Re: [ccp4bb] small lines in diffraction pattern
Gees, I go to Washington DC for a couple of days and then a superbowl party, come back to my stack of emails and find out I missed out on all the fun... again! At first glance I thought it looked like a problem with the CCD detector overloads, but that apparently has been ruled out. Looks like you have already analyzed this, and from what I briefly read it sounds like a superstructure that is commensurate and can be solved by molecular replacement or twinned plates (if the crystals look like plates). Would love to see more of the images though at different distances and delta phi. ** Gloria Borgstahl Eppley Institute for Cancer Research and Allied Diseases 987696 Nebraska Medical Center 10732A Lied Transplant Center Omaha, NE 68198-7696 http://sbl.unmc.edu Office (402) 559-8578 FAX (402) 559-3739 Professor Hobbies: Protein Crystallography, Cancer, Biochemistry, DNA Metabolism, Modulated Crystals, Crystal Perfection, X-ray Topography, Interests: City of Ember, skateboarding, RAGBRAI, basketball, and rollerskating *** James Holton jmhol...@lbl.gov Sent by: CCP4 bulletin board CCP4BB@JISCMAIL.AC.UK 01/28/2009 09:59 PM Please respond to James Holton jmhol...@lbl.gov To CCP4BB@JISCMAIL.AC.UK cc Subject Re: [ccp4bb] small lines in diffraction pattern I'm sure Gloria would be delighted if that were the case, but I don't think this is an incommensurate lattice. These actually don't so much give you diffuse scattering as little satellite spots near the main spots at spacings that don't make any sense given the lattice repeat. My understanding is that these arise from something that is slowly varying from unit cell to unit cell (could be as simple as a side chain waving back and forth) in a repetitive pattern that just doesn't line up in any way with the repeat of the unit cells. Still, I'll ask her. However, I think that the difference is that modulated lattices are gradual changes of structure across many unit cells and what I was talking about is a more simplistic case of two different kinds of unit cells with varying degrees of randomness in their arrangement. That is, using the formalism I described below, a modulated lattice would have unit cells that go: ABCDEFGHGFEDCBABCDEFG... etc. where A is not that different from B, B similar to C, etc., but A and H are very different. -James Holton MAD Scientist Jürgen Bosch wrote: Hi James, what your descriptions aims at is I think shown in this publication Borgstahl, G. E. O. Incommensurate Crystallography by Sander van Smaalen Crystallography reviews 14 , 259-260 (2008). Or am I misunderstanding something here ? Jürgen On 28 Jan 2009, at 12:39, James Holton wrote: I recommend you have a look at a book from OUP called Diffuse X-Ray Scattering and Models of Disorder by T. R. Welberry. The first chapter explains quite well (I think) where all these streaky things come from. It will also make you feel better about having it when you see all the small molecule structures that have horrible diffuse scattering! (such as urea). This looks to me like a fairly classic case of correlated static disorder. Best way to think about it is this: Imagine you have two different kinds of unit cells: A an B. Doesn't really matter what the difference between A and B is, could be a two-headed side chain in conformer A vs conformer B, or it could be as complicated as a domain motion. But, for simplicity, lets assume it is two rotamers of a side chain and also assume that each unit cell in your crystal can only be one or the other (no in betweens). Now, if the arrangement of these unit cells is perfectly correlated and an A always occurs right next door to a B along the c-axis (say), then what you really have is a bigger unit cell than you think. That is, you can draw a unit cell around each A-B pair and call it a supercell with the contents of B as a simple NCS mate of A (with one side chain in a different rotamer). Some people might call this a pseudotranslation. The effect on the diffraction pattern in this case would be the appearance of a very weak spot in between each old spot along your c axis. That is, your supercell is twice as big along c so the reciprocal-space lattice has twice as many spots in it. The new spots are weak because they only correspond to the differences between A and B, which in this case is only a few atoms. Now lets say A and B are not perfectly correlated, but only slightly. That is, in some parts of the crystal A and B are side-by-side, but in other parts you get AAB, ABBA, BABBA, etc. In each of these cases the supercell you must draw is 3, 4 and 5x your original unit cell. Each of these will produce new weak spots with progressively tighter spacings. As the supercell becomes very long
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
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
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] small lines in diffraction pattern
