[ccp4bb] AW: [ccp4bb] cryoprotection
A trick I like is just to freeze the reservoir solution or would-be cryo-solution without a crystal present. If the frozen solution stays clear and does not show ice rings on e.g. a home source, it is worth trying. Otherwise, the solution needs optimization. Herman -Ursprüngliche Nachricht- Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Uday Kumar Gesendet: Freitag, 23. August 2013 19:52 An: CCP4BB@JISCMAIL.AC.UK Betreff: [ccp4bb] cryoprotection Hello Can anyone suggest a cryoprotectant for the following crystallization condition 0.2-0.4M sodium formate ~20% PEG 3350 0-25 mM Nickel 0-100 mM Malonate Thank you with regards uday
Re: [ccp4bb] Resolution, R factors and data quality
Excellent point about R-factors. Indeed, at this resolution they should be quite lower than what you have. Did you: - model solvent? - use anisotropic ADPs? - add H (this alone can drop R by 1-2%)? - model alternative conformations? - How R-factors (Rwork) look in resolution? Pavel On Mon, Aug 26, 2013 at 10:47 PM, Emily Golden 10417...@student.uwa.edu.auwrote: Thanks Yuriy and Pavel, at this resolution one would expect R/Rfree to be ~ 10-11%/12-13% assuming you applied anisotropic B-factor refinement ( and probably having a low symmetry SG). R merge of 80% may be OK if I/sig for high res shell is 2. Yes, I used anisotropic Bfactors and the space group is P1 21 1. However, the I/sig is only 1.5 in the highest shell. Cutting the data such that the I/sig is 2 has improved the R factors. Thank you. Maps get worse Could it be when you use all resolution range you get 59% of missing reflections in highest resolution shell filled in with DFc for the purpose of map calculation? Yes! the map that I was looking at was filled. Emily On 27 August 2013 09:49, Emily Golden 10417...@student.uwa.edu.au wrote: Hi All, I have collected diffraction images to 1 Angstrom resolution to the edge of the detector and 0.9A to the corner.I collected two sets, one for low resolution reflections and one for high resolution reflections. I get 100% completeness above 1A and 41% completeness in the 0.9A-0.95A shell. However, my Rmerge in the highest shelll is not good, ~80%. The Rfree is 0.17 and Rwork is 0.16 but the maps look very good. If I cut the data to 1 Angstrom the R factors improve but I feel the maps are not as good and I'm not sure if I can justify cutting data. So my question is, should I cut the data to 1Angstrom or should I keep the data I have? Also, taking geometric restraints off during refinement the Rfactors improve marginally, am I justified in doing this at this resolution? Thank you, Emily
Re: [ccp4bb] Resolution, R factors and data quality
Maybe a few remarks might help: Ad a) R merge of 80% may be OK if I/sig for high res shell is 2. What rationale is that statement based upon and what is the exact meaning of this statement? Is an Rmerge of 80% not ok when I/sigi is say 1.5? Or would 80% be ok if the i/sigI is 3.0? Why should an R-merge of 80% be (too) high in the first place? b) there is no statistical justification whatsoever for the I/sigI cutoff of 2 for refinement. This has been discussed @CCP4bb multiple times, for good reason. In this particular case, the (in)completeness appears to be the dominating factor. c) as Pavel notes, the R-value improvement means nil when truncating data - try to refine from 8 to 2 A and Rs might be even lower (abuse we engaged in ages ago when we did not know better and no ML) d) absolute values of refinement Rs vs (historic) expectation values cannot be judged without complete and detailed knowledge of the refinement protocol. The ultimate question is whether your model improves with inclusion of more data or not. Kay Diederichs has a few papers to this effect that make good reading. And CC1/2 seems to provide statistically justifiable limits for cut-off of (reasonably complete) high resolution shells. LG, BR From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Emily Golden Sent: Dienstag, 27. August 2013 07:48 To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Resolution, R factors and data quality Thanks Yuriy and Pavel, at this resolution one would expect R/Rfree to be ~ 10-11%/12-13% assuming you applied anisotropic B-factor refinement ( and probably having a low symmetry SG). R merge of 80% may be OK if I/sig for high res shell is 2. Yes, I used anisotropic Bfactors and the space group is P1 21 1. However, the I/sig is only 1.5 in the highest shell. Cutting the data such that the I/sig is 2 has improved the R factors. Thank you. Maps get worse Could it be when you use all resolution range you get 59% of missing reflections in highest resolution shell filled in with DFc for the purpose of map calculation? Yes! the map that I was looking at was filled. Emily On 27 August 2013 09:49, Emily Golden 10417...@student.uwa.edu.au wrote: Hi All, I have collected diffraction images to 1 Angstrom resolution to the edge of the detector and 0.9A to the corner.I collected two sets, one for low resolution reflections and one for high resolution reflections. I get 100% completeness above 1A and 41% completeness in the 0.9A-0.95A shell. However, my Rmerge in the highest shelll is not good, ~80%. The Rfree is 0.17 and Rwork is 0.16 but the maps look very good. If I cut the data to 1 Angstrom the R factors improve but I feel the maps are not as good and I'm not sure if I can justify cutting data. So my question is, should I cut the data to 1Angstrom or should I keep the data I have? Also, taking geometric restraints off during refinement the Rfactors improve marginally, am I justified in doing this at this resolution? Thank you, Emily
