Re: [ccp4bb] Real Space Refine Zone for Ligand
Hi Dipankar you need to pit a unique identifier for each of your ligands, eg phenix.elbow --smiles=cyclopiazonic_acid.smi --id=CZA --output=cza --opt best Preben On 4/16/12 4:52 AM, Dipankar Manna wrote: Dear Crystallographers, After one round of refinement (restrained refinement) with ligand, I inport the .cif file through '-Import CIF Dictionary' into Coot. But when I am going for '-Real Space Refine Zone' for the ligand, its showing Refinement set up failure. Failed to find restrained for: LIG. Please suggest. Thanks and Regards, Dipankar This e-mail and any files transmitted with it are for the sole use of the intended recipient(s) and may contain confidential and privileged information.If you are not the intended recipient, please contact the sender by reply e-mail and destroy all copies of the original message.Any unauthorized review, use, disclosure, dissemination, forwarding,printing or copying of this email or any action taken in reliance on this e-mail is strictly prohibited and may be unlawful. Visit us at http://www.aurigene.com -- J. Preben Morth, Ph.D Group Leader Membrane Transport Group Nordic EMBL Partnership Centre for Molecular Medicine Norway (NCMM) University of Oslo P.O.Box 1137 Blindern 0318 Oslo, Norway Email: j.p.mo...@ncmm.uio.no Tel: +47 2284 0794 http://www.jpmorth.dk
[ccp4bb] Off-topic-Cleavage with TEV protease
Dear all I want to digest a tagged protein with TEV protease, it has disulfide bridges. Is there any way of doing cleavage without DTT? Thank you. Theresa
Re: [ccp4bb] Off-topic-Cleavage with TEV protease
-BEGIN PGP SIGNED MESSAGE- Hash: SHA1 Dear Theresa, I was not aware you need DTT for TEV protease activity. People do on-column digestion, and as far as I remember, a Ni-column would turn really uglily brown of you used DTT on those columns. Have you tried to leave out DTT and the cleavage did not work? Cheers, Tim On 04/16/12 09:31, Theresa Hsu wrote: Dear all I want to digest a tagged protein with TEV protease, it has disulfide bridges. Is there any way of doing cleavage without DTT? Thank you. Theresa - -- - -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A -BEGIN PGP SIGNATURE- Version: GnuPG v1.4.12 (GNU/Linux) Comment: Using GnuPG with Mozilla - http://enigmail.mozdev.org/ iD8DBQFPi+H1UxlJ7aRr7hoRAjguAKCxEGcV2i6r8C31HBPIinJUI+PaogCgoBNb TrcS2lfQ753D4LmZ3tGNYok= =adb3 -END PGP SIGNATURE-
Re: [ccp4bb] Off-topic-Cleavage with TEV protease
I want to digest a tagged protein with TEV protease, it has disulfide bridges. Is there any way of doing cleavage without DTT? Yes, no problem. TEV is slowly inactivated oxidation of the active site cysteine but that's about it. If you absolutely must have no reducer during cleavage, simply up the amount of the enzyme. But it's almost certain that most proteins with S-S bridges will be perfectly happy at low reducer concentration (0.2 mM TCEP, 1 mM DTT or something along these lines; particularly if there is more than one bridge - mass action is a powerful thing and, e.g., IgG has no problem at 10 mM DTT). So I wouldn't worry about it - just add enough TEV under conditions that make your protein happy. Dima
[ccp4bb] sodium vs water near
--- On Mon, 24/1/11, Eleanor Dodson c...@ysbl.york.ac.uk wrote: From: Eleanor Dodson c...@ysbl.york.ac.uk Subject: Re: [ccp4bb] Merging statistics and systematic absences To: CCP4BB@JISCMAIL.AC.UK Date: Monday, 24 Jan
Re: [ccp4bb] bulk solvent treatment inside protein cavities
Dear Allister Could you please update refmac version. In the version you it seems that bulk solvent mask calculation has some problems. New version (at the moment) can be downloaded from this site: http://www.ysbl.york.ac.uk/refmac/data/refmac_experimental/refmac5.7_linux.tar.gz There is a mac version also. regards Garib On 16 Apr 2012, at 11:37, Allister Crow wrote: Board members, I have a couple of questions regarding how to improve the solvent model as applied to solvent-filled cavities inside proteins. I am currently nearing the end of refinement of a protein structure at 2.8 A resolution. I recently switched Refmac versions, upon doing this I noticed a modest improvement in R factors, but I also notice some new features in the difference maps. These features don't show up in the sigma-weighted 2Fo-Fc maps and are unlikely to be 'ligands' of any form. In fact, I suspect that the appearance of these features (which are all located in solvent channels within cavities inside the protein) are probably due to some difference in how the bulk solvent contribution has been applied. I've attached a picture of one such feature showing the difference between Refmac 5.5 and 5.6. (Both difference maps are contoured at 3 sigma- both using the same model and refinement parameters). My questions are therefore: 1) has something substantial changed in the bulk solvent treatment between Refmac versions 5.5 and 5.6? 2) How can I go about changing the bulk solvent treatment to better account for solvent contribution inside the protein cavities? Best wishes, and thanks in advance for all your help, - Allister Crow bulk_solvent_inside_cavities.png Dr Garib N Murshudov Group Leader, 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] bulk solvent treatment inside protein cavities
Oh dear - this is the version of Refmac in the latest ccp4 release - can this be updated on the web site as soon as possible ? Eleanor On 16 April 2012 12:02, Garib N Murshudov ga...@mrc-lmb.cam.ac.uk wrote: Dear Allister Could you please update refmac version. In the version you it seems that bulk solvent mask calculation has some problems. New version (at the moment) can be downloaded from this site: http://www.ysbl.york.ac.uk/refmac/data/refmac_experimental/ refmac5.7_linux.tar.gzhttp://www.ysbl.york.ac.uk/refmac/data/refmac_experimental/refmac5.7_linux.tar.gz There is a mac version also. regards Garib On 16 Apr 2012, at 11:37, Allister Crow wrote: Board members, I have a couple of questions regarding how to improve the solvent model as applied to solvent-filled cavities inside proteins. I am currently nearing the end of refinement of a protein structure at 2.8 A resolution. I recently switched Refmac versions, upon doing this I noticed a modest improvement in R factors, but I also notice some new features in the difference maps. These features don't show up in the sigma-weighted 2Fo-Fc maps and are unlikely to be 'ligands' of any form. In fact, I suspect that the appearance of these features (which are all located in solvent channels within cavities inside the protein) are probably due to some difference in how the bulk solvent contribution has been applied. I've attached a picture of one such feature showing the difference between Refmac 5.5 and 5.6. (Both difference maps are contoured at 3 sigma- both using the same model and refinement parameters). My questions are therefore: 1) has something substantial changed in the bulk solvent treatment between Refmac versions 5.5 and 5.6? 