Margriet This looks like stacking or shift disorder which can occur when perfect 3 dimensional order breaks down. For example one can have a situation where the lattice is preserved in 2 dimensions but the planes can slide with respect to one another destroying the order in the 3rd dimension, thereby giving rods in reciprocal space rather than spots. Preservation of order in 1 dimension but with the 1 D lattices shifted with respect to each other gives diffuse planes in reciprocal space. If displacements are non random, than the diffuse scatter might only occur for certain reflections. As sharp spots are also present, it is likely that the 3D order is preserved over much of the sample. To analyse properly one would have to record diffraction over small angular increments (similar to 3D reciprocal space mapping) and perhaps identify the hkl value of reflections which exhibit strong, weak or absent effects of this type. The reason for the behaviour can become clear when the structure is known and weak lattice interactions identified. However, if these effects are preventing you determining the structure then this statement is not particularly useful. If really interested you can of course google for such types of disorders and find examples with similar types of streaking (e.g. in J. Appl. Cryst. (1971). 4, 329 http://journals.iucr.org/j/issues/1971/04/00/a08549/a08549.pdf http://journals.iucr.org/j/issues/1971/04/00/a08549/a08549.pdf ). Alternatively read ancient text books such as Hosemann and Bagchi. Others will be far better at suggesting means of preventing the effects, which I suspect is what you would really like to know! Colin From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Margriet Ovaere Sent: 28 January 2009 13:52 To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] small lines in diffraction pattern Dear all, In the diffraction pattern of crystals of an RNA decamer, small lines appeared (see pictures attached). We've tried different crystals but they all showed the same small lines. Has anybody seen this phenomena before and has got an explanation for it please..? Many thanks Margriet Ovaere 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
Hi Margriet It's almost certainly due to diffuse scattering as a result of correlated atomic displacements. See this: http://www.nature.com/nsmb/journal/v1/n2/pdf/nsb0294-124.pdf . Are they lines or sheets, in other words do they appear only on one image, or are they also on adjacent images, i.e. are you looking at a slice through more extensive diffuse scattering? According to the above paper there are at least 3 types of DS: haloes around each Bragg spot (thermal diffuse or acoustic scattering) due to long range displacements correlated over different unit cells (this is very common); DS located along reciprocal lattice planes which results from anisotropic intermolecular displacements correlated over a few unit cells (which is I think what you are seeing), and low-intensity very diffuse background patches (optic scattering), but I don't see much of that. -- Ian -Original Message- From: owner-ccp...@jiscmail.ac.uk [mailto:owner-ccp...@jiscmail.ac.uk] On Behalf Of Margriet Ovaere Sent: 28 January 2009 13:52 To: ccp...@dl.ac.uk Subject: small lines in diffraction pattern Dear all, In the diffraction pattern of crystals of an RNA decamer, small lines appeared (see pictures attached). We've tried different crystals but they all showed the same small lines. Has anybody seen thisphenomenabefore and has got an explanation for it please..? Many thanks Margriet Ovaere 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
Couldn't the lines be explained most simply by extreme mosaicity, perhaps severely anisotropic? If not, why not? What were the values obtained in integration? 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 *** - Original Message - From: Margriet Ovaere To: CCP4BB@JISCMAIL.AC.UK Sent: Wednesday, January 28, 2009 7:51 AM Subject: [ccp4bb] small lines in diffraction pattern Dear all, In the diffraction pattern of crystals of an RNA decamer, small lines appeared (see pictures attached). We've tried different crystals but they all showed the same small lines. �Has anybody seen this�phenomena�before and has got an explanation for it please..? Many thanks Margriet Ovaere -- -- Margriet Ovaere Chemistry Department K.U.Leuven Biomolecular Architecture Celestijnenlaan 200 F B-3001 Heverlee (Leuven) Tel: +32(0)16327477 -- Dear all, In the diffraction pattern of crystals of an RNA decamer, small lines appeared (see pictures attached). We've tried different crystals but they all showed the same small lines. Has anybody seen this phenomena before and has got an explanation for it please..? Many thanks Margriet Ovaere  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
I recommend you have a look at a book from OUP called Diffuse X-Ray Scattering and Models of Disorder by T. R. Welberry. The first chapter explains quite well (I think) where all these streaky things come from. It will also make you feel better about having it when you see all the small molecule structures that have horrible diffuse scattering! (such as urea). This looks to me like a fairly classic case of correlated static disorder. Best way to think about it is this: Imagine you have two different kinds of unit cells: A an B. Doesn't really matter what the difference between A and B is, could be a two-headed side chain in conformer A vs conformer B, or it could be as complicated as a domain motion. But, for simplicity, lets assume it is two rotamers of a side chain and also assume that each unit cell in your crystal can only be one or the other (no in betweens). Now, if the arrangement of these unit cells is perfectly correlated and an A always occurs right next door to a B along the c-axis (say), then what you really have is a bigger unit cell than you think. That is, you can draw a unit cell around each A-B pair and call it a supercell with the contents of B as a simple NCS mate of A (with one side chain in a different rotamer). Some people might call this a pseudotranslation. The effect on the diffraction pattern in this case would be the appearance of a very weak spot in between each old spot