To add to Matt's last paragraph, you might also consider beamline 6-2 at SSRL (http://www-ssrl.slac.stanford.edu/beamlines/bl6-2/), as they do a fair amount of work with proteins and have an X-ray Raman setup. I think the beamline scientist occasionally posts to this list. Good luck, Erik -- Erik Farquhar, Ph.D. Case Center for Synchrotron Biosciences Brookhaven National Lab NSLS, Building 725A-X3 Upton, NY  11973 efarquhar@bnl.gov +1-631-344-8174 ________________________________ From: Matt Newville To: XAFS Analysis using Ifeffit Sent: Tuesday, December 18, 2012 7:52 AM Subject: Re: [Ifeffit] Beryllium and EXAFS Hi Gina, On Mon, Dec 17, 2012 at 5:23 PM, Clayton, Gina wrote:

Hi there

I am trying to find out more about EXAFS. I am a protein crystallographer.

I want to identify the environment of a beryllium ion in the protein, I work with,  of which I have a crystal structure.  EXAFS was recommended as a good technique for identification of the beryllium site and species. The beryllium is an acidic pocket of the protein surrounded by oxygens from the protein, likely covalently bound. However I have had  zero result finding a beam line that can tune their setups to the Be edge at 111 eV. Also some EXAFS users say that EXAFS technique would be good for working out the environment of the Be but some say it would not . In particular  I have been advised that  Be EXAFS would not be useful since  the Be  EXAFS signal would have unacceptable interference from the carbon K-edge at 285 eV and the oxygen K edge signal at 535 eV.

Would EXAFS work or something in a similar vein? Or, for instance,  could the acidic pocket be identified using EXAFS, by measuring the oxygens (instead of the Beryllium) using the protein with and without beryllium?

Thank you so much for any advice.

Gina

As you and as the person who advised you suspect, you face a number of challenges: 1.  The extremely low energy (111 eV).  You might be able to find a UV source or x-ray beamline that can reach this energy , but often such work is done in vacuo, and so may not be suitable for proteins.  Using electrons (from a TEM, say) might be possible too -- these can sometimes do C and N edges either directly or looking at Electron Energy Loss (send in an electron of high energy E1, and look for electrons of energy E1 - E, and scan the analyzer for the loss energy E through the absorption edge).  Again, electron probes may not be what you want for your proteins. 2. The C K-edge will limit your signal to "only" 175 eV.  I would say to not worry so much about this limitation.  You'll really be measuring NEXAFS (near-edge X-ray absorption ....)  but that's OK. People measure the B, C, N, and O K edges, all facing the same problem, especially for the O K edge: our planet is more or less covered with the stuff ;).  These spectra tend to be very rich and sensitive to ligands. 3. Not much (zero?? I can almost believe that!) literature on Be XAFS. This is unfortunate because most NEXAFS is measure and compare to known spectra.  I think that might mean you have to spend effort to building up a spectral library for Be in organic phases. I think trying to measure the O K edge with and without Be is nearly hopeless.  All the X-ray spectroscopies average equally over all the atoms of the absorbing species.  In addition all the spectra are grossly the same (a step function from 0 below the edge to 1 above the edge).  These make it difficult to see changes below about 1%.    So I think you would just have too high of an O::Be ratio to see anything useful. One option to consider is using X-ray Raman scattering (http://en.wikipedia.org/wiki/X-ray_Raman_scattering).  Here, one hits the sample with high-energy X-rays (say, 10 keV), which are highly penetrating for reasonably sized protein sample.  One analyzes an energy loss (again, E1 - E, where here E1 is around 10 keV) to get the edge of interest.  I'm familiar with (been involved with) studies on B, C, and O using this technique (all in solids, mostly at high pressure).  See, for example,  S. K. Lee, et al "Probing of bonding changes in B2O3 glasses at high pressure with inelastic X-ray scattering",  Nature Materials 4, pp 851-854 (2005), and also the work of Simo Huotari.    I'm not aware of anyone doing Be K-edge this way, but it should definitely be possible. For the work I've been involved with, Be would be challenging because there are other excitations in solids that appear around 100 eV.  I believe that effect would be far suppressed in a protein, though I'm not sure. That's somewhat more exotic than straightfoward XAFS,, but it might be the best chance for measuring Be in a protein.    I'm not sure I know any beamlines that do X-ray Raman on proteins, but I'd suggest APS beamline 20-ID or ESRF beamline as places to start.    The next questions would be "how many Be atoms are in your protein?", and "how much material do you have?".... Hope that helps, --Matt Newville _______________________________________________ Ifeffit mailing list Ifeffit@millenia.cars.aps.anl.gov http://millenia.cars.aps.anl.gov/mailman/listinfo/ifeffit

