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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