along your c axis. That is, your supercell is twice as big along c so the reciprocal-space lattice has twice as many spots in it. The new spots are weak because they only correspond to the differences between A and B, which in this case is only a few atoms. Now lets say A and B are not perfectly correlated, but only slightly. That is, in some parts of the crystal A and B are side-by-side, but in other parts you get AAB, ABBA, BABBA, etc. In each of these cases the supercell you must draw is 3, 4 and 5x your original unit cell. Each of these will produce new weak spots with progressively tighter spacings. As the supercell becomes very long, these rows of tight spots will become a streak. The streaks are particularly prominent if the A-B disorder is along only one axis. In that case, you must have a whole a-b layer of A and other whole a-b layers of B, and the ordering of these layers along c is fairly random. Colin just described this as a stacking disorder which is probably a good name for it. The final case is when A and B are completely uncorrelated and occur absolutely at random locations in your crystal. In this case the supercell can be anything and the streaks are in every direction (including every diagonal) so they simply show up as increased background. Every crystal does this. In fact, this is the origin of the B-factor as no two unit cells are exactly alike. Ever wonder where those photons go that scatter off protein atoms but don't go into spots? They go into the background. Now, since these streaks represent correlations of neighboring unit cells this means that the diffuse scattering can tell you something about how your molecule moves. There is something about your structure that forces its neighbors to be the same in at least one direction. There are a class of people who study this for a living. I am not one of them. BTW. This is definitely NOT a mosaic spread. Mosaicity occurs on length scales thousands of times larger than this. By definition, a mosaic spread is the width of the distribution of relative rotation angles of mosaic domains and these domains all scatter independently of each other. An infinite mosaic spread (or at least 180 degrees) corresponds to a powder diffraction pattern, and the fact that powder lines are sharp demonstrates how mosaicity cannot smear spots in anything but the tangential direction. That is, no rotation can change the d-spacing of a spot. Changes in unit cell size can do this, but that is a very different phenomenon than mosaic spread as mosaic domains are much much bigger than unit cells. The good news is, it is highly unlikely that this will prevent you from solving the structure. Indeed I think there are many structures in the PDB that had streaks in their diffraction pattern like this. The reason it won't hurt you is that the intensity of the Bragg peaks is the same in the perfectly-correlated, partially-correlated and completely uncorrelated cases. The electron density will simply have a two-headed side chain in it. So, I would suggest doing what most crystallographers do and completely ignore any potentially informative weirdness along the way and sally forth. But save these pictures (and the above book) for when your reviewer tells you your R-merge is too high. -James Holton MAD Scientist Margriet Ovaere wrote: Dear all, In the diffraction pattern of crystals of an RNA decamer, small lines appeared (see pictures attached). We've
Re: [ccp4bb] small lines in diffraction pattern
Jacob Traditional mosaic spread (ordered mosaic blocks imperfectly aligned with respect to one another) gives spherical caps in reciprocal space. These would appear as arcs on a single crystal rotation photograph. If anisotropic, the arcs would be more extensive in some directions. The patterns do not appear to fit this case. Different integration software could have different models for mosaicity but they often simply ensure that most of the intensity is captured in the integration volume by choosing some appropriate reflection range for the reflection. This could capture broadening of the reflections due to many types of disorder, including some of the ones discussed by Ian and myself. However, for the case discussed here, I think most integration programs would struggle. Regards Colin From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Jacob Keller Sent: 28 January 2009 17:05 To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] small lines in diffraction pattern Couldn't the lines be explained most simply by extreme mosaicity, perhaps severely anisotropic? If not, why not? What were the values obtained in integration? 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 *** - Original Message - From: Margriet Ovaere mailto:margriet.ova...@chem.kuleuven.be To: CCP4BB@JISCMAIL.AC.UK Sent: Wednesday, January 28, 2009 7:51 AM Subject: [ccp4bb] small lines in diffraction pattern Dear all, In the diffraction pattern of crystals of an RNA decamer, small lines appeared (see pictures attached). We've tried different crystals but they all showed the same small lines. �Has anybody seen this�phenomena�before and has got an explanation for it please..? Many thanks Margriet Ovaere Margriet Ovaere Chemistry Department K.U.Leuven Biomolecular Architecture Celestijnenlaan 200 F B-3001 Heverlee (Leuven) Tel: +32(0)16327477 Dear all, In the diffraction pattern of crystals of an RNA decamer, small lines appeared (see pictures attached). We've tried different crystals but they all showed the same small lines. Has anybody seen this phenomena before and has got an explanation for it please..? Many thanks Margriet Ovaere  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 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
