Re: [ccp4bb] saxs on xtals
Dear Anna, Very interesting diffraction pattern. Any chance of measuring to higher resolution? Ie to try and capture the higher order rings, which presumably are there. Also interesting that these rings seem quite weak ie the ferritin perhaps not fully loaded? Best wishes, John Prof John R Helliwell DSc FInstP CPhys FRSC CChem F Soc Biol. Chair School of Chemistry, University of Manchester, Athena Swan Team. http://www.chemistry.manchester.ac.uk/aboutus/athena/index.html On 8 May 2012, at 16:54, anna anna marmottalb...@gmail.com wrote: Dear all, first of all I want to thank you for your attention and all your brilliant suggestions that really cleared my head!!! Thanks to you (or because of you!!) now I have many ideas and very much to do. Colin, I was just re-considering my diffraction images. Who knows if they are single xtals indeed! Let's see if I understood your point. Assuming that they are single xtals, if they are located at independent positions in the protein-cage it would be like powder diffraction, with rings at diffraction angles corresponding to magnetite lattice. If they are ordered they should give a diffraction pattern. The corresponding lattice can differ from the protein lattice, do you agree? If this is true, what would I see? Two superimposed diffraction patterns? Actually, I am not able to evaluate it... I attached one of the diffraction images. It seems to me that there are two diffused rings at about 2.5 and 2.9 A. Michael, I just read your reply. I think that the eventual periodicity of the partcles can't be completely independent of the protein periodicity (I attached a hypotethical scheme), as you suggest I will try P1. Once I tryed a naive version of what you suggest: I put a magnet over the xtallization plate. All my collegues made fun of me... :) !! I will check the literature that you all quoted (hard work!) Thank you again, new suggestions will be really appreciated. Cheers, anna 2012/5/8 R. M. Garavito rmgarav...@gmail.com Dear Anna, I know that you already have gotten replies from some top experts, but your intriguing problem brought up some issues I have run across in the past. First, from you experience with single crystal diffraction, your results are not that much different from those seen in virus structures where the nucleic acid structure is averaged out. As the nucleic acid doesn't (and mostly can't) adopt the symmetry of the protein shell, the crystallization process alone does the averaging. Just because that ferritin and magnetite have cubic symmetry elements, if they don't line up, the magnetite structure can be averaged out upon crystallization. So, working at lower symmetry may not help, unless there is some directional correlation of the magnetite symmetry and position with the crystal axes. But try P1 and see what happens. A second comment is why not try neutron scattering (SANS or single crystal neutron diffraction), particularly as you can match out the protein with D2O and see just the magnetite. While the same concerns apply for single crystal neutron diffraction, you see more clearly regions of higher average density inside the protein shell. And lastly, have you tried crystallizing your ferritin/nanoparticle complexes in the presence of a magnetic field? It would be a neat trick, and people have tried such things in the past, such as for orienting biomolecules. Some even used old NMR magnets. Would be wild, if it worked. Good luck, Michael R. Michael Garavito, Ph.D. Professor of Biochemistry Molecular Biology 603 Wilson Rd., Rm. 513 Michigan State University East Lansing, MI 48824-1319 Office: (517) 355-9724 Lab: (517) 353-9125 FAX: (517) 353-9334Email: rmgarav...@gmail.com On May 7, 2012, at 12:30 PM, anna anna wrote: Dear all, I'd like some suggestions/opinions about the sense of an experiment proposed by a collaborator expert in saxs. In few words, he wants to collect SAXS data on a suspension of protein xtals to investigate low resolution periodicity of the xtal (more details below). The experiment requires a very huge number of xtals to obtain the circles typical of saxs and it is very time-consuming to me (I know nothing about saxs, I have only to prepare the sample). I proposed to measure a single rotating xtal (like in XRD) but he told they don't have a goniometer on saxs beamline. Here is my concern: does it make sense to measure many xtals together? Don't we lose information with respect to single xtal? And, most of all, what can I see by saxs that I can't see by waxs?? Sorry for the almost off-topic question but I think that only someone who knows both the techniques can help me!! Some detail for who is
Re: [ccp4bb] saxs on xtals
Thanks to all again! Find below my answers/comments to all your replies. Colin Nave, I'll certainly collect higher resolution dataset to look for more diagnostic rings. Apo-ferritin xtallizes in the same conditions with the same cell (I know it from literature), I'll measure it too, to look for differences. Jacob Keller, we loaded ferritin with Fe and Co and oxidized them obtaining CoxFe(3-x)O4/Fe2O3. About the brillant spots, by eye I attributed them to eventual ammonium sulfate xtals (2M in xtallization conditions) but it could be something else, compare to apo-ferritin will help. James Holton, I didn't think about it! Actually it's the same as nuclear spin in NMR: the orientation is random until an external magnetic field is applied. It would be very very interesting to collect diffraction under this condition, do you know an equipped beamline? Allister Crow, thanks for encouragement! I knew that someone had already studied it... your paper is very usefull. Myabe my case is different because I have Fe3O4 and an external magnetic field could induce order, I hope I can do this kind of experiment. John Hellywell, I'll certainly measure at higer resolution. Ferritin should be fully loaded since there are about 3500 metal atoms per protein shell. I'll keep you updated on my future results!