2) How can I go about changing the bulk solvent treatment to better account for solvent contribution inside the protein cavities? Best wishes, and thanks in advance for all your help, - Allister Crow bulk_solvent_inside_cavities.png Dr Garib N Murshudov Group Leader, MRC Laboratory of Molecular Biology Hills Road Cambridge CB2 0QH UK Email: ga...@mrc-lmb.cam.ac.uk jen...@mrc-lmb.cam.ac.uk Web http://www.mrc-lmb.cam.ac.uk
[ccp4bb] iMosflm version 1.0.6 Mosflm version 7.0.8
Dear all We are pleased to announce the public release of new versions of iMosflm and Mosflm. We have addressed many bugs and performance issues, and also tidied things up in some of the tasks. If you are using a previous version we strongly recommend that you upgrade. In brief - Improved processing for Pilatus images (both 6M and 2M) Faster processing for large datasets in iMosflm More reliability in indexing, refinement integration Better feedback Easier multi-crystal strategy Available from our website - http://www.mrc-lmb.cam.ac.uk/harry/mosflm == In more detail (see http://www.mrc-lmb.cam.ac.uk/harry/mosflm/ver708/Release_notes for more) - Mosflm == Non-Bragg spots (eg zingers and hot pixels) and ice spots are now automatically removed when forming the standard profiles. Improvements in mosaicity estimation and refinement. Improvements in standard deviation estimates. Improved spot finding parameters for in-house diffraction images and for the very small spots that can be obtained from room temperature (in situ screening) crystals. More robust integration of images with very small spot separation. iMosflm == The colour of the predicted reflections can be changed to improve visibility on weak diffraction images. Improved selection of the correct indexing solution in cases of pseudosymmetry. Scaling and merging now performed with AIMLESS rather than SCALA. Release Notes for iMosflm version 1.0.6 New features The speed of the interface has been much improved when processing a large number of images in the Integration pane. The largest speed-up came from not refreshing the profile displays afresh with every new image integrated. A greatly improved multi-crystal strategy option is available that makes it much simpler to calculate a data collection strategy when multiple crystals or multiple segments from one crystal are required (see below, and also see the new iMosflm tutorial for details). Bad spots identified during processing and whose numbers are plotted during integration can now be displayed in the Image display window. New mosaicity estimation will produce successive plots up to 8 degrees if required. Space group validation has been improved wherever a symbol can also be typed in editable, pull-down lists (Indexing and Strategy panes). The multi-sector, pull-down sectors list used in the Integration pane has been adopted in the Indexing and Cell refinement panes. Initial detector and crystal parameters are saved before refinement and checked that they have not refined to unreasonable values. Users may accept the warnings ( reset the parameters) or ignore them. New items added to the Advanced integration tab of Processing options includes provision for outliers affected by ice rings. The maximum number of images in an integration block has been increased from 20 to 200. The FindHKL function has been placed within the Image display window's toolbar. Smaller rotation increments have been added to the Rotation: setting in the Auto-complete menu in the Strategy pane. The editable 'pie' widget displayed at the bottom of the Strategy pane for any sector can now be adjusted in single degree steps (previously only 5 degree changes were possible). Individual images can now be deleted from the Images pane by selecting them (left-click) and then right-clicking on the selected image. This was previously only available for complete sectors. Scala has been replaced by Aimless as the default scaling program used by the QuickScale command button. Scala can be chosen from the Processing options-Advanced integration tab under the Pointless Aimless/Scala switches. Files containing lists of spots can be read into iMosflm and displayed on the appropriate image. Files can be read from the main Session menu item 'Read spots file...' and from the 'Read spots file...' button (with an open file icon) next to the 'Index' button in the Autoindexing pane. Harry -- Dr Harry Powell, MRC Laboratory of Molecular Biology, MRC Centre, Hills Road, Cambridge, CB2 0QH
Re: [ccp4bb] Off-topic-Cleavage with TEV protease
I have used 5 mM beta-Mercaptoethanol, which is a weaker reducing agent than DTT, and that keeps TEV happy as well. Cheers Florian Am 16.04.2012 um 09:31 schrieb Theresa Hsu: Dear all I want to digest a tagged protein with TEV protease, it has disulfide bridges. Is there any way of doing cleavage without DTT? Thank you. Theresa - Dr. Florian Brückner Biomolecular Research Laboratory OFLG/102 Paul Scherrer Institut CH-5232 Villigen PSI Switzerland Tel.: +41-(0)56-310-2332 Email: florian.brueck...@psi.ch
Re: [ccp4bb] Real Space Refine Zone for Ligand
On 16/04/12 03:52, Dipankar Manna wrote: Dear Crystallographers, After one round of refinement (restrained refinement) with ligand, I inport the .cif file through '-Import CIF Dictionary' into Coot. But when I am going for '-Real Space Refine Zone' for the ligand, its showing Refinement set up failure. Failed to find restrained for: LIG. Please suggest. If you are not using refmac-like restraints and not using pre-release Coot then this can happen :-( (my oversight). The work-around is to stop, and on restarting, first read in your LIG dictionary then read in the rest of the files. Fixed in 0.7-pre (and will be in 0.7+ of course). Cheers, Paul.