Re: [ccp4bb] Resolution, R factors and data quality
The question you should ask yourself is why would omitting data improve my model? Phil On 27 Aug 2013, at 02:49, Emily Golden 10417...@student.uwa.edu.au wrote: Hi All, I have collected diffraction images to 1 Angstrom resolution to the edge of the detector and 0.9A to the corner.I collected two sets, one for low resolution reflections and one for high resolution reflections. I get 100% completeness above 1A and 41% completeness in the 0.9A-0.95A shell. However, my Rmerge in the highest shelll is not good, ~80%. The Rfree is 0.17 and Rwork is 0.16 but the maps look very good. If I cut the data to 1 Angstrom the R factors improve but I feel the maps are not as good and I'm not sure if I can justify cutting data. So my question is, should I cut the data to 1Angstrom or should I keep the data I have? Also, taking geometric restraints off during refinement the Rfactors improve marginally, am I justified in doing this at this resolution? Thank you, Emily
[ccp4bb] Last call for MX-beamtime proposals at HZB BESSY II, deadline September 1. 2013 is approaching
Next MX-proposal application deadline: September 1, 2013 is approaching The Pilatus 6M is operational at BL14.1, which leads to a substantial performance increase of this station Single 8h beamtime bookings are now possible We kindly invite new MX-proposals for beamtime applications for the next beamtime period. In order to apply for beamtime, please register at the HZB on-line access tool GATE (https://www.helmholtz-berlin.de/pubbin/hzbgate) and submit a new beamtime application proposal. Please note: Your old accounts are not valid for the new system GATE. Proposals cannot be submitted via BOAT (GATE-Photons) HZB provides beamtime at the MX-beamlines 14.1, 14.2 and 14.3. The requested beamtime is granted based on the reviewed proposal and reports from previous research activities. Reported results from previous beamtimes stated in the Experimental Reports form will affect the chances for future beamtimes significantly. Please make sure to include them if available. Experimental setup: BL14.1 setup: - Photon flux: 1.4x10¹¹ Phot/sx100mAx0.05%BW at sample position (0.1-1 sec exposure time per frame) - User defined beam shaping from 30µm-100µm diameter possible - Pilatus 6M detector, 164mm-680mm max. distance from the sample - Microdiffractometer (MD2) with Mini-kappa goniometer MK3 (STAC controlled) - Automatic sample changer (CATS), 90 sample storage capacity (SPINE-Pin EMBL sample magazine compatibility) - 96-well crystallization plate scanning operational upon request - 32-core XEON-CPU server, with 10Gb uplink to Pilatus 6M - EDNA installed and available - Common MX-software installed including XDS, IMOSFLM, CCP4, Phenix, SHELXC-E, etc. - Automated data processing with XDSAPP available - Remotely controlled cryo-shutter for crystal annealing - Bruker AXS X-Flash XRF detector We are offering the hard- and software environment for carrying out: - UVRIP experiments at BL14.1. For further information, please visit: http://www.helmholtz-berlin.de/forschung/funkma/soft-matter/forschung/bessy-mx/ancillary-facilities/uvrip_en.html - In situ crystal screening experiments, please visit: http://www.helmholtz-berlin.de/forschung/funkma/soft-matter/forschung/bessy-mx/ancillary-facilities/insitu-screening_en.html BL14.2 setup: - Photon flux: 1.9x10¹¹ Phot/sx100mAx0.05%BW at sample position (3-20 sec exposure time per frame) - MX-225 X-ray detector, 45mm-380mm distance from the sample, 30 deg 2-Theta possible - mardtb goniometer installed - 40-core XEON-CPU server, with fibre channel SAN up-link data processing environment - EDNA installed and available - Common MX-software installed including XDS, IMOSFLM, CCP4, Phenix, SHELXC-E, etc. - Automated data processing with XDSAPP available - Remotely controlled cryo-shutter for crystal annealing - Amptek XRF detector - Pressure chamber for noble gas derivatization (Xe, Kr available upon request) - Ultra high performance stereo microscope Leica M205A, 20-255x zoom, 8 Mpixel CCD-camera BL14.3 setup: - Photon flux: 4x10¹0 Phot/sx100mAx0.05%BW at sample position (3-20 sec exposure time per frame) - MX-225 X-ray detector, 45mm-380mm distance from the sample, 30 deg 2-Theta possible - mardtb goniometer installed - 40-core XEON-CPU server, with fibre channel SAN up-link