Re: [ccp4bb] Crystal Imaging Systems - possibilities and recommendations
We are currently contemplating the acquisition of an automated imaging system for crystallization screen plates (96-well). Thanks to Zolt, Renaud and Catherine from Sanofi-Strasbourg, we bought last year a Formulatrix Rock Imager. The system is composed of a big box, containing your 96 wells microplates, a computer and a barcode printer, When you want to add a new plate, you define it into the software, a lot of commercials screens are already predefined. You print the barcode and put the plate in a empty place of the container, Pictures are taken on different focusing levels then merged to have a single image. Visualizing your drops is easy, full screen, and the picture is really clean, you score then with the numpad of the computer. You know instantly the condition, and you can see the growth of the crystal (or more useful, proove the dissolution of a crystal in the weekend). If you're not sure you can see and change the focusing parameters and the polarizer in live. By default the image taken is the whole drop, but when a crystal is identified you can draw a rectangle on the image and a optical zoomed image will be take at each next occurrence of the schedule. /Pros/ -Really easy to use, and a real *gain of time* and *comfort*, you can eventually access via a web browser to see and score your plates (you don't need to be on the computer who control the formulatrix). -No vibration (Pelletier). - advanced users can do sql-like request on the imaging database. /Cons/ - The graphical interface is rich and you can easily be lost at the beginning. So for large institutes, it will probably an issue, and someone who know the software will probably prepared the barcodes for everyone.. -The worst is that the temperature is regulated, but not cooled. We where expected to put it inside at 22'C and ask a 4'C temperature, it's not possible, to have a 4'C inside the container you need to put the formulatrix inside a 4'C room (or at least 8'C). - You can't (or probably in option) put inside 24 well plates, only robot's plates. Regards, -- Watier Yves PhD Student, European Synchrotron Radiation Facility (ESRF) Experiments Division / Materials Science Group ID31 high resolution powder diffraction beamline. 6 Rue Jules Horowitz, BP 220, 38043 Grenoble, Cedex 9, France. Office: 10.01.06 Tel. (+)33(0)4.76.88.29.67 Fax. (+)33(0)4.76.88.27.07 http://www.esrf.fr/UsersAndScience/Experiments/MaterialsScience/ID31/
Re: [ccp4bb] protein expression problem
I have been trying to express a rat protein in bacteria. The MBP-fusion expressed at very high level (~ 40 mg/L) while the GST-fusion and His-tag only gave inclusion bodies. The problem is that all protein runs in the void volume on a size-exclusion column (s-200, hepes, pH 7.4, 200 mM NaCl), no matter it is the intact MBP-fusion or cleaved sample. There is no Cys on this protein so there is unlikely any disulfide bond related problem. Anything I can do before I throw away this construct and try insect or mammalian cells? Thanks. First of all, using a carrying protein (like GST, MBP) can be disconcerting. These proteins are very soluble and can solubilize an insoluble protein in testing condition. So you have something soluble but your protein of interest can be misfolded or can precipitate when the carrying protein was cleaved. So keep in mind that a soluble carried protein is not always a good protein. After this consideration you have a wide range of conditions to test. - Solubility of your MBP-fusion: ultracentrifugation, thermal shift assay (in different pH, salt, salt concentration), micro-dialysis - Folding of your MBP-fusion: circular dichroism, 1D NMR (if you can, compare with MBP alone) - Aggregation/monodispersity of your MBP-fusion: DLS (in different pH, salt, salt concentration) - For gel filtration assays don't forget that MBP can dimerize The second part of your tests can be expression conditions. Sometimes low but native expression is better than high but carried or insoluble expression. - Medium - Temperature - Host cells (different E. coli, yeast, insect cells...) - Inducing strength - Co-expression with ligands or chaperones And the last but not the least part of your tests can be refolding. Inclusion body expression is the first step of your purification. If you have his-tagged protein in inclusion body a one step purification can be performed in denaturing conditions. A wide range of refolding conditions can be tested: - Flash dilution - Dialysis - Refolding by slow gradient on an Ni column (if you have an his tag) - pH, salt, detergent conditions - Chaperones OD at 340 nm can monitor the refolding efficiency. Good luck Michel
Re: [ccp4bb] CCP4 for bioinformatics?