Re: [ccp4bb] saxs on xtals
Anna Are the nanoparticles expected to be single crystals? Magnetite has Fd3m space group with a 8.4A lattice (just looked it up). This should give some diffraction features such as broad spots (broadened because of the small particle size) or rings (if there is no alignment between the nanoparticles). Have you looked for such effects in the diffraction patterns which you already have from the single crystals? Colin From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of anna anna Sent: 07 May 2012 17:30 To: ccp4bb Subject: [ccp4bb] saxs on xtals Dear all, I'd like some suggestions/opinions about the sense of an experiment proposed by a collaborator expert in saxs. In few words, he wants to collect SAXS data on a suspension of protein xtals to investigate low resolution periodicity of the xtal (more details below). The experiment requires a very huge number of xtals to obtain the circles typical of saxs and it is very time-consuming to me (I know nothing about saxs, I have only to prepare the sample). I proposed to measure a single rotating xtal (like in XRD) but he told they don't have a goniometer on saxs beamline. Here is my concern: does it make sense to measure many xtals together? Don't we lose information with respect to single xtal? And, most of all, what can I see by saxs that I can't see by waxs?? Sorry for the almost off-topic question but I think that only someone who knows both the techniques can help me!! Some detail for who is intrigued by my story: we prepared doped magnetite nanoparticles using ferritin as bioreactor. I crystallized this spheres filled with metal and solved the structure at 3.7A but I can see only the protein shell while there is no density inside, even if I know that the nanoparticles are there. A simple explanation is that the particles are free to move in the cavity(note that the diameter of the nanoparticle is shorter then the inner diameter of the protein shell), ie are disordered, and do not contribute to diffraction, in fact, to my knowledge, nobody have ever seen the metal core inside ferritin or dps proteins. However, since they are magnetic particles they must see each other through the protein wall, ie they can't be completely free to move in the cavity. Maybe, but this is just my opinion, I don't see the particle because the period of the particle in the xtal is different/longer than the period of the protein shell. Anyway, we are interested in the relative distance between the magnetic particles in the xtal to study the effects of magnetostatic interactions in nanoparticles 3D arrays. We are going to do this by saxs since, they told me, lower resolution is useful in studying this long range periodicity (the diameter of ferritin is about 120A) but it seems fool to me using a suspension of so many xtals to obtain a scattering curve while I could collect diffraction images from a single xtal!!! I know that saxs is used when you don't have xtals but if you have xtals, ie your system is ordered, xtallography is much more powerful!! Another question: how can I handle my diffraction data at 3.7A resolution to look for nanoparticles? Should I try a lower symmetry? Maybe the anomalous signal? Have you any reference for a similar case? Thank you very much!! anna
Re: [ccp4bb] saxs on xtals
Dear Anna, I know that you already have gotten replies from some top experts, but your intriguing problem brought up some issues I have run across in the past. First, from you experience with single crystal diffraction, your results are not that much different from those seen in virus structures where the nucleic acid structure is averaged out. As the nucleic acid doesn't (and mostly can't) adopt the symmetry of the protein shell, the crystallization process alone does the averaging. Just because that ferritin and magnetite have cubic symmetry elements, if they don't line up, the magnetite structure can be averaged out upon crystallization. So, working at lower symmetry may not help, unless there is some directional correlation of the magnetite symmetry and position with the crystal axes. But try P1 and see what happens. A second comment is why not try neutron scattering (SANS or single crystal neutron diffraction), particularly as you can match out the protein with D2O and see just the magnetite. While the same concerns apply for single crystal neutron diffraction, you see more clearly regions of higher average density inside the protein shell. And lastly, have you tried crystallizing your ferritin/nanoparticle complexes in the presence of a magnetic field? It would be a neat trick, and people have tried such things in the past, such as for orienting biomolecules. Some even used old NMR magnets. Would be wild, if it worked. Good luck, Michael R. Michael Garavito, Ph.D. Professor of Biochemistry Molecular Biology 603 Wilson Rd., Rm. 513 Michigan State University East Lansing, MI 48824-1319 Office: (517) 355-9724 Lab: (517) 353-9125 FAX: (517) 353-9334Email: rmgarav...