data processing environment - EDNA installed and available - Common MX software installed including XDS, IMOSFLM, CCP4, Phenix, SHELXC-E, etc. - Automated data processing with ixds and xdsi available - Remotely controlled cryo-shutter for crystal annealing - HC-1c dehydration device installed, HC1-beamtime upon request - Pressure chamber for noble gas derivatization (Xe, Kr available upon request) - Ultra high performance stereo microscope Leica M205A, 20-255x zoom, 8 Mpixel CCD-camera S1-biolab facilities: - Protein production and purification - Nanoliter 96 well crystallization plate formulation and storage at 5 °C and 20 °C - Biophysical characterization with real time PCR (thermofluor assay) - New fragment screening facility open for collaborations Please visit our web page www.helmholtz-berlin.de/bessy-mxhttp://www.helmholtz-berlin.de/bessy-mx to gain updated information about our experimental setup and other requirements. Uwe Mueller, Manfred Weiss and the HZB BESSY-MX group Dr. Uwe Mueller Soft Matter and Functional Materials Macromolecular Crystallography (BESSY-MX) | Group leader Elektronenspeicherring BESSY II Albert-Einstein-Str. 15, D-12489 Berlin, Germany Fon: +49 30 8062 14974 Fax: +49 30 8062 14975 url: www.helmholtz-berlin.de/bessy-mxhttp://www.helmholtz-berlin.de/bessy-mx email:u...@helmholtz-berlin.demailto:u...@helmholtz-berlin.de Helmholtz-Zentrum Berlin für Materialien und Energie GmbH Mitglied der Hermann von Helmholtz-Gemeinschaft Deutscher Forschungszentren e.V. Aufsichtsrat: Vorsitzender Prof. Dr. Dr. h.c. mult. Joachim Treusch, stv. Vorsitzende Dr. Beatrix Vierkorn-Rudolph Geschäftsführung: Prof. Dr. Anke
[ccp4bb] R factor geeting stuck
Hi ccp4 experts, I have collected diffraction images to 0.96 Angstrom resolution to the edge of the detector. One data set give me the full completeness and below i have pasted my statistic values got from XDS. I have cut off data at 1.12 A which seems to be quite nice regarding all necessary parameter to consider. But the problem is the Rfree is 0.176 and Rwork is 0.151 but the maps look very good. Even If I cut off the data to 1.15 Angstrom the R factors not improved. The space group of my complex is P21212. I used anisotropic Bfactors, add Hydrogen on and also used TLS but unfortunately i can not reduce able to reduce R factor in a good way. so, could you people kindly give me some suggestion how can i improve my data quality. RESOLUTION NUMBER OF REFLECTIONS COMPLETENESS R-FACTOR R-FACTOR COMPARED I/SIGMA R-meas CC(1/2) Anomal SigAno Nano LIMIT OBSERVED UNIQUE POSSIBLE OF DATA observed expected Corr 1.12 104562 21143 21268 99.4% 37.0% 37.8% 103982 4.04 41.4% 91.8* 0 0.767 15016 1.04 86588 21777 23100 94.3% 98.4% 95.8% 84962 1.44 113.0% 74.1* -1 0.700 10431 I would be thankful for your valuable comments AFSHAN
Re: [ccp4bb] Low 280 absorbance imidazole?
Hi Phoebe, Could you please explain me how do you stain the piece of paper? Thank you Armando El 21/08/2013, a las 17:03, Phoebe A. Rice escribió: Hi Bernhard, If your cheap imidazole works fine aside from the absorption problem, there's always my favorite stone-age detection method: pencil a numbered grid onto a piece of filter paper, spot the fractions on it, and stain with coomassie. It'll tell you which fractions to load on a gel, and it goes easy on the budget as well. Phoebe ++ Phoebe A. Rice Dept. of Biochemistry Molecular Biology The University of Chicago 773 834 1723; pr...@uchicago.edu http://bmb.bsd.uchicago.edu/Faculty_and_Research/ http://www.rsc.org/shop/books/2008/9780854042722.asp From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Bernhard Rupp [hofkristall...@gmail.com] Sent: Wednesday, August 21, 2013 9:33 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Low 280 absorbance imidazole? Hi Fellows, could someone please point me towards the source of a known high purity imidazole with low absorbance at 280 nm? I am facing the problem of detecting a low absorption protein in high imidazole background after IMAC gradient elution. In the UV spectra of the 2 imidazoles I checked there is some contaminant that absorbs at 280… Thx, BR Bernhard Rupp Marie Curie Incoming International Fellow Innsbruck Medical University Schöpfstrasse 41 A 6020 Innsbruck – Austria +43 (676) 571-0536 bernhard.r...@i-med.ac.at Dept. of Forensic Crystallography k.-k. Hofkristallamt Vista, CA 92084 001 (925) 209-7429 b...@ruppweb.org b...@hofkristallamt.org http://www.ruppweb.org/ ---
Re: [ccp4bb] AW: [ccp4bb] cryoprotection