Hi, You can use the on-line software EMBOSS. You can use it at the following site : http://bips.u-strasbg.fr/EMBOSS/ enjoy it ! Claudine Jacob Keller wrote: Dear Crystallographers, Does anyone know of a bioinformatics counterpart of ccp4? It seems like there should really be such an entity, so that folks would not have to write scripts, reinventing the wheel all of the time. I am trying right now to manipulate some sequences into various forms, and I was imagining a moleman homolog for bioinformatics (perhaps seqman?). Regards, Jacob Keller *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program Dallos Laboratory F. Searle 1-240 2240 Campus Drive Evanston IL 60208 lab: 847.491.2438 cel: 773.608.9185 email: [EMAIL PROTECTED] *** -- * Dr Claudine MAYER MCF Université Paris 6 LRMA Equipe 12 UMR S 872 Laboratoire de Bactériologie (5ème étage gauche) Faculté de Médecine PITIE-SALPETRIERE 91 bd de l'Hopital 75634 PARIS Cedex 13 tel : 01 40 77 95 56 fax : 01 45 82 75 77 mobil: 06 07 23 51 16 [EMAIL PROTECTED] *
Re: [ccp4bb] differences between Rsym and Rmerge
I thought that as author of Scala I might put in my 2 penn'th to this discussion, FWIW 1. I've never been able to find any useful distinction between Rsym Rmerge, and when filling in the PDBs request for both (undefined by them and irritatingly restricted to 0.99, at least in Autodep) I put the same number in both places. I endorse Manfred's point that the multiplicity-weighed version Rmeas aka Rrim is a better measure than Rmerge As Eleanor, pointed out, the definitions used in Scala are in a CCP4 study weekend article Acta Cryst./ (2006). D*62*, 72-82 [ doi:10.1107/S0907444905036693 ] but note that the printers managed to lose a Sqrt in the definition of Rmeas and Rpim, in the terms (n/n-1) or (1/n-1) Writing the j'th observation of reflection h as Ihj Rmerge = Sum(h) [Sum(j) [I(hj) - Ih] / Sum(hj) Ih Rmeas = Sum(h) [ Sqrt(n/(n-1)) Sum(j) [I(hj) - Ih] / Sum(hj) Ih Rpim = Sum(h) [ Sqrt(1/(n-1)) Sum(j) [I(hj) - Ih] / Sum(hj) Ih where n is the number of observation of reflection h (ie j=1,n) 2. The I/sigma definition used in Scala, labelled Mn(I/sd) in the table) is calculated as follows:- (a) apply correction Sdfac, SdB, Sdfac to the individual estimated sigma(Ihj) to get sigma'(Ihj) (b) get weighted mean for reflection h Ih and its estimated sd sigma(Ih) from sigma'(Ihj) (c) average [ih/sigma(Ih)] in resolution shells, Ih/ sigma(Ih == Mn(I/sd) Note that this is only really useful if the estimated sigmas are valid estimates of the true error, and this is very difficult to do properly. The latest pre-release versions of Scala do have a more automated estimation of the SD correction (or fudge...) factors, which I'm still trying to improve, but it is important to realise that all these statistics, including Rfactors, measure internal consistency rather than absolute accuracy. The more general point is why do we want to look at these statistics? What is the question? (i) I have several datasets from different crystals: which is the best? Judged on Rfactor, I/sigma, completeness (multiplicity improves Rmeas I/sigma). (ii) Should this dataset be thrown away? Not if it's the best you have (iii) What is the resolution of the dataset? Where should we cut it? This is a difficult question - it depends on what you are going to use it for. It is affected by anisotropy (which is treated badly or not at all by most current programs). If I add another shell of data, is it adding any useful information? However, in most cases it isn't critical except for referees (1.99Å resolution, anyone?) I could go on, but back to work trying to improve the programs ... Phil On 18 Jan 2008, at 20:10, Edwin Pozharski wrote: Chris Putnam wrote: I won't belabor this point (or defend this view) any further, though I will repeat my surprise at the lack of a clear consensus for what Rsym and Rmerge actually mean, as opposed to things like I/sigma, for example. I/sigma is also open to interpretation. Is it I/sigma or I/ sigma (averaged over all the reflection in a given resolution shell)?
Re: [ccp4bb] CCP4 for bioinformatics?
A newer one for Ruby is (of course) BioRuby, which I use for simple
manipulation of sequences or chopping up structures.
It's quite nice for this, but I don't think it's as feature-rich as
Bio{Perl,Python,Java} though.
http://dev.bioruby.org/wiki/en/?cmd=viewp=How+do+I+get+the+sequence+of+a+chain%3Fkey=PDB
http://dev.bioruby.org/wiki/en/?cmd=viewp=How+do+I+find+specific+atoms%2C+residues%2C+chains+or+models%3Fkey=PDB
http://dev.bioruby.org/wiki/en/?cmd=viewp=How+do+I+write+a+structure+file%3Fkey=PDB
Mark
On 22/01/2008, Claudine MAYER [EMAIL PROTECTED] wrote:
Hi,
You can use the on-line software EMBOSS.
You can use it at the following site :
http://bips.u-strasbg.fr/EMBOSS/
enjoy it !
Claudine
Jacob Keller wrote:
Dear Crystallographers,
Does anyone know of a bioinformatics counterpart of ccp4? It seems
like there should really be such an entity, so that folks would not
have to write scripts, reinventing the wheel all of the time. I am
trying right now to manipulate some sequences into various forms, and
I was imagining a moleman homolog for bioinformatics (perhaps
seqman?).
Regards,
Jacob Keller
***
Jacob Pearson Keller
Northwestern University
Medical Scientist Training Program
Dallos Laboratory
F. Searle 1-240
2240 Campus Drive
Evanston IL 60208
lab: 847.491.2438
cel: 773.608.9185
email: [EMAIL PROTECTED]
***
--
*
Dr Claudine MAYER
MCF Université Paris 6
LRMA Equipe 12 UMR S 872
Laboratoire de Bactériologie (5ème étage gauche)
Faculté de Médecine PITIE-SALPETRIERE
91 bd de l'Hopital
75634 PARIS Cedex 13
tel : 01 40 77 95 56
fax : 01 45 82 75 77
mobil: 06 07 23 51 16
[EMAIL PROTECTED]
*
--
Mark BROOKS
Telephone: 0169157968
Fax: 0169853715
INSTITUT de BIOCHIMIE et de BIOPHYSIQUE MOLECULAIRE et CELLULAIRE
UMR8619 - Bât 430 - Université de Paris-Sud
91405 ORSAY CEDEX
[ccp4bb] X-ray kit available
Hello, We have some X-ray kit that is now surplus to requirements. We appreciate that it is not state-of-the-art, but if anyone would like it they are very welcome to come and pick it up: The generator is a Nonius FR-591 system with a Enraf-Nonius DIP2000 detector. The MAC X-ray Optical System has double mirror focusing optics consisting of a 80mm long Pt mirror and a 160mm long Ni mirror. If you are interested, get in touch, Cheers, Nick -- NM Burton, Department of Biochemistry, University of Bristol, BS8 1TD UK [EMAIL PROTECTED] +44 117 3312149
[ccp4bb] Organic solvent resistent crystallisation plates
Dear all, we are looking to crystallise some hydrophobic peptides in different organic solvents, but find polystyrene plates are not sufficiently resistent. Also, it is not always obvious from which plastic plates are made. In other words, anyone knows of: - cleared polypropylene sitting drop plates (sealable with resistent cover slips) - cleared polypropylene Terazaki plates (we had some old ones which were resistent but then newly bought ones weren't, unfortunately we have no way to identify the supplier of the old ones) - cleared polypropylene Linbro type plates with idem microbridges (sealable with resistent cover slips)? Note: Unfortunately ordering from Hampton Research is not an option for us, too much hassle with lost international payments and in situ payment of import taxes, and absence of university support to do this for us, so if you can indicate European suppliers or resellers, great. Please reply to me personally and I will of course include non- European suppliers in the summary, which I will prepare for the list. 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/
Re: [ccp4bb] protein expression problem
I wholly agree with the below. I am not sure how well E.coli can correctly fold snaky misfolded/unfolded protein that are chaperoned by folded tags! Not to rule out that tags do it sometimes... Folded by association for insoluble proteins has often not worked well for me. Sometimes, when it 'works' for me and my colleagues, removal of the tag leads to insoluble protein/aggregation etc. I am dealing with SUMO tagged proteins that have enhanced solubility but severe degradation issues. Screening for pH, buffers and all the good-old stuff folks have suggested here is a good approach. Sometimes autoinduction protocols, which keep from 'overexpression' of protein in the cell might be an approach to explore after the above tests. Good luck! Raji First of all, using a carrying protein (like GST, MBP) can be disconcerting. These proteins are very soluble and can solubilize an insoluble protein in testing condition. So you have something soluble but your protein of interest can be misfolded or can precipitate when the carrying protein was cleaved. So keep in mind that a soluble carried protein is not always a good protein.