An interesting possible model compound, basic beryllium acetate: http://en.wikipedia.org/wiki/Basic_beryllium_acetate Unusual mu-4 oxo connecting 4 Be ions. Crystal structure's been done - twice - once by Bragg, once by Pauling. Good luck, Mike B. ________________________________ From: ifeffit-bounces@millenia.cars.aps.anl.gov [ifeffit-bounces@millenia.cars.aps.anl.gov] on behalf of Matthew Marcus [mamarcus@lbl.gov] Sent: Tuesday, December 18, 2012 1:39 PM To: Erik Farquhar; XAFS Analysis using Ifeffit Subject: Re: [Ifeffit] Beryllium and EXAFS Just to stir the pot a little more: 1. ALS has some beamlines which can go down as far as Li. Work has been done on Li, but only on concentrated stuff. 2. The sample is probably really dilute in Be. The signal will be pathetic. 3. Radiation damage will be a major issue, especially with X-ray Raman, which is a highly inefficient technique. Even LHe cooling doesn't solve that problem. 4. Are there model compounds available for proposed local structures of the Be center? You'd need that to have any hope of making sense out of Be XANES. 5. Is there such a thing as neutron PX? That might see the Be. mam ----- Original Message ----- From: Erik Farquharmailto:efarq@yahoo.com To: XAFS Analysis using Ifeffitmailto:ifeffit@millenia.cars.aps.anl.gov Sent: Tuesday, December 18, 2012 5:32 AM Subject: Re: [Ifeffit] Beryllium and EXAFS To add to Matt's last paragraph, you might also consider beamline 6-2 at SSRL (http://www-ssrl.slac.stanford.edu/beamlines/bl6-2/), as they do a fair amount of work with proteins and have an X-ray Raman setup. I think the beamline scientist occasionally posts to this list. Good luck, Erik -- Erik Farquhar, Ph.D. Case Center for Synchrotron Biosciences Brookhaven National Lab NSLS, Building 725A-X3 Upton, NY 11973 efarquhar@bnl.govmailto:efarquhar@bnl.gov +1-631-344-8174 ________________________________ From: Matt Newville To: XAFS Analysis using Ifeffit Sent: Tuesday, December 18, 2012 7:52 AM Subject: Re: [Ifeffit] Beryllium and EXAFS Hi Gina, On Mon, Dec 17, 2012 at 5:23 PM, Clayton, Gina mailto:ClaytonG@njhealth.org> wrote:

Hi there

I am trying to find out more about EXAFS. I am a protein crystallographer.

I want to identify the environment of a beryllium ion in the protein, I work with, of which I have a crystal structure. EXAFS was recommended as a good technique for identification of the beryllium site and species. The beryllium is an acidic pocket of the protein surrounded by oxygens from the protein, likely covalently bound. However I have had zero result finding a beam line that can tune their setups to the Be edge at 111 eV. Also some EXAFS users say that EXAFS technique would be good for working out the environment of the Be but some say it would not . In particular I have been advised that Be EXAFS would not be useful since the Be EXAFS signal would have unacceptable interference from the carbon K-edge at 285 eV and the oxygen K edge signal at 535 eV.