Herman, The trick you suggest is not as valid as you may think. The ice rings can originate from the crystal itself. If you crystallize in a high concentration PEG precipitant you will avoid ice rings, but if you transfer or soak your crystals in the same solution the high molecular weight PEG will not enter the crystal lattice and you will still get ice rings. I have a picture of this in: Vera, L., Stura, E. A. (2013) Strategies for protein cryocrystallography. Crystal Growth Design, e-print http://pubs.acs.org/doi/full/10.1021/cg301531f PDF: Figure3 Page G. So cryoprotectants need to penetrate the crystal lattice to prevent ice rings, but even in the presence of ice rings the data can be used. Regarding optimization: The main problem you encounter in cryoprotection is that some compounds like glycerol and ethylene glycol solubilize protein crystals, but if you create a mixture of various compounds that is precipitation-solubilization neutral, then there is no real need for optimization. Enrico. On Tue, 27 Aug 2013 08:30:28 +0200, herman.schreu...@sanofi.com wrote: A trick I like is just to freeze the reservoir solution or would-be cryo-solution without a crystal present. If the frozen solution stays clear and does not show ice rings on e.g. a home source, it is worth trying. Otherwise, the solution needs optimization. Herman -Ursprüngliche Nachricht- Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Uday Kumar Gesendet: Freitag, 23. August 2013 19:52 An: CCP4BB@JISCMAIL.AC.UK Betreff: [ccp4bb] cryoprotection Hello Can anyone suggest a cryoprotectant for the following crystallization condition 0.2-0.4M sodium formate ~20% PEG 3350 0-25 mM Nickel 0-100 mM Malonate Thank you with regards uday -- 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 http://www-dsv.cea.fr/ibitecs/simopro/ltmb/cristallogenese LTMB, SIMOPRO, IBiTec-S, CE Saclay, 91191 Gif-sur-Yvette, FRANCE http://scholar.google.com/citations?hl=enuser=Kvm06WIoPAsCpagesize=100sortby=pubdate 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] R factor geeting stuck
that doesnt seem too bad an Rfactor to me! What do you expect? Eleanor On 27 August 2013 09:59, Afshan Begum afshan...@yahoo.com wrote: Hi ccp4 experts, I have collected diffraction images to 0.96 Angstrom resolution to the edge of the detector. One data set give me the full completeness and below i have pasted my statistic values got from XDS. I have cut off data at 1.12 A which seems to be quite nice regarding all necessary parameter to consider. But the problem is the Rfree is 0.176 and Rwork is 0.151 but the maps look very good. Even If I cut off the data to 1.15 Angstrom the R factors not improved. The space group of my complex is P21212. I used anisotropic Bfactors, add Hydrogen on and also used TLS but unfortunately i can not reduce able to reduce R factor in a good way. so, could you people kindly give me some suggestion how can i improve my data quality. RESOLUTION NUMBER OF REFLECTIONSCOMPLETENESS R-FACTOR R-FACTOR COMPARED I/SIGMA R-meas CC(1/2) Anomal SigAno Nano LIMIT OBSERVED UNIQUE POSSIBLE OF DATA observed expected Corr 1.12 104562 21143 21268 99.4% 37.0% 37.8% 103982 4.0441.4%91.8* 00.767 15016 1.04 86588 21777 23100 94.3% 98.4% 95.8%84962 1.44 113.0%74.1*-10.700 10431 I would be thankful for your valuable comments AFSHAN
[ccp4bb] AW: [ccp4bb] AW: [ccp4bb] cryoprotection
Dear Enrico, You are right that the trick has its limitations and I am aware of it. However, it might not be as bad as you think. Fiddling with the crystallization buffer or transferring crystals to different buffers also causes stress to the crystals and in many cases loss of resolution. If it turns out that the reservoir solution freezes ok (or the crystallization drop itself if that is feasible), I would risk, as I said, trying to freeze a crystal directly from the drop without any further manipulations. Why try to cryoprotect a crystal when it is not necessary? If that does not work, I would go for more elaborate protocols, which, as far as I know, also do not have 100% guarantee of success. In that case I would also consult the literature like the excellent paper you mention. Best, Herman -Ursprüngliche Nachricht- Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Enrico Stura Gesendet: Dienstag, 27. August 2013 11:58 An: CCP4BB@JISCMAIL.AC.UK Betreff: Re: [ccp4bb] AW: [ccp4bb] cryoprotection Herman, The trick you suggest is not as valid as you may think. The ice rings can originate from the crystal itself. If you crystallize in a high concentration PEG precipitant you will avoid ice rings, but if you transfer or soak your crystals in the same solution the high molecular weight PEG will not enter the crystal lattice and you will still get ice rings. I have a picture of this in: Vera, L., Stura, E. A. (2013) Strategies for protein cryocrystallography. Crystal Growth Design, e-print http://pubs.acs.org/doi/full/10.1021/cg301531f PDF: Figure3 Page G. So cryoprotectants need to penetrate the crystal lattice to prevent ice rings, but even in the presence of ice rings the data can be used. Regarding optimization: The main problem you encounter in cryoprotection is that some compounds like glycerol and ethylene glycol solubilize protein crystals, but if you create a mixture of various compounds that is precipitation-solubilization neutral, then there is no real need for optimization. Enrico. On Tue, 27 Aug 2013 08:30:28 +0200, herman.schreu...