HiI'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 beinaccurate, 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 wasespecially 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 andmaking precise measurements of crystal dimensions.Scaling programs are now more extensive, and include options to calculatea 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 moleculecrystal data collection nowadays, it would not hurt to have the crystalcompletely 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 forradiation damage by extrapolating back to zero dose in those days. Jim -----Original Message----- From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Robert Sweet Sent: Friday, November 23, 2007 4:08 PM To: [email protected] Subject: [ccp4bb] To bathe or not to bathe. Jorge, You said,I remember one former good (small molecule ?) crystallography bookwith words a kind of this "the crystals should be completely bathed bythe x-ray beam during the whole data collection" ...The original motive for bathing the whole crystal was to assure that therelative intensity of the data on each successive film pack was verynearly constant. This was possible (one might say "necessary") in the old days because the laboratory sources were very stable and the intensity waslow 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 tomake 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, thenon-uniformity of many synchrotron beams, and the systematic damageto crystals that we observe now with synchrotron sources cause serioussystematic errors. We're forced to depend on good scaling and gooddetectors to get accurate measurements. Making the measurements in manydifferent crystal orientations (redundancy) helps to smooth out these systematic errors.Nonetheless, it will always pay you to watch for EACH of these sources oferror and to minimize them as best you can. Bob===================================================================== ====Robert M. Sweet E-Dress: [EMAIL PROTECTED] Group Leader, PXRR: Macromolecular ^ (that's LCrystallography 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
