Re: [ccp4bb] small lines in diffraction pattern
Hi Stephan, If there were overflows on the detector, which cause lines due to the spill over of the wells of the CCD, the lines would be visible in the readout direction of the CCD detector, which generally is from bottom to top or top to bottom. You can also see this with your own (pocket) camera if you point it towards a bright light source, before you take the actual picture. The lines cannot be in a skew direction and will always be connected to very strong reflections. As you can see in the images Margriet sent, the lines are parallel to one of the reciprocal planes and there are plenty of lines in the image that are not connected to reflections. With very (and I mean very) strong reflections you will sometimes see an speckled arc around the reflection. This is diffraction of the beryllium window of the detector, where the reflected beam acts as the primary beam (see attached picture.) Bram Stephan Ginell wrote: Hi Such line can occur on a CCD detector if reflection are saturating a pixel and is generally in the direction of the detector readout. 1) what detector are you using, 2) are reflections in the black center saturated i.e. Greater than the dynamic range of the detector. 3) what is your exposure time, 4) do you see such streaks on short / attenuated exposures? 5 do you see such streaks on dark images of (no x-rays) but same time. Steve *Bram Schierbeek* Application Scientist Structural Biology Bruker AXS B.V. Oostsingel 209, P.O.Box 811 2600 AV Delft, the Netherlands Tel.: +31 (15) 2152508 Fax:+31 (15)2152599 bram.schierb...@bruker-axs.nl _www.bruker-axs.com_ www.bruker-axs.de
Re: [ccp4bb] Small lines in diffraction pattern (more info)
Dear all, There were some comments about detector issues, but these can be ruled out, to my opinion, since the lines appeared on different beamlines. Default settings of mosflm (spot picking) finds the cell 34 34 34 90 90 90 (pointless indicating P41212) Structure was solved by SAD phasing on the phosphates in this space group. Double helices stack in continuous helices, the backbone is well defined in the (refined) density maps but the individual bases are messy (purines and pyrimidines seemed to overlap) + obviously not all spots were covered and the duplex does not fit in the A.U. For this reason the integration was repeated in the higher cell 34 34 170 Space group most probably P212121, but solutions can be found in P41212 as well (still disordered bases) There are also indications that the 41 screw axis is rather a pseudo axis than a pure crystallographic one, also in the small cell Reindexing the cell to 34 34 340 also gives a solution, which supports the theory of Holton Rmerg is around 5% for the small cell, about 8% for the 170Å cell (both in P41212) Which refinement procedure would be best to follow? kind regards Margriet Margriet Ovaere Chemistry Department K.U.Leuven Biomolecular Architecture Celestijnenlaan 200 F B-3001 Heverlee (Leuven) Tel: +32(0)16327477 Disclaimer: http://www.kuleuven.be/cwis/email_disclaimer.htm
Re: [ccp4bb] small lines in diffraction pattern
Hi Nice of Jacob to mention the paper below but I don't think it is relevant to these patterns (well it might not be relevant to anything!). I think James has given the most likely explanation. The AB type stacking disorder he mentioned is similar to the type in the paper I referenced. I think James is also right in saying the intensities of the preserved sharp spots can still be used. The point Jacob and others made about the repeats of strong intensity (e.g. every 5 spots in one direction) must be relevant. Can we have unit cell dimensions and any other details.? Cheers Colin From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Jacob Keller Sent: 28 January 2009 18:05 To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] small lines in diffraction pattern Acta Cryst. (1998). D54, 848-853[ doi:10.1107/S0907444998001875 http://dx.doi.org/10.1107/S0907444998001875 ] A Description of Imperfections in Protein Crystals C. Nave http://scripts.iucr.org/cgi-bin/citedin?search_on=nameauthor_name=Nave ,%20C. Abstract: An analysis is given of the contribution of various crystal imperfections to the rocking widths of reflections and the divergence of the diffracted beams. The crystal imperfections are the angular spread of the mosaic blocks in the crystal, the size of the mosaic blocks and the variation in cell dimensions between blocks. The analysis has implications for improving crystal perfection, defining data-collection requirements and for data-processing procedures. Measurements on crystals of tetragonal lysozyme at room temperature and 100 K were made in order to illustrate how parameters describing the crystal imperfections can be obtained. At 100 K, the dominant imperfection appeared to be a variation in unit-cell dimensions in the crystal. *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program Dallos Laboratory F. Searle 1-240 2240 Campus Drive Evanston IL 60208 lab: 847.491.2438 cel: 773.608.9185 email: j-kell...@northwestern.edu mailto:j-kell...@northwestern.edu *** - Original Message - From: Jacob Keller mailto:j-kell...@md.northwestern.edu To: CCP4BB@JISCMAIL.AC.UK mailto:CCP4BB@JISCMAIL.AC.UK Sent: Wednesday, January 28, 2009 11:57 AM Subject: Re: [ccp4bb] small lines in diffraction pattern I had thought that in a previous thread, we had all come to a consensus that actually the largest source of what is normally explained as mosaicity is really differences in unit cell size, due perhaps to uneven shrinkage in crystals upon freezing or otherwise. I believe that there was actually an acta cryst paper which investigated all of the various ingredients of mosaicity which supports this (this is why I said it.) Jacob *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program Dallos Laboratory F. Searle 1-240 2240 Campus Drive Evanston IL 60208 lab: 847.491.2438 cel: 773.608.9185 email: j-kell...@northwestern.edu mailto:j-kell...@northwestern.edu *** DIVFONT size=1 color=grayThis e-mail and any attachments may contain confidential, copyright and or privileged material, and are for the use of the intended addressee only. If you are not the intended addressee or an authorised recipient of the addressee please notify us of receipt by returning the e-mail and do not use, copy, retain, distribute or disclose the information in or attached to the e-mail. Any opinions expressed within this e-mail are those of the individual and not necessarily of Diamond Light Source Ltd. Diamond Light Source Ltd. cannot guarantee that this e-mail or any attachments are free from viruses and we cannot accept liability for any damage which you may sustain as a result of software viruses which may be transmitted in or with the message. Diamond Light Source Limited (company no. 4375679). Registered in England and Wales with its registered office at Diamond House, Harwell Science and Innovation Campus, Didcot, Oxfordshire, OX11 0DE, United Kingdom /FONT/DIV -- Scanned by iCritical.
Re: [ccp4bb] Small lines in diffraction pattern (more info)
The 34-34-34 cell does not predict all the spots, does it? from the diffraction pattern it seems only the 34-34-170 or 34-34-340 cell can predict all spots, so the structure should be solved in the one that predicts all spots. The procedure I would use is to take a 180º dataset, sacrificing some resolution if necessary, then integrate in P1 and solve the structure by MR or if possible by just placing the double helices in the cell. If you then still have disorder of the bases this means that the dsRNA can fit in the density in several ways, either up and down, or, less likely, if the ends of the 10 bp duplexes are not clear, even displaced by one of more bases along the long axis. In principle there is no reason why the differences in sequence (inside structure) always have to lead to significant differences in the outside structure (the phosphates) and thus affect the crystal packing. They usually do, but not always (some dimeric transcription factors crystallised with asymmetric dsDNA duplexes have disordered bases, as long as the bases with which the protein interacts are the same the protein doesn't necessarily care that much about the rest...). Once you have your structure clear in P1, you can introduce the possible symmetry axes one by one; and once you have your spacegroup clear, collect as high as possible resolution data with the minimal wedge necessary and as high a dose as possible without inducing radation damage. If your duplexes fit in two ways, up and down, refinement should be possible with both at partial occupancy. Mark J. van Raaij Dpto de Bioquímica, Facultad de Farmacia Universidad de Santiago 15782 Santiago de Compostela Spain http://web.usc.es/~vanraaij/ On 29 Jan 2009, at 10:45, Margriet Ovaere wrote: Dear all, There were some comments about detector issues, but these can be ruled out, to my opinion, since the lines appeared on different beamlines. Default settings of mosflm (spot picking) finds the cell 34 34 34 90 90 90 (pointless indicating P41212) Structure was solved by SAD phasing on the phosphates in this space group. Double helices stack in continuous helices, the backbone is well defined in the (refined) density maps but the individual bases are messy (purines and pyrimidines seemed to overlap) + obviously not all spots were covered and the duplex does not fit in the A.U. For this reason the integration was repeated in the higher cell 34 34 170 Space group most probably P212121, but solutions can be found in P41212 as well (still disordered bases) There are also indications that the 41 screw axis is rather a pseudo axis than a pure crystallographic one, also in the small cell Reindexing the cell to 34 34 340 also gives a solution, which supports the theory of Holton Rmerg is around 5% for the small cell, about 8% for the 170Å cell (both in P41212) Which refinement procedure would be best to follow? kind regards Margriet Margriet Ovaere Chemistry Department K.U.Leuven Biomolecular Architecture Celestijnenlaan 200 F B-3001 Heverlee (Leuven) Tel: +32(0)16327477 Disclaimer: http://www.kuleuven.be/cwis/email_disclaimer.htm for more information.