Re: [ccp4bb] bulk solvent treatment inside protein cavities
Dear Garib, Is there REFMAC option to output solvent mask information (e.g. Fmask and PHImask in mtz to check with Coot)? I tried to generate it by subtracting (FC, PHIC) from (FC_ALL,PHIC_ALL). But I'm not sure that FC_ALL = FC + FMASK is correct or not. Keitaro 2012/4/16 Garib N Murshudov ga...@mrc-lmb.cam.ac.uk: Dear Allister Could you please update refmac version. In the version you it seems that bulk solvent mask calculation has some problems. New version (at the moment) can be downloaded from this site: http://www.ysbl.york.ac.uk/refmac/data/refmac_experimental/refmac5.7_linux.tar.gz There is a mac version also. regards Garib On 16 Apr 2012, at 11:37, Allister Crow wrote: Board members, I have a couple of questions regarding how to improve the solvent model as applied to solvent-filled cavities inside proteins. I am currently nearing the end of refinement of a protein structure at 2.8 A resolution. I recently switched Refmac versions, upon doing this I noticed a modest improvement in R factors, but I also notice some new features in the difference maps. These features don't show up in the sigma-weighted 2Fo-Fc maps and are unlikely to be 'ligands' of any form. In fact, I suspect that the appearance of these features (which are all located in solvent channels within cavities inside the protein) are probably due to some difference in how the bulk solvent contribution has been applied. I've attached a picture of one such feature showing the difference between Refmac 5.5 and 5.6. (Both difference maps are contoured at 3 sigma- both using the same model and refinement parameters). My questions are therefore: 1) has something substantial changed in the bulk solvent treatment between Refmac versions 5.5 and 5.6? 2) How can I go about changing the bulk solvent treatment to better account for solvent contribution inside the protein cavities? Best wishes, and thanks in advance for all your help, - Allister Crow bulk_solvent_inside_cavities.png Dr Garib N Murshudov Group Leader, 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] bulk solvent treatment inside protein cavities
Yes there is. If you use command line (it is not available on the ccp4i yet). If you run with command lines refmac5 all others like hklin, xyzin etc mskout mask file name eof all options you want eof Then there will be a map and you can visualise it using coot. regards Garib On 16 Apr 2012, at 15:09, Keitaro Yamashita wrote: Dear Garib, Is there REFMAC option to output solvent mask information (e.g. Fmask and PHImask in mtz to check with Coot)? I tried to generate it by subtracting (FC, PHIC) from (FC_ALL,PHIC_ALL). But I'm not sure that FC_ALL = FC + FMASK is correct or not. Keitaro 2012/4/16 Garib N Murshudov ga...@mrc-lmb.cam.ac.uk: Dear Allister Could you please update refmac version. In the version you it seems that bulk solvent mask calculation has some problems. New version (at the moment) can be downloaded from this site: http://www.ysbl.york.ac.uk/refmac/data/refmac_experimental/refmac5.7_linux.tar.gz There is a mac version also. regards Garib On 16 Apr 2012, at 11:37, Allister Crow wrote: Board members, I have a couple of questions regarding how to improve the solvent model as applied to solvent-filled cavities inside proteins. I am currently nearing the end of refinement of a protein structure at 2.8 A resolution. I recently switched Refmac versions, upon doing this I noticed a modest improvement in R factors, but I also notice some new features in the difference maps. These features don't show up in the sigma-weighted 2Fo-Fc maps and are unlikely to be 'ligands' of any form. In fact, I suspect that the appearance of these features (which are all located in solvent channels within cavities inside the protein) are probably due to some difference in how the bulk solvent contribution has been applied. I've attached a picture of one such feature showing the difference between Refmac 5.5 and 5.6. (Both difference maps are contoured at 3 sigma- both using the same model and refinement parameters). My questions are therefore: 1) has something substantial changed in the bulk solvent treatment between Refmac versions 5.5 and 5.6? 2) How can I go about changing the bulk solvent treatment to better account for solvent contribution inside the protein cavities? Best wishes, and thanks in advance for all your help, - Allister Crow bulk_solvent_inside_cavities.png Dr Garib N Murshudov Group Leader, 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 Dr Garib N Murshudov Group Leader, 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] bulk solvent treatment inside protein cavities
A follow up: In the new version there is FC_ALL_LS, PHIC_ALL_LS That should be FC_ALL_LS = FC + FMASK. I have not tried but if you can use vector difference map then it should be: FMASK = FC_ALL_LS - FC But it is after scaling. If you write out mask map then it is just 0 1 map (0 inside protein and 1 outside), except values are not 0 1 but 0 and some constant Regards Garib On 16 Apr 2012, at 15:09, Keitaro Yamashita wrote: Dear Garib, Is there REFMAC option to output solvent mask information (e.g. Fmask and PHImask in mtz to check with Coot)? I tried to generate it by subtracting (FC, PHIC) from (FC_ALL,PHIC_ALL). But I'm not sure that FC_ALL = FC + FMASK is correct or not. Keitaro 2012/4/16 Garib N Murshudov ga...