@gmail.com On May 7, 2012, at 12:30 PM, anna anna wrote: Dear all, I'd like some suggestions/opinions about the sense of an experiment proposed by a collaborator expert in saxs. In few words, he wants to collect SAXS data on a suspension of protein xtals to investigate low resolution periodicity of the xtal (more details below). The experiment requires a very huge number of xtals to obtain the circles typical of saxs and it is very time-consuming to me (I know nothing about saxs, I have only to prepare the sample). I proposed to measure a single rotating xtal (like in XRD) but he told they don't have a goniometer on saxs beamline. Here is my concern: does it make sense to measure many xtals together? Don't we lose information with respect to single xtal? And, most of all, what can I see by saxs that I can't see by waxs?? Sorry for the almost off-topic question but I think that only someone who knows both the techniques can help me!! Some detail for who is intrigued by my story: we prepared doped magnetite nanoparticles using ferritin as bioreactor. I crystallized this spheres filled with metal and solved the structure at 3.7A but I can see only the protein shell while there is no density inside, even if I know that the nanoparticles are there. A simple explanation is that the particles are free to move in the cavity(note that the diameter of the nanoparticle is shorter then the inner diameter of the protein shell), ie are disordered, and do not contribute to diffraction, in fact, to my knowledge, nobody have ever seen the metal core inside ferritin or dps proteins. However, since they are magnetic particles they must see each other through the protein wall, ie they can't be completely free to move in the cavity. Maybe, but this is just my opinion, I don't see the particle because the period of the particle in the xtal is different/longer than the period of the protein shell. Anyway, we are interested in the relative distance between the magnetic particles in the xtal to study the effects of magnetostatic interactions in nanoparticles 3D arrays. We are going to do this by saxs since, they told me, lower resolution is useful in studying this long range periodicity (the diameter of ferritin is about 120A) but it seems fool to me using a suspension of so many xtals to obtain a scattering curve while I could collect diffraction images from a single xtal!!! I know that saxs is used when you don't have xtals but if you have xtals, ie your system is ordered, xtallography is much more powerful!! Another question: how can I handle my diffraction data at 3.7A resolution to look for nanoparticles? Should I try a lower symmetry? Maybe the anomalous signal? Have you any reference for a similar case? Thank you very much!! anna
Re: [ccp4bb] saxs on xtals
Dear Colin, the table you gave seems to have been from Fe2+Fe3+2O4 or from Fe3-xTixO4. I am curious what the nature of the Fe inside the ferritin is (I don't think it has Ti in it, though...). Is it elemental iron? Also, to Anna: can you send a picture of that diffraction pattern with spot predictions superimposed? There seem to be some really bright spots which are outliers, and maybe multiple lattices. JPK On Tue, May 8, 2012 at 12:21 PM, Colin Nave colin.n...@diamond.ac.ukwrote: Anna Yes, you have understood the suggestion. Could be the 220 and 311 reflections. See for example http://rruff.info/magnetite/R080025 and http://rruff.info/repository/sample_child_record_powder/by_minerals/Magnetite__R080025-1__Powder__DIF_File__9448.txt Trying to index powder patterns from 2 rings is risky and the intensities don't seem to agree. I guess you don't have higher angle data. Should be able to evaluate a particle size from the breadth of the rings though. For example a 57A crystal examined with 1A radiation would give broadening of about a degree. There do seem to be other spots - I guess these are ice rings but you should check. Also be nice to know if apoferritin crystallised under the same conditions (if it can be) shows these rings Regards Colin From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of anna anna Sent: 08 May 2012 16:55 To: ccp4bb Subject: Re: [ccp4bb] saxs on xtals Dear all, first of all I want to thank you for your attention and all your brilliant suggestions that really cleared my head!!! Thanks to you (or because of you!!) now I have many ideas and very much to do. Colin, I was just re-considering my diffraction images. Who knows if they are single xtals indeed! Let's see if I understood your point. Assuming that they are single xtals, if they are located at independent positions in the protein-cage it would be like powder diffraction, with rings at diffraction angles corresponding to magnetite lattice. If they are ordered they should give a diffraction pattern. The corresponding lattice can differ from the protein lattice, do you agree? If this is true, what would I see? Two superimposed diffraction patterns? Actually, I am not able to evaluate it... I attached one of the diffraction images. It seems to me that there are two diffused rings at about 2.5 and 2.9 A. Michael, I just read your reply. I think that the eventual periodicity of the partcles can't be completely independent of the protein periodicity (I attached a hypotethical scheme), as you suggest I will try P1. Once I tryed a naive version of what you suggest: I put a magnet over the xtallization plate. All my collegues made fun of me... :) !! I will check the literature that you all quoted (hard work!) Thank you again, new suggestions will be really appreciated. Cheers, anna 2012/5/8 R. M. Garavito rmgarav...@gmail.commailto:rmgarav...