Re: [ccp4bb] protein expression problem
Chen and David, Before adding detergent, be forewarned that the MPB in many fusions will not bind to an amylose column in the presence of most detergents, particularly maltoside detergents. It has been the bane to us so we have engineered MBP vectors with His tags to deal with this. What you might try, as suggested, the NDSBs or the addition of glycylglycine (to make 0.5-1.0M) to the growth media just before innoculation (don't worry about sterility with good antibiotic selection; autoclaving will just make a brown mess). The glycylglycine trick can reduce aggregation. Cheers, 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 Jan 22, 2008, at 2:23 AM, David Briggs wrote: Hi Chen, You could try adding some detergent or other solubilising agent (eg NDSBs) to your buffer. Have you tried other pHs? If you are sat near to or on the pI of your protein, it will be at its least soluble and more likely to aggregate. I've had protein behave like yours at pH 7.5 but behave perfectly (i.e. monodisperse) at pH 5.5. As you can get you protein in inclusion bodies, have you considered doing an inclusion body prep (using 'bugbuster' or something similar) and then trying some refolding protocols? Jungbauer A, Kaar W. Current status of technical protein refolding.J Biotechnol. 2007 Feb 20;128(3):587-96. Some people have had success with SUMO tags as well. HTH, Cheers, David On 22/01/2008, Daniel Jin [EMAIL PROTECTED] wrote: Hi, I have been trying to express a rat protein in bacteria. The MBP- fusion expressed at very high level (~ 40 mg/L) while the GST- fusion and His-tag only gave inclusion bodies. The problem is that all protein runs in the void volume on a size-exclusion column (s-200, hepes, pH 7.4, 200 mM NaCl), no matter it is the intact MBP-fusion or cleaved sample. There is no Cys on this protein so there is unlikely any disulfide bond related problem. Anything I can do before I throw away this construct and try insect or mammalian cells? Thanks. Best, Chen Never miss a thing. Make Yahoo your homepage. -- David C. Briggs PhD Father Crystallographer http://www.dbriggs.talktalk.net AIM ID: dbassophile
[ccp4bb] Characterization of common salt crystal forms?
Salt crystals are common in macromolecular crystallography. Has anyone tried to tabulate salt crystal forms that commonly occur? I just identified a salt crystal as Mirabilite, made of Na2SO4·10H2O. The high water content makes them rather soft, and may not be recognized as salt right away. In this case, it probably happened because the buffer was made with Na·Citrate + HCl instead of citric acid, while trying to optimize conditions. So, characterization of salt crystals can help to avoid the conditions that cause them. There is probably a reasonably small number of salt crystal forms that are very common in crystallization trials. Maybe it would be useful to tabulate common salt crystals to help guide optimization experiments. Has anyone else tried to use salt crystal information beyond ensuring that it is not protein? Joe Krahn
Re: [ccp4bb] protein expression problem
Hi Chen, Since you recognize that this is a protein expression problem, the best way to get return of your investment of efforts is to get soluble expression instead of trying to solubilize the protein down stream. Many good suggestions have been proposed. I just want to add one more thing for you to try. Lower the induction temperature to 16C (or any temperature between 10-30C). With all the tricks to make protein soluble in E. coli, low induction temperature is the only universal method that works. Accelagen has made all kinds of proteins and we have systematically tried many variables. Only induction temperature matters for solubility, everything else just gives you more or less proteins. If your promoter is not tightly controlled, you may just let the E. coli grow to saturation without induction, at low temperature of course. It worked well for pGEX based vectors. Many proteins give little soluble expression in E. coli. In those cases, Baculovirus expression system is a great alternative. Once you have your gene in a transfer vector, it takes about 3 weeks to know the soluble expression level in insect cells and another 3 weeks to harvest 10 L cell pellets. Currently technology allows you to scale up to 100s L in an additional couple weeks. The material cost is actually similar to E. coli expression. It's faster than trying to figure out how to solubilize a protein. Good luck. Chun Chun Luo, Ph.D. The Protein Expert Accelagen, Inc. 11585 Sorrento Valley Road, Suite 107 San Diego, CA 92121 TeL: 858-350-8085 ext 111 Fax: 858-350-8001 mailto:[EMAIL PROTECTED] [EMAIL PROTECTED] www.accelagen.com _ From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Daniel Jin Sent: Monday, January 21, 2008 10:56 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] protein expression problem Hi, I have been trying to express a rat protein in bacteria. The MBP-fusion expressed at very high level (~ 40 mg/L) while the GST-fusion and His-tag only gave inclusion bodies. The problem is that all protein runs in the void volume on a size-exclusion column (s-200, hepes, pH 7.4, 200 mM NaCl), no matter it is the intact MBP-fusion or cleaved sample. There is no Cys on this protein so there is unlikely any disulfide bond related problem. Anything I can do before I throw away this construct and try insect or mammalian cells? Thanks. Best, Chen _ Never miss a thing. Make Yahoo http://us.rd.yahoo.com/evt=51438/*http:/www.yahoo.com/r/hs your homepage.