Re: [ccp4bb] Small lines in diffraction pattern (more info)
Dear Margriet, From your description and what James Holton wrote, it seems that you have 2 types of unit cells: A: with the sense strand in position 1 and the antisense strand in position 2 B: with the antisense strand in position 1 and the sense strand in position 2 If the crystal contacts are mainly via the backbone, your crystal may contain a random distribution of both and the electron density you see is a superposition of both and for the crystal packing, both chains are identical. This situation is similar to the situation when an asymmetric inhibitor is bound to a dimeric, symmetric molecule like e.g. HIV protease. In this case, both orientations are deconvoluted using detwinning methods for perfect twinning (see e.g. Proc Natl Acad Sci U S A. 2002 November 12; 99(23): 14664-14669). The 34,34,34 cell is definitively too small, so I would process in the 34,34,170 cell and detwin. You molecular replacement solutions should tell you which twinning operator to use. Best regards, Herman From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Margriet Ovaere Sent: Thursday, January 29, 2009 10:45 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Small lines in diffraction pattern (more info) Dear all, There were some comments about detector issues, but these can be ruled out, to my opinion, since the lines appeared on different beamlines. Default settings of mosflm (spot picking) finds the cell 34 34 34 90 90 90 (pointless indicating P41212) Structure was solved by SAD phasing on the phosphates in this space group. Double helices stack in continuous helices, the backbone is well defined in the (refined) density maps but the individual bases are messy (purines and pyrimidines seemed to overlap) + obviously not all spots were covered and the duplex does not fit in the A.U. For this reason the integration was repeated in the higher cell 34 34 170 Space group most probably P212121, but solutions can be found in P41212 as well (still disordered bases) There are also indications that the 41 screw axis is rather a pseudo axis than a pure crystallographic one, also in the small cell Reindexing the cell to 34 34 340 also gives a solution, which supports the theory of Holton Rmerg is around 5% for the small cell, about 8% for the 170Å cell (both in P41212) Which refinement procedure would be best to follow? kind regards Margriet Margriet Ovaere Chemistry Department K.U.Leuven Biomolecular Architecture Celestijnenlaan 200 F B-3001 Heverlee (Leuven) Tel: +32(0)16327477 Disclaimer: http://www.kuleuven.be/cwis/email_disclaimer.htm for more information.
Re: [ccp4bb] Small lines in diffraction pattern (more info)
Hi Herman Aren't detwinning methods appropriate only in the case of true twin domains which are larger than the X-ray photon correlation length in order for the assumption to be valid that |F|^2 from each domain can be summed? This wouldn't give rise to the apparent 'diffuse scatter' phenomenon. However if what you are describing is rather static disorder of unit cells, which would give rise to diffuse scatter, where A B type cells are randomly mixed (so a domain is only one or at most a few unit cells), as opposed to being confined to A B type domains, then detwinning would not be appropriate. Cheers -- Ian -Original Message- From: owner-ccp...@jiscmail.ac.uk [mailto:owner-ccp...@jiscmail.ac.uk] On Behalf Of herman.schreu...@sanofi-aventis.com Sent: 29 January 2009 11:19 To: margriet.ova...@chem.kuleuven.be; CCP4BB@JISCMAIL.AC.UK Subject: RE: [ccp4bb] Small lines in diffraction pattern (more info) Dear Margriet, From your description and what James Holton wrote, it seems that you have 2 types of unit cells: A: with the sense strand in position 1 and the antisense strand in position 2 B: with the antisense strand in position 1 and the sense strand in position 2 If the crystal contacts are mainly via the backbone, your crystal may contain a random distribution of both and the electron density you see is a superposition of both and for the crystal packing, both chains are identical. This situation is similar to the situation when an asymmetric inhibitor is bound to a dimeric, symmetric molecule like e.g. HIV protease. In this case, both orientations are deconvoluted using detwinning methods for perfect twinning (see e.g. Proc Natl Acad Sci U S A. 2002 November 12; 99(23): 14664-14669). The 34,34,34 cell is definitively too small, so I would process in the 34,34,170 cell and detwin. You molecular replacement solutions should tell you which twinning operator to use. Best regards, Herman From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Margriet Ovaere Sent: Thursday, January 29, 2009 10:45 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Small lines in diffraction pattern (more info) Dear all, There were some comments about detector issues, but these can be ruled out, to my opinion, since the lines appeared on different beamlines. Default settings of mosflm (spot picking) finds the cell 34 34 34 90 90 90 (pointless indicating P41212) Structure was solved by SAD phasing on the phosphates in this space group. Double helices stack in continuous helices, the backbone is well defined in the (refined) density maps but the individual bases are messy (purines and pyrimidines seemed to overlap) + obviously not all spots were covered and the duplex does not fit in the A.U. For this reason the integration was repeated in the higher cell 34 34 170 Space group most probably P212121, but solutions can be found in P41212 as well (still disordered bases) There are also indications that the 41 screw axis is rather a pseudo axis than a pure crystallographic one, also in the small cell Reindexing the cell to 34 34 340 also gives a solution, which supports the theory of Holton Rmerg is around 5% for the small cell, about 8% for the 170Å cell (both in P41212) Which refinement procedure would be best to follow? kind regards Margriet Margriet Ovaere Chemistry Department K.U.Leuven Biomolecular Architecture Celestijnenlaan 200 F B-3001 Heverlee (Leuven) Tel: +32(0)16327477 Disclaimer: http://www.kuleuven.be/cwis/email_disclaimer.htm for more information. Disclaimer This communication is confidential and may contain privileged information intended solely for the named addressee(s). It may not be used or disclosed except for the purpose for which it has been sent. If you are not the intended recipient you must not review, use, disclose, copy, distribute or take any action in reliance upon it. If you have received this communication in error, please notify Astex Therapeutics Ltd by emailing i.tic...@astex-therapeutics.com and destroy all copies of the message and any attached documents. Astex Therapeutics Ltd monitors, controls and protects all its messaging traffic in compliance with its corporate email policy. The Company accepts no liability or responsibility for any onward transmission or use of emails and attachments having left the Astex Therapeutics domain. Unless expressly stated, opinions in this message are those of the individual sender and not of Astex Therapeutics Ltd. The recipient should check this email and any attachments for the presence of computer viruses. Astex
Re: [ccp4bb] Small lines in diffraction pattern (more info)
Dear Ian and Margriet, You are right, the correction needs to be done on F, not on |F|^2. If I recall correctly (I did not do it myself), the assumption was that Fobs = 0.5*Fobs(A) + 0.5*Fobs(B), so Fcorrected(A) = 2*Fobs - Fcalc(B) where A and B are the two orientations. Since one does not have an observed phase, one would have to take calculated phases. I am unsure though, if that was done in practise and one did not just subtract the absolute values. Since the inhibitor is usually only a small part of the total scattering mass, the phases might not differ too much and therefore the error would not be too big. In case of superposition of base pairs, I guess that the differences in scattering between the different base-pairs is not too much, so one might also be able to get away with not using phases, but here you are the expert. Using this method, one could much better interpret the convoluted electron density, but one has to be very careful not introducing severe model bias. I would look in the literature in detail, what people from the HIV protease field had done to solve this problem. Cheers, Herman -Original Message- From: Ian Tickle [mailto:i.tic...@astex-therapeutics.com] Sent: Thursday, January 29, 2009 12:53 PM To: Schreuder, Herman RD/DE; margriet.ova...@chem.kuleuven.be; CCP4BB@JISCMAIL.AC.UK Subject: RE: [ccp4bb] Small lines in diffraction pattern (more info) Hi Herman Aren't detwinning methods appropriate only in the case of true twin domains which are larger than the X-ray photon correlation length in order for the assumption to be valid that |F|^2 from each domain can be summed? This wouldn't give rise to the apparent 'diffuse scatter' phenomenon. However if what you are describing is rather static disorder of unit cells, which would give rise to diffuse scatter, where A B type cells are randomly mixed (so a domain is only one or at most a few unit cells), as opposed to being confined to A B type domains, then detwinning would not be appropriate. Cheers -- Ian -Original Message- From: owner-ccp...