Re: [ccp4bb] Merging hi and low res data
Dear Nick, If you're happy to keep on using xia2, you can just put both of the data sets in a single directory and run xia2 -3dii /heres/where/the/data/went And wait a little while. To comment on your analysis of the statistics: inside xia2 the scaling is switched off as far as possible in the XDS CORRECT step and is instead done with XSCALE, which will scale several data sets at the same time. The data are then merged with Scala. I have found that data scaled with XSCALE will always give lower residuals than the same data (say from INTEGRATE.HKL) scaled with Scala or Aimless. I suspect that this relates to the parametrisations used - Scala (and I assume Aimless) use an analytical model with a small number of parameters, XDS and XSCALE use a numerical approach with rather more parameters. I will leave discussion of which way is best to the experts :o) On the subject of overloads, the 2^20 limit is an absolute limit - however there is a more real (and lower) limit which is written in the image headers, which relates to the count rate dead time correction - if the pixel count exceeds this threshold then the correction becomes less reliable. Typically though this is quite a large number, and is usually not met, though I should include this in the xia2 / XDS running code. If you'd like any help specifically with fiddling the xia2 run please feel free to contact me off list - at Diamond we also have facilities for remotely reprocessing the data which may help to make things a little quicker and would save moving the data around. Best wishes, Graeme On 7 February 2012 17:36, Nicholas Keep n.k...@mail.cryst.bbk.ac.uk wrote: We have a high and low res pass on a crystal that is going to around 1.2 A on the pilatus at Diamond. We tried to scale them together using the xds CORRECT files from the xia2 -3dii runs (gives best results as far as we can see comparing xia2.html files) through aimless via ccp4i interface. The combined dataset looks worse (based on merging statistics at lower resolutions) than the individual particular at low res.(Compared to scala xia2 runs). XDS does not record the pilatus as having overloads even on the high res pass using the correct detector parameters http://xds.mpimf-heidelberg.mpg.de/html_doc/detectors.html What I wanted to do was limit the resolution of the two runs ie to use low res between 100-2.5 beyond which completeness plummets and then the highres between 8 and 1.2. Aimless does not seem to allow me to do that at least by the ccp4i interface- is this then bad practice? I guess I could run a utility to remove the resolutions I don't want. The one effect that makes me wonder if the lowres is better data, and worth combining, ie there is some overload in the high res, is that it goes from 30-8 mean I/SIGI whereas the high res has I/SIGI of 15 in most of the low res shells and then slowly falls off to 2.0 at 1.2 angstrom. Am intending to carry on just using the hires pass. Any comments? I think Phil is in NZ and not looking at email hence putting this to the whole community -- Prof Nicholas H. Keep Executive Dean of School of Science Professor of Biomolecular Science Crystallography, Institute for Structural and Molecular Biology, Department of Biological Sciences Birkbeck, University of London, Malet Street, Bloomsbury LONDON WC1E 7HX email n.k...@mail.cryst.bbk.ac.uk Telephone 020-7631-6852 (Room G57 Office) 020-7631-6800 (Department Office) Fax 020-7631-6803 If you want to access me in person you have to come to the crystallography entrance and ring me or the department office from the internal phone by the door
Re: [ccp4bb] pH optimisation for crystallisation
Hello Sreetama: I think for crystallization, everything is hard to say. But if you find your crystal is sensitive to the pH, you certainly can optimize the pH value but it is better not to deviate a lot. For example you can make 0.2 unit interval (for example: pH value 4.5, 4.7, 4.9...etc which are closed to your original pH value ). For the buffer, you can change or not. Another thing is that, you can also incorporate bis-tris in your last purification, since you find your crystal in this buffer. When you do additive screen, the drops which is clear, also can give you important information. You might find a compont which can inhibit crystal formation. You can use it to slow down the crystal formation to get a big or single crystal.However, you see, sometimes, this optimization is time consuming. I suggest you to try seeding. It can give you a big surprise, sometimes. Yu Xiaodi Date: Wed, 8 Feb 2012 11:56:30 +0530 From: somon_...@yahoo.co.in Subject: [ccp4bb] pH optimisation for crystallisation To: CCP4BB@JISCMAIL.AC.UK Dear all, I have a 17 KDa protein that gives crystals in a condition that has 0.1M bis-tris pH 6.5. The crystals are thin needle clusters and do not diffract. I have tried additives, but they haven't improved the crystals. I intend to vary the pH of the condition. My questions are-1. should the buffer be kept the same or can it also be changed (as long as the desired pH is within the range of both the buffers)?2. in case of a different buffer, should its molarity be the same as that of the original one in the crystallization condition? regards,sreetama
[ccp4bb] rosetta fragment viewer
Dear all, Is there any program that can view the fragment files generated from Rosetta Fragment library? For example, I want to build a model myself, and I know the secondary structure of the target protein, so I will dock the 9-fragment myself. But, at the first step, I have to choose one fragment from the Rosetta fragment library generated. So, I wish if I can find any program that integrate the 200 9ers at specific position together, and then choose a good model by the shape I wanted. Did anyone here know such a program? Best Regards, Yuan SHANG
Re: [ccp4bb] pH optimisation for crystallisation