@gmail.com Dear Anna, I know that you already have gotten replies from some top experts, but your intriguing problem brought up some issues I have run across in the past. First, from you experience with single crystal diffraction, your results are not that much different from those seen in virus structures where the nucleic acid structure is averaged out. As the nucleic acid doesn't (and mostly can't) adopt the symmetry of the protein shell, the crystallization process alone does the averaging. Just because that ferritin and magnetite have cubic symmetry elements, if they don't line up, the magnetite structure can be averaged out upon crystallization. So, working at lower symmetry may not help, unless there is some directional correlation of the magnetite symmetry and position with the crystal axes. But try P1 and see what happens. A second comment is why not try neutron scattering (SANS or single crystal neutron diffraction), particularly as you can match out the protein with D2O and see just the magnetite. While the same concerns apply for single crystal neutron diffraction, you see more clearly regions of higher average density inside the protein shell. And lastly, have you tried crystallizing your ferritin/nanoparticle complexes in the presence of a magnetic field? It would be a neat trick, and people have tried such things in the past, such as for orienting biomolecules. Some even used old NMR magnets. Would be wild, if it worked. Good luck, Michael R. Michael Garavito, Ph.D. Professor of Biochemistry Molecular Biology 603 Wilson Rd., Rm. 513 Michigan State University East Lansing, MI 48824-1319 Office: (517) 355-9724tel:%28517%29%20355-9724 Lab: (517) 353-9125 tel:%28517%29%20353-9125 FAX: (517) 353-9334tel:%28517%29%20353-9334Email: rmgarav...@gmail.commailto:garav...@gmail.com On May 7, 2012, at 12:30 PM
Re: [ccp4bb] saxs on xtals
Jacob, Anna There are 5 magnetite examples in http://rruff.info/magnetite/ with different elemental compositions. All similar cells but the data for some is very noisy. R06 is the least noisy - contains nickel. Isn't iron stored as ferrihydrite in normal ferritin? Would be interesting to see whether this gives any similar diffuse rings in ferritin single crystal diffraction patterns. People must have checked after all these years. There is even the powder diffraction pattern in Wikipedia (http://en.wikipedia.org/wiki/Ferrihydrite) The bright spots you refer might be the ones I was fretting about. All diffraction features (rings or spots) should be indexed otherwise the job is incomplete! Colin From: Jacob Keller [mailto:j-kell...@fsm.northwestern.edu] Sent: 08 May 2012 18:29 To: Nave, Colin (DLSLtd,RAL,DIA) Cc: ccp4bb Subject: Re: [ccp4bb] saxs on xtals Dear Colin, the table you gave seems to have been from Fe2+Fe3+2O4 or from Fe3-xTixO4. I am curious what the nature of the Fe inside the ferritin is (I don't think it has Ti in it, though...). Is it elemental iron? Also, to Anna: can you send a picture of that diffraction pattern with spot predictions superimposed? There seem to be some really bright spots which are outliers, and maybe multiple lattices. JPK On Tue, May 8, 2012 at 12:21 PM, Colin Nave colin.n...@diamond.ac.ukmailto:colin.n...@diamond.ac.uk wrote: Anna Yes, you have understood the suggestion. Could be the 220 and 311 reflections. See for example http://rruff.info/magnetite/R080025 and http://rruff.info/repository/sample_child_record_powder/by_minerals/Magnetite__R080025-1__Powder__DIF_File__9448.txt Trying to index powder patterns from 2 rings is risky and the intensities don't seem to agree. I guess you don't have higher angle data. Should be able to evaluate a particle size from the breadth of the rings though. For example a 57A crystal examined with 1A radiation would give broadening of about a degree. There do seem to be other spots - I guess these are ice rings but you should check. Also be nice to know if apoferritin crystallised under the same conditions (if it can be) shows these rings Regards Colin From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of anna anna Sent: 08 May 2012 16:55 To: ccp4bb Subject: Re: [ccp4bb] saxs on xtals Dear all, first of all I want to thank you for your attention and all your brilliant suggestions that really cleared my head!!! Thanks to you (or because of you!!) now I have many ideas and very much to do. Colin, I was just re-considering my diffraction images. Who knows if they are single xtals indeed! Let's see if I understood your point. Assuming that they are single xtals, if they are located at independent positions in the protein-cage it would be like powder diffraction, with rings at diffraction angles corresponding to magnetite lattice. If they are ordered they should give a diffraction pattern. The corresponding lattice can differ from the protein lattice, do you agree? If this is true, what would I see? Two superimposed diffraction patterns? Actually, I am not able to evaluate it... I attached one of the diffraction images. It seems to me that there are two diffused rings at about 2.5 and 2.9 A. Michael, I just read your reply. I think that the eventual periodicity of the partcles can't be completely independent of the protein periodicity (I attached a hypotethical scheme), as you suggest I will try P1. Once I tryed a naive version of what you suggest: I put a magnet over the xtallization plate. All my collegues made fun of me... :) !! I will check the literature that you all quoted (hard work!) Thank you again, new suggestions will be really appreciated. Cheers, anna 2012/5/8 R. M. Garavito rmgarav...@gmail.commailto:rmgarav...@gmail.commailto:rmgarav...@gmail.commailto:rmgarav...@gmail.com Dear Anna, I know that you already have gotten replies from some top experts, but your intriguing problem brought up some issues I have run across in the past. First, from you experience with single crystal diffraction, your results are not that much different from those seen in virus structures where the nucleic acid structure is averaged out. As the nucleic acid doesn't (and mostly can't) adopt the symmetry of the protein shell, the crystallization process alone does the averaging. Just because that ferritin and magnetite have cubic symmetry elements, if they don't line up, the magnetite structure can be averaged out upon crystallization. So, working at lower symmetry may not help, unless there is some directional correlation of the magnetite symmetry and position with the crystal axes. But try P1 and see what happens. A second comment is why not try neutron scattering (SANS or single crystal neutron diffraction), particularly as you can match out the protein with D2O and see just the magnetite. While the same concerns apply
