Mark may or may not have meant what I think he meant, but it did remind
me of the following passage from Blundell and Johnson (1976):
"The chain reaction initiated by fee radical formation probably accounts
for the common observation that radiation damage effects in protein
crystals continue, even after the X-ray shutter has been closed."
Perhaps for no other reason than it appears in The Book, this
expectation of "dark progression" seems to continue to this day?
However, I was recently challenged by a colleague to find a paper or a
system that demonstrated dark progression of damage at "room"
temperature, and I have to admit I can't find one. Any help?
I myself once convinced myself that I had a crystal that had "healed"
after sitting under lN2 for a week. I had been "burning" it in a rad
dam study, but ran out of time and had to dismount it and continue the
experiment on my next staff shift. I was quite excited to see that it
diffracted better the second time! Unfortunately, after repeating this
experiment several times and making sure that I had the beam hitting
exactly the same part of the crystal after the "delay" I was
disappointed to find that the damage always "picked up where it left
off". So, the most likely explanation for my crystal "healing" was that
my alignment was slightly off the second time and I was shooting some
"fresh" crystal that had remained outside the beam for the first round
of "burning". I never did publish anything about that. In fact, I'm
not sure I could publish it if I tried!
I have now heard three anecdotal stories about "attenuation" making
crystals endure more dose before it "dies", but all of these seem to
have arisen from an error in the attenuation factor. Attenuators not
only absorb photons, but they can also blow up the beam size, making the
photons/area smaller than the "% transmittance" in the GUI would lead
you to believe. Best way to check that the attenuation is what you
think it is is to look at the scale factors when scaling the two runs
together, they should be ~1 if the integrated photons/area, crystal
volume, wavelength, etc. was the same.
However, that is a "dose rate effect", and although "dark progression"
implies a dose-rate effect, the converse is not necessarily true. There
is much debate about this in the rad dam field, but it seems whenever a
traditionally-held belief like "dark progression" is challenged, the
rest of the MX community seems to dismiss it as "oh, you're just working
on lysozyme". Well, what should we be working on?
-James Holton
MAD Scientist
On 11/20/2011 5:22 PM, Sanishvili, Ruslan wrote:
Hi James,
I don't think the comment you referenced meant to imply "dark progression of radiation
damage. If I remember from the recent thread, it was to say that if you can only collect few
(3?) shots from one crystal before it's "too dead" and you use 1st of these shots to
devise the strategy, then you are wasting your crystals and will never get you data. Of
course, you don't have to use so much flux for the image which is meant only for defining the
orientation but it was omitted from that comment.
Now back to the rest of your message. I can add another warning observation:
If a cryo-cooled crystal was exposed long enough (i.e. for data collection) then stored
(by a robot) and then mounted again, some times one sees that it had
"exploded". Such an explosion, presumably a hydrogen gas escape, can be seen
almost always if a crystal is wormed up after long data collection. The fact that
robot-stored crystals sometimes display same behavior, indicates that a crystal in the
arms of the robot can worm up somewhat. Therefore, comparing diffraction before and after
storage is not always valid.
Also beware of comparing diffraction quality from different parts of the
crystal as large crystals are almost never homogeneous.
Cheers,
Nukri
-----Original Message-----
From: CCP4 bulletin board on behalf of James Holton
Sent: Sun 11/20/2011 2:31 PM
To: [email protected]
Subject: [ccp4bb] dark progression of radiation damage
Mark's comment below reminded me of a quandary that is starting to
develop in the rad dam field. The idea of the "free radical cascade"
continuing to damage protein crystals even after the beam has been
turned off seems to have originated on page 253 of Blundell and Johnson
(1976), and I think most of us have had the unpleasant experience of
loosing diffraction after a "delay" in data collection. However, can
one be sure that the incident beam alignment was the same if the "delay
in data collection" was due to a storage ring dump, or a filament
change? Can one be sure that a crystal stored under cryo never ever got
warmed up (like during mounts and dismounts, or perhaps a colleague
making an undocumented late-night rummage through the storage dewar)?
Can one be sure that a crystal at room temperature wasn't just drying
up? Can one be sure that the damage didn't all occur during the first
shot (and the image we saw is just the sum over the decay)?
I ask because many systematic studies have now been made to try and
quantify the "dark progression" phenomenon, only to find it doesn't seem
to really exist, either under cryo (Garman& McSweeney, 2007; Sliz et
al., 2003; Leiros et al., 2006; Owen et al., 2006), or at room
temperature (Southworth-Davies et al. Structure 2007; Warkentin et al.
Acta D 2011), except at temperatures that are almost never used for data
collection (Warkentin et al. Acta D 2011). Now, there are observations
of radiochemical reactions progressing for several minutes "in the dark"
(Weik et al., 2002, Southworth-Davies& Gaman Acta D 2007 McGeehen et
al., 2009 ), but I don't personally know of anyone (other than Warkentin
et al. 2011) who has demonstrated that _diffraction_ continues to decay
in the dark.
So, my question is: does anyone out there have an example system where
one can reproducibly demonstrate "dark progression" of diffraction spot
fading? That is, you can mount the crystal, store it in its "mount" for
at least a few days (to prove that its not just drying up), take at
least two low-dose shots to get an idea of the expected rate of decay,
then wait for "a while" and start shooting again. Do you see
significantly worse diffraction?
-James Holton
MAD Scientist
On 11/18/2011 1:50 AM, Mark J van Raaij wrote:
I.e. if you collect one image and then wait until the orientation and
strategy is calculated, the crystal is probably already dead.