[ccp4bb] .cif file
Hi all I am using COOT for model building and refinement. i have to introducea ligand in my model i have downloaded .cif file from RCSB. However while importing the cif file i m getting the warning message of having No restraints in the CIF file is there any problem in the format of the following cif file data_SPM # _chem_comp.idSPM _chem_comp.name SPERMINE _chem_comp.type NON-POLYMER _chem_comp.pdbx_type HETAI _chem_comp.formula C10 H26 N4 _chem_comp.mon_nstd_parent_comp_id ? _chem_comp.pdbx_synonyms ? _chem_comp.pdbx_formal_charge0 _chem_comp.pdbx_initial_date 1999-07-08 _chem_comp.pdbx_modified_date2007-08-16 _chem_comp.pdbx_ambiguous_flag N _chem_comp.pdbx_release_status REL _chem_comp.pdbx_replaced_by ? _chem_comp.pdbx_replaces ? _chem_comp.formula_weight202.340 _chem_comp.one_letter_code ? _chem_comp.three_letter_code SPM _chem_comp.pdbx_model_coordinates_details? _chem_comp.pdbx_model_coordinates_missing_flag N _chem_comp.pdbx_ideal_coordinates_details? _chem_comp.pdbx_ideal_coordinates_missing_flag N _chem_comp.pdbx_model_coordinates_db_code1DPL _chem_comp.pdbx_processing_site ? # loop_ _chem_comp_atom.comp_id _chem_comp_atom.atom_id _chem_comp_atom.alt_atom_id _chem_comp_atom.type_symbol _chem_comp_atom.charge _chem_comp_atom.pdbx_align _chem_comp_atom.pdbx_aromatic_flag _chem_comp_atom.pdbx_leaving_atom_flag _chem_comp_atom.pdbx_stereo_config _chem_comp_atom.model_Cartn_x _chem_comp_atom.model_Cartn_y _chem_comp_atom.model_Cartn_z _chem_comp_atom.pdbx_model_Cartn_x_ideal _chem_comp_atom.pdbx_model_Cartn_y_ideal _chem_comp_atom.pdbx_model_Cartn_z_ideal _chem_comp_atom.pdbx_ordinal SPM N1 N1 N 0 1 N N N -1.386 5.560 25.140 -0.292 0.012 7.961 1 SPM C2 C2 C 0 1 N N N -2.248 4.344 25.282 0.514 -0.023 6.734 2 SPM C3 C3 C 0 1 N N N -1.844 3.214 24.376 -0.408 0.014 5.515 3 SPM C4 C4 C 0 1 N N N -2.834 2.063 24.495 0.432 -0.022 4.237 4 SPM N5 N5 N 0 1 N N N -2.313 0.871 23.720 -0.453 0.013 3.066 5 SPM C6 C6 C 0 1 N N N -3.235 -0.310 23.760 0.411 -0.024 1.880 6 SPM C7 C7 C 0 1 N N N -2.669 -1.391 22.871 -0.451 0.011 0.617 7 SPM C8 C8 C 0 1 N N N -3.306 -2.753 23.219 0.450 -0.028 -0.617 8 SPM C9 C9 C 0 1 N N N -2.886 -3.777 22.188 -0.412 0.006 -1.880 9 SPM N10 N10 N 0 1 N N N -3.212 -5.172 22.585 0.453 -0.031 -3.066 10 SPM C11 C11 C 0 1 N N N -2.084 -5.778 23.391 -0.433 0.005 -4.237 11 SPM C12 C12 C 0 1 N N N -2.443 -7.162 23.912 0.407 -0.032 -5.515 12 SPM C13 C13 C 0 1 N N N -1.448 -8.158 23.367 -0.515 0.005 -6.734 13 SPM N14 N14 N 0 1 N N N -0.553 -8.726 24.427 0.292 -0.029 -7.961 14 SPM HN11 1HN1 H 0 0 N N N -1.660 6.326 25.754 -0.739 0.916 7.988 15 SPM HN12 2HN1 H 0 0 N N N -1.355 5.869 24.168 0.354 -0.014 8.735 16 SPM H21 1H2 H 0 1 N N N -2.281 4.005 26.343 1.181 0.838 6.713 17 SPM H22 2H2 H 0 1 N N N -3.322 4.602 25.135 1.105 -0.939 6.715 18 SPM H31 1H3 H 0 1 N N N -1.722 3.550 23.320 -1.074 -0.847 5.536 19 SPM H32 2H3 H 0 1 N N N -0.796 2.883 24.565 -0.999 0.930 5.534 20 SPM H41 1H4 H 0 1 N N N -3.061 1.809 25.556 1.098 0.839 4.215 21 SPM H42 2H4 H 0 1 N N N -3.862 2.355 24.178 1.023 -0.938 4.217 22 SPM HN5 HN5 H 0 1 N N N -1.378 0.612 24.037 -0.976 -0.849 3.071 23 SPM H61 1H6 H 0 1 N N N -3.427 -0.667 24.798 1.078 0.838 1.889 24 SPM H62 2H6 H 0 1 N N N -4.284 -0.042 23.493 1.002 -0.940 1.891 25 SPM H71 1H7 H 0 1 N N N -2.780 -1.142 21.789 -1.117 -0.851 0.608 26 SPM H72 2H7 H 0 1 N N N -1.555 -1.427 22.916 -1.042 0.927 0.607 27 SPM H81 1H8 H 0 1 N N N -3.068 -3.077 24.258 1.117 0.833 -0.608 28 SPM H82 2H8 H 0 1 N N N -4.414 -2.687 23.323 1.041 -0.944 -0.607 29 SPM H91 1H9 H 0 1 N N N -3.319 -3.536 21.189 -1.079 -0.855 -1.889 30 SPM H92 2H9 H 0 1 N N N -1.802 -3.675 21.945 -1.003 0.922 -1.891 31 SPM HN0 HN0 H 0 1 N N N -3.451 -5.748 21.778 0.976 0.831 -3.071 32 SPM H111 1H11 H 0 0 N N N -1.133 -5.797 22.808 -1.099 -0.857 -4.215 33 SPM H112 2H11 H 0 0 N N N -1.767 -5.101 24.219 -1.023 0.921 -4.217 34 SPM H121 1H12 H 0 0 N N N -2.512 -7.194 25.024 1.074 0.830 -5.536 35 SPM H122 2H12 H 0 0 N N N -3.497 -7.443 23.683 0.998 -0.948 -5.534 36 SPM H131 1H13 H 0 0 N N N -1.964 -8.969 22.803 -1.181 -0.856 -6.713 37 SPM H132 2H13 H 0 0 N N N -0.851 -7.712 22.537 -1.105 0.921 -6.714 38 SPM HN41 1HN4 H 0 0 N N N 0.119 -9.398 24.058 -0.354 -0.003 -8.735 39 SPM HN42 2HN4 H 0 0 N N N -1.099 -9.133 25.186 0.814 0.833 -7.990 40 # loop_ _chem_comp_bond.comp_id _chem_comp_bond.atom_id_1 _chem_comp_bond.atom_id_2