Re: [ccp4bb] small lines in diffraction pattern
Hi, it's clearly not a detector problem (at least not an obvious one!), since the 'lines' clearly follow the *curved* path of the reciprocal lattice lines as they are projected from the Ewald sphere onto the detector. A saturated pixel would presumably affect other pixels only in the same row and would appear in *straight* lines unrelated to the reciprocal lattice. I'm sticking with diffuse scattering! Cheers -- Ian -Original Message- From: owner-ccp...@jiscmail.ac.uk [mailto:owner-ccp...@jiscmail.ac.uk] On Behalf Of Stephan Ginell Sent: 28 January 2009 17:43 To: Margriet Ovaere; CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] small lines in diffraction pattern 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 Stephan L. Ginell, Ph.D. Coordinator, SBC User Program Biosciences Argonne National Laboratory 9700 S. Cass Ave Argonne, IL 60439 (630)252-3972 office (630)218-8122 pager (630)252-6126 Fax gin...@anl.gov Email On 1/28/09 7:51 AM, Margriet Ovaere margriet.ova...@chem.kuleuven.be wrote: Dear all, In the diffraction pattern of crystals of an RNA decamer, small lines appeared (see pictures attached). We've tried different crystals but they all showed the same small lines. Has anybody seen this phenomena before and has got an explanation for it please..? Many thanks Margriet Ovaere Margriet Ovaere Chemistry Department K.U.Leuven Biomolecular Architecture Celestijnenlaan 200 F B-3001 Heverlee (Leuven) Tel: +32(0)16327477 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
Re: [ccp4bb] small lines in diffraction patternI 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 ***
Re: [ccp4bb] small lines in diffraction pattern
Re: [ccp4bb] small lines in diffraction patternActa Cryst. (1998). D54, 848-853 [ doi:10.1107/S0907444998001875 ] A Description of Imperfections in Protein Crystals C. Nave 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 *** - Original Message - From: Jacob Keller To: 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 ***
Re: [ccp4bb] small lines in diffraction pattern
There is something in the unit cell, aligned with the long axis of the cell, with a periodicity corresponding to ~1/5 of the long axis. This can be seen as greater intensities along the long axis every fifth spot. Without knowing the unit cell parameters, I would guess it is either the interplanar spacings of the nucleotides (probably this is too small) or the periodic twist of the helix itself. Interesting that the RNA is a decamer ( = 2 x 5). I would be curious to know what the unit cell parameters are, or more generally, what is causing that noticeable periodicity... 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 *** - Original Message - From: James Holton jmhol...@lbl.gov To: CCP4BB@JISCMAIL.AC.UK Sent: Wednesday, January 28, 2009 12:20 PM Subject: Re: [ccp4bb] small lines in diffraction pattern Hmm. I don't remember that thread. However, I personally think it is a good idea to keep the mosaic crystal as Ewald and Darwin defined it. Just because current integration software lumps things together into a mosaicity does not mean that every mechanism contributing to the rocking width of a spot should be given the same name. Especially when it is difficult to describe the mosaic crystal using any other words. Perhaps Colin could come up with a cool word for unit cell non-uniformity? Or is he waiting for us to name it after him? Nonuniform Anisotropic Variance of Elasticity? or Cells Of Loose INdex? Comments and suggestions are welcome. -James Holton MAD Scientist Jacob Keller wrote: 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 ***
Re: [ccp4bb] small lines in diffraction pattern
I'm coming in late here, having only now found time to look at the images. It's facinating, isn't it? Since the lines are not arcs centered on the origin, this isn't mosaic spread. For those who haven't seen the image and the zoom, the diffraction pattern clearly shows one very long axis and a couple of much shorter ones. The rotation image is taken rotating around the long one. The small lines are perpendicular to the long axis, and run fairly uniformly, narrowly and evenly spaced at the intervals of the reflections along this axis, throughout the diffraction pattern. Also it appears that this is a rotation of about a degree; Margriet doesn't give us clues for any of this. I'm guessing that whoever said it's a diffuse scatter effect is close to the mark. I think diffuse scatter comes from the contents of each unit cell being essentially identical, but that within the molecule things are waving around a bit (where are Don Caspar and George Phillips when we need them?). I'll go a touch farther and suggest that it's really disorder -- each unit cell is well aligned to the others, but each one is different in some way. I'll guess that the RNA decamer is aligned along this long unit cell axis, but in some way either there's an opportunity for the register along the RNA axis to slip from one unit cell to the other or each is rotated slightly. On the other hand, the fact that there's a wide distribution of intensities in the Bragg spots, which are quite sharp, is confusing -- there must be a lot of contrast in the averaged structure for this to be true. Ok, it's interesting, but I have no idea. Bob On Wed, 28 Jan 2009, James Holton wrote: Hmm. I don't remember that thread. However, I personally think it is a good idea to keep the mosaic crystal as Ewald and Darwin defined it. Just because current integration software lumps things together into a mosaicity does not mean that every mechanism contributing to the rocking width of a spot should be given the same name. Especially when it is difficult to describe the mosaic crystal using any other words. Perhaps Colin could come up with a cool word for unit cell non-uniformity? Or is he waiting for us to name it after him? Nonuniform Anisotropic Variance of Elasticity? or Cells Of Loose INdex? Comments and suggestions are welcome. -James Holton MAD Scientist Jacob Keller wrote: 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 ***