@jiscmail.ac.uk [mailto:owner-ccp...@jiscmail.ac.uk] On Behalf Of herman.schreu...@sanofi-aventis.com Sent: 29 January 2009 11:19 To: margriet.ova...@chem.kuleuven.be; CCP4BB@JISCMAIL.AC.UK Subject: RE: [ccp4bb] Small lines in diffraction pattern (more info) Dear Margriet, From your description and what James Holton wrote, it seems that you have 2 types of unit cells: A: with the sense strand in position 1 and the antisense strand in position 2 B: with the antisense strand in position 1 and the sense strand in position 2 If the crystal contacts are mainly via the backbone, your crystal may contain a random distribution of both and the electron density you see is a superposition of both and for the crystal packing, both chains are identical. This situation is similar to the situation when an asymmetric inhibitor is bound to a dimeric, symmetric molecule like e.g. HIV protease. In this case, both orientations are deconvoluted using detwinning methods for perfect twinning (see e.g. Proc Natl Acad Sci U S A. 2002 November 12; 99(23): 14664-14669). The 34,34,34 cell is definitively too small, so I would process in the 34,34,170 cell and detwin. You molecular replacement solutions should tell you which twinning operator to use. Best regards, Herman From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Margriet Ovaere Sent: Thursday, January 29, 2009 10:45 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Small lines in diffraction pattern (more info) Dear all, There were some comments about detector issues, but these can be ruled out, to my opinion, since the lines appeared on different beamlines. Default settings of mosflm (spot picking) finds the cell 34 34 34 90 90 90 (pointless indicating P41212) Structure was solved by SAD phasing on the phosphates in this space group. Double helices stack in continuous helices, the backbone is well defined in the (refined) density maps but the individual bases are messy (purines and pyrimidines seemed to overlap) + obviously not all spots were covered and the duplex does not fit in the A.U. For this reason the integration was repeated in the higher cell 34 34 170 Space group most probably P212121, but solutions can be found in P41212 as well (still disordered bases) There are also indications that the 41 screw axis is rather a pseudo axis than a pure crystallographic one, also in the small cell Reindexing the cell to 34 34 340 also gives a solution, which supports the theory of Holton Rmerg is around 5% for the small cell, about 8% for the 170Å cell (both in P41212) Which refinement procedure would be best to follow? kind
Re: [ccp4bb] Small lines in diffraction pattern (more info)
For this discussion another relevant reference might be: The 1.8 A crystal structure of a statically disordered 17 base-pair RNA duplex: principles of RNA crystal packing and its effect on nucleic acid structure. Shah SA, Brunger AT. J Mol Biol. 1999 Jan 29;285(4):1577-88. -tommi On Jan 29, 2009, at 1:53 PM, Ian Tickle wrote: Hi Herman Aren't detwinning methods appropriate only in the case of true twin domains which are larger than the X-ray photon correlation length in order for the assumption to be valid that |F|^2 from each domain can be summed? This wouldn't give rise to the apparent 'diffuse scatter' phenomenon. However if what you are describing is rather static disorder of unit cells, which would give rise to diffuse scatter, where A B type cells are randomly mixed (so a domain is only one or at most a few unit cells), as opposed to being confined to A B type domains, then detwinning would not be appropriate. Cheers -- Ian -Original Message- From: owner-ccp...@jiscmail.ac.uk [mailto:owner-ccp...@jiscmail.ac.uk] On Behalf Of herman.schreu...@sanofi-aventis.com Sent: 29 January 2009 11:19 To: margriet.ova...@chem.kuleuven.be; CCP4BB@JISCMAIL.AC.UK Subject: RE: [ccp4bb] Small lines in diffraction pattern (more info) Dear Margriet, From your description and what James Holton wrote, it seems that you have 2 types of unit cells: A: with the sense strand in position 1 and the antisense strand in position 2 B: with the antisense strand in position 1 and the sense strand in position 2 If the crystal contacts are mainly via the backbone, your crystal may contain a random distribution of both and the electron density you see is a superposition of both and for the crystal packing, both chains are identical. This situation is similar to the situation when an asymmetric inhibitor is bound to a dimeric, symmetric molecule like e.g. HIV protease. In this case, both orientations are deconvoluted using detwinning methods for perfect twinning (see e.g. Proc Natl Acad Sci U S A. 2002 November 12; 99(23): 14664-14669). The 34,34,34 cell is definitively too small, so I would process in the 34,34,170 cell and detwin. You molecular replacement solutions should tell you which twinning operator to use. Best regards, Herman From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Margriet Ovaere Sent: Thursday, January 29, 2009 10:45 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Small lines in diffraction pattern (more info) Dear all, There were some comments about detector issues, but these can be ruled out, to my opinion, since the lines appeared on different beamlines. Default settings of mosflm (spot picking) finds the cell 34 34 34 90 90 90 (pointless indicating P41212) Structure was solved by SAD phasing on the phosphates in this space group. Double helices stack in continuous helices, the backbone is well defined in the (refined) density maps but the individual bases are messy (purines and pyrimidines seemed to overlap) + obviously not all spots were covered and the duplex does not fit in the A.U. For this reason the integration was repeated in the higher cell 34 34 170 Space group most probably P212121, but solutions can be found in P41212 as well (still disordered bases) There are also indications that the 41 screw axis is rather a pseudo axis than a pure crystallographic one, also in the small cell Reindexing the cell to 34 34 340 also gives a solution, which supports the theory of Holton Rmerg is around 5% for the small cell, about 8% for the 170Å cell (both in P41212) Which refinement procedure would be best to follow? kind regards Margriet Margriet Ovaere Chemistry Department K.U.Leuven Biomolecular Architecture Celestijnenlaan 200 F B-3001 Heverlee (Leuven) Tel: +32(0)16327477 Disclaimer: http://www.kuleuven.be/cwis/email_disclaimer.htm for more information. Disclaimer This communication is confidential and may contain privileged information intended solely for the named addressee(s). It may not be used or disclosed except for the purpose for which it has been sent. If you are not the intended recipient you must not review, use, disclose, copy, distribute or take any action in reliance upon it. If you have received this communication in error, please notify Astex Therapeutics Ltd by emailing i.tic...@astex-therapeutics.com and destroy all copies of the message and any attached documents. Astex Therapeutics Ltd monitors, controls and protects all its messaging traffic in compliance with its corporate email policy. The Company accepts no liability or responsibility for any onward transmission or use of emails and attachments having left
[ccp4bb] Fobs - Fobs
Hi, I was wondering if anyone knows what programme I need to use to subtract the Fobs of two different crystals from each other. Regards, Rana _ Invite your mail contacts to join your friends list with Windows Live Spaces. It's easy! http://spaces.live.com/spacesapi.aspx?wx_action=createwx_url=/friends.aspxmkt=en-us
Re: [ccp4bb] Fobs - Fobs
Hi Rana, You probably have multiple options suggested to you. One is sftools using the CALC command. If the subtraction includes a phase then sftools can also do the calculation on the full structure factor. Plain subtraction of amplitudes ensuring the result is = 0 READ yourfile.mtz CALC col fnew = col f1 col f2 - abs WRITE yournewfile.mtz Subtraction of structure factors CALC (col fnew pnew) = (col f1 p1) (col f2 p1) - f1, p1 etc are the column labels for amplitude and phase columns respectively. I recommend to run the program interactively and you can use CALC help to get more help. Bart Rana Refaey wrote: Hi, I was wondering if anyone knows what programme I need to use to subtract the Fobs of two different crystals from each other. Regards, Rana Invite your mail contacts to join your friends list with Windows Live Spaces. It's easy! Try it! http://spaces.live.com/spacesapi.aspx?wx_action=createwx_url=/friends.aspxmkt=en-us
Re: [ccp4bb] Fobs - Fobs
You can use the CCP4 program sftools. You will need to use the program twice if you want to keep the differences positive ie F1 - F2 if F1F2 and F2 - F1 if F2F1 Adam On Thu, 29 Jan 2009, Rana Refaey wrote: Hi, I was wondering if anyone knows what programme I need to use to subtract the Fobs of two different crystals from each other. Regards, Rana _ Invite your mail contacts to join your friends list with Windows Live Spaces. It's easy! http://spaces.live.com/spacesapi.aspx?wx_action=createwx_url=/friends.aspxmkt=en-us