[ccp4bb] saxs on xtals
Dear all, I'd like some suggestions/opinions about the sense of an experiment proposed by a collaborator expert in saxs. In few words, he wants to collect SAXS data on a suspension of protein xtals to investigate low resolution periodicity of the xtal (more details below). The experiment requires a very huge number of xtals to obtain the circles typical of saxs and it is very time-consuming to me (I know nothing about saxs, I have only to prepare the sample). I proposed to measure a single rotating xtal (like in XRD) but he told they don't have a goniometer on saxs beamline. Here is my concern: does it make sense to measure many xtals together? Don't we lose information with respect to single xtal? And, most of all, what can I see by *s*axs that I can't see by* w*axs?? Sorry for the almost off-topic question but I think that only someone who knows both the techniques can help me!! Some detail for who is intrigued by my story: we prepared doped magnetite nanoparticles using ferritin as bioreactor. I crystallized this spheres filled with metal and solved the structure at 3.7A but I can see only the protein shell while there is no density inside, even if I know that the nanoparticles are there. A simple explanation is that the particles are free to move in the cavity(note that the diameter of the nanoparticle is shorter then the inner diameter of the protein shell), ie are disordered, and do not contribute to diffraction, in fact, to my knowledge, nobody have ever seen the metal core inside ferritin or dps proteins. However, since they are magnetic particles they must see each other through the protein wall, ie they can't be completely free to move in the cavity. Maybe, but this is just my opinion, I don't see the particle because the period of the particle in the xtal is different/longer than the period of the protein shell. Anyway, we are interested in the relative distance between the magnetic particles in the xtal to study the effects of magnetostatic interactions in nanoparticles 3D arrays. We are going to do this by saxs since, they told me, lower resolution is useful in studying this long range periodicity (the diameter of ferritin is about 120A) but it seems fool to me using a suspension of so many xtals to obtain a scattering curve while I could collect diffraction images from a single xtal!!! I know that saxs is used when you don't have xtals but if you have xtals, ie your system is ordered, xtallography is much more powerful!! Another question: how can I handle my diffraction data at 3.7A resolution to look for nanoparticles? Should I try a lower symmetry? Maybe the anomalous signal? Have you any reference for a similar case? Thank you very much!! anna
Re: [ccp4bb] saxs on xtals
That sounds like powder diffraction. On 05/07/12 12:30, anna anna wrote: Dear all, I'd like some suggestions/opinions about the sense of an experiment proposed by a collaborator expert in saxs. In few words, he wants to collect SAXS data on a suspension of protein xtals to investigate low resolution periodicity of the xtal (more details below). The experiment requires a very huge number of xtals to obtain the circles typical of saxs and it is very time-consuming to me (I know nothing about saxs, I have only to prepare the sample). I proposed to measure a single rotating xtal (like in XRD) but he told they don't have a goniometer on saxs beamline. Here is my concern: does it make sense to measure many xtals together? Don't we lose information with respect to single xtal? And, most of all, what can I see by /s/axs that I can't see by/w/axs?? Sorry for the almost off-topic question but I think that only someone who knows both the techniques can help me!! Some detail for who is intrigued by my story: we prepared doped magnetite nanoparticles using ferritin as bioreactor. I crystallized this spheres filled with metal and solved the structure at 3.7A but I can see only the protein shell while there is no density inside, even if I know that the nanoparticles are there. A simple explanation is that the particles are free to move in the cavity(note that the diameter of the nanoparticle is shorter then the inner diameter of the protein shell), ie are disordered, and do not contribute to diffraction, in fact, to my knowledge, nobody have ever seen the metal core inside ferritin or dps proteins. However, since they are magnetic particles they must see each other through the protein wall, ie they can't be completely free to move in the cavity. Maybe, but this is just my opinion, I don't see the particle because the period of the particle in the xtal is different/longer than the period of the protein shell. Anyway, we are interested in the relative distance between the magnetic particles in the xtal to study the effects of magnetostatic interactions in nanoparticles 3D arrays. We are going to do this by saxs since, they told me, lower resolution is useful in studying this long range periodicity (the diameter of ferritin is about 120A) but it seems fool to me using a suspension of so many xtals to obtain a scattering curve while I could collect diffraction images from a single xtal!!! I know that saxs is used when you don't have xtals but if you have xtals, ie your system is ordered, xtallography is much more powerful!! Another question: how can I handle my diffraction data at 3.7A resolution to look for nanoparticles? Should I try a lower symmetry? Maybe the anomalous signal? Have you any reference for a similar case? Thank you very much!! anna -- === All Things Serve the Beam === David J. Schuller modern man in a post-modern world MacCHESS, Cornell University schul...@cornell.edu
