A few follow-up questions I got out-of-band:
how did you get to the 1:1 relationship between Bijvoet ratio and dose?
I got this from fitting a straight line to Table 1 of Banu's 2004 paper:
10.1107/S0907444904007917
Is this a rough estimate based on a singular result? Of course it is!
This is how we roll in radiation damage research.
Comment: with the more modern pixel array detectors (e.g. Eiger), you
can slice your dose even more finely than 0.1s, and not worry about
the readout time.
yes.
With a bit of a caveat on how many photons/pixel you need for stable
background subtraction. XDS starts having issues around 1 photon/pixel
or less, and DIALS claims to be able to get to 0.01 photons/pixel, but I
have not personally pushed it that far. Not yet.
I have a plan to try and push zero-dose extrapolation to the
one-photon-per-image level, but that is on another thread.
is it better to collect 360 or 720º at half the dose
Nothing wrong with going longer than 360, especially if you want to do
zero-dose extrapolation, because it is only by repeating the same phi
range (and everything else) exactly that you get a genuinely "same"
increment in dose.
However, once you go past 360 the "multiplicity" you gain starts turning
into what you might call a "redundancy". What I mean by that is that in
the first 360 each spot and its symmetry mates generally show up on
different pixels. Each pixel has about 1% to 3% calibration error
associated with it (depending on the detector). So, for the 2nd 360 you
will re-measure all the same spots with the same pixels again, repeating
a systematic error. You will also have the same sample self-absorption,
etc. But, the pixel calibration error starts to really matter for
anomalous at high "redundancy". To put it another way, if a particular
pixel has 1% error, then counting more than 10,000 photons with it is a
waste, because the systematic error of 1% will start to dominate the
total error at higher photon counts. So, for anomalous especially, I
recommend moving the detector between 360s. Sliding it horizontally is
best. Or you can use 2theta. But, a small change in detector distance
can usually do it and is almost always an available option.
The only problem with all this "dose slicing" is the images get very
very weak.
And that brings us back to the "weak image limit". What if instead of
images we just collected a list of x-y coordinates of photon hits vs
time? Anyone have a suggestion for the name to give to the program that
can process such data?
-James Holton
MAD Scientist
On 5/15/2024 3:28 PM, James Holton wrote:
Thank you to all who provided helpful suggestions so far.
A few things I'd recommend for this particular beamline (which I have
been running for 20+ years)
Do NOT collect one wavelength at a time. This was a good strategy on
old beamlines with noisy detectors and slow, drifty monochromators.
This is not the case at any of the ALS beamlines today. With modern
zero-read-noise detectors there is no penalty to spreading your
photons over a lot more images, and round-robin changes between at
least two wavelengths will double your phasing power for the same
dose. With 8.3.1's monochromator, wavelength changes take about 1
second and are reproducible to well within the intrinsic width of the
Se peak. So you don't need to worry about missing or drifting off the
peak or inflection. The only thing you need to worry about is
over-cooking your crystal before you get all the data you need.
No matter what beamline you use the number of photons your crystal
will give off before it dies is a fixed number. All you get to do is
decide how to spread them over the images. Doing two wavelengths
within this photon budget doesn't hurt. You can always scale and merge
them together. But keeping them separate gives you both kinds of
anomalous differences, which are 90 deg apart. So, when one zigs the
other zags. It is like having twice as many sites without the extra
damage you would get from them. Also, by taking shorter/weaker
exposures you maximize your chances of winning over radiation damage
"in-post" by cutting off images that degrade your signal.
And before anybody says it: NO! Collecting fainter images does NOT
degrade your resolution. I don't know where this idea comes from, but
it never seems to die. It was true with film and image plates, but
with pixel arrays and modern CCDs there is no penalty to weak images.
Don't believe me? Read the manual for your detector. Modern PADs
actually sum a bunch of weak images internally before writing them to
disk. You can do the same "in post" if you want to.
Yes, there are many cases where SAD is good enough, but my advice is
never to tempt fate.
What I recommend is:
1) collect two wavelengths: remote, and halfway between the peak and
inflection.
this will maximize both kinds of anomalous differences
2) calculate your Bijvoet ratio here:
https://bl831.als.lbl.gov/xtalsize.html
3) convert this into MGy. I.E. if your Bijvoet ratio is 3%, then 3 MGy
is the max dose to avoid.
4) do a strategy and start at the recommended phi value
5) set delta-phi to be 1/3 of your estimated mosaic spread, or 0.2
deg, whichever is lower
this is all done automatically by the "index" program at 8.3.1
6) set your exposure time to be 0.1 s or more.
This is because the Pilatus M 6M has a 1 ms read-out and you want
that to be 1% of the exposure.
7) attenuate the beam so that you will get complete data in less than
1/2 your Bijvoet ratio in MGy.
This is handled by the exposure_time program at 8.3.1
8) collect data in inverse beam and round-robin for both wavelengths
(45 deg wedges)
In BLU-ICE, just enter the wavelengths into the list on the
Collect tab
9) keep collecting until you get 360 deg for both wavelengths
10) move the detector up by ~5 mm, this puts the next sphere of spots
onto new pixels
11) multiply your exposure or de-attenuate by a factor of 4
12) goto 8
When the diffraction image is noticeably damaged, you are done with
this crystal. If it is bigger than the beam, move to a fresh spot and
do this again. When the crystal is all burnt up, mount the next one
and do this again.
If you're lucky, the automatic processing will finish before you mount
your next crystal and you can try SHELXC/D/E on the 448-core
shared-memory computer we have for doing such things. I expect it
might be faster than the cloud.
Sorry if any of this sounds gruff, I don't mean to shout down on
anyone, but I want the message to be clear. This is something Gerard B
and I have struggled to communicate for decades:
Collecting one wavelength at a time is not MAD, but rather M-SAD.
Multiple, non-isomorphous SAD data sets.
-James Holton
MAD Scientist
On 5/13/2024 10:23 PM, dbellini wrote:
Hi Marco,
A few suggestions that I like to follow for MAD experiments:
Before everything, check you have at least about 1 SeMet per 100
residues
Then before crystallisation check by MassSpec that SeMet is properly
incorporated in your protein
After crystallisation collect first on the peak with (very) high
redundancy and as little/gentle dose as possible
Collecting the other wavelengths should give you better starting
phases/maps, which might be very helpful at your resolution of 2.8
(especially if it is a very anisotropic 2.8...)
Automated pipelines are so good nowadays, if you collect good data
they should solve it without problems (as long as your crystal is not
suffering from other pathologies like twinning or pseudosymmtries).
Good luck!
D
On 2024-05-14 01:17, Marco Bravo wrote:
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Hello all,
I have a data collection trip next week and plan to collect data on
selenomethionine derivative crystals at the al831 beamline. Are there
any resources, tips, tutorials, literature etc. That you can recommend
to help me prepare for these experiments. Also is there a way to plug
in the experimental data into ccp4 cloud to do the automatic structure
solution? Do I need native and derivative data to solve the structure?
Last trip I collected a seemingly 2.8 angstrom resolution data on a
crystal of the native protein but could not get a solution depsite
extensive molecular replacement attempts. It seems that assigning a
space group for the crystals has been troublesome as well. here is my
last thread I posted about the issue for reference.
https://www.jiscmail.ac.uk/cgi-bin/wa-jisc.exe?A2=ind2402&L=CCP4BB&O=D&X=CCE6DFA19FA3D40346&Y=mbrav005%40ucr.edu&P=112302
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