@mrc-lmb.cam.ac.uk: Dear Allister Could you please update refmac version. In the version you it seems that bulk solvent mask calculation has some problems. New version (at the moment) can be downloaded from this site: http://www.ysbl.york.ac.uk/refmac/data/refmac_experimental/refmac5.7_linux.tar.gz There is a mac version also. regards Garib On 16 Apr 2012, at 11:37, Allister Crow wrote: Board members, I have a couple of questions regarding how to improve the solvent model as applied to solvent-filled cavities inside proteins. I am currently nearing the end of refinement of a protein structure at 2.8 A resolution. I recently switched Refmac versions, upon doing this I noticed a modest improvement in R factors, but I also notice some new features in the difference maps. These features don't show up in the sigma-weighted 2Fo-Fc maps and are unlikely to be 'ligands' of any form. In fact, I suspect that the appearance of these features (which are all located in solvent channels within cavities inside the protein) are probably due to some difference in how the bulk solvent contribution has been applied. I've attached a picture of one such feature showing the difference between Refmac 5.5 and 5.6. (Both difference maps are contoured at 3 sigma- both using the same model and refinement parameters). My questions are therefore: 1) has something substantial changed in the bulk solvent treatment between Refmac versions 5.5 and 5.6? 2) How can I go about changing the bulk solvent treatment to better account for solvent contribution inside the protein cavities? Best wishes, and thanks in advance for all your help, - Allister Crow bulk_solvent_inside_cavities.png Dr Garib N Murshudov Group Leader, 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 Dr Garib N Murshudov Group Leader, 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] iMosflm version 1.0.6 Mosflm version 7.0.8
Where can one find a discussion of the differences between Aimless and Scala? JPK On Mon, Apr 16, 2012 at 8:28 AM, Harry Powell ha...@mrc-lmb.cam.ac.ukwrote: Dear all We are pleased to announce the public release of new versions of iMosflm and Mosflm. We have addressed many bugs and performance issues, and also tidied things up in some of the tasks. If you are using a previous version we strongly recommend that you upgrade. In brief - Improved processing for Pilatus images (both 6M and 2M) Faster processing for large datasets in iMosflm More reliability in indexing, refinement integration Better feedback Easier multi-crystal strategy Available from our website - http://www.mrc-lmb.cam.ac.uk/harry/mosflm == In more detail (see http://www.mrc-lmb.cam.ac.uk/harry/mosflm/ver708/Release_notes for more) - Mosflm == Non-Bragg spots (eg zingers and hot pixels) and ice spots are now automatically removed when forming the standard profiles. Improvements in mosaicity estimation and refinement. Improvements in standard deviation estimates. Improved spot finding parameters for in-house diffraction images and for the very small spots that can be obtained from room temperature (in situ screening) crystals. More robust integration of images with very small spot separation. iMosflm == The colour of the predicted reflections can be changed to improve visibility on weak diffraction images. Improved selection of the correct indexing solution in cases of pseudosymmetry. Scaling and merging now performed with AIMLESS rather than SCALA. Release Notes for iMosflm version 1.0.6 New features The speed of the interface has been much improved when processing a large number of images in the Integration pane. The largest speed-up came from not refreshing the profile displays afresh with every new image integrated. A greatly improved multi-crystal strategy option is available that makes it much simpler to calculate a data collection strategy when multiple crystals or multiple segments from one crystal are required (see below, and also see the new iMosflm tutorial for details). Bad spots identified during processing and whose numbers are plotted during integration can now be displayed in the Image display window. New mosaicity estimation will produce successive plots up to 8 degrees if required. Space group validation has been improved wherever a symbol can also be typed in editable, pull-down lists (Indexing and Strategy panes). The multi-sector, pull-down sectors list used in the Integration pane has been adopted in the Indexing and Cell refinement panes. Initial detector and crystal parameters are saved before refinement and checked that they have not refined to unreasonable values. Users may accept the warnings ( reset the parameters) or ignore them. New items added to the Advanced integration tab of Processing options includes provision for outliers affected by ice rings. The maximum number of images in an integration block has been increased from 20 to 200. The FindHKL function has been placed within the Image display window's toolbar. Smaller rotation increments have been added to the Rotation: setting in the Auto-complete menu in the Strategy pane. The editable 'pie' widget displayed at the bottom of the Strategy pane for any sector can now be adjusted in single degree steps (previously only 5 degree changes were possible). Individual images can now be deleted from the Images pane by selecting them (left-click) and then right-clicking on the selected image. This was previously only available for complete sectors. Scala has been replaced by Aimless as the default scaling program used by the QuickScale command button. Scala can be chosen from the Processing options-Advanced integration tab under the Pointless Aimless/Scala switches. Files containing lists of spots can be read into iMosflm and displayed on the appropriate image. Files can be read from the main Session menu item 'Read spots file...' and from the 'Read spots file...' button (with an open file icon) next to the 'Index' button in the Autoindexing pane. Harry -- Dr Harry Powell, MRC Laboratory of Molecular Biology, MRC Centre, Hills Road, Cambridge, CB2 0QH -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program email: j-kell...@northwestern.edu ***
