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