If you find yourself in the situation where the buffer you started with is out of range of the pH you would like to attain, there are sets of buffers you can use that contain most of the standard buffers that will give you a fairly linear response across ~4-10, as described by Newman, Acta Cryst D, 2004, v60, pp 610-612. cheers, tom From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] On Behalf Of Xiaodi Yu [uppsala@hotmail.com] Sent: Wednesday, February 08, 2012 8:06 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] pH optimisation for crystallisation Hello Sreetama: I think for crystallization, everything is hard to say. But if you find your crystal is sensitive to the pH, you certainly can optimize the pH value but it is better not to deviate a lot. For example you can make 0.2 unit interval (for example: pH value 4.5, 4.7, 4.9...etc which are closed to your original pH value ). For the buffer, you can change or not. Another thing is that, you can also incorporate bis-tris in your last purification, since you find your crystal in this buffer. When you do additive screen, the drops which is clear, also can give you important information. You might find a compont which can inhibit crystal formation. You can use it to slow down the crystal formation to get a big or single crystal. However, you see, sometimes, this optimization is time consuming. I suggest you to try seeding. It can give you a big surprise, sometimes. Yu Xiaodi Date: Wed, 8 Feb 2012 11:56:30 +0530 From: somon_...@yahoo.co.in Subject: [ccp4bb] pH optimisation for crystallisation To: CCP4BB@JISCMAIL.AC.UK Dear all, I have a 17 KDa protein that gives crystals in a condition that has 0.1M bis-tris pH 6.5. The crystals are thin needle clusters and do not diffract. I have tried additives, but they haven't improved the crystals. I intend to vary the pH of the condition. My questions are- 1. should the buffer be kept the same or can it also be changed (as long as the desired pH is within the range of both the buffers)? 2. in case of a different buffer, should its molarity be the same as that of the original one in the crystallization condition? regards, sreetama
Re: [ccp4bb] Generating parameters/cif files for macrocyclic ligands
Hi Joel, The way I solved this problem was by generating a linear peptide and then connecting the ends using a LINK card in the header of the pdb. Good luck! Herman From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Joel Tyndall Sent: Tuesday, February 07, 2012 10:44 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Generating parameters/cif files for macrocyclic ligands Hi folks, I have an intriguing problem. I'm trying to generate a cif file for a macrocyclic peptide (of the likes in pdb1d4k http://www.rcsb.org/pdb/explore/explore.do?structureId=1d4k ). They are cyclic tripeptides units. I can generate a pdb or mol2 file easily. I have used PRODRG to generate a .cif file and Coot read thjis in nicely. However, as it is cyclic one cannot adjust the dihedral angles. I have previously done this using CNS where you can break the tricyclic peptide into residues and generate parameters to specify bonds/links between the residues (which allows this kind of movement). I can't come up with a way to do this without using CNS. I have looked ta J-ligand which allows for one link between two separate residues which precludes a macrocycle. I have looked at sketcher within CCP4 which reads the pdb files but I don't believe this can be done here. Within Coot I can refine the whole ligand but not certain components. Any suggestions greatly appreciated . ( I may stick to coot refinement with fixed atoms at this stage) Regards Joel _ Joel Tyndall, PhD Senior Lecturer in Medicinal Chemistry National School of Pharmacy University of Otago PO Box 56 Dunedin 9054 New Zealand Skype: jtyndall http://www.researcherid.com/rid/C-2803-2008 Pukeka Matua Te Kura Taiwhanga Putaiao Te Whare Wananga o Otago Pouaka Poutapeta 56 Otepoti 9054 Aotearoa Ph / Waea +64 3 4797293 Fax / Waeawhakaahua +64 3 4797034
Re: [ccp4bb] Generating parameters/cif files for macrocyclic ligands
Hi Joel Herman is right: If you are refining cyclic peptides then the easiest way is to use link record linking C-terminus with N terminus. the name of the link should be TRANS. Here is an example: LINK ALA S 21 ASN S 1TRANS It will force ALA 21 to be linked (with torsion, angles, planes, bonds etc) to ASN 1 of chain S. This way you do not have to create description for large molecule. If you still want to create one molecule and you have mol2 file with coordinates then you can use libcheck to generate full dictionary using following commands libcheck file_mol mol_file_name nodist y It should generate fdescription. However I would prefer using link record. this way you keep amino acid names etc intact. If you amino acids are not among existing then you will need to create their description first and declare them peptide. regards Garib On 8 Feb 2012, at 10:33, herman.schreu...@sanofi.com wrote: Hi Joel, The way I solved this problem was by generating a linear peptide and then connecting the ends using a LINK card in the header of the pdb. Good luck! Herman From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Joel Tyndall Sent: Tuesday, February 07, 2012 10:44 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Generating parameters/cif files for macrocyclic ligands Hi folks, I have an intriguing problem. I’m trying to generate a cif file for a macrocyclic peptide (of the likes in pdb1d4k). They are cyclic tripeptides units. I can generate a pdb or mol2 file easily. I have used PRODRG to generate a .cif file and Coot read thjis in nicely. However, as it is cyclic one cannot adjust the dihedral angles. I have previously done this using CNS where you can break the tricyclic peptide into residues and generate parameters to specify bonds/links between the residues (which allows this kind of movement). I can’t come up with a way to do this without using CNS. I have looked ta J-ligand which allows for one link “between” two separate residues which precludes a macrocycle. I have looked at sketcher within CCP4 which reads the pdb files but I don’t believe this can be done here. Within Coot I can refine the whole ligand but not certain components. Any suggestions greatly appreciated . ( I may stick to coot refinement with fixed atoms at this stage) Regards Joel _ Joel Tyndall, PhD Senior Lecturer in Medicinal Chemistry National School of Pharmacy University of Otago PO Box 56 Dunedin 9054 New Zealand Skype: jtyndall http://www.researcherid.com/rid/C-2803-2008 Pukeka Matua Te Kura Taiwhanga Putaiao Te Whare Wananga o Otago Pouaka Poutapeta 56 Otepoti 9054 Aotearoa Ph / Waea +64 3 4797293 Fax / Waeawhakaahua +64 3 4797034 Garib N Murshudov Structural Studies Division MRC Laboratory of Molecular Biology Hills Road Cambridge CB2 0QH UK Email: ga...@mrc-lmb.cam.ac.uk Web http://www.mrc-lmb.cam.ac.uk
Re: [ccp4bb] Freezing crystal
A little off from the original question. Why don't small crystals dissolve to make a bigger crystal, especially when the small ones grow on top of each other? Can the clustered 3D crystals (I think it is called macroscopic twin) be used for full data collection? Again, thank you. Theresa
[ccp4bb] Collecting small-molecule diffraction on a Macromolecular xtallography beam line
Hello, I have some interesting small molecule xtals. I was wondering if it is possible to collect a small molecule data-set using a sincrotron macromolecular xtallography beam line, maybe with a very low beam intensity and moving the detector as close as possible? Has anybody experienced that? And if I get the images back home, can I process them using standard macromolecular software or do I need ab-initio special programs? Will MR work for phasing? Thanks in advance, Giorgio
Re: [ccp4bb] Collecting small-molecule diffraction on a Macromolecular xtallography beam line