Re: [ccp4bb] Fobs - Fobs
Rana, to a large extent this depends on what you what to do with the delta-F's afterwards (you didn't say). If all you want to do is calculate a map, Patterson or Fourier, (which is probably what we do most of the time with delta-F's) then you don't need to calculate the difference as a separate step, the FFT input routine will do it for you, also applying an appropriate phase if required. Or even if you want to say calculate normalised moduli of the differences for direct methods, again the ECALC program will calculate the difference before doing the normalisation. It goes without saying that subtracting F's is pretty meaningless if they are not on the same scale, and any relative B factor has been taken out as well. -- Ian -Original Message- From: owner-ccp...@jiscmail.ac.uk [mailto:owner-ccp...@jiscmail.ac.uk] On Behalf Of Rana Refaey Sent: 29 January 2009 14:36 To: ccp4bb@jiscmail.ac.uk Subject: Fobs - Fobs Hi, I was wondering if anyone knows what programme I need to use to subtract the Fobs of two different crystals from each other. Regards, Rana Invite your mail contacts to join your friends list with Windows Live Spaces. It's easy! Try it! http://spaces.live.com/spacesapi.aspx?wx_action=createwx_url =/friends.aspxmkt=en-us Disclaimer This communication is confidential and may contain privileged information intended solely for the named addressee(s). It may not be used or disclosed except for the purpose for which it has been sent. If you are not the intended recipient you must not review, use, disclose, copy, distribute or take any action in reliance upon it. If you have received this communication in error, please notify Astex Therapeutics Ltd by emailing i.tic...@astex-therapeutics.com and destroy all copies of the message and any attached documents. Astex Therapeutics Ltd monitors, controls and protects all its messaging traffic in compliance with its corporate email policy. The Company accepts no liability or responsibility for any onward transmission or use of emails and attachments having left the Astex Therapeutics domain. Unless expressly stated, opinions in this message are those of the individual sender and not of Astex Therapeutics Ltd. The recipient should check this email and any attachments for the presence of computer viruses. Astex Therapeutics Ltd accepts no liability for damage caused by any virus transmitted by this email. E-mail is susceptible to data corruption, interception, unauthorized amendment, and tampering, Astex Therapeutics Ltd only send and receive e-mails on the basis that the Company is not liable for any such alteration or any consequences thereof. Astex Therapeutics Ltd., Registered in England at 436 Cambridge Science Park, Cambridge CB4 0QA under number 3751674
Re: [ccp4bb] Small lines in diffraction pattern (more info)
Ian and Herman, does one want to convolute the electron density at all? I was under the impression that current thinking favors convolution of the model instead, i.e. placing both the helices in both orientations at partial occupancy and letting the refinement program figure things out? Andreas herman.schreu...@sanofi-aventis.com wrote: Dear Ian and Margriet, You are right, the correction needs to be done on F, not on |F|^2. If I recall correctly (I did not do it myself), the assumption was that Fobs = 0.5*Fobs(A) + 0.5*Fobs(B), so Fcorrected(A) = 2*Fobs - Fcalc(B) where A and B are the two orientations. Since one does not have an observed phase, one would have to take calculated phases. I am unsure though, if that was done in practise and one did not just subtract the absolute values. Since the inhibitor is usually only a small part of the total scattering mass, the phases might not differ too much and therefore the error would not be too big. In case of superposition of base pairs, I guess that the differences in scattering between the different base-pairs is not too much, so one might also be able to get away with not using phases, but here you are the expert. Using this method, one could much better interpret the convoluted electron density, but one has to be very careful not introducing severe model bias. I would look in the literature in detail, what people from the HIV protease field had done to solve this problem. Cheers, Herman -- Andreas Förster, Research Associate Paul Freemont Xiaodong Zhang Labs Department of Biochemistry, Imperial College London
Re: [ccp4bb] Small lines in diffraction pattern (more info)
aa8297f160771e4da7fba29578ebaba2018ba...@nt-mail3.astex-technology.com 6d56f0ec21490b4db31a8ff1f0cb2914014ce...@ffpw10.f2.enterprise 4981d8a7.6000...@gmail.com From: Ian Tickle i.tic...@astex-therapeutics.com To: =?iso-8859-1?Q?Andreas_Förster?= docandr...@gmail.com, herman.schreu...@sanofi-aventis.com Cc: CCP4BB@JISCMAIL.AC.UK Return-Path: i.tic...@astex-therapeutics.com X-OriginalArrivalTime: 29 Jan 2009 16:27:43.0581 (UTC) FILETIME=[831110D0:01C9822E] Yes I agree with that! -- Ian -Original Message- From: Andreas Förster [mailto:docandr...@gmail.com] Sent: 29 January 2009 16:26 To: herman.schreu...@sanofi-aventis.com; Ian Tickle Cc: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Small lines in diffraction pattern (more info) Ian and Herman, does one want to convolute the electron density at all? I was under the impression that current thinking favors convolution of the model instead, i.e. placing both the helices in both orientations at partial occupancy and letting the refinement program figure things out? Andreas herman.schreu...@sanofi-aventis.com wrote: Dear Ian and Margriet, You are right, the correction needs to be done on F, not on |F|^2. If I recall correctly (I did not do it myself), the assumption was that Fobs = 0.5*Fobs(A) + 0.5*Fobs(B), so Fcorrected(A) = 2*Fobs - Fcalc(B) where A and B are the two orientations. Since one does not have an observed phase, one would have to take calculated phases. I am unsure though, if that was done in practise and one did not just subtract the absolute values. Since the inhibitor is usually only a small part of the total scattering mass, the phases might not differ too much and therefore the error would not be too big. In case of superposition of base pairs, I guess that the differences in scattering between the different base-pairs is not too much, so one might also be able to get away with not using phases, but here you are the expert. Using this method, one could much better interpret the convoluted electron density, but one has to be very careful not introducing severe model bias. I would look in the literature in detail, what people from the HIV protease field had done to solve this problem. Cheers, Herman -- Andreas Förster, Research Associate Paul Freemont Xiaodong Zhang Labs Department of Biochemistry, Imperial College London Disclaimer This communication is confidential and may contain privileged information intended solely for the named addressee(s). It may not be used or disclosed except for the purpose for which it has been sent. If you are not the intended recipient you must not review, use, disclose, copy, distribute or take any action in reliance upon it. If you have received this communication in error, please notify Astex Therapeutics Ltd by emailing i.tic...@astex-therapeutics.com and destroy all copies of the message and any attached documents. Astex Therapeutics Ltd monitors, controls and protects all its messaging traffic in compliance with its corporate email policy. The Company accepts no liability or responsibility for any onward transmission or use of emails and attachments having left the Astex Therapeutics domain. Unless expressly stated, opinions in this message are those of the individual sender and not of Astex Therapeutics Ltd. The recipient should check this email and any attachments for the presence of computer viruses. Astex Therapeutics Ltd accepts no liability for damage caused by any virus transmitted by this email. E-mail is susceptible to data corruption, interception, unauthorized amendment, and tampering, Astex Therapeutics Ltd only send and receive e-mails on the basis that the Company is not liable for any such alteration or any consequences thereof. Astex Therapeutics Ltd., Registered in England at 436 Cambridge Science Park, Cambridge CB4 0QA under number 3751674
[ccp4bb] X-ray photon correlation length
I always wondered - how is the X-ray photon correlation length defined and where do I find it? This is not the interaction length, I assume. So, to the physicists: How large is the 'X-ray photon correlation length' for a given wavelength in a given material? I had the impression that the term photon correlation refers to the time correlation of the scattering such as in photon correlation spectroscopy. Best regards, BR
Re: [ccp4bb] X-ray photon correlation length