@sanofi.com wrote: A trick I like is just to freeze the reservoir solution or would-be cryo-solution without a crystal present. If the frozen solution stays clear and does not show ice rings on e.g. a home source, it is worth trying. Otherwise, the solution needs optimization. Herman -Ursprüngliche Nachricht- Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Uday Kumar Gesendet: Freitag, 23. August 2013 19:52 An: CCP4BB@JISCMAIL.AC.UK Betreff: [ccp4bb] cryoprotection Hello Can anyone suggest a cryoprotectant for the following crystallization condition 0.2-0.4M sodium formate ~20% PEG 3350 0-25 mM Nickel 0-100 mM Malonate Thank you with regards uday -- 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 http://www-dsv.cea.fr/ibitecs/simopro/ltmb/cristallogenese LTMB, SIMOPRO, IBiTec-S, CE Saclay, 91191 Gif-sur-Yvette, FRANCE http://scholar.google.com/citations?hl=enuser=Kvm06WIoPAsCpagesize=100sortby=pubdate 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] Resolution, R factors and data quality
Hi all, does this not again bring up the still prevailing adherence to R factors and not a shift to correlation coefficients ( CC1/2 and CC*) ? (as Dr. Phil Evans has indicated).? The way we look at data quality ( by we I mean the end users ) needs to be altered, I guess. best, Arka Chakraborty On Tue, Aug 27, 2013 at 9:50 AM, Phil Evans p...@mrc-lmb.cam.ac.uk wrote: The question you should ask yourself is why would omitting data improve my model? Phil On 27 Aug 2013, at 02:49, Emily Golden 10417...@student.uwa.edu.au wrote: Hi All, I have collected diffraction images to 1 Angstrom resolution to the edge of the detector and 0.9A to the corner.I collected two sets, one for low resolution reflections and one for high resolution reflections. I get 100% completeness above 1A and 41% completeness in the 0.9A-0.95A shell. However, my Rmerge in the highest shelll is not good, ~80%. The Rfree is 0.17 and Rwork is 0.16 but the maps look very good. If I cut the data to 1 Angstrom the R factors improve but I feel the maps are not as good and I'm not sure if I can justify cutting data. So my question is, should I cut the data to 1Angstrom or should I keep the data I have? Also, taking geometric restraints off during refinement the Rfactors improve marginally, am I justified in doing this at this resolution? Thank you, Emily -- *Arka Chakraborty* *ibmb (Institut de Biologia Molecular de Barcelona)** **BARCELONA, SPAIN** *
Re: [ccp4bb] Insertion of a Tag protein in of the molecules of a Dimer protein complex
Anindito, First off, I have never tried this, but here are a few initial thoughts on how I might approach this. But first a question, how tight is the dimer form? if you mixed dimers with tags and dimers without tags, would you get any exchange? If so, you might be able to use this to your advantage. But for now, i'll ignore this. Now for how i was thinking of possibly accomplishing the goal you want... 1- use two vectors or a DUET vector to express two versions of your protein, (1) with N-term cleavable (TEV site?) HIS tag and your fusion protein, and (2) with N-term cleavable (TEV site?) GST tag only. 2- express your protein as normal and then purify by (1) running the GSH column to capture all GST tagged proteins, followed by a (2) Ni-NTA column to capture those with HIS tags. If all works as planned, you would end up with one monomer having only HIS-fusion protein tags and one having GST tag. You could use SDS-PAGE/Western blot analysis to verify this, since the monomers will run at different sizes (you might have to modify your gel system to get the best separationi recommend 5-7% gels for this size in MES) AND should be reactive to antiHIS and antiGST antibodies in needed. Further, you could get an idea for the ratios of each by comparing the overall intensity of the bands under normal staining methods. 3- to remove the HIS and GST tags, just treat with TEV protease to yield your dimer with only one of the monomers containing your fusion protein. I am sure i likely overlooked something in my haste, but you get the idea, basically a two step purification with two tags. Again, I haven't tried this but seems like it should work to me. hope this helps, good luck! Cheers, Nick [ Nicholas Noinaj ] the Buchanan Lab Laboratory of Molecular Biology LMB-NIDDK, NIH 50 South Drive, Room 4505 Bethesda, MD 20892-8030 1-301-594-9230 (lab) 1-859-893-4789 (cell) noin...@niddk.nih.gov [ the Buchanan Lab ] http://www-mslmb.niddk.nih.gov/buchanan/ From: Anindito Sen [andysen.to...@gmail.com] Sent: Monday, August 26, 2013 11:26 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Insertion of a Tag protein in of the molecules of a Dimer protein complex Dear All, I need to insert a tag protein of ~5 kDa in one of the molecules of a dimer protein (size is ~100kDa). To be more precise - The tag need to get itself attached only on one of the dimer molecules. My expertise are not in Cell biology and therefore any suggestion in this regard will be of great help. Thanks Best Wishes Dr. Anindito Sen (Ph.D) Structural Biology Graduate School of Medicine University of Tokyo 7-3-1 Hongo. Bunkyo-ku. Tokyo 113-0033. Japan Tel Fax: +81-3-5841-3339