-BEGIN PGP SIGNED MESSAGE- Hash: SHA1 Hello Giorgio, most synchrotron beamlines should have a resolution limit beyond or near 1A resolution which is sufficient for solving the structure with direct methods. At least XDS has no problems with non-chiral space groups and can be used to process the data. Since with a small cell you are going to have only few spots per image, make sure you increase the DELPHI parameter to 30 or 60, as Kay Diederichs pointed out to me. XDS_ASCII.HKL can be read into xprep which, if you keep on hitting enter and provide it with the chemical composition when it asks you to, is going to prepare a shelxd input file that can be used to solving the structure with shelxd. Its output .res-file is the starting point for refining the structure with e.g. shelxl. So: yes, you can process the data with [sparkle ;-)] standard macromolecular software [/sparkle] Cheers, Tim On 02/08/2012 12:41 PM, Giorgio Giardina wrote: Hello, I have some interesting small molecule xtals. I was wondering if it is possible to collect a small molecule data-set using a sincrotron macromolecular xtallography beam line, maybe with a very low beam intensity and moving the detector as close as possible? Has anybody experienced that? And if I get the images back home, can I process them using standard macromolecular software or do I need ab-initio special programs? Will MR work for phasing? Thanks in advance, Giorgio - -- - -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A -BEGIN PGP SIGNATURE- Version: GnuPG v1.4.10 (GNU/Linux) Comment: Using GnuPG with Mozilla - http://enigmail.mozdev.org/ iD8DBQFPMmgSUxlJ7aRr7hoRAtb7AKDhMgf+ImXfp/qR4WwR/AhCM1Wj6ACgh93K jpXsZgiGY9CfBHmMHd+L928= =dW6B -END PGP SIGNATURE-
[ccp4bb] Dye for protein affinity measurement
Dear all, I am looking for some kind of dye for protein affinity comparison, but do not know which to choose. I know protein A can contact B to form a complex,now I hope to find something simiar with A to act as an inhibitor to block the process of A-B complex formation. Maybe a short peptide, a segment of protein A or even some organic molecule. Because here is a poor access to ITC nor Biacore, I can only rely on some dye to check the competence between A and inhibitor candidates. If any one can offer any suggestions. That would be so grateful! Any way,thank kind-hearted people in advance! Regards Jiahong
Re: [ccp4bb] Dye for protein affinity measurement
Jiahong Thermo sells a series of kits called DyLight Fluor for fluorescent labelling of antibodies or other proteins. They have everything you need and they're very convenient and easy to use. You can pick the excitation and emission wavelength. If you label both A and B (or C) with different colors you will be able to see if both are in your crystals (assuming crystallization is part of your approach). You need only label a small percentage of your protein or peptide to see whether the protein is present in a crystal. Patrick http://en.wikipedia.org/wiki/DyLight_Fluor Forsythe, E.L., Achari, A., and Pusey, Marc L. (2006), Trace Fluorescent Labeling for High Throughput Crystallography, Acta Cryst. D62, 339-346. We used DyLight 350 NHS Ester to check we had protein crystals - see methods section of *Cryst. Growth Des.*, 2011, *11* (8), pp 3432–3441 2012/2/8 Jiang Jiahong jiang_jiah...@126.com Dear all, I am looking for some kind of dye for protein affinity comparison, but do not know which to choose. I know protein A can contact B to form a complex,now I hope to find something simiar with A to act as an inhibitor to block the process of A-B complex formation. Maybe a short peptide, a segment of protein A or even some organic molecule. Because here is a poor access to ITC nor Biacore, I can only rely on some dye to check the competence between A and inhibitor candidates. If any one can offer any suggestions. That would be so grateful! Any way,thank kind-hearted people in advance! Regards Jiahong -- 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] What happened to JAXA
I notice links in various places to JAXA Cryoprotectant Database for protein crystals http://idb.exst.jaxa.jp/db_data/protein/200304E02478000.html But when I follow the links, I get: Forbidden - You dont have permission to access... Is this data base still available? Is there another good summary of cryoprotectants available? I would want a list of what cryoprotectants have worked, how many cases they have been successful, and what amounts are necessary for ice prevention. Thanks. -- === All Things Serve the Beam === David J. Schuller modern man in a post-modern world MacCHESS, Cornell University schul...@cornell.edu
Re: [ccp4bb] Dye for protein affinity measurement
Hello Jiahong: If I understand correctly that you want to test protein-protein interaction or inhibition study in solution, maybe you can try something like ELISA to test protein-protein interaction. Or if your B protein has 6 histag, you can use Ni-NTA agrose beads to test inhibition or binding depending on your purpose. And another option (a little dangerous ), is using radio active to label one of your protein. Yu Xiaodi Date: Wed, 8 Feb 2012 14:17:47 + From: patr...@douglas.co.uk Subject: Re: [ccp4bb] Dye for protein affinity measurement To: CCP4BB@JISCMAIL.AC.UK Jiahong Thermo sells a series of kits called DyLight Fluor for fluorescent labelling of antibodies or other proteins. They have everything you need and they're very convenient and easy to use. You can pick the excitation and emission wavelength. If you label both A and B (or C) with different colors you will be able to see if both are in your crystals (assuming crystallization is part of your approach). You need only label a small percentage of your protein or peptide to see whether the protein is present in a crystal. Patrick http://en.wikipedia.org/wiki/DyLight_Fluor Forsythe, E.L., Achari, A., and Pusey, Marc L. (2006), Trace Fluorescent Labeling for High Throughput Crystallography, Acta Cryst. D62, 339-346. We used DyLight 350 NHS Ester to check we had protein crystals - see methods section of Cryst. Growth Des., 2011, 11 (8), pp 3432–3441 2012/2/8 Jiang Jiahong jiang_jiah...@126.com Dear all, I am looking for some kind of dye for protein affinity comparison, but do not know which to choose. I know protein A can contact B to form a complex,now I hope to find something simiar with A to act as an inhibitor to block the process of A-B complex formation. Maybe a short peptide, a segment of protein A or even some organic molecule. Because here is a poor access to ITC nor Biacore, I can only rely on some dye to check the competence between A and inhibitor candidates. If any one can offer any suggestions. That would be so grateful! Any way,thank kind-hearted people in advance! Regards Jiahong -- 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
Re: [ccp4bb] On pKa of Aspartic acid
Roger Rowlett wrote:
No. Kw = [H3O+][OH-] = 1 x 10^-14 at 25 deg C.