Re: [ccp4bb] saxs on xtals
It might be that the bulk solvent correction is nullifying the interior of the ferritin structure, and there should be a way to tell the refinement software not to treat the interior as solvent. Perhaps then you might find your Fe? Also, I would think there should be some powder-like diffraction rings in the background of the ferritin crystal diffraction corresponding to the scattering from the jumbled but crystalline Fe in the inside--did you see any extra rings in the background of your original diffraction data? Not sure what the Fe-Fe distance is in this case in particular, but there should be a ring (or rings) corresponding to that (those) distance(s) JPK On Mon, May 7, 2012 at 11:30 AM, anna anna marmottalb...@gmail.com wrote: Dear all, I'd like some suggestions/opinions about the sense of an experiment proposed by a collaborator expert in saxs. In few words, he wants to collect SAXS data on a suspension of protein xtals to investigate low resolution periodicity of the xtal (more details below). The experiment requires a very huge number of xtals to obtain the circles typical of saxs and it is very time-consuming to me (I know nothing about saxs, I have only to prepare the sample). I proposed to measure a single rotating xtal (like in XRD) but he told they don't have a goniometer on saxs beamline. Here is my concern: does it make sense to measure many xtals together? Don't we lose information with respect to single xtal? And, most of all, what can I see by *s*axs that I can't see by* w*axs?? Sorry for the almost off-topic question but I think that only someone who knows both the techniques can help me!! Some detail for who is intrigued by my story: we prepared doped magnetite nanoparticles using ferritin as bioreactor. I crystallized this spheres filled with metal and solved the structure at 3.7A but I can see only the protein shell while there is no density inside, even if I know that the nanoparticles are there. A simple explanation is that the particles are free to move in the cavity(note that the diameter of the nanoparticle is shorter then the inner diameter of the protein shell), ie are disordered, and do not contribute to diffraction, in fact, to my knowledge, nobody have ever seen the metal core inside ferritin or dps proteins. However, since they are magnetic particles they must see each other through the protein wall, ie they can't be completely free to move in the cavity. Maybe, but this is just my opinion, I don't see the particle because the period of the particle in the xtal is different/longer than the period of the protein shell. Anyway, we are interested in the relative distance between the magnetic particles in the xtal to study the effects of magnetostatic interactions in nanoparticles 3D arrays. We are going to do this by saxs since, they told me, lower resolution is useful in studying this long range periodicity (the diameter of ferritin is about 120A) but it seems fool to me using a suspension of so many xtals to obtain a scattering curve while I could collect diffraction images from a single xtal!!! I know that saxs is used when you don't have xtals but if you have xtals, ie your system is ordered, xtallography is much more powerful!! Another question: how can I handle my diffraction data at 3.7A resolution to look for nanoparticles? Should I try a lower symmetry? Maybe the anomalous signal? Have you any reference for a similar case? Thank you very much!! anna -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program email: j-kell...@northwestern.edu ***
Re: [ccp4bb] saxs on xtals
Dear Anna, I once modified CNS to refine two solvent regions of ferritin, one inside and one outside the shell. Perhaps this can be done in Phenix now. If you want to locate magnetite particles in this way, you should collect data to as low a resolution as you can (may need to move backstop), as this is where the solvent has most effect. I would also collect control data with apo and holo ferritin to compare. However a much easier way may be to directly visualise the particles in cryoEM. I have seen very nice micrographs of particles inside ferritin (can't remember ref now). best wishes James -- Dr. James W. Murray David Phillips Research Fellow Division of Molecular Biosciences Imperial College, LONDON Tel: +44 (0)20 759 48895 From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of anna anna [marmottalb...