Re: [ccp4bb] .cif file
Your cif-file contains coordinates, but as far as I could see it does not contain bond lengths, and angles and their deviations. That is what coot complains about. Unless your cif-file describes a modified spermine you could use the spermine already described in the refmac5 library: In coot, go to the File-Get Monomer menu and enter the three letter code SPM. It should load a spermine together with geometric restrains (have a look at $CLIB/data/monomers/s/SPM.cif to compare with your cif-file and see that it contains bond length information etc.). Tim -- Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A On Tue, 22 Jan 2008, Vineet Gaur wrote: Hi all I am using COOT for model building and refinement. i have to introducea ligand in my model i have downloaded .cif file from RCSB. However while importing the cif file i m getting the warning message of having No restraints in the CIF file is there any problem in the format of the following cif file data_SPM # _chem_comp.idSPM _chem_comp.name SPERMINE _chem_comp.type NON-POLYMER _chem_comp.pdbx_type HETAI _chem_comp.formula C10 H26 N4 _chem_comp.mon_nstd_parent_comp_id ? _chem_comp.pdbx_synonyms ? _chem_comp.pdbx_formal_charge0 _chem_comp.pdbx_initial_date 1999-07-08 _chem_comp.pdbx_modified_date2007-08-16 _chem_comp.pdbx_ambiguous_flag N _chem_comp.pdbx_release_status REL _chem_comp.pdbx_replaced_by ? _chem_comp.pdbx_replaces ? _chem_comp.formula_weight202.340 _chem_comp.one_letter_code ? _chem_comp.three_letter_code SPM _chem_comp.pdbx_model_coordinates_details? _chem_comp.pdbx_model_coordinates_missing_flag N _chem_comp.pdbx_ideal_coordinates_details? _chem_comp.pdbx_ideal_coordinates_missing_flag N _chem_comp.pdbx_model_coordinates_db_code1DPL _chem_comp.pdbx_processing_site ? # loop_ _chem_comp_atom.comp_id _chem_comp_atom.atom_id _chem_comp_atom.alt_atom_id _chem_comp_atom.type_symbol _chem_comp_atom.charge _chem_comp_atom.pdbx_align _chem_comp_atom.pdbx_aromatic_flag _chem_comp_atom.pdbx_leaving_atom_flag _chem_comp_atom.pdbx_stereo_config _chem_comp_atom.model_Cartn_x _chem_comp_atom.model_Cartn_y _chem_comp_atom.model_Cartn_z _chem_comp_atom.pdbx_model_Cartn_x_ideal _chem_comp_atom.pdbx_model_Cartn_y_ideal _chem_comp_atom.pdbx_model_Cartn_z_ideal _chem_comp_atom.pdbx_ordinal SPM N1 N1 N 0 1 N N N -1.386 5.560 25.140 -0.292 0.012 7.961 1 SPM C2 C2 C 0 1 N N N -2.248 4.344 25.282 0.514 -0.023 6.734 2 SPM C3 C3 C 0 1 N N N -1.844 3.214 24.376 -0.408 0.014 5.515 3 SPM C4 C4 C 0 1 N N N -2.834 2.063 24.495 0.432 -0.022 4.237 4 SPM N5 N5 N 0 1 N N N -2.313 0.871 23.720 -0.453 0.013 3.066 5 SPM C6 C6 C 0 1 N N N -3.235 -0.310 23.760 0.411 -0.024 1.880 6 SPM C7 C7 C 0 1 N N N -2.669 -1.391 22.871 -0.451 0.011 0.617 7 SPM C8 C8 C 0 1 N N N -3.306 -2.753 23.219 0.450 -0.028 -0.617 8 SPM C9 C9 C 0 1 N N N -2.886 -3.777 22.188 -0.412 0.006 -1.880 9 SPM N10 N10 N 0 1 N N N -3.212 -5.172 22.585 0.453 -0.031 -3.066 10 SPM C11 C11 C 0 1 N N N -2.084 -5.778 23.391 -0.433 0.005 -4.237 11 SPM C12 C12 C 0 1 N N N -2.443 -7.162 23.912 0.407 -0.032 -5.515 12 SPM C13 C13 C 0 1 N N N -1.448 -8.158 23.367 -0.515 0.005 -6.734 13 SPM N14 N14 N 0 1 N N N -0.553 -8.726 24.427 0.292 -0.029 -7.961 14 SPM HN11 1HN1 H 0 0 N N N -1.660 6.326 25.754 -0.739 0.916 7.988 15 SPM HN12 2HN1 H 0 0 N N N -1.355 5.869 24.168 0.354 -0.014 8.735 16 SPM H21 1H2 H 0 1 N N N -2.281 4.005 26.343 1.181 0.838 6.713 17 SPM H22 2H2 H 0 1 N N N -3.322 4.602 25.135 1.105 -0.939 6.715 18 SPM H31 1H3 H 0 1 N N N -1.722 3.550 23.320 -1.074 -0.847 5.536 19 SPM H32 2H3 H 0 1 N N N -0.796 2.883 24.565 -0.999 0.930 5.534 20 SPM H41 1H4 H 0 1 N N N -3.061 1.809 25.556 1.098 0.839 4.215 21 SPM H42 2H4 H 0 1 N N N -3.862 2.355 24.178 1.023 -0.938 4.217 22 SPM HN5 HN5 H 0 1 N N N -1.378 0.612 24.037 -0.976 -0.849 3.071 23 SPM H61 1H6 H 0 1 N N N -3.427 -0.667 24.798 1.078 0.838 1.889 24 SPM H62 2H6 H 0 1 N N N -4.284 -0.042 23.493 1.002 -0.940 1.891 25 SPM H71 1H7 H 0 1 N N N -2.780 -1.142 21.789 -1.117 -0.851 0.608 26 SPM H72 2H7 H 0 1 N N N -1.555 -1.427 22.916 -1.042 0.927 0.607 27 SPM H81 1H8 H 0 1 N N N -3.068 -3.077 24.258 1.117 0.833 -0.608 28 SPM H82 2H8 H 0 1 N N N -4.414 -2.687 23.323 1.041 -0.944 -0.607 29 SPM H91 1H9 H 0 1 N N N -3.319 -3.536 21.189 -1.079 -0.855 -1.889 30 SPM H92 2H9 H 0 1 N N N -1.802 -3.675 21.945 -1.003 0.922 -1.891 31 SPM HN0 HN0 H 0