So at pH 7.0, you have 10^-7 M each at equilibrium no matter how you slice it
or whatever
else is in solution. If equilibrium [H3O+] goes up [OH-] goes down
commensurately.
The pKa of water as an acid is based on Kw and water's effective
concentration of 55 M
in pure water. This pKa is used to compare the instrinsic acidity of water to
other weak
acids. Water is an exceptionally weak acid or base.
And note that Kw, like other physical constants, depends on temperature,
ionic strength, etc. Therefore neutrality, defined as the concentration
where H+ = OH- , which is half of pKw*, is not exactly 7.0, but varies.
A lot of students come out of first-year chemistry with the idea that
pH 7. is by definition neutral.
(*That is using the old Kw definition where {H2O} is taken as 1)
The pK at 14 (or 14+log(55)) is for H2O - OH-, H+
i.e. pH where H2O = OH-
The pK at 0 (or -log 55)) is for H3O+ - H2O, H+
i.e. pH ph when H3O+ = H2O
But isn't H+ = H30? then when H3O+ = H2O, [H3O] = 55/2,
pH would be -log (55/2)
pH is log of activity of water, or whatever the glass electrode measure.
Or- assuming [H2O] always unity, when H2O = H3O+, H3O+ = 1, pH=0
assuming [H2O] always 55, extrapolate to where H3O+ = H2O while keeping H20=55,
then pH would be -log 55
[ccp4bb] Fwd: Re: [ccp4bb] Why don't small crystals dissolve
On 02/08/12 06:49, Enrico Stura wrote: On Wed, 08 Feb 2012 12:08:23 +0100, Theresa H. Hsu theresah...@live.com wrote: A little off from the original question. Why don't small crystals dissolve to make a bigger crystal, especially when the small ones grow on top of each other? Sometimes they do. Key phrase: Ostwald ripening. http://xray.bmc.uu.se/terese/crystallization/tutorials/tutorial6.html -- === All Things Serve the Beam === David J. Schuller modern man in a post-modern world MacCHESS, Cornell University schul...@cornell.edu
Re: [ccp4bb] Why don't small crystals dissolve
Hi Enricho, The scenario of streak seeding follows Ostwald ripening but will this happen in other situations as follows But in a special case where you have some crystals that appear as large rods which dissolved when taken out of the incubator (or) during the observation( these were antibody-complex crystals which were grown in bicelles(DMPC:CHAPSO and detergent mixtures and cholestrol, conditions citric acid pH 4.5, with 2.4 M ammonium sulfate). The crystals re-apparered in a day over noght incuabtion as heavy showers of needles with heavy precipitate around. Very hard to reproduce the conditions. Still trying to work around these conditions. Would like to know your thoughts if this is against the laws small crystals to large crystals (energetically favoured) conditions. Also any suggestions welcome for improvements. Pius The answer to your question is very simple. Small crystals will dissolve when the degree of saturation of the solution becomes too low to support their relatively high surface to volume ratio. The larger crystals will still continue to grow because of their higher surface/volume ratio but will do so slowly. I have achieved the dissolving of small crystals in favour of large ones only once with 10 µl drops. While it is difficult to achieve this with spontaneously nucleated crystals, with seeding thing are very different. This phenomenon is an every day observation if you use streak seeding on drops that have been equilibrated for different amount of time against different concentrations of precipitant and you can also add an additional variable by using different ratios of protein to precipitant in the drop. The goal is to seed at a low degree of superstauration. The small seeds will be visible along the streak immediately after seeding. When you look later on you will see only the bigger crystals. Streak seeding is great if you want to play this game. Enrico. On Wed, 08 Feb 2012 12:08:23 +0100, Theresa H. Hsu theresah...@live.com wrote: A little off from the original question. Why don't small crystals dissolve to make a bigger crystal, especially when the small ones grow on top of each other? Can the clustered 3D crystals (I think it is called macroscopic twin) be used for full data collection? Again, thank you. Theresa -- 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, CE Saclay, 91191 Gif-sur-Yvette, FRANCE http://www-dsv.cea.fr/en/institutes/institute-of-biology-and-technology-saclay-ibitec-s/unites-de-recherche/department-of-molecular-engineering-of-proteins-simopro/molecular-toxinology-and-biotechnology-laboratory-ltmb/crystallogenesis-e.-stura http://www.chem.gla.ac.uk/protein/mirror/stura/index2.html e-mail: est...@cea.fr Fax: 33 (0)1 69 08 90 71 -- Pius S Padayatti,PhD, Phone: 216-658-4528
Re: [ccp4bb] Collecting small-molecule diffraction on a Macromolecular xtallography beam line
I collected GTP/Mg2+ crystal on SSRL beamline 9-1 before. The images was processed by Mosflm and structure was solved by Shelx as usual. Kevin On Wed, Feb 8, 2012 at 3:41 AM, Giorgio Giardina giorgio.giard...@uniroma1.it wrote: Hello, I have some interesting small molecule xtals. I was wondering if it is possible to collect a small molecule data-set using a sincrotron macromolecular xtallography beam line, maybe with a very low beam intensity and moving the detector as close as possible? Has anybody experienced that? And if I get the images back home, can I process them using standard macromolecular software or do I need ab-initio special programs? Will MR work for phasing? Thanks in advance, Giorgio
Re: [ccp4bb] Collecting small-molecule diffraction on a Macromolecular xtallography beam line