Re: [ccp4bb] small lines in diffraction pattern
Hi James, what your descriptions aims at is I think shown in this publication Borgstahl, G. E. O. Incommensurate Crystallography by Sander van Smaalen Crystallography reviews 14 , 259-260 (2008). Or am I misunderstanding something here ? Jürgen On 28 Jan 2009, at 12:39, James Holton wrote: I recommend you have a look at a book from OUP called Diffuse X-Ray Scattering and Models of Disorder by T. R. Welberry. The first chapter explains quite well (I think) where all these streaky things come from. It will also make you feel better about having it when you see all the small molecule structures that have horrible diffuse scattering! (such as urea). This looks to me like a fairly classic case of correlated static disorder. Best way to think about it is this: Imagine you have two different kinds of unit cells: A an B. Doesn't really matter what the difference between A and B is, could be a two-headed side chain in conformer A vs conformer B, or it could be as complicated as a domain motion. But, for simplicity, lets assume it is two rotamers of a side chain and also assume that each unit cell in your crystal can only be one or the other (no in betweens). Now, if the arrangement of these unit cells is perfectly correlated and an A always occurs right next door to a B along the c-axis (say), then what you really have is a bigger unit cell than you think. That is, you can draw a unit cell around each A-B pair and call it a supercell with the contents of B as a simple NCS mate of A (with one side chain in a different rotamer). Some people might call this a pseudotranslation. The effect on the diffraction pattern in this case would be the appearance of a very weak spot in between each old spot along your c axis. That is, your supercell is twice as big along c so the reciprocal-space lattice has twice as many spots in it. The new spots are weak because they only correspond to the differences between A and B, which in this case is only a few atoms. Now lets say A and B are not perfectly correlated, but only slightly. That is, in some parts of the crystal A and B are side-by-side, but in other parts you get AAB, ABBA, BABBA, etc. In each of these cases the supercell you must draw is 3, 4 and 5x your original unit cell. Each of these will produce new weak spots with progressively tighter spacings. As the supercell becomes very long, these rows of tight spots will become a streak. The streaks are particularly prominent if the A-B disorder is along only one axis. In that case, you must have a whole a-b layer of A and other whole a-b layers of B, and the ordering of these layers along c is fairly random. Colin just described this as a stacking disorder which is probably a good name for it. The final case is when A and B are completely uncorrelated and occur absolutely at random locations in your crystal. In this case the supercell can be anything and the streaks are in every direction (including every diagonal) so they simply show up as increased background. Every crystal does this. In fact, this is the origin of the B-factor as no two unit cells are exactly alike. Ever wonder where those photons go that scatter off protein atoms but don't go into spots? They go into the background. Now, since these streaks represent correlations of neighboring unit cells this means that the diffuse scattering can tell you something about how your molecule moves. There is something about your structure that forces its neighbors to be the same in at least one direction. There are a class of people who study this for a living. I am not one of them. BTW. This is definitely NOT a mosaic spread. Mosaicity occurs on length scales thousands of times larger than this. By definition, a mosaic spread is the width of the distribution of relative rotation angles of mosaic domains and these domains all scatter independently of each other. An infinite mosaic spread (or at least 180 degrees) corresponds to a powder diffraction pattern, and the fact that powder lines are sharp demonstrates how mosaicity cannot smear spots in anything but the tangential direction. That is, no rotation can change the d-spacing of a spot. Changes in unit cell size can do this, but that is a very different phenomenon than mosaic spread as mosaic domains are much much bigger than unit cells. The good news is, it is highly unlikely that this will prevent you from solving the structure. Indeed I think there are many structures in the PDB that had streaks in their diffraction pattern like this. The reason it won't hurt you is that the intensity of the Bragg peaks is the same in the perfectly-correlated, partially-correlated and completely uncorrelated cases. The electron density will simply have a two- headed side chain in it. So, I would suggest doing what most crystallographers do and completely ignore any potentially informative weirdness along the way and sally forth.