@gmail.com] Sent: Monday, May 07, 2012 5:30 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] saxs on xtals Dear all, I'd like some suggestions/opinions about the sense of an experiment proposed by a collaborator expert in saxs. In few words, he wants to collect SAXS data on a suspension of protein xtals to investigate low resolution periodicity of the xtal (more details below). The experiment requires a very huge number of xtals to obtain the circles typical of saxs and it is very time-consuming to me (I know nothing about saxs, I have only to prepare the sample). I proposed to measure a single rotating xtal (like in XRD) but he told they don't have a goniometer on saxs beamline. Here is my concern: does it make sense to measure many xtals together? Don't we lose information with respect to single xtal? And, most of all, what can I see by saxs that I can't see by waxs?? Sorry for the almost off-topic question but I think that only someone who knows both the techniques can help me!! Some detail for who is intrigued by my story: we prepared doped magnetite nanoparticles using ferritin as bioreactor. I crystallized this spheres filled with metal and solved the structure at 3.7A but I can see only the protein shell while there is no density inside, even if I know that the nanoparticles are there. A simple explanation is that the particles are free to move in the cavity(note that the diameter of the nanoparticle is shorter then the inner diameter of the protein shell), ie are disordered, and do not contribute to diffraction, in fact, to my knowledge, nobody have ever seen the metal core inside ferritin or dps proteins. However, since they are magnetic particles they must see each other through the protein wall, ie they can't be completely free to move in the cavity. Maybe, but this is just my opinion, I don't see the particle because the period of the particle in the xtal is different/longer than the period of the protein shell. Anyway, we are interested in the relative distance between the magnetic particles in the xtal to study the effects of magnetostatic interactions in nanoparticles 3D arrays. We are going to do this by saxs since, they told me, lower resolution is useful in studying this long range periodicity (the diameter of ferritin is about 120A) but it seems fool to me using a suspension of so many xtals to obtain a scattering curve while I could collect diffraction images from a single xtal!!! I know that saxs is used when you don't have xtals but if you have xtals, ie your system is ordered, xtallography is much more powerful!! Another question: how can I handle my diffraction data at 3.7A resolution to look for nanoparticles? Should I try a lower symmetry? Maybe the anomalous signal? Have you any reference for a similar case? Thank you very much!! anna
Re: [ccp4bb] saxs on xtals
Hi Anna, There has been some nice single crystal SAXS work done, check out J. Mol. Biol (1998) 284, 1439-1452 Imaging RNA and Dynamic Protein Segments with Low-resolution Virus Crystallography: Experimental Design, Data Processing and Implications of Electron Density Maps by Tsuruta et al. There is the capability to study single crystals on SAXS lines - in this case 4-2 at SSRL. However if you desperately need to use crystals, you may be better served with a single one by moving the beamstop back, choosing a lower energy and trying to collect all the low resolution diffraction information from a single crystal. You can truncate the high resolution data and see if you start to see information from the particles if there is some ordering. Even better if you can compare it from a crystal without them if it's in the same space group. Have you thought about the possibility of anomalous SAXS with and without doped ferritin? Note that you may be inducing interparticle effects which will complicate the interpretation of any SAXS data, solution or suspended crystals. Cheers,, Eddie Edward Snell Ph.D. Assistant Prof. Department of Structural Biology, SUNY Buffalo, Senior Scientist, Hauptman-Woodward Medical Research Institute 700 Ellicott Street, Buffalo, NY 14203-1102 Phone: (716) 898 8631 Fax: (716) 898 8660 Skype: eddie.snell Email: esn...@hwi.buffalo.edu Telepathy: 42.2 GHz Heisenberg was probably here! From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of anna anna Sent: Monday, May 07, 2012 12:30 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] saxs on xtals Dear all, I'd like some suggestions/opinions about the sense of an experiment proposed by a collaborator expert in saxs. In few words, he wants to collect SAXS data on a suspension of protein xtals to investigate low resolution periodicity of the xtal (more details below). The experiment requires a very huge number of xtals to obtain the circles typical of saxs and it is very time-consuming to me (I know nothing about saxs, I have only to prepare the sample). I proposed to measure a single rotating xtal (like in XRD) but he told they don't have a goniometer on saxs beamline. Here is my concern: does it make sense to measure many xtals together? Don't we lose information with respect to single xtal? And, most of all, what can I see by saxs that I can't see by waxs?? Sorry for the almost off-topic question but I think that only someone who knows both the techniques can help me!! Some detail for who is intrigued by my story: we prepared doped magnetite nanoparticles using ferritin as bioreactor. I crystallized this spheres filled with metal and solved the structure at 3.7A but I can see only the protein shell while there is no density inside, even if I know that the nanoparticles are there. A simple explanation is that the particles are free to move in the cavity(note that the diameter of the nanoparticle is shorter then the inner diameter of the protein shell), ie are disordered, and do not contribute to diffraction, in fact, to my knowledge, nobody have ever seen the metal core inside ferritin or dps proteins. However, since they are magnetic particles they must see each other through the protein wall, ie they can't be completely free to move in the cavity. Maybe, but this is just my opinion, I don't see the particle because the period of the particle in the xtal is different/longer than the period of the protein shell. Anyway, we are interested in the relative distance between the magnetic particles in the xtal to study the effects of magnetostatic interactions in nanoparticles 3D arrays. We are going to do this by saxs since, they told me, lower resolution is useful in studying this long range periodicity (the diameter of ferritin is about 120A) but it seems fool to me using a suspension of so many xtals to obtain a scattering curve while I could collect diffraction images from a single xtal!!! I know that saxs is used when you don't have xtals but if you have xtals, ie your system is ordered, xtallography is much more powerful!! Another question: how can I handle my diffraction data at 3.7A resolution to look for nanoparticles? Should I try a lower symmetry? Maybe the anomalous signal? Have you any reference for a similar case? Thank you very much!! anna