Would EXAFS work or something in a similar vein? Or, for instance, could the acidic pocket be identified using EXAFS, by measuring the oxygens (instead of the Beryllium) using the protein with and without beryllium?

Thank you so much for any advice.

Gina

As you and as the person who advised you suspect, you face a number of challenges: 1. The extremely low energy (111 eV). You might be able to find a UV source or x-ray beamline that can reach this energy , but often such work is done in vacuo, and so may not be suitable for proteins. Using electrons (from a TEM, say) might be possible too -- these can sometimes do C and N edges either directly or looking at Electron Energy Loss (send in an electron of high energy E1, and look for electrons of energy E1 - E, and scan the analyzer for the loss energy E through the absorption edge). Again, electron probes may not be what you want for your proteins. 2. The C K-edge will limit your signal to "only" 175 eV. I would say to not worry so much about this limitation. You'll really be measuring NEXAFS (near-edge X-ray absorption ....) but that's OK. People measure the B, C, N, and O K edges, all facing the same problem, especially for the O K edge: our planet is more or less covered with the stuff ;). These spectra tend to be very rich and sensitive to ligands. 3. Not much (zero?? I can almost believe that!) literature on Be XAFS. This is unfortunate because most NEXAFS is measure and compare to known spectra. I think that might mean you have to spend effort to building up a spectral library for Be in organic phases. I think trying to measure the O K edge with and without Be is nearly hopeless. All the X-ray spectroscopies average equally over all the atoms of the absorbing species. In addition all the spectra are grossly the same (a step function from 0 below the edge to 1 above the edge). These make it difficult to see changes below about 1%. So I think you would just have too high of an O::Be ratio to see anything useful. One option to consider is using X-ray Raman scattering (http://en.wikipedia.org/wiki/X-ray_Raman_scattering). Here, one hits the sample with high-energy X-rays (say, 10 keV), which are highly penetrating for reasonably sized protein sample. One analyzes an energy loss (again, E1 - E, where here E1 is around 10 keV) to get the edge of interest. I'm familiar with (been involved with) studies on B, C, and O using this technique (all in solids, mostly at high pressure). See, for example, S. K. Lee, et al "Probing of bonding changes in B2O3 glasses at high pressure with inelastic X-ray scattering", Nature Materials 4, pp 851-854 (2005), and also the work of Simo Huotari. I'm not aware of anyone doing Be K-edge this way, but it should definitely be possible. For the work I've been involved with, Be would be challenging because there are other excitations in solids that appear around 100 eV. I believe that effect would be far suppressed in a protein, though I'm not sure. That's somewhat more exotic than straightfoward XAFS,, but it might be the best chance for measuring Be in a protein. I'm not sure I know any beamlines that do X-ray Raman on proteins, but I'd suggest APS beamline 20-ID or ESRF beamline as places to start. The next questions would be "how many Be atoms are in your protein?", and "how much material do you have?".... Hope that helps, --Matt Newville _______________________________________________ Ifeffit mailing list Ifeffit@millenia.cars.aps.anl.govmailto:Ifeffit@millenia.cars.aps.anl.gov http://millenia.cars.aps.anl.gov/mailman/listinfo/ifeffit ________________________________ _______________________________________________ Ifeffit mailing list Ifeffit@millenia.cars.aps.anl.gov http://millenia.cars.aps.anl.gov/mailman/listinfo/ifeffit

