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 <jmhol...@lbl.gov<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 <jmhol...@lbl.gov><mailto:jmhol...@lbl.gov> Sent: Thursday, August 29, 2019 10:50 AM To: Kimberly Stanek <ksta...@uci.edu><mailto:ksta...@uci.edu>; CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK> <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
