Re: [ccp4bb] Semet derivative dying almost immediately in beam
If it is any of these things, then we want to know about it! Miss-alignment I doubt because the cameras on 8.2.2 are all co-axial (looking down the beam). The beam size is about 1/5 the field of view, and the sample stays visible during the exposure. It would be hard not to notice the crystal moving out of the beam. I also doubt fluorescence scans damaging the crystal because the general ALS fluorescence scan protocol attenuates the incident beam by a factor of 1000 or more. Few fluorescence scans give the crystal more dose than a single screening shot. The cryo can be a problem, but I also doubt it in this case. Over 20 years, I have seen maybe 3 instances of a cryo system "burping". These problems are extremely difficult to detect and even more difficult to solve. Hence the concern here. Usually this happens if the neck of the exhaust vent gets full of ice. Checking that is routine PM. I just double-checked myself and its clean. All of the above things are very unlikely to happen twice in a row. Hopefully Kimberly can grow more crystals and we can try again (with more than a few eyes watching everything very carefully). But right now I'd bet even money on equipment vs the sample. -James Holton MAD Scientist On 8/30/2019 11:48 AM, Green, Todd Jason wrote: Certainly not trying to be insulting by the suggestions but could it be as simple an alignment issue with the crystal, issues with the beam alignment or crystal damage following a fluorescence scan? The cryo setup? Like James, I’ve seen crystals decay quickly in high salts at longer wavelengths. Good luck! - Todd Sent from my iPhone On Aug 30, 2019, at 1:41 PM, James Holton <mailto:jmhol...@lbl.gov>> wrote: That is absolutely mind-blowing. ALS 8.2.2 has a nominal dose rate of 95 kGy/s, and you are seeing global damage go to completion in ~0.4s, or 40 kGy. This is 750 times faster than expected. I have never heard of a cryo-cooled crystal decaying that fast. This may be a new world record! I know that is probably not what you wanted, but it is exciting to people like me. Being at 4.5A is actually a good thing for radiation damage because features at poor resolution tend to evolve more slowly with dose than fine details (high-angle spots). That is probably also not what you wanted, but it makes such a fast decay rate even more unusual. I have encountered reports of extra-sensitive proteins before, but they always turn out to be due to something predictable, like having a super-high concentration of heavy atom. For example, having 200 mM sodium iodide in your solvent channels will cut a typical crystal lifetime in half. 4 M NaI will cut it by a factor of 20, but to get 1000x the usual decay rate you would need something like 10 M Ta6Br12. And Ta6Br12 is only soluble to 2 mM. Yes, you have Se atoms in there, but even if you have 1 in 10 residues containing an Se atom at 0% solvent that is still only 1.0 M Se in the crystal. Only enough for about a factor of 10. I have also seen errors in beam size and flux lead to apparently abnormal radiation damage rates. These can easily be as large as a factor of two, especially with very small beams, but it is hard to imagine how one could get a factor of 1000. ALS 8.2.2 is only a few meters from where I'm sitting, and I'm pretty sure the flux density is accurate to within 20%, probably better. I also just checked out the cryo stream, and it has none of the usual warning signs. So, that leaves either a genuinely ultra-sensitive protein, or some kind of undetected equipment malfunction. Either way, we at the ALS are very interested in this result. Would you mind trying again? And let me know when you are coming? -James Holton MAD Scientist On 8/29/2019 11:07 AM, Kimberly Stanek wrote: ALS 822. I tried as low as 0.2 sec exposure but it didn't seem to help much. Kimberly Stanek Postdoctoral Researcher, Goulding Lab Co-chair, UCI Postdoctoral Association University of California, Irvine Natural Science I, Room 2302 (949) 824-0014 *From:* James Holton *Sent:* Thursday, August 29, 2019 10:50 AM *To:* Kimberly Stanek ; CCP4BB@JISCMAIL.AC.UK *Subject:* Re: [ccp4bb] Semet derivative dying almost immediately in beam What exposure time are you using? And which beamline? -James Holton MAD Scientist On 8/29/2019 10:26 AM, Kimberly Stanek wrote: Hi folks, We have a protein that we have been trying to solve the structure of for a while now but so far haven't been able to get any diffraction better than ~4.5A. I was able to collect a full 360 degrees of data and index, but MR is failing so we have turned to de novo phasing. Recently we prepared crystals of the Semet derivative under the same condition. While these crystals diffracted to about the same resolution, I found they were dying after just
Re: [ccp4bb] Semet derivative dying almost immediately in beam