Re: [ccp4bb] Insertion of a Tag protein in of the molecules of a Dimer protein complex
Nicholas is right that it depends a lot on the dimer you are working with. I worked on a dimeric tRNA synthetase and was able to make heterodimers in a couple of ways. The most effective was to make a bicistronic version of the ORFs each with their own S-D. Each had a tag for tandem affinity purification as Nick suggests. In my case, using two plasmids or even putting the two ORFs with separate promoters did not result in heterodimers (probably due to folding/dimerization during translation?). I was able to express to the two dimeric versions separately, mix, dilute extensively (which ultimately resulted in a big hit in yield), concentrate, and purify through the tandem approach. I eventually noticed that even though it was slow, there was dimer exchange occurring at intermediate concentrations. I ended up engineering a disulfide at the dimer interface to minimize that. --paul On 08/27/2013 01:10 PM, Noinaj, Nicholas (NIH/NIDDK) [F] wrote: Anindito, First off, I have never tried this, but here are a few initial thoughts on how I might approach this. But first a question, how tight is the dimer form? if you mixed dimers with tags and dimers without tags, would you get any exchange? If so, you might be able to use this to your advantage. But for now, i'll ignore this. Now for how i was thinking of possibly accomplishing the goal you want... 1- use two vectors or a DUET vector to express two versions of your protein, (1) with N-term cleavable (TEV site?) HIS tag and your fusion protein, and (2) with N-term cleavable (TEV site?) GST tag only. 2- express your protein as normal and then purify by (1) running the GSH column to capture all GST tagged proteins, followed by a (2) Ni-NTA column to capture those with HIS tags. If all works as planned, you would end up with one monomer having only HIS-fusion protein tags and one having GST tag. You could use SDS-PAGE/Western blot analysis to verify this, since the monomers will run at different sizes (you might have to modify your gel system to get the best separationi recommend 5-7% gels for this size in MES) AND should be reactive to antiHIS and antiGST antibodies in needed. Further, you could get an idea for the ratios of each by comparing the overall intensity of the bands under normal staining methods. 3- to remove the HIS and GST tags, just treat with TEV protease to yield your dimer with only one of the monomers containing your fusion protein. I am sure i likely overlooked something in my haste, but you get the idea, basically a two step purification with two tags. Again, I haven't tried this but seems like it should work to me. hope this helps, good luck! Cheers, Nick [ Nicholas Noinaj ] the Buchanan Lab Laboratory of Molecular Biology LMB-NIDDK, NIH 50 South Drive, Room 4505 Bethesda, MD 20892-8030 1-301-594-9230 (lab) 1-859-893-4789 (cell) noin...@niddk.nih.gov [ the Buchanan Lab ] http://www-mslmb.niddk.nih.gov/buchanan/ From: Anindito Sen [andysen.to...@gmail.com] Sent: Monday, August 26, 2013 11:26 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Insertion of a Tag protein in of the molecules of a Dimer protein complex Dear All, I need to insert a tag protein of ~5 kDa in one of the molecules of a dimer protein (size is ~100kDa). To be more precise - The tag need to get itself attached only on one of the dimer molecules. My expertise are not in Cell biology and therefore any suggestion in this regard will be of great help. Thanks Best Wishes Dr. Anindito Sen (Ph.D) Structural Biology Graduate School of Medicine University of Tokyo 7-3-1 Hongo. Bunkyo-ku. Tokyo 113-0033. Japan Tel Fax: +81-3-5841-3339
Re: [ccp4bb] Insertion of a Tag protein in of the molecules of a Dimer protein complex
Hello Anindito, While Nick and Paul have thrown you some great ideas around the TAP-tag theme (although GST may not be ideal as it dimerises itself), I feel that I've read your initial e-mail differently from them. Maybe you could clarify? It sounds like you have a specific tag of ~5kDa in mind, presumably for a specific purpose? It's not the case that any old tag will suffice? Are you producing the dimeric protein recombinantly? If so, you have lots of options. If not, you're going to need a chemical modification post-purification (or to establish a recombinant system). Does the insertion of the tag need to be at a specific site within the one protein? Would one of the termini do? Critically, is your dimer a homodimer? If it's a heterodimer, things could be quite easy. All the best, Will. On 27 August 2013 18:50, Paul Paukstelis shocksofmig...