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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. >