Re: [ccp4bb] small lines in diffraction pattern
I'm coming in late here, having only now found time to look at the images. It's facinating, isn't it? Since the lines are not arcs centered on the origin, this isn't mosaic spread. For those who haven't seen the image and the zoom, the diffraction pattern clearly shows one very long axis and a couple of much shorter ones. The rotation image is taken rotating around the long one. The small lines are perpendicular to the long axis, and run fairly continuously, narrowly and evenly spaced at the intervals of the reflections along this axis, throughout the diffraction pattern. They're all faint and about the same intensity, and modulated along their length only slightly. Also it appears that this is a rotation of about one degree; Margriet doesn't give us clues for any of this. I'm guessing that whoever said it's a diffuse scatter effect is close to the mark. I think diffuse scatter comes from the contents of each unit cell being essentially identical, but that within the molecule things are waving around a bit (where are Don Caspar and George Phillips when we need them?), that is, different in each unit cell. I'll go a touch farther and suggest that it's really disorder -- each unit cell is well aligned to the others, but each one is different in a more significant way. I'll guess that the RNA decamer is aligned along this long unit cell axis, but in some way either there's an opportunity for the register along the RNA axis to slip from one unit cell to the other or each is rotated slightly. On the other hand, the fact that there's a wide distribution of intensities in the Bragg spots, which are quite sharp, is confusing -- there must be a lot of contrast in the averaged structure for this to be true. Ok, it's interesting, but I have no idea. Bob On Wed, 28 Jan 2009, Jacob Keller wrote: There is something in the unit cell, aligned with the long axis of the cell, with a periodicity corresponding to ~1/5 of the long axis. This can be seen as greater intensities along the long axis every fifth spot. Without knowing the unit cell parameters, I would guess it is either the interplanar spacings of the nucleotides (probably this is too small) or the periodic twist of the helix itself. Interesting that the RNA is a decamer ( = 2 x 5). I would be curious to know what the unit cell parameters are, or more generally, what is causing that noticeable periodicity... 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 *** - Original Message - From: James Holton jmhol...@lbl.gov To: CCP4BB@JISCMAIL.AC.UK Sent: Wednesday, January 28, 2009 12:20 PM Subject: Re: [ccp4bb] small lines in diffraction pattern Hmm. I don't remember that thread. However, I personally think it is a good idea to keep the mosaic crystal as Ewald and Darwin defined it. Just because current integration software lumps things together into a mosaicity does not mean that every mechanism contributing to the rocking width of a spot should be given the same name. Especially when it is difficult to describe the mosaic crystal using any other words. Perhaps Colin could come up with a cool word for unit cell non-uniformity? Or is he waiting for us to name it after him? Nonuniform Anisotropic Variance of Elasticity? or Cells Of Loose INdex? Comments and suggestions are welcome. -James Holton MAD Scientist Jacob Keller wrote: 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 ***
Re: [ccp4bb] small lines in diffraction pattern (fwd)
I'm coming in late here, having only now found time to look at the images. It's facinating, isn't it? Since the lines are not arcs centered on the origin, this isn't mosaic spread. For those who haven't seen the image and the zoom, the diffraction pattern clearly shows one very long axis and a couple of much shorter ones. The rotation image is taken rotating around the long one. The small lines are perpendicular to the long axis, and run fairly continuously, narrowly and evenly spaced at the intervals of the reflections along this axis, throughout the diffraction pattern. They're all faint and about the same intensity, and modulated along their length only slightly. Also it appears that this is a rotation of about one degree; Margriet doesn't give us clues for any of this. I'm guessing that whoever said it's a diffuse scatter effect is close to the mark. I think diffuse scatter comes from the contents of each unit cell being essentially identical, but that within the molecule things are waving around a bit (where are Don Caspar and George Phillips when we need them?), that is, different in each unit cell. I'll go a touch farther and suggest that it's really disorder -- each unit cell is well aligned to the others, but each one is different in a more significant way. I'll guess that the RNA decamer is aligned along this long unit cell axis, but in some way either there's an opportunity for the register along the RNA axis to slip from one unit cell to the other or each is rotated slightly. On the other hand, the fact that there's a wide distribution of intensities in the Bragg spots, which are quite sharp, is confusing -- there must be a lot of contrast in the averaged structure for this to be true. Ok, it's interesting, but I have no idea. Bob On Wed, 28 Jan 2009, Jacob Keller wrote: There is something in the unit cell, aligned with the long axis of the cell, with a periodicity corresponding to ~1/5 of the long axis. This can be seen as greater intensities along the long axis every fifth spot. Without knowing the unit cell parameters, I would guess it is either the interplanar spacings of the nucleotides (probably this is too small) or the periodic twist of the helix itself. Interesting that the RNA is a decamer ( = 2 x 5). I would be curious to know what the unit cell parameters are, or more generally, what is causing that noticeable periodicity... 