Hi Lindsey,
My couple cents:
1) Make sure to use "Auto-corrections" option for scaling with HKL. This
option is great for extracting the anomalous signal.
2) Im also with Andreas and Nukri on collecting 360 at lower dose and
leaving the inverse beam for later.
Best,
Ivan
With best regards,
Ivan Shabalin, Ph.D.
Research Scientist,
Department of Molecular Physiology and Biological Physics,
University of Virginia,
1340 Jefferson Park Avenue, Pinn Hall,Room 4223,
Charlottesville, VA 22908
https://www.linkedin.com/in/shabalinig/
https://minorlab.org/person/ivan_s/
On 8/27/19 03:21, Andreas Förster wrote:
Dear Lindsey,
I'm all with Nukri on this one. 12 Se atoms (whose incorporation you've
shown) should be plenty to give you enough anomalous signal to phase the
data and solve the structure. That's why I would do the simplest
experiment first. Collect 360° of data at the peak energy, maybe a bit
finer sliced than you have now (0.1° per image) and with fewer X-rays.
Reduce exposure fivefold (just a guess, but most people tend to
overexpose - see Winter et al., Acta D 75:242, 2019) and see what you
can get from this one dataset after processing with FRIEDEL'S_LAW= FALSE.
Look at the table "SUBSET OF INTENSITY DATA WITH SIGNAL/NOISE >= -3.0 AS
FUNCTION OF RESOLUTION" in CORRECT.LP and see how far the anomalous
signal extends (Anomal Corr values above 15 to 20, highlighted by XDS
with an asterisk) and how strong it is (SigAno values, above 3 gives you
confidence, though I have solved structures with much less signal).
If the anomalous signal isn't enough for structure solution, collect
more data from the same crystal. This is one reason why you reduced the
exposure in the first place. Move the detector in or out depending on
the processing of the first 360° (How far does diffraction extend?) and
collect another 360°. Or change chi/kappa. (See Basu et al., Acta D
75:262, 2019 for inspiration.) Or - and this is the best but unlikely
to be necessary with your crystals - do two-wavelength MAD. This
shouldn't be an afterthought to the SAD experiment, though, but properly
planned as one comprehensive experiment. Using the inflection and
remote energies might be a good idea because it maximizes the
differences between the two datasets.
Inverse beam can be powerful but also poses challenges. I use this
technique only if I fail otherwise.
All best.
Andreas
On Tue, Aug 27, 2019 at 3:55 AM Nukri Sanishvili <[email protected]
<mailto:[email protected]>> wrote:
Hi Lindsey,
Obviously, one would need a lot more information to properly
diagnose the problem and I am sure much smarter people them me will
ask you for that. But just to move the task by couple of steps, I
want to point out couple of things.
1. Trivial question: did you have the anomalous option turned on
during data processing? (Just like from the IT help - is your
computer turned on?)
2. How much data did you collect for each half of the inverse beam
geometry? If you have enough, try phasing with only one half. When
done properly, inverse beam experiment is great but it can easily
get tricky introducing systematic errors and thus swamping anomalous
signal. If you redo the inverse beam, use little wider wedges, say,
5-10 degrees.
3. I thought an example of diffraction image would not give any
useful information but... Judging by how smooth the background is on
your Pilatus image, I am guessing you have used a lot of exposure.
Can you calculate how much dose did you put in your crystal? If you
are going to re-do the experiment, I would suggest reducing the
exposure level and collecting more data.
4. Because you are not showing f' and f" plots, I am guessing that
you are doing SAD. If it fails and you end up redoing your
experiment and you have crystals for it, you might want to try
2-wavelength MAD but for that you would need to know exactly where
is inflection point and collect one of the datasets there.
Good luck!
Nukri
On Mon, Aug 26, 2019 at 5:45 PM L. Doyle <[email protected]
<mailto:[email protected]>> wrote:
I have some Seleno-Methionine protein crystals (12 SeMet of 211
amino acids, incorporation verified by Mass Spec). I've already
collected several datasets (ALS BL5.0.2) but I seem to be losing
(rejecting?) a lot of anomalous signal during data processing.
I'm most familiar with HKL2000, but I have tried XDS and DIALS
auto-processing. Here is a scan: https://ibb.co/LZqm33p and here
is an example of a frame: https://ibb.co/gR3ZR47. Each frame is
0.25° and I'm using inverse beam with wedge size 1°. Maybe I
need to adjust my collection strategy? All previous datasets
have been in space group P 21 with dimensions of approx. 24.5Å,
85Å, 40Å, 90°, 96.5°, 90°. I'm sure there are additional things
I can be doing in HKL but I've run out of ideas. Any advice or
recommendations would be appreciated. Please let me know if you
need additional information.
Thank you,
Lindsey
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