A bit unrelated to the question at hand, but regarding #5, yes, people do neutron PX.  The focus is almost entirely on locating hydrogen atoms (e.g. for protonation state), so I wonder what the neutron PX guys would say about looking for a Be atom. The downside is that you usually need quite large crystals compared to X-ray PX and long data collection times.   Erik ________________________________ From: Matthew Marcus To: Erik Farquhar ; XAFS Analysis using Ifeffit Sent: Tuesday, December 18, 2012 1:39 PM Subject: Re: [Ifeffit] Beryllium and EXAFS Just to stir the pot a little more: 1.    ALS has some beamlines which can go down as far as Li.  Work has been done on Li, but only on concentrated stuff. 2.    The sample is probably really dilute in Be.  The signal will be pathetic. 3.    Radiation damage will be a major issue, especially with X-ray Raman, which is a highly inefficient technique.  Even LHe cooling doesn't solve that problem. 4.    Are there model compounds available for proposed local structures of the Be center?  You'd need that to have any hope of making sense out of Be XANES. 5.    Is there such a thing as neutron PX?  That might see the Be.     mam   ----- Original Message -----

From: Erik Farquhar To: XAFS Analysis using Ifeffit Sent: Tuesday, December 18, 2012 5:32 AM Subject: Re: [Ifeffit] Beryllium and EXAFS

To add to Matt's last paragraph, you might also consider beamline 6-2 at SSRL (http://www-ssrl.slac.stanford.edu/beamlines/bl6-2/), as they do a fair amount of work with proteins and have an X-ray Raman setup. I think the beamline scientist occasionally posts to this list.

Good luck, Erik

-- Erik Farquhar, Ph.D. Case Center for Synchrotron Biosciences Brookhaven National Lab NSLS, Building 725A-X3 Upton, NY  11973 efarquhar@bnl.gov +1-631-344-8174

________________________________ From: Matt Newville To: XAFS Analysis using Ifeffit Sent: Tuesday, December 18, 2012 7:52 AM Subject: Re: [Ifeffit] Beryllium and EXAFS

Hi Gina,

On Mon, Dec 17, 2012 at 5:23 PM, Clayton, Gina wrote:

...

Hi there

I am trying to find out more about EXAFS. I am a protein crystallographer.

I want to identify the environment of a beryllium ion in the protein, I work with,  of which I have a crystal structure.  EXAFS was recommended as a good technique for identification of the beryllium site and species. The beryllium is an acidic pocket of the protein surrounded by oxygens from the protein, likely covalently bound. However I have had  zero result finding a beam line that can tune their setups to the Be edge at 111 eV. Also some EXAFS users say that EXAFS technique would be good for working out the environment of the Be but some say it would not . In particular  I have been advised that  Be EXAFS would not be useful since  the Be  EXAFS signal would have unacceptable interference from the carbon K-edge at 285 eV and the oxygen K edge signal at 535 eV.

Would EXAFS work or something in a similar vein? Or, for instance,  could the acidic pocket be identified using EXAFS, by measuring the oxygens (instead of the Beryllium) using the protein with and without beryllium?

Thank you so much for any advice.

Gina

As you and as the person who advised you suspect, you face a number of challenges:

1.  The extremely low energy (111 eV).  You might be able to find a UV source or x-ray beamline that can reach this energy , but often such work is done in vacuo, and so may not be suitable for proteins.  Using electrons (from a TEM, say) might be possible too -- these can sometimes do C and N edges either directly or looking at Electron Energy Loss (send in an electron of high energy E1, and look for electrons of energy E1 - E, and scan the analyzer for the loss energy E through the absorption edge).  Again, electron probes may not be what you want for your proteins.

2. The C K-edge will limit your signal to "only" 175 eV.  I would say to not worry so much about this limitation.  You'll really be measuring NEXAFS (near-edge X-ray absorption ....)  but that's OK. People measure the B, C, N, and O K edges, all facing the same problem, especially for the O K edge: our planet is more or less covered with the stuff ;).  These spectra tend to be very rich and sensitive to ligands.

3. Not much (zero?? I can almost believe that!) literature on Be XAFS. This is unfortunate because most NEXAFS is measure and compare to known spectra.  I think that might mean you have to spend effort to building up a spectral library for Be in organic phases.

