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Re: [ccp4bb] To bathe or not to bathe. |
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CCP4bb navigationCCP4bb <-- 1999 <-- November 1999 <-- 30 November 1999Subject: Re: To bathe or not to bathe. From: harry powell harry {- at -} MRC-LMB {- dot -} CAM {- dot -} AC {- dot -} UK Date: 2007-11-24 Hi I'm not convinced that the first sentence here has much to do with the second (although both might be true). > The main reason was related to absorption. If you didn't completely > bathe > the crystal in the xray beam, then the diffracting volume of the > crystal > would be different during the data collection, and thus, scaling > would be > inaccurate, especially when there was radiation damage. Absorption can be a real problem when the path length through the crystal differs significantly, and is often not closely related to the diffracting volume - think of different paths through a flat plate or a needle. This is the main reason why "old-fashioned" crystallographers in days of yore used to grind their crystals into a sphere. Absorption problems are exacerbated when you have atoms present in your sample that absorb heavily (this should not be a surprise...). As a former small-molecule crystallographer, I always made sure the crystal was bathed in the beam (so that the diffracting volume was the same), and usually tried to make sure the the whole crystal was in the central part of the beam (to try to make sure I was using the more uniform part of the beam). When I moved to macromolecular work, I found that most people seemed to prefer to get the whole beam going through the crystal, and not worry too much about bits of crystal "hanging off" outside the beam. There are, of course, reasons for this, among which is that small molecule crystallographers are often spoilt for choice when it comes to picking out the right crystal, and protein crystallographers aren't (at least when I made the switch); they often needed all the crystals they could get in order to get a single dataset - this was in the days before cryo was standard, and room temperature data collection was de rigueur. While practising small molecule crystallography, I still needed to apply absorption corrections to the data, especially where I had loads of strongly absorbing atoms (e.g. in third-row transition metal clusters), in order to both solve and refine the structures. The absorption corrections I used were based on different methods (psi- scans, analytical corrections based on the making precise measurements of the crystal itself, and the (ahem) wonderful Walker & Stuart DIFABS (which could turn a pig's ear into a silk purse, in spite of what the purists might say about what it was actually doing to the data!)). just my two ha'porth... > This was > especially true when you weren't sure that the crystal was well- > centered > in the xray beam (in a cryostat, and therefore not visible). We > typically > collected highly redundant data to help compensate for this. We > also used > to correct for absorption by assigning Bragg indices to the crystal > and > making precise measurements of crystal dimensions. > > Scaling programs are now more extensive, and include options to > calculate > a pseudo-absorption surface. In principle, if you have a beam that is > ALWAYS smaller than the crystal, then the same crystal volume is > illuminated by the xray beam, and will minimize scaling errors. > > Bernie Santarsiero > > > > On Fri, November 23, 2007 4:34 pm, Jim Pflugrath wrote: >> It probably goes back to the days of using a single-counter >> diffractometer >> where one didn't have multiple Bragg reflections on an image or >> film pack. >> That is, each reflection was collected by itself. Even in a small >> molecule >> crystal data collection nowadays, it would not hurt to have the >> crystal >> completely bathed in the beam. >> >> Also in the old days (let's say pre-cryo), there was plenty of >> radiation >> damage going on even with a sealed-tube source. We always >> corrected for >> radiation damage by extrapolating back to zero dose in those days. >> >> Jim >> >> -----Original Message----- >> From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of >> Robert >> Sweet >> Sent: Friday, November 23, 2007 4:08 PM >> To: CCP4BB@JISCMAIL.AC.UK >> Subject: [ccp4bb] To bathe or not to bathe. >> >> Jorge, >> >> You said, >> >>> I remember one former good (small molecule ?) crystallography book >>> with words a kind of this "the crystals should be completely >>> bathed by >>> the x-ray beam during the whole data collection" ... >> >> The original motive for bathing the whole crystal was to assure >> that the >> relative intensity of the data on each successive film pack was very >> nearly constant. This was possible (one might say "necessary") in >> the old >> days because the laboratory sources were very stable and the >> intensity was >> low enough that there wasn't a lot of x-ray damage to the crystals. >> There were a couple of other good reasons to pay attention to >> details like >> this. One was that methods for scaling images together were not >> quite as >> good as now, and another was that film data were relatively very >> much less >> accurate than what is achievable now with excellent detectors and >> brighter >> sources. To combat all of that, we tried to do everything >> possible to >> make things better. >> >> These days scaling algorithms are good, the detectors are >> excellent, and >> very often it pays to employ a beam smaller than the x-tal. This, >> the >> non-uniformity of many synchrotron beams, and the systematic damage >> to crystals that we observe now with synchrotron sources cause >> serious >> systematic errors. We're forced to depend on good scaling and good >> detectors to get accurate measurements. Making the measurements >> in many >> different crystal orientations (redundancy) helps to smooth out these >> systematic errors. >> >> Nonetheless, it will always pay you to watch for EACH of these >> sources of >> error and to minimize them as best you can. >> >> Bob >> >> ===================================================================== >> ==== >> Robert M. Sweet E-Dress: sweet@bnl.gov >> Group Leader, PXRR: Macromolecular ^ (that's L >> Crystallography Research Resource at NSLS >> not 1) >> http://px.nsls.bnl.gov/ >> Biology Dept >> Brookhaven Nat'l Lab. Phones: >> Upton, NY 11973 631 344 3401 (Office) >> U.S.A. 631 344 2741 (Facsimile) >> ===================================================================== >> ==== >> Harry -- Dr Harry Powell, MRC Laboratory of Molecular Biology, MRC Centre, Hills Road, Cambridge, CB2 2QH CCP4bb navigationCCP4bb <-- 1999 <-- November 1999 <-- 30 November 1999 |
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