Hi Giorgio, there are beamlines dedicated to small molecule crystallography at synchrotrons as well. I can suggest I19 at Diamond (obviously) but there are others! http://www.diamond.ac.uk/Home/Beamlines/I19.html Martin -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Giorgio Giardina Sent: 08 February 2012 11:42 To: ccp4bb Subject: [ccp4bb] Collecting small-molecule diffraction on a Macromolecular xtallography beam line Hello, I have some interesting small molecule xtals. I was wondering if it is possible to collect a small molecule data-set using a sincrotron macromolecular xtallography beam line, maybe with a very low beam intensity and moving the detector as close as possible? Has anybody experienced that? And if I get the images back home, can I process them using standard macromolecular software or do I need ab-initio special programs? Will MR work for phasing? Thanks in advance, Giorgio
[ccp4bb] Post-doctoral position available
Dear All, The Ho lab at the Institute of Biological Chemistry of Academia Sinca in Taiwan seeks to recruit a motivated structural biologist at post-doctoral level with interest in structure and activity studies of pharmaceutically important enzymes. Applicant should have a strong background in protein expression and purification. Some experience in protein crystallography is required. Candidate possessing experience in high-throughput protein production, protein thermal assay, or fragment-based screening are preferred. This position provides the opportunity or broad training in protein expression, crystallization, small anlge X-ray scattering, enzymology and protein-protein interaction. Applicants should submit the curriculum vitae and the names and contact information of three professional references via email to: sbddintai...@gmail.com For more information about the lab, please visit http://www.ibc.sinica.edu.tw/ho We are looking forward to receiving your application! Joseph Ho
[ccp4bb] Off topic: PyRosetta unrecognized aa Ur
Hello everybody, This is slightly off-topic but I still hope there might be somebody in the crowd with (Py)Rosetta experience. I successfully tried protein_protein docking before, but now I am trying to dock a RNA into a protein using PyRosetta v.1.1, which, as you can imagine, fails in a new an unusual way ;) I used the dna_dock.py script provided. When I run the script it finishes with following error message: ERROR: unrecognized aa Ur ERROR:: Exit from: src/core/io/pdb/file_data.cc line: 629 Traceback (most recent call last): File rna_dock.py, line 6, in module pose_from_pdb(p, rnadock.pdb) RuntimeError: unidentifiable C++ exception Obviously PyRosetta doesn't recognize the RNA specific residues and is still in DNA-mode. Looking fot hints in the internet took me to this website (http://www.rosettacommons.org/manuals/archive/rosetta3.2.1_user_guide/RNA_p rotein_changes.html), I followed the instructions changing files and pathes but still I receive the same error message. Below I posted the respective changes that I made according to the recommendations in /PyRosetta/rosetta_database/chemical/residue_type_sets/fa_standard: My residue_types.txt ## Nucleic Acid Types #residue_types/nucleic/ADE.params #residue_types/nucleic/THY.params #residue_types/nucleic/CYT.params #residue_types/nucleic/GUA.params residue_types/nucleic/RAD.params residue_types/nucleic/RCY.params residue_types/nucleic/RGU.params residue_types/nucleic/URA.params #residue_types/nucleic/GNP.params #residue_types/nucleic/GDP.params #residue_types/nucleic/GTP.params My patches.txt: patches/CtermProteinFull.txt patches/Cterm_amidation.txt patches/NtermProteinFull.txt #patches/LowerDNA.txt #patches/UpperDNA.txt patches/LowerRNA.txt patches/UpperRNA.txt patches/SpecialRotamer.txt patches/protein_cutpoint_upper.txt patches/protein_cutpoint_lower.txt patches/VirtualBB.txt patches/ShoveBB.txt patches/VirtualDNAPhosphate.txt patches/VirtualNterm.txt patches/RepulsiveOnly_fa.txt patches/VirtualProteinResidue.txt Do you have any ideas what could have gone wrong or what else I need to do in order to make it work? Many thanks in advance and best regards Eike
Re: [ccp4bb] What happened to JAXA
It worked just as you said, but I don't read Japanese so it wasn't particularly helpful. Thanks. On 02/08/12 11:37, Clemens Vonrhein wrote: Hi, the waybackmachine at http://www.archive.org/ helps here. Just type http://idb.exst.jaxa.jp/ into the box and hit Take me back. It then presents you with a timeline and some dates (bold), e.g. March 27 2009. Then completing the URL to read (according to your post below): http://web.archive.org/web/20090327100225/http://idb.exst.jaxa.jp/db_data/protein/200304E02478000.html seems to give me 'something' ... does that work for you? Cheers Clemens On Wed, Feb 08, 2012 at 09:42:25AM -0500, David Schuller wrote: I notice links in various places to JAXA Cryoprotectant Database for protein crystals http://idb.exst.jaxa.jp/db_data/protein/200304E02478000.html But when I follow the links, I get: Forbidden - You dont have permission to access... Is this data base still available? Is there another good summary of cryoprotectants available? I would want a list of what cryoprotectants have worked, how many cases they have been successful, and what amounts are necessary for ice prevention. Thanks. -- === All Things Serve the Beam === David J. Schuller modern man in a post-modern world MacCHESS, Cornell University schul...@cornell.edu -- === All Things Serve the Beam === David J. Schuller modern man in a post-modern world MacCHESS, Cornell University schul...@cornell.edu
Re: [ccp4bb] What happened to JAXA