Re: [ccp4bb] bulk solvent treatment inside protein cavities
Hi there! I think some of you have given my email for correspondence or CC, please I do not have to do anything related to your conversation so please delete my email address from your correspondence I shall be grateful THANKS REGARDS! Tallat Hussain Ghazi IT Manager / Head of Web Graphics Depts. ITSS (Pvt) Ltd. Email: tal...@itssols.com Website: www.itssols.com Mob: +92 (0)323 992 Tel: +92 (0)42 5694723-25 Fax: +92 (0)42 5694727 -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Keitaro Yamashita Sent: Monday, April 16, 2012 7:09 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] bulk solvent treatment inside protein cavities Dear Garib, Is there REFMAC option to output solvent mask information (e.g. Fmask and PHImask in mtz to check with Coot)? I tried to generate it by subtracting (FC, PHIC) from (FC_ALL,PHIC_ALL). But I'm not sure that FC_ALL = FC + FMASK is correct or not. Keitaro 2012/4/16 Garib N Murshudov ga...@mrc-lmb.cam.ac.uk: Dear Allister Could you please update refmac version. In the version you it seems that bulk solvent mask calculation has some problems. New version (at the moment) can be downloaded from this site: http://www.ysbl.york.ac.uk/refmac/data/refmac_experimental/refmac5.7_l inux.tar.gz There is a mac version also. regards Garib On 16 Apr 2012, at 11:37, Allister Crow wrote: Board members, I have a couple of questions regarding how to improve the solvent model as applied to solvent-filled cavities inside proteins. I am currently nearing the end of refinement of a protein structure at 2.8 A resolution. I recently switched Refmac versions, upon doing this I noticed a modest improvement in R factors, but I also notice some new features in the difference maps. These features don't show up in the sigma-weighted 2Fo-Fc maps and are unlikely to be 'ligands' of any form. In fact, I suspect that the appearance of these features (which are all located in solvent channels within cavities inside the protein) are probably due to some difference in how the bulk solvent contribution has been applied. I've attached a picture of one such feature showing the difference between Refmac 5.5 and 5.6. (Both difference maps are contoured at 3 sigma- both using the same model and refinement parameters). My questions are therefore: 1) has something substantial changed in the bulk solvent treatment between Refmac versions 5.5 and 5.6? 2) How can I go about changing the bulk solvent treatment to better account for solvent contribution inside the protein cavities? Best wishes, and thanks in advance for all your help, - Allister Crow bulk_solvent_inside_cavities.png Dr Garib N Murshudov Group Leader, 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] bulk solvent treatment inside protein cavities
Dear Garib, Thank you very much for your quick reply. I tried mskout option and the output looked almost the same as the map generated by FC_ALL - FC. By the way, when mskout option is specified, refmac stops before CGMAT cycles. Is there any way to do refinement with mskout option? I have not tried but if you can use vector difference map then it should be: FMASK = FC_ALL_LS - FC What is FC_ALL in the new version? Thanks, Keitaro 2012/4/16 Garib N Murshudov ga...@mrc-lmb.cam.ac.uk: A follow up: In the new version there is FC_ALL_LS, PHIC_ALL_LS That should be FC_ALL_LS = FC + FMASK. I have not tried but if you can use vector difference map then it should be: FMASK = FC_ALL_LS - FC But it is after scaling. If you write out mask map then it is just 0 1 map (0 inside protein and 1 outside), except values are not 0 1 but 0 and some constant Regards Garib On 16 Apr 2012, at 15:09, Keitaro Yamashita wrote: Dear Garib, Is there REFMAC option to output solvent mask information (e.g. Fmask and PHImask in mtz to check with Coot)? I tried to generate it by subtracting (FC, PHIC) from (FC_ALL,PHIC_ALL). But I'm not sure that FC_ALL = FC + FMASK is correct or not. Keitaro 2012/4/16 Garib N Murshudov ga...@mrc-lmb.cam.ac.uk: Dear Allister Could you please update refmac version. In the version you it seems that bulk solvent mask calculation has some problems. New version (at the moment) can be downloaded from this site: http://www.ysbl.york.ac.uk/refmac/data/refmac_experimental/refmac5.7_linux.tar.gz There is a mac version also. regards Garib On 16 Apr 2012, at 11:37, Allister Crow wrote: Board members, I have a couple of questions regarding how to improve the solvent model as applied to solvent-filled cavities inside proteins. I am currently nearing the end of refinement of a protein structure at 2.8 A resolution. I recently switched Refmac versions, upon doing this I noticed a modest improvement in R factors, but I also notice some new features in the difference maps. These features don't show up in the sigma-weighted 2Fo-Fc maps and are unlikely to be 'ligands' of any form. In fact, I suspect that the appearance of these features (which are all located in solvent channels within cavities inside the protein) are probably due to some difference in how the bulk solvent contribution has been applied. I've attached a picture of one such feature showing the difference between Refmac 5.5 and 5.6. (Both difference maps are contoured at 3 sigma- both using the same model and refinement parameters). My questions are therefore: 1) has something substantial changed in the bulk solvent treatment between Refmac versions 5.5 and 5.6? 2) How can I go about changing the bulk solvent treatment to better account for solvent contribution inside the protein cavities? Best wishes, and thanks in advance for all your help, - Allister Crow bulk_solvent_inside_cavities.png Dr Garib N Murshudov Group Leader, 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 Dr Garib N Murshudov Group Leader, 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] iMosflm version 1.0.6 Mosflm version 7.0.8