Certainly not trying to be insulting by the suggestions but could it be as simple an alignment issue with the crystal, issues with the beam alignment or crystal damage following a fluorescence scan? The cryo setup? Like James, I’ve seen crystals decay quickly in high salts at longer wavelengths. Good luck! - Todd Sent from my iPhone On Aug 30, 2019, at 1:41 PM, James Holton mailto:jmhol...@lbl.gov>> wrote: That is absolutely mind-blowing. ALS 8.2.2 has a nominal dose rate of 95 kGy/s, and you are seeing global damage go to completion in ~0.4s, or 40 kGy. This is 750 times faster than expected. I have never heard of a cryo-cooled crystal decaying that fast. This may be a new world record! I know that is probably not what you wanted, but it is exciting to people like me. Being at 4.5A is actually a good thing for radiation damage because features at poor resolution tend to evolve more slowly with dose than fine details (high-angle spots). That is probably also not what you wanted, but it makes such a fast decay rate even more unusual. I have encountered reports of extra-sensitive proteins before, but they always turn out to be due to something predictable, like having a super-high concentration of heavy atom. For example, having 200 mM sodium iodide in your solvent channels will cut a typical crystal lifetime in half. 4 M NaI will cut it by a factor of 20, but to get 1000x the usual decay rate you would need something like 10 M Ta6Br12. And Ta6Br12 is only soluble to 2 mM. Yes, you have Se atoms in there, but even if you have 1 in 10 residues containing an Se atom at 0% solvent that is still only 1.0 M Se in the crystal. Only enough for about a factor of 10. I have also seen errors in beam size and flux lead to apparently abnormal radiation damage rates. These can easily be as large as a factor of two, especially with very small beams, but it is hard to imagine how one could get a factor of 1000. ALS 8.2.2 is only a few meters from where I'm sitting, and I'm pretty sure the flux density is accurate to within 20%, probably better. I also just checked out the cryo stream, and it has none of the usual warning signs. So, that leaves either a genuinely ultra-sensitive protein, or some kind of undetected equipment malfunction. Either way, we at the ALS are very interested in this result. Would you mind trying again? And let me know when you are coming? -James Holton MAD Scientist On 8/29/2019 11:07 AM, Kimberly Stanek wrote: ALS 822. I tried as low as 0.2 sec exposure but it didn't seem to help much. Kimberly Stanek Postdoctoral Researcher, Goulding Lab Co-chair, UCI Postdoctoral Association University of California, Irvine Natural Science I, Room 2302 (949) 824-0014 From: James Holton <mailto:jmhol...@lbl.gov> Sent: Thursday, August 29, 2019 10:50 AM To: Kimberly Stanek <mailto:ksta...@uci.edu>; CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK> <mailto:CCP4BB@JISCMAIL.AC.UK> Subject: Re: [ccp4bb] Semet derivative dying almost immediately in beam What exposure time are you using? And which beamline? -James Holton MAD Scientist On 8/29/2019 10:26 AM, Kimberly Stanek wrote: Hi folks, We have a protein that we have been trying to solve the structure of for a while now but so far haven't been able to get any diffraction better than ~4.5A. I was able to collect a full 360 degrees of data and index, but MR is failing so we have turned to de novo phasing. Recently we prepared crystals of the Semet derivative under the same condition. While these crystals diffracted to about the same resolution, I found they were dying after just one or two snaps, even with increased beam attenuation and decreased exposure time. I am wondering if anyone has experienced anything like this before and had any suggestions on what to do about it. Thank you, Kimberly Stanek Postdoctoral Researcher, Goulding Lab Co-chair, UCI Postdoctoral Association University of California, Irvine Natural Science I, Room 2302 (949) 824-0014 To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1 To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1 To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1 To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1
Re: [ccp4bb] Semet derivative dying almost immediately in beam
That is absolutely mind-blowing. ALS 8.2.2 has a nominal dose rate of 95 kGy/s, and you are seeing global damage go to completion in ~0.4s, or 40 kGy. This is 750 times faster than expected. I have never heard of a cryo-cooled crystal decaying that fast. This may be a new world record! I know that is probably not what you wanted, but it is exciting to people like me. Being at 4.5A is actually a good thing for radiation damage because features at poor resolution tend to evolve more slowly with dose than fine details (high-angle spots). That is probably also not what you wanted, but it makes such a fast decay rate even more unusual. I have encountered reports of extra-sensitive proteins before, but they always turn out to be due to something predictable, like having a super-high concentration of heavy atom. For example, having 200 mM sodium iodide in your solvent channels will cut a typical crystal lifetime in half. 4 M NaI will cut it by a factor of 20, but to get 1000x the usual decay rate you would need something like 10 M Ta6Br12. And Ta6Br12 is only soluble to 2 mM. Yes, you have Se atoms in there, but even if you have 1 in 10 residues containing an Se atom at 0% solvent that is still only 1.0 M Se in the crystal. Only enough for about a factor of 10. I have also seen errors in beam size and flux lead to apparently abnormal radiation damage rates. These can easily be as large as a factor of two, especially with very small beams, but it is hard to imagine how one could get a factor of 1000. ALS 8.2.2 is only a few meters from where I'm sitting, and I'm pretty sure the flux density is accurate to within 