and Rfree only. The P1-ness of this structure is dubious, however. Pointless is 99.6% sure it's orthorhombic and puts out an mtz file in P212121 containing I SIGI BATCH M/ISYM where the batch numbers are all 1 and ISYM runs from 1 to 8. So far so good, but now I'm stuck. I can't persuade Scala or Aimless to merge the symmetry mates and report a merging R factor.Is there a trick to this? Some other program sequence? Ethan