Aimless is a complete rewrite of Scala, but does essentially the same task. I haven't yet written it up properly, but there is a program document at ftp://ftp.mrc-lmb.cam.ac.uk/pub/pre/aimless.html A rough flow of the program is as follows:- Read file from eg POINTLESS, sort data if necessary (SORTMTZ no longer needed) Initial scale estimates from average intensities First round scaling with small selection of strong reflections, chosen on I/sigI First outlier rejection Optimise the combination of profile-fitted (for weak spots) or summation integration intensities (for strong spots) from MOSFLM First optimisation of sig(I) estimates Main scaling on relatively strong reflections, chosen on normalised intensity E^2 (eg 0.8 E^2 5) Second outlier rejection Final optimisation of sig(I) estimates Final outlier rejection Final statistics Output of merged or unmerged data (MTZ or Scalepack format, or both) Phil On 16 Apr 2012, at 15:42, Jacob Keller wrote: Where can one find a discussion of the differences between Aimless and Scala? JPK On Mon, Apr 16, 2012 at 8:28 AM, Harry Powell ha...@mrc-lmb.cam.ac.uk wrote: Dear all We are pleased to announce the public release of new versions of iMosflm and Mosflm. We have addressed many bugs and performance issues, and also tidied things up in some of the tasks. If you are using a previous version we strongly recommend that you upgrade. In brief - Improved processing for Pilatus images (both 6M and 2M) Faster processing for large datasets in iMosflm More reliability in indexing, refinement integration Better feedback Easier multi-crystal strategy Available from our website - http://www.mrc-lmb.cam.ac.uk/harry/mosflm == In more detail (see http://www.mrc-lmb.cam.ac.uk/harry/mosflm/ver708/Release_notes for more) - Mosflm == Non-Bragg spots (eg zingers and hot pixels) and ice spots are now automatically removed when forming the standard profiles. Improvements in mosaicity estimation and refinement. Improvements in standard deviation estimates. Improved spot finding parameters for in-house diffraction images and for the very small spots that can be obtained from room temperature (in situ screening) crystals. More robust integration of images with very small spot separation. iMosflm == The colour of the predicted reflections can be changed to improve visibility on weak diffraction images. Improved selection of the correct indexing solution in cases of pseudosymmetry. Scaling and merging now performed with AIMLESS rather than SCALA. Release Notes for iMosflm version 1.0.6 New features The speed of the interface has been much improved when processing a large number of images in the Integration pane. The largest speed-up came from not refreshing the profile displays afresh with every new image integrated. A greatly improved multi-crystal strategy option is available that makes it much simpler to calculate a data collection strategy when multiple crystals or multiple segments from one crystal are required (see below, and also see the new iMosflm tutorial for details). Bad spots identified during processing and whose numbers are plotted during integration can now be displayed in the Image display window. New mosaicity estimation will produce successive plots up to 8 degrees if required. Space group validation has been improved wherever a symbol can also be typed in editable, pull-down lists (Indexing and Strategy panes). The multi-sector, pull-down sectors list used in the Integration pane has been adopted in the Indexing and Cell refinement panes. Initial detector and crystal parameters are saved before refinement and checked that they have not refined to unreasonable values. Users may accept the warnings ( reset the parameters) or ignore them. New items added to the Advanced integration tab of Processing options includes provision for outliers affected by ice rings. The maximum number of images in an integration block has been increased from 20 to 200. The FindHKL function has been placed within the Image display window's toolbar. Smaller rotation increments have been added to the Rotation: setting in the Auto-complete menu in the Strategy pane. The editable 'pie' widget displayed at the bottom of the Strategy pane for any sector can now be adjusted in single degree steps (previously only 5 degree changes were possible). Individual images can now be deleted from the Images pane by selecting them (left-click) and then right-clicking on the selected image. This was previously only available for complete sectors. Scala has been replaced by Aimless as the default scaling program used by the QuickScale command button. Scala can be chosen from the Processing options-Advanced integration tab under the
Re: [ccp4bb] bulk solvent treatment inside protein cavities
