Yafang,
I'm afraid that just because you still have spots at the end of your
dataset does not mean radiation damage was "not a problem". The
reactions that disorder your heavy atom sites go to completion at doses
that can be as little as 1/30th of the dose required to noticeably fade
your spots. There are a number of nice reviews written about this:
http://dx.doi.org/10.1107/S0909049509004361
http://dx.doi.org/10.1107/S0909049512050418
http://dx.doi.org/10.1107/S0909049506048898
http://dx.doi.org/10.1107/S0907444907019580
Also, If your datasets were collected one wavelength at a time, such as
a complete dataset at the peak, then another complete dataset at the
inflection, and then, after all that, you collect the "reference"
dataset at the remote, then what you have is not a MAD dataset. This is
a series of SAD datasets (M-SAD). Of these three SAD datasets only the
"peak" is at the optimum energy for anomalous, and also has the least
radiation damage, so that one will work better than the other two. I
use the term M-SAD instead of MAD because you are effectively using a
different crystal for each wavelength, and that means the
inter-wavelength differences are dominated by non-isomorphism.
Non-isomorphism can easily bury an anomalous signal, and radiation
damage is a pretty efficient way to make a crystal non-isomorphous with
its former self.
By looking at examples in the literature, (such as Banumathi et al.
2004) one can guestimate that the degree of non-isomorphism induced by
radiation damage is about 1% per MGy of dose. You can look up the
nominal dose rate of the beamline you collected these data at here:
http://bl831.als.lbl.gov/damage_rates.pdf
I try to keep the numbers in this document up to date, but most
beamlines are attenuated to the point where they deliver about 1 MGy per
minute of shutter-open time. That's for a crystal with < ~20 mM heavy
atoms, and unattenuated beam.
So, if the dispersive signal you are looking for is 3%, then once your
crystal has endured more than ~3 minutes of shutter-open time, the
non-isomorphism will start to overwhelm that signal, and then trying to
use dispersive (inter-wavelength) differences becomes
counterproductive. This is because the software is trying to reconcile
all the observed differences in terms of heavy-atom positions, and when
half the differences are coming from non-isomorphism, the equations all
fall apart. This is probably why treating your M-SAD dataset as a MAD
experiment fails. Anomalous (Bijvoet) differences, however, tend to
come up fairly close together in "phi" because once a spot passes
through the Ewald sphere its Friedel mate will generally pop up on the
opposite side of the beamstop a few degrees later. Basically, if you're
measuring a difference, it is best to measure the two numbers you are
going to subtract as close together in time as possible. This is why
"inverse beam" with "round robin" wavelength changes is the approach
that is most robust to damage effects. Yes, you still get damage, but
at least the differences you are subtracting are close together, and
therefore comparing "apples to apples".
I suppose it was the advent of saggital-focusing monochromators that
made wavelength changes more difficult and more recently the advent of
so-called "shutterless" data collection has led to more and more M-SAD
data collections than MAD. This is a pity, really, because as George
has already said, MAD gives you significantly better phases than SAD.
It just requires a little more patience to collect it properly.
-James Holton
MAD Scientist
On Tue, Aug 20, 2013 at 2:05 PM, Yafang Chen <yafangche...@gmail.com
<mailto:yafangche...@gmail.com>> wrote:
Hi All,
I have three datasets of SeMet-incorporated protein at peak, infl
and high wavelength respectively. SAD with peak dataset works well
to solve the phase problem. However, MAD with all three datasets
didn't work at all. The completeness of all three datasets are more
than 99%. So I think radiation damage should not be a problem. Does
anyone have any idea about the possible reasons that MAD didn't work
in this case? Thank you so much for any of your help!
Best,
Yafang
--
Yafang Chen
Graduate Research Assistant
Mesecar Lab
Department of Biological Sciences
Purdue University
Hockmeyer Hall of Structural Biology
240 S. Martin Jischke Drive
West Lafayette, IN 47907