I think trying to measure the O K edge with and without Be is nearly hopeless.  All the X-ray spectroscopies average equally over all the atoms of the absorbing species.  In addition all the spectra are grossly the same (a step function from 0 below the edge to 1 above the edge).  These make it difficult to see changes below about 1%.    So I think you would just have too high of an O::Be ratio to see anything useful.

One option to consider is using X-ray Raman scattering (http://en.wikipedia.org/wiki/X-ray_Raman_scattering).  Here, one hits the sample with high-energy X-rays (say, 10 keV), which are highly penetrating for reasonably sized protein sample.  One analyzes an energy loss (again, E1 - E, where here E1 is around 10 keV) to get the edge of interest.  I'm familiar with (been involved with) studies on B, C, and O using this technique (all in solids, mostly at high pressure).  See, for example,  S. K. Lee, et al "Probing of bonding changes in B2O3 glasses at high pressure with inelastic X-ray scattering",  Nature Materials 4, pp 851-854 (2005), and also the work of Simo Huotari.    I'm not aware of anyone doing Be K-edge this way, but it should definitely be possible. For the work I've been involved with, Be would be challenging because there are other excitations in solids that appear around 100 eV.  I believe that effect would be far suppressed in a protein, though I'm not sure.

That's somewhat more exotic than straightfoward XAFS,, but it might be the best chance for measuring Be in a protein.    I'm not sure I know any beamlines that do X-ray Raman on proteins, but I'd suggest APS beamline 20-ID or ESRF beamline as places to start.    The next questions would be "how many Be atoms are in your protein?", and "how much material do you have?"....

Hope that helps,

--Matt Newville

_______________________________________________ Ifeffit mailing list Ifeffit@millenia.cars.aps.anl.gov http://millenia.cars.aps.anl.gov/mailman/listinfo/ifeffit

________________________________ _______________________________________________ Ifeffit mailing list Ifeffit@millenia.cars.aps.anl.gov http://millenia.cars.aps.anl.gov/mailman/listinfo/ifeffit

Thank you so much for the discussion it's been really helpful i'm going to try to pursue some of the suggestions. To answer Matt's last few question and perhaps obtain any opinion, we have a reasonable amount of protein, its not highly expressed (one of "those" ones) but it is very stable especially when Be is added so we tend to mix protein batches together. How much would we need? However one further complication is that Be will, in theory, tend to bind any part of the protein where there are oxygens in the right configuration. I assume 2.5 angstroms from one oxygen to the other in near tetrahedral arrangement. However that particular binding may be variable throughout the protein population. The part of the protein that does the business when Be is bound, is on a surface cleft inside an acidic pocket and liganded by oxygens from the peptide and oxygens from the peptide binding groove. This feature is what allows a T cell Recepetor to bind to the Be bound protein, activating the immune system and causing a nasty disease. My worry is would this site be discernible in a similar way to how we can see it in the crystal structure. Gina On Dec 18, 2012, at 7:29 AM, Jeff Terry wrote:

To add to what Matt has suggested, If your protein is stable under vacuum, beamline 8-1 at SSRL would be able to do the NEXAFS measurement. You don't need to worry about carbon as it can't reach the carbon edge.

I left a vacuum flange mounted, low energy fluorescence detector at SSRL many years ago, it may still be around or I can tell you how I made it. It worked very well at the low energy edges or you can use electron yield detection.

It is fairly easy to get time on 8-1 and worth trying. I would probably try this before exploring x-ray Raman. Of course, I can't say how happy safety people will be with a proposal to put uncontained Beryllium in a vacuum chamber but that is another issue you will have to overcome.

I'd feel better about x-ray Raman if you could find some standards measured with absorption to compare to the Raman data, just so you could be sure you are not seeing the effects Matt talked about below.

Jeff

Sent from my iPad

On Dec 18, 2012, at 6:52 AM, Matt Newville wrote:

...

Hi Gina,

On Mon, Dec 17, 2012 at 5:23 PM, Clayton, Gina wrote:

...

Hi there

I am trying to find out more about EXAFS. I am a protein crystallographer.