Dear Ketaro At the moment mskout option is a signal that the program should stop. Obviously I can add an option to continue. However if you have mskout option it is likely that you want to check what is going on with the mask. If you want to compare starting and final mask then you could run refmac with mskout in the beginning and after refinement. If you need it urgently then I can add continuation of refinement with mskout option. FC_ALL is ML scaled FC+FMASK Sometime it may be different from least-squares scaled FC+FMASK regards Garib On 16 Apr 2012, at 16:01, Keitaro Yamashita wrote: Dear Garib, Thank you very much for your quick reply. I tried mskout option and the output looked almost the same as the map generated by FC_ALL - FC. By the way, when mskout option is specified, refmac stops before CGMAT cycles. Is there any way to do refinement with mskout option? I have not tried but if you can use vector difference map then it should be: FMASK = FC_ALL_LS - FC What is FC_ALL in the new version? Thanks, Keitaro 2012/4/16 Garib N Murshudov ga...@mrc-lmb.cam.ac.uk: A follow up: In the new version there is FC_ALL_LS, PHIC_ALL_LS That should be FC_ALL_LS = FC + FMASK. I have not tried but if you can use vector difference map then it should be: FMASK = FC_ALL_LS - FC But it is after scaling. If you write out mask map then it is just 0 1 map (0 inside protein and 1 outside), except values are not 0 1 but 0 and some constant Regards Garib On 16 Apr 2012, at 15:09, Keitaro Yamashita wrote: Dear Garib, Is there REFMAC option to output solvent mask information (e.g. Fmask and PHImask in mtz to check with Coot)? I tried to generate it by subtracting (FC, PHIC) from (FC_ALL,PHIC_ALL). But I'm not sure that FC_ALL = FC + FMASK is correct or not. Keitaro 2012/4/16 Garib N Murshudov ga...@mrc-lmb.cam.ac.uk: Dear Allister Could you please update refmac version. In the version you it seems that bulk solvent mask calculation has some problems. New version (at the moment) can be downloaded from this site: http://www.ysbl.york.ac.uk/refmac/data/refmac_experimental/refmac5.7_linux.tar.gz There is a mac version also. regards Garib On 16 Apr 2012, at 11:37, Allister Crow wrote: Board members, I have a couple of questions regarding how to improve the solvent model as applied to solvent-filled cavities inside proteins. I am currently nearing the end of refinement of a protein structure at 2.8 A resolution. I recently switched Refmac versions, upon doing this I noticed a modest improvement in R factors, but I also notice some new features in the difference maps. These features don't show up in the sigma-weighted 2Fo-Fc maps and are unlikely to be 'ligands' of any form. In fact, I suspect that the appearance of these features (which are all located in solvent channels within cavities inside the protein) are probably due to some difference in how the bulk solvent contribution has been applied. I've attached a picture of one such feature showing the difference between Refmac 5.5 and 5.6. (Both difference maps are contoured at 3 sigma- both using the same model and refinement parameters). My questions are therefore: 1) has something substantial changed in the bulk solvent treatment between Refmac versions 5.5 and 5.6? 2) How can I go about changing the bulk solvent treatment to better account for solvent contribution inside the protein cavities? Best wishes, and thanks in advance for all your help, - Allister Crow bulk_solvent_inside_cavities.png Dr Garib N Murshudov Group Leader, 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 Dr Garib N Murshudov Group Leader, 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 Dr Garib N Murshudov Group Leader, 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] bulk solvent treatment inside protein cavities
Dear Garib, I think it is better if refmac outputs final solvent mask when mskout specified. If one just wanted to calculate the mask, NCYC 0 should be specified. I hope my suggestion would be accepted, but I'm not in a hurry. FC_ALL is ML scaled FC+FMASK Sometime it may be different from least-squares scaled FC+FMASK For what purpose is FC_ALL_LS written? Can I check something by comparing FC_ALLto FC_ALL_LS? I found somewhat large difference between FC_ALL_LS map and FC_ALL map in Se position. I used SAD function. What does it mean? Cheers, Keitaro 2012/4/17 Garib N Murshudov ga...@mrc-lmb.cam.ac.uk: Dear Ketaro At the moment mskout option is a signal that the program should stop. Obviously I can add an option to continue. However if you have mskout option it is likely that you want to check what is going on with the mask. If you want to compare starting and final mask then you could run refmac with mskout in the beginning and after refinement. If you need it urgently then I can add continuation of refinement with mskout option. FC_ALL is ML scaled FC+FMASK Sometime it may be different from least-squares scaled FC+FMASK regards Garib On 16 Apr 2012, at 16:01, Keitaro Yamashita wrote: Dear Garib, Thank you very much for your quick reply. I tried mskout option and the output looked almost the same as the map generated by FC_ALL - FC. By the way, when mskout option is specified, refmac stops before CGMAT cycles. Is there any way to do refinement with mskout option? I have not tried but if you can use vector difference map then it should be: FMASK = FC_ALL_LS - FC What is FC_ALL in the new version? Thanks, Keitaro 2012/4/16 Garib N Murshudov ga...@mrc-lmb.cam.ac.uk: A follow up: In the new version there is FC_ALL_LS, PHIC_ALL_LS That should be FC_ALL_LS = FC + FMASK. I have not tried but if you can use vector difference map then it should be: FMASK = FC_ALL_LS - FC But it is after scaling. If you write out mask map then it is just 0 1 map (0 inside protein and 1 outside), except values are not 0 1 but 0 and some constant Regards Garib On 16 Apr 2012, at 15:09, Keitaro Yamashita wrote: Dear Garib, Is there REFMAC option to output solvent mask information (e.g. Fmask and PHImask in mtz to check with Coot)? I tried to generate it by subtracting (FC, PHIC) from (FC_ALL,PHIC_ALL). But I'm not sure that FC_ALL = FC + FMASK is correct or not. Keitaro 2012/4/16 Garib N Murshudov ga...