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 *** - Original Message - From: James Holton jmhol...@lbl.gov To: CCP4BB@JISCMAIL.AC.UK Sent: Wednesday, January 28, 2009 12:20 PM Subject: Re: [ccp4bb] small lines in diffraction pattern Hmm. I don't remember that thread. However, I personally think it is a good idea to keep the mosaic crystal as Ewald and Darwin defined it. Just because current integration software lumps things together into a mosaicity does not mean that every mechanism contributing to the rocking width of a spot should be given the same name. Especially when it is difficult to describe the mosaic crystal using any other words. Perhaps Colin could come up with a cool word for unit cell non-uniformity? Or is he waiting for us to name it after him? Nonuniform Anisotropic Variance of Elasticity? or Cells Of Loose INdex? Comments and suggestions are welcome. -James Holton MAD Scientist Jacob Keller wrote: 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 ***
Re: [ccp4bb] small lines in diffraction pattern
I'm sure Gloria would be delighted if that were the case, but I don't think this is an incommensurate lattice. These actually don't so much give you diffuse scattering as little satellite spots near the main spots at spacings that don't make any sense given the lattice repeat. My understanding is that these arise from something that is slowly varying from unit cell to unit cell (could be as simple as a side chain waving back and forth) in a repetitive pattern that just doesn't line up in any way with the repeat of the unit cells. Still, I'll ask her. However, I think that the difference is that modulated lattices are gradual changes of structure across many unit cells and what I was talking about is a more simplistic case of two different kinds of unit cells with varying degrees of randomness in their arrangement. That is, using the formalism I described below, a modulated lattice would have unit cells that go: ABCDEFGHGFEDCBABCDEFG... etc. where A is not that different from B, B similar to C, etc., but A and H are very different. -James Holton MAD Scientist Jürgen Bosch wrote: Hi James, what your descriptions aims at is I think shown in this publication Borgstahl, G. E. O. Incommensurate Crystallography by Sander van Smaalen Crystallography reviews 14 , 259-260 (2008). Or am I misunderstanding something here ? Jürgen On 28 Jan 2009, at 12:39, James Holton wrote: I recommend you have a look at a book from OUP called Diffuse X-Ray Scattering and Models of Disorder by T. R. Welberry. The first chapter explains quite well (I think) where all these streaky things come from. It will also make you feel better about having it when you see all the small molecule structures that have horrible diffuse scattering! (such as urea). This looks to me like a fairly classic case of correlated static disorder. Best way to think about it is this: Imagine you have two different kinds of unit cells: A an B. Doesn't really matter what the difference between A and B is, could be a two-headed side chain in conformer A vs conformer B, or it could be as complicated as a domain motion. But, for simplicity, lets assume it is two rotamers of a side chain and also assume that each unit cell in your crystal can only be one or the other (no in betweens). Now, if the arrangement of these unit cells is perfectly correlated and an A always occurs right next door to a B along the c-axis (say), then what you really have is a bigger unit cell than you think. That is, you can draw a unit cell around each A-B pair and call it a supercell with the contents of B as a simple NCS mate of A (with one side chain in a different rotamer). Some people might call this a pseudotranslation. The effect on the diffraction pattern in this case would be the appearance of a very weak spot in between each old spot along your c axis. That is, your supercell is twice as big along c so the reciprocal-space lattice has twice as many spots in it. The new spots are weak because they only correspond to the differences between A and B, which in this case is only a few atoms. Now lets say A and B are not perfectly correlated, but only slightly. That is, in some parts of the crystal A and B are side-by-side, but in other parts you get AAB, ABBA, BABBA, etc. In each of these cases the supercell you must draw is 3, 4 and 5x your original unit cell. Each of these will produce new weak spots with progressively tighter spacings. As the supercell becomes very long, these rows of tight spots will become a streak. The streaks are particularly prominent if the A-B disorder is along only one axis. In that case, you must have a whole a-b layer of A and other whole a-b layers of B, and the ordering of these layers along c is fairly random. Colin just described this as a stacking disorder which is probably a good name for it. The final case is when