From memory, correlation length is the length during which the phase of the electric field is preserved. It's typically computed by applying time (pulse width)- frequency (converted to length) uncertainty principle. V. Nagarajan JAN Scientific, Inc. -Original Message- From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Bernhard Rupp Sent: Thursday, January 29, 2009 9:51 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] X-ray photon correlation length I always wondered - how is the X-ray photon correlation length defined and where do I find it? This is not the interaction length, I assume. So, to the physicists: How large is the 'X-ray photon correlation length' for a given wavelength in a given material? I had the impression that the term photon correlation refers to the time correlation of the scattering such as in photon correlation spectroscopy. Best regards, BR
Re: [ccp4bb] Small lines in diffraction pattern (more info)
So it would seem that the five-fold periodicity in the spot intensities along the long axis is probably due to a short-range, 34 Ang pseudo-repeat (the decamer) along the true 170 Ang cell axis (170 = 34 x 5). I do not think that the streaks are helical layer lines, because the spacing on the detector is too small, indicating that the layers are really ~170 Ang apart. If the decamer's helical repeats are really ~34 Ang each, this wold not work out--the lines would be much further apart. Rather, it could be that: 1. The long axis consists of five RNA decamers in a line (as Margriet informs us), constituted of random orientations, which reunite strictly at every fifth position, to form a more-ordered superlattice (170 Ang), which is significant enough to make spots. 2. Alternatively, it might be that there are fibers with strict fivefold (170 Ang) periodicity which are translated by n x 34 Ang with respect to each other. In both cases, the variety of distances in between the fibers due to the variety of combinations of neighbors might be enough to make the streaks. I think this combinatorial distance variety is what James was proposing before to explain the streaks. In terms of modelling the structure, I do not think it matters whether the reality is 1 or 2, although both have unlikely features to them, realistically. Jacob *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program Dallos Laboratory F. Searle 1-240 2240 Campus Drive Evanston IL 60208 lab: 847.491.2438 cel: 773.608.9185 email: j-kell...@northwestern.edu ***
Re: [ccp4bb] X-ray photon correlation length
Bernard I guess this came from Aren't detwinning methods appropriate only in the case of true twin domains which are larger than the X-ray photon correlation length in order for the assumption to be valid that |F|^2 from each domain can be summed? This wouldn't give rise to the apparent 'diffuse scatter' phenomenon. I think this is normally called coherence length. Probably best not to think of photons at all but waves (though there is an equivalent quantum mechanical treatment based, as V Nagarajan says, on the uncertainty principle). I don't think the domains have to be larger then the correlation (sorry coherence) length of the incident x-rays in any case. They have to be large enough to give an intensity which can be integrated. If smaller domains are present, the intensity just spread out a bit more.When the domains are very large, the size of the spots would be determined by the incident beam properties. The article cited some years ago on CCP4BB gives a primer on all this J. Phys.: Condens. Matter 16 (2004) 5003-5030 PII: S0953-8984(04)75896-8. Coherent x-ray scattering Friso van der Veen1,2 and Franz Pfeiffer1 http://www.iop.org/EJ/article/0953-8984/16/28/020/cm4_28_020.pdf?request -id=8848d3f0-5a4b-4ffe-8ea4-c1eabfaf1657 Cheers Colin From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Bernhard Rupp Sent: 29 January 2009 17:51 To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] X-ray photon correlation length I always wondered - how is the X-ray photon correlation length defined and where do I find it? This is not the interaction length, I assume. So, to the physicists: How large is the 'X-ray photon correlation length' for a given wavelength in a given material? I had the impression that the term photon correlation refers to the time correlation of the scattering such as in photon correlation spectroscopy... Best regards, BR DIVFONT size=1 color=grayThis e-mail and any attachments may contain confidential, copyright and or privileged material, and are for the use of the intended addressee only. If you are not the intended addressee or an authorised recipient of the addressee please notify us of receipt by returning the e-mail and do not use, copy, retain, distribute or disclose the information in or attached to the e-mail. Any opinions expressed within this e-mail are those of the individual and not necessarily of Diamond Light Source Ltd. Diamond Light Source Ltd. cannot guarantee that this e-mail or any attachments are free from viruses and we cannot accept liability for any damage which you may sustain as a result of software viruses which may be transmitted in or with the message. Diamond Light Source Limited (company no. 4375679). Registered in England and Wales with its registered office at Diamond House, Harwell Science and Innovation Campus, Didcot, Oxfordshire, OX11 0DE, United Kingdom /FONT/DIV -- Scanned by iCritical.
Re: [ccp4bb] X-ray photon correlation length
Ok, following seems to be correct: a) interaction length = mean free path : relevant for absorption b) correlation length = time correlation between photons : relevant for multi-photon scattering c) coherence length = longitudinal coherence length : relevant for single photon scattering. It follows from Heisenberg for a Lorentzian source (anode) with natural emisson line width per formula on p 5007 of Colin's ref Lc=(2/pi)lambda**2/delLambda Using 8084 eV and 2.1 eV respectively for Cu, I obtain ~3800 A coherence length for a Cu (anode) X-ray photon The pre-factor is different for other source types like synchrotron. In any case I would accept the vague term of 'a few 1000 A' or 'several 1000 A' as a general statement for coherence length in materials where the interaction length is larger (practically always). Does this sound reasonable? BR From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Nave, C (Colin) Sent: Thursday, January 29, 2009 10:14 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] X-ray photon correlation length Bernard I guess this came from Aren't detwinning methods appropriate only in the case of true twin domains which are larger than the X-ray photon correlation length in order for the assumption to be valid that |F|^2 from each domain can be summed? This wouldn't give rise to the apparent 'diffuse scatter' phenomenon. I think this is normally called coherence length. Probably best not to think of photons at all but waves (though there is an equivalent quantum mechanical treatment based, as V Nagarajan says, on the uncertainty principle). I don't think the domains have to be larger then the correlation (sorry coherence) length of the incident x-rays in any case. They have to be large enough to give an intensity which can be integrated. If smaller domains are present, the intensity just spread out a bit more.When the domains are very large, the size of the spots would be determined by the incident beam properties. The article cited some years ago on CCP4BB gives a primer on all this J. Phys.: Condens. Matter 16 (2004) 5003-5030 PII: S0953-8984(04)75896-8. Coherent x-ray scattering Friso van der Veen1,2 and Franz Pfeiffer1 http://www.iop.org/EJ/article/0953-8984/16/28/020/cm4_28_020.pdf?request-id= 8848d3f0-5a4b-4ffe-8ea4-c1eabfaf1657 Cheers Colin _ From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Bernhard Rupp Sent: 29 January 2009 17:51 To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] X-ray photon correlation length I always wondered - how is the X-ray photon correlation length defined and where do I find it? This is not the interaction length, I assume. So, to the physicists: How large is the 'X-ray photon correlation length' for a given wavelength in a given material? I had the impression that the term photon correlation refers to the time correlation of the scattering such as in photon correlation spectroscopy. Best regards, BR This e-mail and any attachments may contain confidential, copyright and or privileged material, and are for the use of the intended addressee only. If you are not the intended addressee or an authorised recipient of the addressee please notify us of receipt by returning the e-mail and do not use, copy, retain, distribute or disclose the information in or attached to the e-mail. Any opinions expressed within this e-mail are those of the individual and not necessarily of Diamond Light Source Ltd. Diamond Light Source Ltd. cannot guarantee that this e-mail or any attachments are free from viruses and we cannot accept liability for any damage which you may sustain as a result of software viruses which may be transmitted in or with the message. Diamond Light Source Limited (company no. 4375679). Registered in England and Wales with its registered office at Diamond House, Harwell Science and Innovation Campus, Didcot, Oxfordshire, OX11 0DE, United Kingdom _ Scanned by iCritical.