@mrc-lmb.cam.ac.uk: Dear Allister Could you please update refmac version. In the version you it seems that bulk solvent mask calculation has some problems. New version (at the moment) can be downloaded from this site: http://www.ysbl.york.ac.uk/refmac/data/refmac_experimental/refmac5.7_linux.tar.gz There is a mac version also. regards Garib On 16 Apr 2012, at 11:37, Allister Crow wrote: Board members, I have a couple of questions regarding how to improve the solvent model as applied to solvent-filled cavities inside proteins. I am currently nearing the end of refinement of a protein structure at 2.8 A resolution. I recently switched Refmac versions, upon doing this I noticed a modest improvement in R factors, but I also notice some new features in the difference maps. These features don't show up in the sigma-weighted 2Fo-Fc maps and are unlikely to be 'ligands' of any form. In fact, I suspect that the appearance of these features (which are all located in solvent channels within cavities inside the protein) are probably due to some difference in how the bulk solvent contribution has been applied. I've attached a picture of one such feature showing the difference between Refmac 5.5 and 5.6. (Both difference maps are contoured at 3 sigma- both using the same model and refinement parameters). My questions are therefore: 1) has something substantial changed in the bulk solvent treatment between Refmac versions 5.5 and 5.6? 2) How can I go about changing the bulk solvent treatment to better account for solvent contribution inside the protein cavities? Best wishes, and thanks in advance for all your help, - Allister Crow bulk_solvent_inside_cavities.png Dr Garib N Murshudov Group Leader, 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 Dr Garib N Murshudov Group Leader, 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 Dr Garib N Murshudov Group Leader, 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] software to generate protein topology diagram like those available at PDBsum webserver
Dear All, Guillaume Ponchel referred me to ProOrigami (which can be installed locally) to generate protein topology diagrams: http://munk.csse.unimelb.edu.au/pro-origami/ Thanks again. Best wishes, Partha On Mon, Apr 16, 2012 at 12:12 AM, Parthasarathy Sampathkumar spart...@gmail.com wrote: Dear All, Thanks to Herb, John, Simanshu, and Anu for link: http://www.ebi.ac.uk/thornton-srv/databases/pdbsum/Generate.html to generate PDBsum like topology diagrams. Partha On Sun, Apr 15, 2012 at 8:08 PM, Parthasarathy Sampathkumar spart...@gmail.com wrote: Dear All, I would like to generate a topology diagram similar to the ones available for deposited entries at the PDBsum webpage (attached one such diagram here for reference) for a new structure. Is this program is available for download? I am aware of TopDraw. However, I am trying to analyze a ~850 a.a. structure. So, some automation would be helpful. Thanks in advance for your help. Best wishes, Partha
Re: [ccp4bb] Off-topic-Cleavage with TEV protease
I've run into the same problem, and found David Waugh's FAQ to be a great resource: http://mcl1.ncifcrf.gov/waugh_tech.html They use a 3mM buffer of 10:1 reduced:oxidized glutathione. I've tried that and it cleaves my protein without reducing reducing the disulfide bridges. I'll second someone else's suggestion to add more TEV. That's worked for me as well, as long as the TEV's relatively fresh and there isn't too much reducing agent introduced along with it. Jason
[ccp4bb] Off-pick: suggesiton of puck
Hi guys: Recently we need purchase some puck for Actor, unfortunately we can't find any agency in China. I really appreciate that if anyone can give us some suggestion about that? such like brand, price, and necessary accessory? Thanks for help Best regards Frank
Re: [ccp4bb] Off-topic-Cleavage with TEV protease
Also keep in mind that many of the purchased TEVs are formulated with some reducing agent (e.g. AcTEV comes in a buffer with 5mM DTT, if I recall correctly). So unless the enzyme is buffer exchanged beforehand, there will be some reducing agent introduced alongside it, depending on the dilution. HTH, -Tim On Mon, Apr 16, 2012 at 4:32 PM, Jason Forse fo...@scripps.edu wrote: I've run into the same problem, and found David Waugh's FAQ to be a great resource: http://mcl1.ncifcrf.gov/waugh_tech.html They use a 3mM buffer of 10:1 reduced:oxidized glutathione. I've tried that and it cleaves my protein without reducing reducing the disulfide bridges. I'll second someone else's suggestion to add more TEV. That's worked for me as well, as long as the TEV's relatively fresh and there isn't too much reducing agent introduced along with it. Jason
Re: [ccp4bb] Off-pick: suggesiton of puck
Rigaku ACTOR robots can accept many different styles of pucks depending on the LN2 dewar baseplate that the particular ACTOR is equipped with. Rigaku in China will sell ACTOR pucks, but if you are looking for ALS-style pucks or ESRF-style pucks or Unipucks or ???, then I am not sure where to get them. Anyways, I have forwarded your query to my colleagues in China. Jim From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Frank [fanshil...@hotmail.com] Sent: Monday, April 16, 2012 8:35 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Off-pick: suggesiton of puck Hi guys: Recently we need purchase some puck for Actor, unfortunately we can't find any agency in China. I really appreciate that if anyone can give us some suggestion about that? such like brand, price, and necessary accessory? Thanks for help Best regards Frank