On 02/08/12 12:47, Clemens Vonrhein wrote: On Wed, Feb 08, 2012 at 12:45:35PM -0500, David Schuller wrote: It worked just as you said, but I don't read Japanese so it wasn't particularly helpful. Odd ... at http://web.archive.org/web/20080927013137/http://idb.exst.jaxa.jp/db_data/protein/search-e.php I get a complete English page? Cheers Clemens OK, that gives me English. Thanks. -- === All Things Serve the Beam === David J. Schuller modern man in a post-modern world MacCHESS, Cornell University schul...@cornell.edu
Re: [ccp4bb] Why don't small crystals dissolve
Pius, The situation you describe is an off-equilibrium situation. You have applied a perturbation and that may not be reversible! Enrico. On Wed, 08 Feb 2012 16:35:56 +0100, Pius Padayatti ppadaya...@gmail.com wrote: Hi Enricho, The scenario of streak seeding follows Ostwald ripening but will this happen in other situations as follows But in a special case where you have some crystals that appear as large rods which dissolved when taken out of the incubator (or) during the observation( these were antibody-complex crystals which were grown in bicelles(DMPC:CHAPSO and detergent mixtures and cholestrol, conditions citric acid pH 4.5, with 2.4 M ammonium sulfate). The crystals re-apparered in a day over noght incuabtion as heavy showers of needles with heavy precipitate around. Very hard to reproduce the conditions. Still trying to work around these conditions. Would like to know your thoughts if this is against the laws small crystals to large crystals (energetically favoured) conditions. Also any suggestions welcome for improvements. Pius The answer to your question is very simple. Small crystals will dissolve when the degree of saturation of the solution becomes too low to support their relatively high surface to volume ratio. The larger crystals will still continue to grow because of their higher surface/volume ratio but will do so slowly. I have achieved the dissolving of small crystals in favour of large ones only once with 10 µl drops. While it is difficult to achieve this with spontaneously nucleated crystals, with seeding thing are very different. This phenomenon is an every day observation if you use streak seeding on drops that have been equilibrated for different amount of time against different concentrations of precipitant and you can also add an additional variable by using different ratios of protein to precipitant in the drop. The goal is to seed at a low degree of superstauration. The small seeds will be visible along the streak immediately after seeding. When you look later on you will see only the bigger crystals. Streak seeding is great if you want to play this game. Enrico. On Wed, 08 Feb 2012 12:08:23 +0100, Theresa H. Hsu theresah...@live.com wrote: A little off from the original question. Why don't small crystals dissolve to make a bigger crystal, especially when the small ones grow on top of each other? Can the clustered 3D crystals (I think it is called macroscopic twin) be used for full data collection? Again, thank you. Theresa -- Enrico A. Stura D.Phil. (Oxon) ,Tel: 33 (0)1 69 08 4302 Office Room 19, Bat.152, Tel: 33 (0)1 69 08 9449Lab LTMB, SIMOPRO, IBiTec-S, CE Saclay, 91191 Gif-sur-Yvette, FRANCE http://www-dsv.cea.fr/en/institutes/institute-of-biology-and-technology-saclay-ibitec-s/unites-de-recherche/department-of-molecular-engineering-of-proteins-simopro/molecular-toxinology-and-biotechnology-laboratory-ltmb/crystallogenesis-e.-stura http://www.chem.gla.ac.uk/protein/mirror/stura/index2.html e-mail: est...@cea.fr Fax: 33 (0)1 69 08 90 71 -- Enrico A. Stura D.Phil. (Oxon) ,Tel: 33 (0)1 69 08 4302 Office Room 19, Bat.152, Tel: 33 (0)1 69 08 9449Lab LTMB, SIMOPRO, IBiTec-S, CE Saclay, 91191 Gif-sur-Yvette, FRANCE http://www-dsv.cea.fr/en/institutes/institute-of-biology-and-technology-saclay-ibitec-s/unites-de-recherche/department-of-molecular-engineering-of-proteins-simopro/molecular-toxinology-and-biotechnology-laboratory-ltmb/crystallogenesis-e.-stura http://www.chem.gla.ac.uk/protein/mirror/stura/index2.html e-mail: est...@cea.fr Fax: 33 (0)1 69 08 90 71
Re: [ccp4bb] Collecting small-molecule diffraction on a Macromolecular xtallography beam line
Giorgo, We have done that routinely for quite some time now. We had problems when using a normal CCD detector, where we had to collect two or three sweeps to avoid overloads (see below). Since we have the PILATUS this is not necessary anymore and the data behaves fine. Problems still persisting are: we have only a single axis goniometer, which can lead to low completeness in P1 and P-1. Highest energy (17keV) and closest distance (188mm) at our beamline have many SM crystals (even the ones that don't diffract in house -- that is a 300 or 500 u sealed tube) with an I/sig of 5-10 at the edge of the detector. Crunch, Acorn, ShelxCDE and ShelxS don't have any problem with any of the data we collected to 0.9A resolution. The multipass caused some inexplicable non definite positives during refinement. We haven't tracked that down systematically, so it might just have happened haphazardly. HTH, Jens On Wed, 2012-02-08 at 11:41 +, Giorgio Giardina wrote: Hello, I have some interesting small molecule xtals. I was wondering if it is possible to collect a small molecule data-set using a sincrotron macromolecular xtallography beam line, maybe with a very low beam intensity and moving the detector as close as possible? Has anybody experienced that? And if I get the images back home, can I process them using standard macromolecular software or do I need ab-initio special programs? Will MR work for phasing? Thanks in advance, Giorgio
Re: [ccp4bb] Collecting small-molecule diffraction on a Macromolecular xtallography beam line