20%, probably better. I also just checked out the cryo stream, and it has none of the usual warning signs. So, that leaves either a genuinely ultra-sensitive protein, or some kind of undetected equipment malfunction. Either way, we at the ALS are very interested in this result. Would you mind trying again? And let me know when you are coming? -James Holton MAD Scientist On 8/29/2019 11:07 AM, Kimberly Stanek wrote: ALS 822. I tried as low as 0.2 sec exposure but it didn't seem to help much. Kimberly Stanek Postdoctoral Researcher, Goulding Lab Co-chair, UCI Postdoctoral Association University of California, Irvine Natural Science I, Room 2302 (949) 824-0014 *From:* James Holton *Sent:* Thursday, August 29, 2019 10:50 AM *To:* Kimberly Stanek ; CCP4BB@JISCMAIL.AC.UK *Subject:* Re: [ccp4bb] Semet derivative dying almost immediately in beam What exposure time are you using? And which beamline? -James Holton MAD Scientist On 8/29/2019 10:26 AM, Kimberly Stanek wrote: Hi folks, We have a protein that we have been trying to solve the structure of for a while now but so far haven't been able to get any diffraction better than ~4.5A. I was able to collect a full 360 degrees of data and index, but MR is failing so we have turned to de novo phasing. Recently we prepared crystals of the Semet derivative under the same condition. While these crystals diffracted to about the same resolution, I found they were dying after just one or two snaps, even with increased beam attenuation and decreased exposure time. I am wondering if anyone has experienced anything like this before and had any suggestions on what to do about it. Thank you, Kimberly Stanek Postdoctoral Researcher, Goulding Lab Co-chair, UCI Postdoctoral Association University of California, Irvine Natural Science I, Room 2302 (949) 824-0014 To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1 To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1 To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1
Re: [ccp4bb] Semet derivative dying almost immediately in beam
Ah, low resolution experimental phasing. Fun stuff, seems like a lost art these days. Are the crystals large ? Is it possible to do a (continuous helical data) collection? (Manually or if your beamline permits you to). Granted this is anomalous, is it possible to then merge enough partial datasets together? (Very carefully of course). If not there’s always SIRAS. With this rapid decay, are you using a strategy? (Another lost art these days) Given the difficulty of this project, have you pursued other derivatives (via soaking)? > On Aug 29, 2019, at 11:26 AM, Kimberly Stanek wrote: > > Hi folks, > > We have a protein that we have been trying to solve the structure of for a > while now but so far haven't been able to get any diffraction better than > ~4.5A. I was able to collect a full 360 degrees of data and index, but MR is > failing so we have turned to de novo phasing. > > Recently we prepared crystals of the Semet derivative under the same > condition. While these crystals diffracted to about the same resolution, I > found they were dying after just one or two snaps, even with increased beam > attenuation and decreased exposure time. I am wondering if anyone has > experienced anything like this before and had any suggestions on what to do > about it. > > Thank you, > > > > Kimberly Stanek > Postdoctoral Researcher, Goulding Lab > Co-chair, UCI Postdoctoral Association > University of California, Irvine > Natural Science I, Room 2302 > (949) 824-0014 > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1 To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1
Re: [ccp4bb] Semet derivative dying almost immediately in beam
Their web page indicates they’re running a 315, so there are limits on useful low dose data collection. If you can get to a beamline with a pixel array detector and mount your samples carefully you’ll probably get more from low dose methods. The key thing with this is minimising background from loop, drop etc so the photons you measure are as far as possible from the sample under study. You’ll probably benefit from combining data from multiple samples if they are isomorphous. Obviously your ultimate resolution will be limited by the number of photons your sample can scatter before suffering measurable damage... but combining samples can help you here Best wishes Graeme On 29 Aug 2019, at 19:07, Kimberly Stanek mailto:ksta...@uci.edu>> wrote: ALS 822. I tried as low as 0.2 sec exposure but it didn't seem to help much. Kimberly Stanek Postdoctoral Researcher, Goulding Lab Co-chair, UCI Postdoctoral Association University of California, Irvine Natural Science I, Room 2302 (949) 824-0014 From: James Holton mailto:jmhol...@lbl.gov>> Sent: Thursday, August 29, 2019 10:50 AM To: Kimberly Stanek mailto:ksta...