@gmail.com wrote: Nicholas is right that it depends a lot on the dimer you are working with. I worked on a dimeric tRNA synthetase and was able to make heterodimers in a couple of ways. The most effective was to make a bicistronic version of the ORFs each with their own S-D. Each had a tag for tandem affinity purification as Nick suggests. In my case, using two plasmids or even putting the two ORFs with separate promoters did not result in heterodimers (probably due to folding/dimerization during translation?). I was able to express to the two dimeric versions separately, mix, dilute extensively (which ultimately resulted in a big hit in yield), concentrate, and purify through the tandem approach. I eventually noticed that even though it was slow, there was dimer exchange occurring at intermediate concentrations. I ended up engineering a disulfide at the dimer interface to minimize that. --paul On 08/27/2013 01:10 PM, Noinaj, Nicholas (NIH/NIDDK) [F] wrote: Anindito, First off, I have never tried this, but here are a few initial thoughts on how I might approach this. But first a question, how tight is the dimer form? if you mixed dimers with tags and dimers without tags, would you get any exchange? If so, you might be able to use this to your advantage. But for now, i'll ignore this. Now for how i was thinking of possibly accomplishing the goal you want... 1- use two vectors or a DUET vector to express two versions of your protein, (1) with N-term cleavable (TEV site?) HIS tag and your fusion protein, and (2) with N-term cleavable (TEV site?) GST tag only. 2- express your protein as normal and then purify by (1) running the GSH column to capture all GST tagged proteins, followed by a (2) Ni-NTA column to capture those with HIS tags. If all works as planned, you would end up with one monomer having only HIS-fusion protein tags and one having GST tag. You could use SDS-PAGE/Western blot analysis to verify this, since the monomers will run at different sizes (you might have to modify your gel system to get the best separationi recommend 5-7% gels for this size in MES) AND should be reactive to antiHIS and antiGST antibodies in needed. Further, you could get an idea for the ratios of each by comparing the overall intensity of the bands under normal staining methods. 3- to remove the HIS and GST tags, just treat with TEV protease to yield your dimer with only one of the monomers containing your fusion protein. I am sure i likely overlooked something in my haste, but you get the idea, basically a two step purification with two tags. Again, I haven't tried this but seems like it should work to me. hope this helps, good luck! Cheers, Nick --**-- [ Nicholas Noinaj ] the Buchanan Lab Laboratory of Molecular Biology LMB-NIDDK, NIH 50 South Drive, Room 4505 Bethesda, MD 20892-8030 1-301-594-9230 (lab) 1-859-893-4789 (cell) noin...@niddk.nih.gov [ the Buchanan Lab ] http://www-mslmb.niddk.nih.**gov/buchanan/http://www-mslmb.niddk.nih.gov/buchanan/ __**__ From: Anindito Sen [andysen.to...@gmail.com] Sent: Monday, August 26, 2013 11:26 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Insertion of a Tag protein in of the molecules of a Dimer protein complex Dear All, I need to insert a tag protein of ~5 kDa in one of the molecules of a dimer protein (size is ~100kDa). To be more precise - The tag need to get itself attached only on one of the dimer molecules. My expertise are not in Cell biology and therefore any suggestion in this regard will be of great help. Thanks Best Wishes Dr. Anindito Sen (Ph.D) Structural Biology Graduate School of Medicine University of Tokyo 7-3-1 Hongo. Bunkyo-ku. Tokyo 113-0033. Japan Tel Fax: +81-3-5841-3339
Re: [ccp4bb] Negative IOBS in XDS
Dear Niu, I first would look at the data set statistics of the data set before investigating individual reflections, and also take a look at FRAME.cbf which shows the predicted spots for the last integrated image. Best, Tim On Tue, Aug 27, 2013 at 04:34:11PM -0400, Niu Tou wrote: Dear Colleagues, When I processed one data set with XDS, the index step said there is no sufficient (50%) spots indexed, however after I tried various parameters to get the same cell unit and space group, I chose to ignore this message and wen on to integration. During the integration I had to modify the following keywords to finish as instructed by the manual: REFLECTING_RANGE REFLECTING_RANGE_E.S.D. BEAM_DIVERGENCE= BEAM_DIVERGENCE_E.S.D. Finally I got a scaled file, but there are many negative numbers in the column 4 IOBS, and the SIGMA seems very high, for example: 0 0-6 1.751E+03 9.025E+02 1547.9 1935.5 55.0 0.32158 95 33 -85.79 0 0-6 -1.795E+00 5.171E+02 1547.9 1117.2 22.7 0.32158 100 -2 93.56 0 0 6 1.426E+03 7.902E+02 1550.9 1117.2 58.7 0.32158 100 38 85.86 0 0 6 8.512E+02 5.216E+02 1550.9 1935.5 19.0 0.32158 95 23 -93.63 0 0-7 -4.436E+01 4.495E+02 1547.5 2011.5 54.7 0.37469 100 -11 -85.79 Does this mean the process is wrong? Anything I can do in this case? Best, Niu -- -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A signature.asc Description: Digital signature
Re: [ccp4bb] Resolution, R factors and data quality