Most beamlines have attenuators, so there's little reason to collect multiple sweeps. We always collect 360deg. Since it's a small molecule, and usually fairly large and robust, you can warm it up, nudge it in a different direction with a pin (we use sterile, disposable acupunture needle), and refreeze it in the cryostream. Then do a second sweep in a different orientation. I recommend moving the beam energy to 15.5KeV or higher to compress the diffraction image. Collect with 5-10deg widths. We can typically get the detector to around 70-80mm. You need to get to 0.85A resolution or better for good, stable refinement, and Acta Cryst. requires that resolution for publication. Often you need the low-resolution data and data to better than 1A to help with the sigma2 relationships in direct methods. You see both primary and secondary extinction, and that extinction can be anisotropic, so the SWAT option in SHELX is most useful. Otherwise, the overall scale factor is off, typically overestimated by the strong low-resolution reflection intensities, with the result that the anisotropic Gaussian displacement parameters may become non-positive definate. Bernie On Wed, February 8, 2012 12:46 pm, Jens Kaiser wrote: Giorgo, We have done that routinely for quite some time now. We had problems when using a normal CCD detector, where we had to collect two or three sweeps to avoid overloads (see below). Since we have the PILATUS this is not necessary anymore and the data behaves fine. Problems still persisting are: we have only a single axis goniometer, which can lead to low completeness in P1 and P-1. Highest energy (17keV) and closest distance (188mm) at our beamline have many SM crystals (even the ones that don't diffract in house -- that is a 300 or 500 u sealed tube) with an I/sig of 5-10 at the edge of the detector. Crunch, Acorn, ShelxCDE and ShelxS don't have any problem with any of the data we collected to 0.9A resolution. The multipass caused some inexplicable non definite positives during refinement. We haven't tracked that down systematically, so it might just have happened haphazardly. HTH, Jens On Wed, 2012-02-08 at 11:41 +, Giorgio Giardina wrote: Hello, I have some interesting small molecule xtals. I was wondering if it is possible to collect a small molecule data-set using a sincrotron macromolecular xtallography beam line, maybe with a very low beam intensity and moving the detector as close as possible? Has anybody experienced that? And if I get the images back home, can I process them using standard macromolecular software or do I need ab-initio special programs? Will MR work for phasing? Thanks in advance, Giorgio -- Bernard D. Santarsiero Research Professor Center for Pharmaceutical Biotechnology and the Department of Medicinal Chemistry and Pharmacognosy Center for Structural Biology Center for Clinical and Translational Science University of Illinois at Chicago MC870 3070MBRB 900 South Ashland Avenue Chicago, IL 60607-7173 USA (312) 413-0339 (office) (312) 413-9303 (FAX) http://www.uic.edu/labs/bds
[ccp4bb] Problems with COOT and Pymol stereo
Dear CCP4ers, I'd purchased a stereo monitor from Sharper Technology with a Planar SA2311W stereo 3D LCD and the Nvidia 3D vision kit. However, I'm having a problem with the dual monitor setup. Everything works fine with Pymol and Coot when using a single 3D capable monitor (Planar SA2311W) and NVIDIA 3D Vision system (Quadro 4000). However, whenever I plug in the second monitor the 3D image (Coot and Pymol) becomes unstable (jittery - the molecule actually starts jiggling). Oddly, I don't have the same problem with the example stereo pictures and movies the company sends along. I'm using the MS Windows platform and I use the 2nd monitor for simultaneous word-processing etc. Any ideas for fixes? Best, Elias
Re: [ccp4bb] Generating parameters/cif files for macrocyclic ligands
Hi Garib, Thanks for that (and thanks Herman). How do I declare a non-natural amino acid a peptide? My ligand contains two peptidic cycles (non-N to C) where the side chains are cyclised. I think I'll be able to use several linbk records for the connections but the non-natural amino acid are complicating the issue _ Joel Tyndall, PhD Senior Lecturer in Medicinal Chemistry National School of Pharmacy University of Otago PO Box 56 Dunedin 9054 New Zealand Skype: jtyndall http://www.researcherid.com/rid/C-2803-2008 Pukeka Matua Te Kura Taiwhanga Putaiao Te Whare Wananga o Otago Pouaka Poutapeta 56 Otepoti 9054 Aotearoa Ph / Waea +64 3 4797293 Fax / Waeawhakaahua +64 3 4797034 From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Garib N Murshudov Sent: Wednesday, 8 February 2012 11:56 p.m. To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Generating parameters/cif files for macrocyclic ligands Hi Joel Herman is right: If you are refining cyclic peptides then the easiest way is to use link record linking C-terminus with N terminus. the name of the link should be TRANS. Here is an example: LINK ALA S 21 ASN S 1TRANS It will force ALA 21 to be linked (with torsion, angles, planes, bonds etc) to ASN 1 of chain S. This way you do not have to create description for large molecule. If you still want to create one molecule and you have mol2 file with coordinates then you can use libcheck to generate full dictionary using following commands libcheck file_mol mol_file_name nodist y It should generate fdescription. However I would prefer using link record. this way you keep amino acid names etc intact. If you amino acids are not among existing then you will need to create their description first and declare them peptide. regards Garib On 8 Feb 2012, at 10:33, herman.schreu...@sanofi.commailto:herman.schreu...