Re: [ccp4bb] saxs on xtals
Anna Interesting. Yes, the cryo-em might be the way to go to see if some structures (i.e. not just spheres) within the protein shell are aligned. The SAXS study does make some sense. If the magnetic particles have some alignment this should manifest itself in the SAXS pattern, with the precise effects depending on the correlation length (how far the alignment extends). This really requires SAXS, not WAXS which would not be sensitive to the long range effects you wish to see. As you suggest, it would also be good fun (and perhaps even informative) to do this on single crystals. A coherent x-ray beam, using CDI or ptychography (search for these terms or contact me if you want some details) could be employed if you restrict yourself to crystals of a few microns in size. One can even play around with absorption edges/anomalous dispersion/magnetic resonance (for single crystals or powders) as I understand the interest is in magnetic phenomena. Making resonant soft x-ray scattering measurements at the oxygen K edge have been used for studying magnetite. The polarisation of the x-rays could be exploited. Our neutron friends can also study magnetic effects but I don't know about sample size requirements. Doing the measurements in a strong magnetic field might be interesting. If the samples are easy to prepare, I would start from collecting the lowest hkl reflections from single crystals to check you are not missing anything. Then a low angle SAXS pattern as suggested by your collaborators. After this consider the other suggestions which are rather speculative and not really routine. Good luck Colin -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Murray, James W Sent: 07 May 2012 19:25 To: ccp4bb Subject: Re: [ccp4bb] saxs on xtals Dear Anna, I once modified CNS to refine two solvent regions of ferritin, one inside and one outside the shell. Perhaps this can be done in Phenix now. If you want to locate magnetite particles in this way, you should collect data to as low a resolution as you can (may need to move backstop), as this is where the solvent has most effect. I would also collect control data with apo and holo ferritin to compare. However a much easier way may be to directly visualise the particles in cryoEM. I have seen very nice micrographs of particles inside ferritin (can't remember ref now). best wishes James -- Dr. James W. Murray David Phillips Research Fellow Division of Molecular Biosciences Imperial College, LONDON Tel: +44 (0)20 759 48895 From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of anna anna [marmottalb...@gmail.com] Sent: Monday, May 07, 2012 5:30 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] saxs on xtals Dear all, I'd like some suggestions/opinions about the sense of an experiment proposed by a collaborator expert in saxs. In few words, he wants to collect SAXS data on a suspension of protein xtals to investigate low resolution periodicity of the xtal (more details below). The experiment requires a very huge number of xtals to obtain the circles typical of saxs and it is very time-consuming to me (I know nothing about saxs, I have only to prepare the sample). I proposed to measure a single rotating xtal (like in XRD) but he told they don't have a goniometer on saxs beamline. Here is my concern: does it make sense to measure many xtals together? Don't we lose information with respect to single xtal? And, most of all, what can I see by saxs that I can't see by waxs?? Sorry for the almost off-topic question but I think that only someone who knows both the techniques can help me!! Some detail for who is intrigued by my story: we prepared doped magnetite nanoparticles using ferritin as bioreactor. I crystallized this spheres filled with metal and solved the structure at 3.7A but I can see only the protein shell while there is no density inside, even if I know that the nanoparticles are there. A simple explanation is that the particles are free to move in the cavity(note that the diameter of the nanoparticle is shorter then the inner diameter of the protein shell), ie are disordered, and do not contribute to diffraction, in fact, to my knowledge, nobody have ever seen the metal core inside ferritin or dps proteins. However, since they are magnetic particles they must see each other through the protein wall, ie they can't be completely free to move in the cavity. Maybe, but this is just my opinion, I don't see the particle because the period of the particle in the xtal is different/longer than the period of the protein shell. Anyway, we are interested in the relative distance between the magnetic particles in the xtal to study the effects of magnetostatic interactions in nanoparticles 3D arrays. We are going to do this by saxs since, they told me, lower resolution is useful in studying this long range periodicity