[ccp4bb] Cocrystals - should they pop up under native conditions?
Hello all, I am expecting a somewhat homogeneous reply to this one, but that is fine and welcomed as are anecdotal experiences. I am running co crystallisation experiments and have thus far been trying under oil screens with some success (i.e. various hits in various conditions resulting in crystal growth). These conditions have varied from the native condition up until now. I have recently peered at some fairly old plates I set up under hanging drop under native conditions when running a co crystal trial and have found crystals. Their morphology is very similar to the 'native' form. I bit punier than them if I had to push. So, 1. Should I be surprised that co crystals pop up under native conditions or does it entirely depend on the ligand? 2. Is it probably not going to be a co crystal as usually they don't pop up under native conditions? 3. It totally depends and if I don't like it then I should get out of this beautiful game? I am going to shoot them either way, but just thought I would ask. cheers Brenda
Re: [ccp4bb] .cif file
Hello Vineet, This seems old one and doesn't have all restraints. you may use refmac dictionary for spermine. I am sure this will work in coot also. you may find it at /ccp4-6.0.2/lib/data/monomers/s/SPM.cif thanks Manish Vineet Gaur [EMAIL PROTECTED] wrote: Hi all I am using COOT for model building and refinement. i have to introducea ligand in my model i have downloaded .cif file from RCSB. However while importing the cif file i m getting the warning message of having No restraints in the CIF file is there any problem in the format of the following cif file data_SPM # _chem_comp.idSPM _chem_comp.name SPERMINE _chem_comp.type NON-POLYMER _chem_comp.pdbx_type HETAI _chem_comp.formula C10 H26 N4 _chem_comp.mon_nstd_parent_comp_id ? _chem_comp.pdbx_synonyms ? _chem_comp.pdbx_formal_charge0 _chem_comp.pdbx_initial_date 1999-07-08 _chem_comp.pdbx_modified_date2007-08-16 _chem_comp.pdbx_ambiguous_flag N _chem_comp.pdbx_release_status REL _chem_comp.pdbx_replaced_by ? _chem_comp.pdbx_replaces ? _chem_comp.formula_weight202.340 _chem_comp.one_letter_code ? _chem_comp.three_letter_code SPM _chem_comp.pdbx_model_coordinates_details? _chem_comp.pdbx_model_coordinates_missing_flag N _chem_comp.pdbx_ideal_coordinates_details? _chem_comp.pdbx_ideal_coordinates_missing_flag N _chem_comp.pdbx_model_coordinates_db_code1DPL _chem_comp.pdbx_processing_site ? # loop_ _chem_comp_atom.comp_id _chem_comp_atom.atom_id _chem_comp_atom.alt_atom_id _chem_comp_atom.type_symbol _chem_comp_atom.charge _chem_comp_atom.pdbx_align _chem_comp_atom.pdbx_aromatic_flag _chem_comp_atom.pdbx_leaving_atom_flag _chem_comp_atom.pdbx_stereo_config _chem_comp_atom.model_Cartn_x _chem_comp_atom.model_Cartn_y _chem_comp_atom.model_Cartn_z _chem_comp_atom.pdbx_model_Cartn_x_ideal _chem_comp_atom.pdbx_model_Cartn_y_ideal _chem_comp_atom.pdbx_model_Cartn_z_ideal _chem_comp_atom.pdbx_ordinal SPM N1 N1 N 0 1 N N N -1.386 5.560 25.140 -0.292 0.012 7.961 1 SPM C2 C2 C 0 1 N N N -2.248 4.344 25.282 0.514 -0.023 6.734 2 SPM C3 C3 C 0 1 N N N -1.844 3.214 24.376 -0.408 0.014 5.515 3 SPM C4 C4 C 0 1 N N N -2.834 2.063 24.495 0.432 -0.022 4.237 4 SPM N5 N5 N 0 1 N N N -2.313 0.871 23.720 -0.453 0.013 3.066 5 SPM C6 C6 C 0 1 N N N -3.235 - 0.310 23.760 0.411 -0.024 1.880 6 SPM C7 C7 C 0 1 N N N -2.669 -1.391 22.871 -0.451 0.011 0.617 7 SPM C8 C8 C 0 1 N N N -3.306 -2.753 23.219 0.450 -0.028 -0.617 8 SPM C9 C9 C 0 1 N N N -2.886 -3.777 22.188 -0.412 0.006 -1.880 9 SPM N10 N10 N 0 1 N N N -3.212 -5.172 22.585 0.453 -0.031 -3.066 10 SPM C11 C11 C 0 1 N N N -2.084 -5.778 23.391 -0.433 0.005 -4.237 11 SPM C12 C12 C 0 1 N N N -2.443 -7.162 23.912 0.407 -0.032 -5.515 12 SPM C13 C13 C 0 1 N N N -1.448 -8.158 23.367 -0.515 0.005 -6.734 13 SPM N14 N14 N 0 1 N N N -0.553 -8.726 24.427 0.292 -0.029 -7.961 14 SPM HN11 1HN1 H 0 0 N N N -1.660 6.326 25.754 -0.739 0.916 7.988 15 SPM HN12 2HN1 H 0 0 N N N -1.355 5.869 24.168 0.354 -0.014 8.735 16 SPM H21 1H2 H 0 1 N N N -2.281 4.005 26.343 1.181 0.838 6.713 17 SPM H22 2H2 H 0 1 N N N -3.322 4.602 25.135 1.105 -0.939 6.715 18 SPM H31 1H3 H 0 1 N N N -1.722 3.550 23.320 -1.074 -0.847 5.536 19 SPM H32 2H3 H 0 1 N N N -0.796 2.883 24.565 -0.999 0.930 5.534 20 SPM H41 1H4 H 0 1 N N N -3.061 1.809 25.556 1.098 0.839 4.215 21 SPM H42 2H4 H 0 1 N N N -3.862 2.355 24.178 1.023 -0.938 4.217 22 SPM HN5 HN5 H 0 1 N N N -1.378 0.612 24.037 -0.976 -0.849 3.071 23 SPM H61 1H6 H 0 1 N N N -3.427 -0.667 24.798 1.078 0.838 1.889 24 SPM H62 2H6 H 0 1 N N N -4.284 -0.042 23.493 1.002 -0.940 1.891 25 SPM H71 1H7 H 0 1 N N N -2.780 -1.142 21.789 -1.117 -0.851 0.608 26 SPM H72 2H7 H 0 1 N N N -1.555 -1.427 22.916 -1.042 0.927 0.607 27 SPM H81 1H8 H 0 1 N N N -3.068 -3.077 24.258 1.117 0.833 -0.608 28 SPM H82 2H8 H 0 1 N N N -4.414 -2.687 23.323 1.041 -0.944 -0.607 29 SPM H91 1H9 H 0 1 N N N -3.319 -3.536 21.189 -1.079 -0.855 -1.889 30 SPM H92 2H9 H 0 1 N N N -1.802 -3.675 21.945 -1.003 0.922 -1.891 31 SPM HN0 HN0 H 0 1 N N N -3.451 -5.748 21.778 0.976 0.831 -3.071 32 SPM H111 1H11 H 0 0 N N N -1.133 -5.797 22.808 -1.099 -0.857 -4.215 33 SPM H112 2H11 H 0 0 N N N -1.767 -5.101 24.219 -1.023 0.921 -4.217 34 SPM H121 1H12 H 0 0 N N N -2.512 -7.194 25.024 1.074 0.830 -5.536 35 SPM H122 2H12 H 0 0 N N N -3.497 -7.443 23.683 0.998 -0.948 -5.534
Re: [ccp4bb] Cocrystals - should they pop up under native conditions?
Don't call them co-crystals until you've actually shot them and seen the density. It will depend not only on the ligand but on the ligor(?) as well. You don't mention the identity of your ligand or ligor(?). You mention that some of your screens are under oil, so I will point out that some non-polar ligands might prefer to partition in the oil rather than the mother liquor. That would explain why the crystals look so much like the native. Posting to the BB is always a valuable way to fill time until you can collect the data which will answer the question. Cheers, - === With the single exception of Cornell, there is not a college in the United States where truth has ever been a welcome guest - R.G. Ingersoll === David J. Schuller modern man in a post-modern world MacCHESS, Cornell University [EMAIL PROTECTED] On Tue, 2008-01-22 at 19:33 +, Brenda Patterson wrote: Hello all, I am expecting a somewhat homogeneous reply to this one, but that is fine and welcomed as are anecdotal experiences. I am running co crystallisation experiments and have thus far been trying under oil screens with some success (i.e. various hits in various conditions resulting in crystal growth). These conditions have varied from the native condition up until now. I have recently peered at some fairly old plates I set up under hanging drop under native conditions when running a co crystal trial and have found crystals. Their morphology is very similar to the 'native' form. I bit punier than them if I had to push. So, 1. Should I be surprised that co crystals pop up under native conditions or does it entirely depend on the ligand? 2. Is it probably not going to be a co crystal as usually they don't pop up under native conditions? 3. It totally depends and if I don't like it then I should get out of this beautiful game? I am going to shoot them either way, but just thought I would ask. cheers Brenda
Re: [ccp4bb] antibody crystallization
I want to thank the very many people who responded to my inquiry. I'm overwhelmed and really appreciative. I've still got to read through it all, but a clear (near-unanimous) consensus is emerging. All the best, and again thanks very much for the help. I really appreciate it. Bill
Re: [ccp4bb] Cocrystals - should they pop up under native conditions?