Re: [ccp4bb] Problem running BALBES
Thank you Fei for your input. Your response inspired me and I found the error I was making (a poorly truncated PDB file). However I have another question regarding BALBES for the brains out there. As I mentioned before, I have a two protein complex with a nice homology model for Protein 1 and the structure of Protein 2 is known (we're using only one domain of Protein 2 in our complex). Balbes finds a solution using the homology model of Protein 1 as an input (Rfree ~ .40) , but it stops there. I was under the impression that the program would then go ahead and try to fit Protein 2 using this newly found solution. It doesn't find a solution when using just the known structure of Protein 2 as a model. Is there a way to accomplish this? Sincerely, Jesse On Wed, Jan 28, 2009 at 1:25 PM, Dr. F Long f...@ysbl.york.ac.uk wrote: Dear Jesse, In both cases, there are some log files under, e,g. Test/process_details/ If you can make tar.gz files on these log files and send them to us. We can analyse what happened in your processes. Thanks, Fei Dr Fei Long Structural Biology Laboratory University of York Heslington York YO10 5YW UK
Re: [ccp4bb] X-ray photon correlation length
On Thursday 29 January 2009 10:59:23 Bernhard Rupp wrote: Ok, following seems to be correct: a) interaction length = mean free path : relevant for absorption b) correlation length = time correlation between photons : relevant for multi-photon scattering c) coherence length = longitudinal coherence length : relevant for single photon scattering. It follows from Heisenberg for a Lorentzian source (anode) with natural emisson line width per formula on p 5007 of Colin's ref Lc=(2/pi)lambda**2/delLambda Using 8084 eV and 2.1 eV respectively for Cu, I obtain ~3800 A coherence length for a Cu (anode) X-ray photon The pre-factor is different for other source types like synchrotron. The coherence length for an undulator source is the relativistically contracted length of the undulator. Ref: http://xdb.lbl.gov/Section2/Sec_2-1.html In any case I would accept the vague term of 'a few 1000 A' or 'several 1000 A' as a general statement for coherence length in materials where the interaction length is larger (practically always). Does this sound reasonable? My impression is that the coherence length from synchrotron sources is generally larger than the x-ray path through a protein crystal. But I have not gone through the exercise of plugging in specific storage ring energies and undulator parameters to confirm this impression. Perhaps James Holton will chime in again? Ethan From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Nave, C (Colin) Sent: Thursday, January 29, 2009 10:14 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] X-ray photon correlation length Bernard I guess this came from Aren't detwinning methods appropriate only in the case of true twin domains which are larger than the X-ray photon correlation length in order for the assumption to be valid that |F|^2 from each domain can be summed? This wouldn't give rise to the apparent 'diffuse scatter' phenomenon. I think this is normally called coherence length. Probably best not to think of photons at all but waves (though there is an equivalent quantum mechanical treatment based, as V Nagarajan says, on the uncertainty principle). I don't think the domains have to be larger then the correlation (sorry coherence) length of the incident x-rays in any case. They have to be large enough to give an intensity which can be integrated. If smaller domains are present, the intensity just spread out a bit more.When the domains are very large, the size of the spots would be determined by the incident beam properties. The article cited some years ago on CCP4BB gives a primer on all this J. Phys.: Condens. Matter 16 (2004) 5003-5030 PII: S0953-8984(04)75896-8. Coherent x-ray scattering Friso van der Veen1,2 and Franz Pfeiffer1 http://www.iop.org/EJ/article/0953-8984/16/28/020/cm4_28_020.pdf?request-id= 8848d3f0-5a4b-4ffe-8ea4-c1eabfaf1657 Cheers Colin _ From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Bernhard Rupp Sent: 29 January 2009 17:51 To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] X-ray photon correlation length I always wondered - how is the X-ray photon correlation length defined and where do I find it? This is not the interaction length, I assume. So, to the physicists: How large is the 'X-ray photon correlation length' for a given wavelength in a given material? I had the impression that the term photon correlation refers to the time correlation of the scattering such as in photon correlation spectroscopy. Best regards, BR This e-mail and any attachments may contain confidential, copyright and or privileged material, and are for the use of the intended addressee only. If you are not the intended addressee or an authorised recipient of the addressee please notify us of receipt by returning the e-mail and do not use, copy, retain, distribute or disclose the information in or attached to the e-mail. Any opinions expressed within this e-mail are those of the individual and not necessarily of Diamond Light Source Ltd. Diamond Light Source Ltd. cannot guarantee that this e-mail or any attachments are free from viruses and we cannot accept liability for any damage which you may sustain as a result of software viruses which may be transmitted in or with the message. Diamond Light Source Limited (company no. 4375679). Registered in England and Wales with its registered office at Diamond House, Harwell Science and Innovation Campus, Didcot, Oxfordshire, OX11 0DE, United Kingdom _ Scanned by iCritical. -- Ethan A Merritt Biomolecular Structure Center University of Washington, Seattle 98195-7742
[ccp4bb] mapping sequence conservation metrics on the B factor column ...
Dear all, I was wondering what is the state of the art for this old dark art ... are there any good servers / programs that allow to easily upload your own sequence alignments or create a 'transparent' alignment (I want to see the alignment first and not a total black box) and then allow you to write out sequence conservation based either on identity or in e.g a Dayhoff matrix on the B factor column for displaying it later in eg Pymol? To be clear I do not want a structural alignment, but mapping sequence alignment of eg a family to a single structure of a family member. Thanks in advance, Tassos
Re: [ccp4bb] mapping sequence conservation metrics on the B factor column ...
On Thu, Jan 29, 2009 at 1:25 PM, Anastassis Perrakis a.perra...@nki.nlwrote: Dear all, I was wondering what is the state of the art for this old dark art ... are there any good servers / programs that allow to easily upload your own sequence alignments or create a 'transparent' alignment (I want to see the alignment first and not a total black box) and then allow you to write out sequence conservation based either on identity or in e.g a Dayhoff matrix on the B factor column for displaying it later in eg Pymol? To be clear I do not want a structural alignment, but mapping sequence alignment of eg a family to a single structure of a family member. ConSurf! You can submit your own Clustal alignment (the MSA program MUSCLE will also generate this file format). However, I was never able to visualize the PDB files it generates very well in PyMOL; I ended up extracting a different, simpler type of score (integers 1-9) from the results and applying that to the B-factor column instead of the score ConSurf writes.
Re: [ccp4bb] mapping sequence conservation metrics on the B factor column ...