A and B are completely uncorrelated and occur absolutely at random locations in your crystal. In this case the supercell can be anything and the streaks are in every direction (including every diagonal) so they simply show up as increased background. Every crystal does this. In fact, this is the origin of the B-factor as no two unit cells are exactly alike. Ever wonder where those photons go that scatter off protein atoms but don't go into spots? They go into the background. Now, since these streaks represent correlations of neighboring unit cells this means that the diffuse scattering can tell you something about how your molecule moves. There is something about your structure that forces its neighbors to be the same in at least one direction. There are a class of people who study this for a living. I am not one of them. BTW. This is definitely NOT a mosaic spread. Mosaicity occurs on length scales thousands of times larger than this. By definition, a mosaic spread is the width of the distribution of relative rotation angles of mosaic domains and these domains all scatter
Re: [ccp4bb] small lines in diffraction pattern
Hi Bob et al: I think that this is the convolution of the helical transform with the Bragg diffraction, which you often see in nucleic acid diffraction patters. I think I first saw it, in fact, at your beam-line in 1994, with ribozyme crystals. (Aaron Klug had to explain it to me.) The part that puzzled me was that the dark spots seem to be modulo 10, whereas for A-form RNA it should be 11 (due to 11 bases per helical turn). But then I read the initial email, and it says RNA decamer. So I think what is happening is that the RNA is slightly distorted to 10 bases per turn to accommodate the crystal lattice repeat. The horizontal lines aren't diffuse scatter, but rather the layer line scatter of a helical transform described by increasing order cylindrical Bessel functions (mod 10). All the best, Bill On Jan 28, 2009, at 6:57 PM, Robert Sweet wrote: I'm coming in late here, having only now found time to look at the images. It's facinating, isn't it? Since the lines are not arcs centered on the origin, this isn't mosaic spread. For those who haven't seen the image and the zoom, the diffraction pattern clearly shows one very long axis and a couple of much shorter ones. The rotation image is taken rotating around the long one. The small lines are perpendicular to the long axis, and run fairly continuously, narrowly and evenly spaced at the intervals of the reflections along this axis, throughout the diffraction pattern. They're all faint and about the same intensity, and modulated along their length only slightly. Also it appears that this is a rotation of about one degree; Margriet doesn't give us clues for any of this. I'm guessing that whoever said it's a diffuse scatter effect is close to the mark. I think diffuse scatter comes from the contents of each unit cell being essentially identical, but that within the molecule things are waving around a bit (where are Don Caspar and George Phillips when we need them?), that is, different in each unit cell. I'll go a touch farther and suggest that it's really disorder -- each unit cell is well aligned to the others, but each one is different in a more significant way. I'll guess that the RNA decamer is aligned along this long unit cell axis, but in some way either there's an opportunity for the register along the RNA axis to slip from one unit cell to the other or each is rotated slightly. On the other hand, the fact that there's a wide distribution of intensities in the Bragg spots, which are quite sharp, is confusing -- there must be a lot of contrast in the averaged structure for this to be true. Ok, it's interesting, but I have no idea. Bob On Wed, 28 Jan 2009, Jacob Keller wrote: There is something in the unit cell, aligned with the long axis of the cell, with a periodicity corresponding to ~1/5 of the long axis. This can be seen as greater intensities along the long axis every fifth spot. Without knowing the unit cell parameters, I would guess it is either the interplanar spacings of the nucleotides (probably this is too small) or the periodic twist of the helix itself. Interesting that the RNA is a decamer ( = 2 x 5). I would be curious to know what the unit cell parameters are, or more generally, what is causing that noticeable periodicity... 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 *** - Original Message - From: James Holton jmhol...@lbl.gov To: CCP4BB@JISCMAIL.AC.UK Sent: Wednesday, January 28, 2009 12:20 PM Subject: Re: [ccp4bb] small lines in diffraction pattern Hmm. I don't remember that thread. However, I personally think it is a good idea to keep the mosaic crystal as Ewald and Darwin defined it. Just because current integration software lumps things together into a mosaicity does not mean that every mechanism contributing to the rocking width of a spot should be given the same name. Especially when it is difficult to describe the mosaic crystal using any other words. Perhaps Colin could come up with a cool word for unit cell non-uniformity? Or is he waiting for us to name it after him? Nonuniform Anisotropic Variance of Elasticity? or Cells Of Loose INdex? Comments and suggestions are welcome. -James Holton MAD Scientist Jacob Keller wrote: 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