@sanofi.com wrote: Hi Joel, The way I solved this problem was by generating a linear peptide and then connecting the ends using a LINK card in the header of the pdb. Good luck! Herman From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK]mailto:[mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Joel Tyndall Sent: Tuesday, February 07, 2012 10:44 PM To: CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Generating parameters/cif files for macrocyclic ligands Hi folks, I have an intriguing problem. I'm trying to generate a cif file for a macrocyclic peptide (of the likes in pdb1d4khttp://www.rcsb.org/pdb/explore/explore.do?structureId=1d4k). They are cyclic tripeptides units. I can generate a pdb or mol2 file easily. I have used PRODRG to generate a .cif file and Coot read thjis in nicely. However, as it is cyclic one cannot adjust the dihedral angles. I have previously done this using CNS where you can break the tricyclic peptide into residues and generate parameters to specify bonds/links between the residues (which allows this kind of movement). I can't come up with a way to do this without using CNS. I have looked ta J-ligand which allows for one link between two separate residues which precludes a macrocycle. I have looked at sketcher within CCP4 which reads the pdb files but I don't believe this can be done here. Within Coot I can refine the whole ligand but not certain components. Any suggestions greatly appreciated . ( I may stick to coot refinement with fixed atoms at this stage) Regards Joel _ Joel Tyndall, PhD Senior Lecturer in Medicinal Chemistry National School of Pharmacy University of Otago PO Box 56 Dunedin 9054 New Zealand Skype: jtyndall http://www.researcherid.com/rid/C-2803-2008 Pukeka Matua Te Kura Taiwhanga Putaiao Te Whare Wananga o Otago Pouaka Poutapeta 56 Otepoti 9054 Aotearoa Ph / Waea +64 3 4797293 Fax / Waeawhakaahua +64 3 4797034 Garib N Murshudov Structural Studies Division MRC Laboratory of Molecular Biology Hills Road Cambridge CB2 0QH UK Email: ga...@mrc-lmb.cam.ac.ukmailto:jen...@mrc-lmb.cam.ac.uk Web http://www.mrc-lmb.cam.ac.ukhttp://www.mrc-lmb.cam.ac.uk/
Re: [ccp4bb] Dye for protein affinity measurement
Hi, - If ur protein is making strong complex then You can run Native page with increasing concentration of your inhibitor peptide and decrease in complex band intensity will show you competitive binding of your inhibitor to proteins. - You can do ELISA... by coating one of your protein ad then add second protein and then detect by using antibodies against second protein ... with increasing concentration of peptide or inhibitor signal should go down. - if interaction is weak then design a peptide (part of protein B ) which definitely binds to protein with Alexa , or FITC labelled and do interaction study by observing the change in fluorescence. and if interaction is there do assay with different inhibitors ... On Wed, Feb 8, 2012 at 8:13 PM, Xiaodi Yu uppsala@hotmail.com wrote: Hello Jiahong: If I understand correctly that you want to test protein-protein interaction or inhibition study in solution, maybe you can try something like ELISA to test protein-protein interaction. Or if your B protein has 6 histag, you can use Ni-NTA agrose beads to test inhibition or binding depending on your purpose. And another option (a little dangerous ), is using radio active to label one of your protein. Yu Xiaodi -- Date: Wed, 8 Feb 2012 14:17:47 + From: patr...@douglas.co.uk Subject: Re: [ccp4bb] Dye for protein affinity measurement To: CCP4BB@JISCMAIL.AC.UK Jiahong Thermo sells a series of kits called DyLight Fluor for fluorescent labelling of antibodies or other proteins. They have everything you need and they're very convenient and easy to use. You can pick the excitation and emission wavelength. If you label both A and B (or C) with different colors you will be able to see if both are in your crystals (assuming crystallization is part of your approach). You need only label a small percentage of your protein or peptide to see whether the protein is present in a crystal. Patrick http://en.wikipedia.org/wiki/DyLight_Fluor Forsythe, E.L., Achari, A., and Pusey, Marc L. (2006), Trace Fluorescent Labeling for High Throughput Crystallography, Acta Cryst. D62, 339-346. We used DyLight 350 NHS Ester to check we had protein crystals - see methods section of *Cryst. Growth Des.*, 2011, *11* (8), pp 3432–3441 2012/2/8 Jiang Jiahong jiang_jiah...@126.com Dear all, I am looking for some kind of dye for protein affinity comparison, but do not know which to choose. I know protein A can contact B to form a complex,now I hope to find something simiar with A to act as an inhibitor to block the process of A-B complex formation. Maybe a short peptide, a segment of protein A or even some organic molecule. Because here is a poor access to ITC nor Biacore, I can only rely on some dye to check the competence between A and inhibitor candidates. If any one can offer any suggestions. That would be so grateful! Any way,thank kind-hearted people in advance! Regards Jiahong -- 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 -- Vandana kukshal
Re: [ccp4bb] Collecting small-molecule diffraction on a Macromolecular xtallography beam line
Hi Giorgio, some XDS-related hints can be found at http://strucbio.biologie.uni-konstanz.de/xdswiki/index.php/Small_molecules which I renamed to Difficult datasets since some of the suggestions also apply to those. What is lacking in that article is that you really should specify SENSOR_THICKNESS= and SILICON= . This is already taken care of in the Pilatus XDS.INP templates, but for CCD detectors this has to be specified manually; see hints in the script http://strucbio.biologie.uni-konstanz.de/xdswiki/index.php/Generate_XDS.INP HTH, Kay