AL2CO ( http://www.ncbi.nlm.nih.gov/pubmed/11524371 ) may be what you need. It takes the multiple alignment (made by user, so it isn't a black box) that must contain the sequence of your structure, and maps the positional conservation in the B-factor column of the structure. To see whether this is what you want, try: http://rnajournal.cshlp.org/content/10/11/1698/F1.expansion.html Although the image was created by Bobscript, this also works fine with Pymol. Hope this helps, Mensur At 02:25 PM 1/29/2009, Anastassis Perrakis wrote: Dear all, I was wondering what is the state of the art for this old dark art ... are there any good servers / programs that allow to easily upload your own sequence alignments or create a 'transparent' alignment (I want to see the alignment first and not a total black box) and then allow you to write out sequence conservation based either on identity or in e.g a Dayhoff matrix on the B factor column for displaying it later in eg Pymol? To be clear I do not want a structural alignment, but mapping sequence alignment of eg a family to a single structure of a family member. Thanks in advance, Tassos == | Mensur Dlakic, PhD| Tel: (406) 994-6576| | Department of Microbiology| Fax: (406) 994-4926| | Montana State University | Lab: (406) 994-6237| | 109 Lewis Hall, P.O. Box 173520 | http://myprofile.cos.com/mensur| | Bozeman, MT 59717-3520| E-mail: mdla...@montana.edu| ==
[ccp4bb] difficult MAD dataset
We recently collected a complete 2.5A MAD dataset. However, finding a solution has not been as straightfoward for reasons unclear to us. We would be grateful for any helpful advice or suggestions. The thin plate shaped crystal was grown from a relatively small protein (90 residues). The crystal diffracted well with visible and defined reflections up to ~2.8A range. With both HKL2000 and mosflm, initial indexing indicated orthorhombic unit cell with dimension of 52 x 82 x 100. Systematic absences along a and b axis were observed thus the dataset was scaled in P21212 space group. The unit cell dimension and space group suggested 4 protein chains per ASU. There is a pseudo-translation with 55 % peak at a fractional coordinate of 0.5, 0.5, 0.48. There are 7 methionines per chain. Thus we expect 28 Se per ASU. Mass spectroscopy and fluoresence scan both confirmed successful incorporation of Se-methionine in the crystal. According to xtriage, anomalous signal extended to 3.0A at least in the peak dataset (The table of measurability as a function of resolution is shown below). unused: - 43.0868 [ 0/5 ] bin 1: 43.0868 - 5.3953 [2751/2902] 0.5838 bin 2: 5.3953 - 4.2836 [2893/2905] 0.4575 bin 3: 4.2836 - 3.7425 [2891/2899] 0.2820 bin 4: 3.7425 - 3.4005 [2888/2896] 0.1898 bin 5: 3.4005 - 3.1568 [2864/2898] 0.1222 bin 6: 3.1568 - 2.9708 [2835/2898] 0.0653 bin 7: 2.9708 - 2.8220 [2777/2906] 0.0458 bin 8: 2.8220 - 2.6992 [2714/2902] 0.0226 bin 9: 2.6992 - 2.5953 [2550/2865] 0.0168 bin 10: 2.5953 - 2.5057 [2307/2914] 0.0071 unused: 2.5057 - [ 0/0 ] However, HA search using hkl2map, Autosol, and Autosharp resulted in only 3~4 HA sites. When hkl2map was used, most HA sites had poor CC and Patterson FOM and did not clearly stand out as they normally should. Structural homologs suggest that the protein has a compact single core domain comprised of 4 a-helices. The positions of most HAs are unlikely to be located in the flexible region. If any abnormalies are seen with the dataset, it's during scaling step in HKL2000. Chi2 is unusually high at lower resolution (Chi2 is 3 from 3.5A as shown below) and there is a relatively high percentage of rejections (1.5 %). Shell Lower Upper Average Average Norm. Linear Square limitAngstrom I error stat. Chi**2 R-fac R-fac 50.00 5.38 4299.077.349.0 7.886 0.076 0.085 5.38 4.27 2938.752.735.0 5.843 0.083 0.090 4.27 3.73 2314.245.933.5 3.935 0.082 0.084 3.73 3.39 1245.334.428.9 2.838 0.101 0.094 3.39 3.15 658.628.125.9 1.957 0.132 0.127 3.15 2.96 451.927.125.7 1.322 0.157 0.138 2.96 2.82 307.227.126.3 1.001 0.201 0.169 2.82 2.69 253.128.828.1 0.866 0.225 0.193 2.69 2.59 199.331.531.0 0.801 0.262 0.233 2.59 2.50 159.534.734.4 0.688 0.292 0.261 All reflections 1312.939.031.9 2.748 0.100 0.089 Xtriage also complains that there are abnormal intensities at some resolution ranges. Finally, the crystallization requires CdSO4, so we suspect that cadmium ions are incorporated in the crystal. If so, we suspect there may be weak anomalous signal contribution from Cd as well. In summary, we appear to have a complete dataset that shows strong anomalous signal. However it appears that we have overlooked something or there is an unusual crystallographic issue that we are not aware of. Any suggestions will be very much appreciated. Alison Li Graudate student, Dr. Mark Paetzel's group Simon Fraser University, BC, Canada
Re: [ccp4bb] mapping sequence conservation metrics on the B factor column ...
Dear All, Thanks to all that replied in private and in the bb! Answers are and will be 'permanently' summarized at: http://strucbio.biologie.uni-konstanz.de/ccp4wiki/ http://strucbio.biologie.uni-konstanz.de/ccp4wiki/index.php/Mapping_sequence_alignment_to_a_structure (a nice opportunity to create a missing chapter!) Tassos On 29 Jan 2009, at 22:39, Luca Jovine wrote: On 29 Jan 2009, at 22:25, Anastassis Perrakis wrote: Dear all, I was wondering what is the state of the art for this old dark art ... are there any good servers / programs that allow to easily upload your own sequence alignments or create a 'transparent' alignment (I want to see the alignment first and not a total black box) and then allow you to write out sequence conservation based either on identity or in e.g a Dayhoff matrix on the B factor column for displaying it later in eg Pymol? To be clear I do not want a structural alignment, but mapping sequence alignment of eg a family to a single structure of a family member. Thanks in advance, Tassos Hi Tassos, Consurf will do the job! Have a look at: http://consurf.tau.ac.il HTH, Luca Luca Jovine, Ph.D. Group Leader, Protein Crystallography Unit Karolinska Institutet Department of Biosciences and Nutrition Hälsovägen 7, SE-141 57 Huddinge, Sweden Voice: +46.(0)8.6083-301 FAX: +46.(0)8.6089-290 E-mail: luca.jov...@ki.se W3: http://jovinelab.org
[ccp4bb] Transfer Rfree flag
Hello: Can someone tell me if there is a relatively straightforward way of transferring Free R flags between two CNS format reflection files ? Thanks
Re: [ccp4bb] mapping sequence conservation metrics on the B factor column ...
