Hi there,
1. See attached for life-time of crystals ("How long will my crystal lost?") at various beamlines in the world.
2. The citations for the beauty and quality of the datasets from bending magnet beamlines. Please read most of the papers from Dauter group.
Cheers,
-Babu
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Babu A Manjasetty, Ph.D
Case Center for Proteomics
Center for Synchrotron Biosciences
Upton NY 11973
631-344-2568 (office)
[log in to unmask]
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________________________________
From: CCP4 bulletin board on behalf of Nave, C (Colin)
Sent: Mon 11/26/2007 6:50 AM
To: [log in to unmask]
Subject: Re: [ccp4bb] To bathe or not to bathe.
To bathers and non bathers
This is an interesting discussion with several relevant points. I agree
that, if small beams can pick up the best bits of the crystal that is a
very good reason for using them. The background arguments can be
relevant and having the beam size at the detector matched to the
detector resolution is a good idea to obtain the best signal above the
background for weak spots.
Several have raised the issue of radiation damage. The strategy which
Bob mentions can make sense, ensuring fresh parts of the crystal are
regularly brought in to the beam. I would have thought 5-10 micron beams
were rather too small if your crystal is several times bigger than this.
I think the microbeams can overcook the samples, particularly when
collecting large rotation ranges. The centre part of the crystal (if
centred!) is constantly in the beam in this case.
With a crystal of uniform quality, one should try and make use of the
entire volume if radiation damage is an issue. This could be by using
either using the method outlined by Bob or by bathing the whole crystal
in the beam. For large crystals, matching the beam at the crystal to the
crystal size and that at the detector to the detector resolution can
make a significant difference (see for example W. R. Wikoff, W.
Schildkamp and J. E. Johnson Acta Cryst. (2000). D56, 890-893 ,
http://scripts.iucr.org/cgi-bin/paper?S0907444900005941).
We occasionally hear reports (citations needed!) of better data from
bending magnet beamlines and overcooking part of the crystal on an
undulator may be one of the reasons. Undulator beamlines are great
especially for small crystals. However, the parallel nature of the beam
means that it is not always easy to get a big beam at the crystal and a
smaller one at the detector, though it can be done if the set up is
sufficiently flexible.
Colin
-----Original Message-----
From: CCP4 bulletin board [mailto:[log in to unmask]] On Behalf Of
Robert Sweet
Sent: 26 November 2007 00:28
To: [log in to unmask]
Subject: Re: [ccp4bb] To bathe or not to bathe.
Thanks, Ron,
Regarding the "bathing" question, these days the major source of error
we find in synchrotron-based data is crystal damage. Several groups,
notably the two ID23s (one each of pairs of matched canted undulators at
ESRF and
APS) are producing small x-ray beams, on the order of 5-10 micron
diameters, and are saying that the principal use to which they're put is
to shoot a single larger crystal several times. There's a certain
synergy here -- each new exposure is essentially a new and undamaged
tiny crystal, and the orientations of each succesive spot on the crystal
are essentially identical. The orientations abut nearly precisely and
various data-reduction questions are simplified. These days, where
x-tals tend to be smallish and synchrotron access is pretty available,
this seems to us to be an excellent strategy.
Regarding absorption corrections, here at the PXRR/NSLS a whole lot of
data are taken with wavelengths on the order of one Angstroem. At these
wavelengths the absorption of a typical crystal in its mount, even one
that is, say 3M ammonium sulfate, is nearly negligible. On the other
hand, if one tries to optimize S or I anomalous with 1.7 Angstroem
radiation, all bets are off. Here I'm sure >you< would take excellent
data.
About empirical corrections: to employ Tony North's method in about 1969
I took what are formally psi scans (repeat mesurements on a
diffractometer of one reflection at increments of rotation about one
real-lattice vector) on the trypsin/soybean-trypsin-inhibitor complex.
I was using "Wyckoff"
scans* to do this. The crystal was an orthorhombic needle, and I was
rotating about the needle axis. I observed that the rocking curve
varied from narrow to wide, with the extremes being separated by about
180 deg.
I'd jammed the needle into a tapered capillary and it was slightly
bent(!), giving a focused beam in one direction (sharp) and an unfocused
one in the opposite (broad). I'm told this happens these days with
frozen needles sticking out of the gob of vitreous mother liquor.
* Hal Wyckoff (the inventor of the true Inverse Beam Method**) reasoned
that there were three ways to "integrate" a reflection: one could rotate
the crystal through its rocking curve (the rotation method), one could
use a broad bandpass of wavelengths (the Laue method), or one could use
a widely convergent source (the Wyckoff scan). To do this one increased
the take-off angle of the x-ray tube (Ron knows what this is, but those
of you who don't know should ask the oldest crystallographer nearby) so
that the angle subtended from the crystal would fill the rocking curve.
In principal the single largest measurement would equal the integrated
intensity. To hedge ones bets, one did a five-point scan and summed the
top three. This was how I could see the sharpness of the scans.
** Wyckoff also invented a diffractometer that had two opposing x-ray
generators, literally shooting at the crystal from opposite directions,
and then two opposing detectors, each on its own 2-theta arm. One
detector would measure (h,k,l) at precisely the same time the other
would measure (-h,-k,-l). What we do now, to flip the rotation axis by
180 degrees, seeing the (-h,-k,-l) reflections in a mirror image to the
(h,k,l) ones, would better be termed the Friedel Flip.
Keep those cards and letters coming, folks.
Bob
On Sun, 25 Nov 2007, Ronald E Stenkamp wrote:
> Just a few comments on "consider a crystal bathed in a uniform beam".
>
> I've not fully bought into the idea that it's OK to have the beam
> smaller than the crystal. I learned most of my crystallography in a
> lab dedicated to precise structure determinations, and somewhere along
> the line, I picked up the idea that it's better to remove systematic
> errors experimentally than to correct for them computationally.
> (Maybe that had something to do with the computing power and programs
> available at the time?)
>
> Anyway, I thought the reason people went to smaller beams was that it
> made it possible to resolve the spots on the film or detector. Isn't
> that the main reason for using small beams?
>
> In practice, I guess the change in crystal volume actually diffracting
> hasn't been a big issue and that frame-to-frame scaling deals with the
> problem adequately.
>
> I'm less convinced that frame-to-frame scaling can correct for
> absorption very well. Due to our irregular-shaped protein crystals,
> before the area detectors came along, we'd use an empirical correction
> (one due to North comes to mind) based on rotation about the phi axis
> of a four-circle goniostat. It was clearly an approximation to the
> more detailed calculations of path-lengths available for crystals with
> well-defined faces not surrounded by drops of mother liquor and glass
> capillaries. Has anyone checked to see how frame-to-frame scaling
> matches up with analytical determinations of absorption corrections?
> It'd be interesting to determine the validity of the assumption that
> absorption is simply a function of frame number.
>
> Ron
>
--
========================================================================
=
Robert M. Sweet E-Dress: [log in to unmask]
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)
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