I want to identify the environment of a beryllium ion in the protein, I work with, of which I have a crystal structure. EXAFS was recommended as a good technique for identification of the beryllium site and species. The beryllium is an acidic pocket of the protein surrounded by oxygens from the protein, likely covalently bound. However I have had zero result finding a beam line that can tune their setups to the Be edge at 111 eV. Also some EXAFS users say that EXAFS technique would be good for working out the environment of the Be but some say it would not . In particular I have been advised that Be EXAFS would not be useful since the Be EXAFS signal would have unacceptable interference from the carbon K-edge at 285 eV and the oxygen K edge signal at 535 eV.

Would EXAFS work or something in a similar vein? Or, for instance, could the acidic pocket be identified using EXAFS, by measuring the oxygens (instead of the Beryllium) using the protein with and without beryllium?

Thank you so much for any advice.

Gina

As you and as the person who advised you suspect, you face a number of challenges:

1. The extremely low energy (111 eV). You might be able to find a UV source or x-ray beamline that can reach this energy , but often such work is done in vacuo, and so may not be suitable for proteins. Using electrons (from a TEM, say) might be possible too -- these can sometimes do C and N edges either directly or looking at Electron Energy Loss (send in an electron of high energy E1, and look for electrons of energy E1 - E, and scan the analyzer for the loss energy E through the absorption edge). Again, electron probes may not be what you want for your proteins.

2. The C K-edge will limit your signal to "only" 175 eV. I would say to not worry so much about this limitation. You'll really be measuring NEXAFS (near-edge X-ray absorption ....) but that's OK. People measure the B, C, N, and O K edges, all facing the same problem, especially for the O K edge: our planet is more or less covered with the stuff ;). These spectra tend to be very rich and sensitive to ligands.

3. Not much (zero?? I can almost believe that!) literature on Be XAFS. This is unfortunate because most NEXAFS is measure and compare to known spectra. I think that might mean you have to spend effort to building up a spectral library for Be in organic phases.

I think trying to measure the O K edge with and without Be is nearly hopeless. All the X-ray spectroscopies average equally over all the atoms of the absorbing species. In addition all the spectra are grossly the same (a step function from 0 below the edge to 1 above the edge). These make it difficult to see changes below about 1%. So I think you would just have too high of an O::Be ratio to see anything useful.

One option to consider is using X-ray Raman scattering (http://en.wikipedia.org/wiki/X-ray_Raman_scattering). Here, one hits the sample with high-energy X-rays (say, 10 keV), which are highly penetrating for reasonably sized protein sample. One analyzes an energy loss (again, E1 - E, where here E1 is around 10 keV) to get the edge of interest. I'm familiar with (been involved with) studies on B, C, and O using this technique (all in solids, mostly at high pressure). See, for example, S. K. Lee, et al "Probing of bonding changes in B2O3 glasses at high pressure with inelastic X-ray scattering", Nature Materials 4, pp 851-854 (2005), and also the work of Simo Huotari. I'm not aware of anyone doing Be K-edge this way, but it should definitely be possible. For the work I've been involved with, Be would be challenging because there are other excitations in solids that appear around 100 eV. I believe that effect would be far suppressed in a protein, though I'm not sure.

That's somewhat more exotic than straightfoward XAFS,, but it might be the best chance for measuring Be in a protein. I'm not sure I know any beamlines that do X-ray Raman on proteins, but I'd suggest APS beamline 20-ID or ESRF beamline as places to start. The next questions would be "how many Be atoms are in your protein?", and "how much material do you have?"....

Hope that helps,

--Matt Newville

_______________________________________________ Ifeffit mailing list Ifeffit@millenia.cars.aps.anl.gov http://millenia.cars.aps.anl.gov/mailman/listinfo/ifeffit

_______________________________________________ Ifeffit mailing list Ifeffit@millenia.cars.aps.anl.gov http://millenia.cars.aps.anl.gov/mailman/listinfo/ifeffit

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