I like the consurf coloring scheme the best. I forget things so I had to write down the instructions which I copied off of some website which I can't seem to find at the moment. The other program is ESPript which I've also included my cheat sheet. Mapping conservation onto your structure (ConSurf): Here we will map sequence conservation among species onto a structure so that you can visualize areas of conservation Create alignment file 1. Open your preferred internet browser and go to Clustalw (www.ebi.ac.uk/clustalw) 2. copy sequences into the sequence box in Clustalw The following format (FASTA) MUST be used as an input: yeast (name followed by any text and carriage return) acgtacgtagc (sequence with or without carriage returns.) human (next sequence name - must have unique first words) ataagtac . . . . 3. submit (you can choose to get results emailed back to you or you will be allowed to open a window containing you results) 4. Save the aln file in a place you wont forget Generating pdb file with conservation values replacing B-values Go to http://consurf.tau.ac.il/index.html How to load in pymol explanation http://consurf.tau.ac.il/gallery/1217929900/pyMOL_instructions.html this is from the web site, the key is that you have to download a consurf strip # Download the PDB_FILE updated with ConSurf's colors. (a) PDB_FILE showing Insufficient Data (b) PDB_FILE hiding Insufficient Data # Download the file consurf_new.py. # Start PyMOL. # Load the pdb file; In the PyMOL viewer window type: PyMOLload PDB_FILE (no quotes) and hit return. # Run the script to define ConSurf's color; Type: PyMOLrun consurf_new.py (no quotes) and hit return. # Run the coloring scheme; Type this command: PyMOLcolour_consurf The other one I've used is ESPript Mapping conservation onto your structure (ESPript): Here we will map sequence conservation among species onto a structure so that you can visualize areas of conservation Create alignment file 1. Open your preferred internet browser and go to Clustalw (www.ebi.ac.uk/clustalw) 2. copy sequences into the sequence box in Clustalw (make sure your pdb sequence is the top sequence) The following format (FASTA) MUST be used as an input: yeast (name followed by any text and carriage return) acgtacgtagc (sequence with or without carriage returns.) human (next sequence name - must have unique first words) ataagtac . . . . 3. submit (you can choose to get results emailed back to you or you will be allowed to open a window containing you results) 4. Save the aln file in a place you wont forget Generating pdb file with conservation values replacing B-values 7. open ESPrint 2.2 http://espript.ibcp.fr/ESPript/ESPript/ i. Click on execute ii. Select expert (top EXP) iii. Browse to your aln sequence file (see figure below) iv. Two boxes will be available for you to input a pdb file (see the next page for the second pdb input) v. Browse to your pdb file in the first Aligned Sequences window vi. In addition you need to input the same pdb file into the bottom of the two boxes. This box maps secondary structure onto the aa sequence. If you do not input a pdb, it will map a structure prediction which can be quite useful when analyzing a sequence f an unsolved structure. There are different algorithms for calculations, choose which you think is applicable. For more information click on the question mark to get a small discussion To submit the job, return to the top of the page and click on Submit (MAKE SURE THAT THE POP-UP BLOCKER IS OFF) After a short time a new window will pop up. The BCOL [xx Kb] will have the pdb file now with conservation values in the B-value column (the other files are just your starting files The pdf [xx Kb] file will have the structure alignment in pretty colors with secondary structure predictions 8. save output pdb file (if you look at the text file, it will have conservation mapped in the B-factor column) 9. open output pdb in pymol (again-must be most recent version- 0.97) 10. show surface 11. color by B-factor fore even more fun type in cartoon putty show cartoon On 1/29/09 3:37 PM, Nathaniel Echols nathaniel.ech...@gmail.com wrote: On Thu, Jan 29, 2009 at 1:25 PM, Anastassis Perrakis a.perra...@nki.nl wrote: Dear all, I was wondering what is the state of the art for this old dark art ... are there any good servers / programs that allow to easily upload your own sequence alignments or create a 'transparent' alignment (I want to see the alignment first and not a total black box) and then allow you to write out sequence conservation based either on identity or in e.g a Dayhoff matrix on the B factor column for displaying it later in eg Pymol? To be clear I do not want a structural alignment, but mapping sequence alignment of eg a family to a single structure of a family member. ConSurf! You can submit your own Clustal alignment (the MSA program MUSCLE will also generate this
Re: [ccp4bb] OT: VectorNTI alternatives
Not sure if it's been mentioned, but I personally use EnzymeX(http://mekentosj.com/enzymex/ ) . Also, I find their PDF library organizer Papers (http://mekentosj.com/papers/ ) to be exceptional. Cheers FR On Jan 28, 2009, at 1:47 AM, Darren Hart wrote: Hello, After several years of offering the molecular biology software VectorNTI free to the academic community (their open access program) and building up a huge user base, Invitrogen have suddenly announced that they will no longer renew these free licences and the existing ones will be left to expire within the year. There are heavy renewal fees for anyone wishing to continue use of this software. Can anyone recommend decent alternative PC compatible alternatives? Main uses are construct and primer design, plus simple quick alignments, sequence data analysis etc. The database structure for storing sequences was pretty useful also. Ideally free, otherwise reasonably priced. I've seen CLCbio and Geneious have products, both free and paid. Any experience? Thanks, Darren EMBL Grenoble ps anyone using VNTI might consider a backup of their work by exporting files to .gb format. I don't know if a locked up (expired) version permits this and you will have no notice that it is about to expire. - Francis Reyes M.Sc. 215 UCB University of Colorado at Boulder gpg --keyserver pgp.mit.edu --recv-keys 67BA8D5D 8AE2 F2F4 90F7 9640 28BC 686F 78FD 6669 67BA 8D5D
[ccp4bb] sparse matrix screen design
Hi all, a non-CCP4 question: Are there any good free programs for the design of one's own sparse matrix screens? I am looking for something in the lines of a set of mixtures of what gives you the best coverage of, say, seven variables with five states each in 96 experiments. Thanks, Jose. ** Jose Antonio Cuesta-Seijo Cancer Genomics and Proteomics Ontario Cancer Institute, UHN MaRS TMDT Room 4-902M 101 College Street M5G 1L7 Toronto, ON, Canada Phone: (416)581-7544 Fax: (416)581-7562 email: jcue...@uhnres.utoronto.ca **
Re: [ccp4bb] Problem running BALBES
Dear Jesse, The current version of BALBES can only find MR models of a protein complex from its own internal database. I guess in your case, BALBES did not find the complex models (assembly as shown on the balbes log file). What you expect BALBES to do is under developing within the new version of BALBES. We hope we can deliver that soon. But maybe you can continue your work for now by running some MR programs such as MOLREP, using the solution found by BALBES as the fixed model. Thanks, Fei 2009/1/29 Jesse Sundlov je...@jhu.edu: Thank you Fei for your input. Your response inspired me and I found the error I was making (a poorly truncated PDB file). However I have another question regarding BALBES for the brains out there. As I mentioned before, I have a two protein complex with a nice homology model for Protein 1 and the structure of Protein 2 is known (we're using only one domain of Protein 2 in our complex). Balbes finds a solution using the homology model of Protein 1 as an input (Rfree ~ .40) , but it stops there. I was under the impression that the program would then go ahead and try to fit Protein 2 using this newly found solution. It doesn't find a solution when using just the known structure of Protein 2 as a model. Is there a way to accomplish this? Sincerely, Jesse On Wed, Jan 28, 2009 at 1:25 PM, Dr. F Long f...@ysbl.york.ac.uk wrote: Dear Jesse, In both cases, there are some log files under, e,g. Test/process_details/ If you can make tar.gz files on these log files and send them to us. We can analyse what happened in your processes. Thanks, Fei Dr Fei Long Structural Biology Laboratory University of York Heslington York YO10 5YW UK
Re: [ccp4bb] [OFF TOPIC] his-tag doesn't bind
Dear Fred -- just to check... are you sure you have the His-tag? Might have been cleaved somehow? You might want to increase the number of His in the tag as well. HTH. -- Leo -- On 27 Jan 2009, at 21:00, Fred wrote: Hi ccp4 list, I am trying to purify a his-tag protein by metal affinity chromatography. The protein was expressed in inclusion bodies and its his-tag doesn't bind the Qiagen Ni resin in denatured conditions (urea 8M or GndHCl 6M). Playing with NaCl and detergents didn't help much. Any help is appreciated. Fred Chavas Leonard, Ph.D. @ home Research Associate Marie Curie Actions Fellow Faculty of Life Sciences The University of Manchester The Michael Smith Building Oxford Road Manchester Lancashire M13 9PT Tel: +44(0)161-275-1586 e-mail: leonard.cha...@manchester.ac.uk http://personalpages.manchester.ac.uk/staff/leonard.chavas/
Re: [ccp4bb] Transfer Rfree flag
riya doreen wrote: Hello: Can someone tell me if there is a relatively straightforward way of transferring Free R flags between two CNS format reflection files ? Thanks Dear Riya, You can do it either by using CNS (merge.inp: keep the flags Rfree(A: dataset A), Fobs(B: dataset B) for example) or CCP4 (CNS---MTZ and use cad). David -- David Cobessi Institut de Biologie Structurale 41, Rue Jules Horowitz 38027 Grenoble Cedex-1, France Tel:33(0)438789613 33(0)608164340 Fax:33(0)438785122
