I think I need to clarify couple of things in my recent post about "exploding" crystals during re-mounting by a robot. First, it was a bit over-dramatization - what I meant by "explosion" was actually bubbling often observed when heavily exposed crystal is warmed up. Second, this bubbling was observed only after a HEAVILY OVEREXPOSED crystal was re-mounted and not during a regular screening procedure. Third and more import, as couple of people pointed out to me, the observed bubbling was probably a result of something being wrong in the robot operation, rather than intrinsic feature of that or any other robot. This indeed seems to be the case since the observations I was referring to were made a couple of years ago and have not been seen since. During this time the robot operation underwent several upgrades and tweaks, perhaps fixing whatever problem it might have had resulting in occasional bubbling.
Ruslan Sanishvili (Nukri), Ph.D. GM/CA-CAT Biosciences Division, ANL 9700 S. Cass Ave. Argonne, IL 60439 Tel: (630)252-0665 Fax: (630)252-0667 rsanishv...@anl.gov 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: CCP4BB@JISCMAIL.AC.UK > 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. >> >