@uci.edu>>; CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK> mailto:CCP4BB@JISCMAIL.AC.UK>> Subject: Re: [ccp4bb] Semet derivative dying almost immediately in beam What exposure time are you using? And which beamline? -James Holton MAD Scientist On 8/29/2019 10:26 AM, Kimberly Stanek wrote: Hi folks, We have a protein that we have been trying to solve the structure of for a while now but so far haven't been able to get any diffraction better than ~4.5A. I was able to collect a full 360 degrees of data and index, but MR is failing so we have turned to de novo phasing. Recently we prepared crystals of the Semet derivative under the same condition. While these crystals diffracted to about the same resolution, I found they were dying after just one or two snaps, even with increased beam attenuation and decreased exposure time. I am wondering if anyone has experienced anything like this before and had any suggestions on what to do about it. Thank you, Kimberly Stanek Postdoctoral Researcher, Goulding Lab Co-chair, UCI Postdoctoral Association University of California, Irvine Natural Science I, Room 2302 (949) 824-0014 To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1 To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1 -- This e-mail and any attachments may contain confidential, copyright and or privileged material, and are for the use of the intended addressee only. If you are not the intended addressee or an authorised recipient of the addressee please notify us of receipt by returning the e-mail and do not use, copy, retain, distribute or disclose the information in or attached to the e-mail. Any opinions expressed within this e-mail are those of the individual and not necessarily of Diamond Light Source Ltd. Diamond Light Source Ltd. cannot guarantee that this e-mail or any attachments are free from viruses and we cannot accept liability for any damage which you may sustain as a result of software viruses which may be transmitted in or with the message. Diamond Light Source Limited (company no. 4375679). Registered in England and Wales with its registered office at Diamond House, Harwell Science and Innovation Campus, Didcot, Oxfordshire, OX11 0DE, United Kingdom To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1
Re: [ccp4bb] Semet derivative dying almost immediately in beam
ALS 822. I tried as low as 0.2 sec exposure but it didn't seem to help much. Kimberly Stanek Postdoctoral Researcher, Goulding Lab Co-chair, UCI Postdoctoral Association University of California, Irvine Natural Science I, Room 2302 (949) 824-0014 From: James Holton Sent: Thursday, August 29, 2019 10:50 AM To: Kimberly Stanek ; CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Semet derivative dying almost immediately in beam What exposure time are you using? And which beamline? -James Holton MAD Scientist On 8/29/2019 10:26 AM, Kimberly Stanek wrote: Hi folks, We have a protein that we have been trying to solve the structure of for a while now but so far haven't been able to get any diffraction better than ~4.5A. I was able to collect a full 360 degrees of data and index, but MR is failing so we have turned to de novo phasing. Recently we prepared crystals of the Semet derivative under the same condition. While these crystals diffracted to about the same resolution, I found they were dying after just one or two snaps, even with increased beam attenuation and decreased exposure time. I am wondering if anyone has experienced anything like this before and had any suggestions on what to do about it. Thank you, Kimberly Stanek Postdoctoral Researcher, Goulding Lab Co-chair, UCI Postdoctoral Association University of California, Irvine Natural Science I, Room 2302 (949) 824-0014 To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1 To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1
Re: [ccp4bb] Semet derivative dying almost immediately in beam
What exposure time are you using? And which beamline? -James Holton MAD Scientist On 8/29/2019 10:26 AM, Kimberly Stanek wrote: Hi folks, We have a protein that we have been trying to solve the structure of for a while now but so far haven't been able to get any diffraction better than ~4.5A. I was able to collect a full 360 degrees of data and index, but MR is failing so we have turned to de novo phasing. Recently we prepared crystals of the Semet derivative under the same condition. While these crystals diffracted to about the same resolution, I found they were dying after just one or two snaps, even with increased beam attenuation and decreased exposure time. I am wondering if anyone has experienced anything like this before and had any suggestions on what to do about it. Thank you, Kimberly Stanek Postdoctoral Researcher, Goulding Lab Co-chair, UCI Postdoctoral Association University of California, Irvine Natural Science I, Room 2302 (949) 824-0014 To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1 To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1
[ccp4bb] Semet derivative dying almost immediately in beam
Hi folks, We have a protein that we have been trying to solve the structure of for a while now but so far haven't been able to get any diffraction better than ~4.5A. I was able to collect a full 360 degrees of data and index, but MR is failing so we have turned to de novo phasing. Recently we prepared crystals of the Semet derivative under the same condition. While these crystals diffracted to about the same resolution, I found they were dying after just one or two snaps, even with increased beam attenuation and decreased exposure time. I am wondering if anyone has experienced anything like this before and had any suggestions on what to do about it. Thank you, Kimberly Stanek Postdoctoral Researcher, Goulding Lab Co-chair, UCI Postdoctoral Association University of California, Irvine Natural Science I, Room 2302 (949) 824-0014 To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1