I have to ask flamingly: So what about CC1/2 and CC*? Did we not replace an arbitrary resolution cut-off based on a value of Rmerge with an arbitrary resolution cut-off based on a value of Rmeas already? And now we are going to replace that with an arbitrary resolution cut-off based on a value of CC* or is it CC1/2? I am asked often: What value of CC1/2 should I cut my resolution at? What should I tell my students? I've got a course coming up and I am sure they will ask me again. Jim From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Arka Chakraborty [arko.chakrabort...@gmail.com] Sent: Tuesday, August 27, 2013 7:45 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Resolution, R factors and data quality Hi all, does this not again bring up the still prevailing adherence to R factors and not a shift to correlation coefficients ( CC1/2 and CC*) ? (as Dr. Phil Evans has indicated).? The way we look at data quality ( by we I mean the end users ) needs to be altered, I guess. best, Arka Chakraborty On Tue, Aug 27, 2013 at 9:50 AM, Phil Evans p...@mrc-lmb.cam.ac.ukmailto:p...@mrc-lmb.cam.ac.uk wrote: The question you should ask yourself is why would omitting data improve my model? Phil
Re: [ccp4bb] Resolution, R factors and data quality
Hi Jim, all data is good data - the more data you have the better (that's what they say anyhow) Not everybody is adopting to the Karplus Diederich paper as quickly as you do. And not to be confused with the Diederichs and Karplus paper :-) http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3689524/ http://www.ncbi.nlm.nih.gov/pubmed/22628654 My models get better by including the data I had been omitting before, that's all that counts for me. Jürgen P.S. reminds me somehow of those guys collecting more and more data - PRISM greetings On Aug 27, 2013, at 8:29 PM, Jim Pflugrath wrote: I have to ask flamingly: So what about CC1/2 and CC*? Did we not replace an arbitrary resolution cut-off based on a value of Rmerge with an arbitrary resolution cut-off based on a value of Rmeas already? And now we are going to replace that with an arbitrary resolution cut-off based on a value of CC* or is it CC1/2? I am asked often: What value of CC1/2 should I cut my resolution at? What should I tell my students? I've got a course coming up and I am sure they will ask me again. Jim From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK] on behalf of Arka Chakraborty [arko.chakrabort...@gmail.commailto:arko.chakrabort...@gmail.com] Sent: Tuesday, August 27, 2013 7:45 AM To: CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Resolution, R factors and data quality Hi all, does this not again bring up the still prevailing adherence to R factors and not a shift to correlation coefficients ( CC1/2 and CC*) ? (as Dr. Phil Evans has indicated).? The way we look at data quality ( by we I mean the end users ) needs to be altered, I guess. best, Arka Chakraborty On Tue, Aug 27, 2013 at 9:50 AM, Phil Evans p...@mrc-lmb.cam.ac.ukmailto:p...@mrc-lmb.cam.ac.uk wrote: The question you should ask yourself is why would omitting data improve my model? Phil .. Jürgen Bosch Johns Hopkins University Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Office: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-2926 http://lupo.jhsph.edu
Re: [ccp4bb] Resolution, R factors and data quality
Based on the simulations I've done the data should be cut at CC1/2 = 0. Seriously. Problem is figuring out where it hits zero. Alternately, if French Wilson can be modified so the Wilson plot is always straight, then the data don't need to be cut at all. As for the resolution of the structure I'd say call that where |Fo-Fc| (error in the map) becomes comparable to Sigma(Fo). This is I/Sigma = 2.5 if Rcryst is 20%. That is: |Fo-Fc| / Fo = 0.2, which implies |Io-Ic|/Io = 0.4 or Io/|Io-Ic| = Io/sigma(Io) = 2.5. Makes sense to me... -James Holton MAD Scientist On Aug 27, 2013, at 5:29 PM, Jim Pflugrath jim.pflugr...@rigaku.com wrote: I have to ask flamingly: So what about CC1/2 and CC*? Did we not replace an arbitrary resolution cut-off based on a value of Rmerge with an arbitrary resolution cut-off based on a value of Rmeas already? And now we are going to replace that with an arbitrary resolution cut-off based on a value of CC* or is it CC1/2? I am asked often: What value of CC1/2 should I cut my resolution at? What should I tell my students? I've got a course coming up and I am sure they will ask me again. Jim From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Arka Chakraborty [arko.chakrabort...@gmail.com] Sent: Tuesday, August 27, 2013 7:45 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Resolution, R factors and data quality Hi all, does this not again bring up the still prevailing adherence to R factors and not a shift to correlation coefficients ( CC1/2 and CC*) ? (as Dr. Phil Evans has indicated).? The way we look at data quality ( by we I mean the end users ) needs to be altered, I guess. best, Arka Chakraborty On Tue, Aug 27, 2013 at 9:50 AM, Phil Evans p...@mrc-lmb.cam.ac.uk wrote: The question you should ask yourself is why would omitting data improve my model? Phil