Brenda, Generally speaking it is not abnormal to at least *hope* for co-crystals to appear at or around the condition(s) where the native crystals are grown. Therefore many people in fact begin looking for co-crystals by screening a fine grid around the expected crystallization conditions for the apo- protein. It is useful to remember those early crystallization hits that were never followed up - often the co-crystals will pop up in those, instead of the 'main' crystallization hit, so if you remember these early hits you may be able to spare yourself the expense and tedium of a complete re-screen. Now, having said this I also would like to add that it is also quite likely to get co-crystals to grow in other (sometimes completely unrelated) conditions. Depending on the number of ligands under consideration and on protein supply you may or may not be able to do a full re-screen with each ligand. In pharma we often have to co-crystallize with dozens (and sometimes hundreds) of compounds during the course of a project and we tend to pick up patterns of crystallization preferences based on compound class and/or geometry. Finally, it helps to remember that some protein/ligand combinations may not (easily, or ever) crystallize. In several cases that I can think of we had to use cross-soaking, different protein versions, or analogous (but different!) compounds in order to get the structure. In one recent case I had a situation where the protein would only crystallize if the ligand occupied one of the two binding sites in a homodimer. The other site was occupied by sulfate. This was pure serendipity - by luck my ligand and sulfate concentrations were just right - when I purposefully shifted the ratio either way, the crystals grew worse and eventually did not grow at all. Good luck, Artem -Original Message- From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Brenda Patterson Sent: Tuesday, January 22, 2008 2:33 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Cocrystals - should they pop up under native conditions? Hello all, I am expecting a somewhat homogeneous reply to this one, but that is fine and welcomed as are anecdotal experiences. I am running co crystallisation experiments and have thus far been trying under oil screens with some success (i.e. various hits in various conditions resulting in crystal growth). These conditions have varied from the native condition up until now. I have recently peered at some fairly old plates I set up under hanging drop under native conditions when running a co crystal trial and have found crystals. Their morphology is very similar to the 'native' form. I bit punier than them if I had to push. So, 1. Should I be surprised that co crystals pop up under native conditions or does it entirely depend on the ligand? 2. Is it probably not going to be a co crystal as usually they don't pop up under native conditions? 3. It totally depends and if I don't like it then I should get out of this beautiful game? I am going to shoot them either way, but just thought I would ask. cheers Brenda
[ccp4bb] salt sensitive complex
Dear All: Recently I am pursuing the crystallziation of a complex formd by two individual proteins and I met several interesting problems though they are kind of off-topic. Any suggestions for these problems will be highly appreciated. BIAcore showed about submicromolar affinity(both Kinetic and steady-state fitting) for these two proteins in the complex. However, precipitates immediately appeared when these two proteins were mixed together even at 10uM(0.3mg/ml) concentration in the condition of low salt(less than 20mM NaCl). By the way, these two proteins completely precipitated when the molar ratio is 1:1 in this condition. THerefore, I increased the salt concentraion step by step and finally I can keep both of them soluble in the solution with 25mM Tris(pH8) and 60mM NaCl(the minimum of salt concentration). Wierd thing happened when ITC experiments were carried out to confirm the binding affinity. 20uM in the sample cell and 200uM in the syringe could not give enough heat for a good curve fitting. The optimistic estimation of the affinity is lower than 5uM, which is much lower than the affinity given by BIAcore in the same buffer(25mM Tris plus 150mM NaCl). Now I am suspecting the capability of the interaction between these two proteins. However, I can not explain why these two guys precipitated stoichiometrically if they do not interact with each other. Is the complex salt-sensitive therefore there was just minor binding in the high-salt condition revealed by ITC? I am planning to do the ITC again in the condition of 25mMTris and 60mM NaCl. What if the affinity given by ITC is still much lower than that by BIAcore. Which one should I choose to believe? Are there some better ways that I can validate the binding affinity? Thanks again for your great ideas. Jerry McCully _ Need to know the score, the latest news, or you need your Hotmail®-get your fix. http://www.msnmobilefix.com/Default.aspx
[ccp4bb] Why there are difference density when occupancy is 1.00
Hello Everyone, When I refined a structure, I found strong difference density Fo-Fc at 3 sigma contour for the for five residues which already have occupancy of 1. The density is continuous and so strong as if I did not put the residues there. Why was that? Can I put greater than 1 occupancy for those residues? Thank you very much for your opinions and suggestions! Best wishes, Sun Tang - Never miss a thing. Make Yahoo your homepage.
Re: [ccp4bb] Characterization of common salt crystal forms?
Dear Joe, working with divalent ions, I often get salt crystals (most surely phosphate). I do not have any table available, but can give you the commercial kits numbers going with it. Just let me know. On Jan 22, 2008, at 6:12 PM, Joe Krahn wrote: Salt crystals are common in macromolecular crystallography. Has anyone tried to tabulate salt crystal forms that commonly occur? HTH Kind regards. Leo Chavas Leonard, Ph.D. Research Associate Faculty of Life Sciences The University of Manchester The Michael Smith Building Oxford Road Manchester Lancashire M13 9PT Tel: +44(0)161-275-1586 e-mail: [EMAIL PROTECTED]
Re: [ccp4bb] Cocrystals - should they pop up under native conditions?
Dear Brenda, I am running co crystallisation experiments and have thus far been trying under oil screens with some success (i.e. various hits in various conditions resulting in crystal growth). These conditions have varied from the native condition up until now. Do you already have the structure of the native form? If so, you can look at the putative binding region of your ligand, and check whereas it is involved in crystal packing or not. In any case, you'll have to shoot them and look at the density for confirmation; but just to have an idea of what worth can be expected. HTH Kind regards. Leo Chavas Leonard, Ph.D. Research Associate Faculty of Life Sciences The University of Manchester The Michael Smith Building Oxford Road Manchester Lancashire M13 9PT Tel: +44(0)161-275-1586 e-mail: [EMAIL PROTECTED]
