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: [log in to unmask]
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.
>
|