There are a few things that synchrotron beamlines generally do better
than "home sources", but the most important are flux, collimation and
absorption.
Flux is in photons/s and simply scales down the amount of time it takes
to get a given amount of photons onto the crystal. Contrary to popular
belief, there is nothing "magical" about having more photons/s: it does
not somehow make your protein molecules "behave" and line up in a more
ordered way. However, it does allow you to do the equivalent of a
24-hour exposure in a few seconds (depending on which beamline and which
home source you are comparing), so it can be hard to get your brain
around the comparison.
Collimation, in a nutshell, is putting all the incident photons through
the crystal, preferably in a straight line. Illuminating anything that
isn't the crystal generates background, and background buries weak
diffraction spots (also known as high-resolution spots). Now, when I
say "crystal" I mean the thing you want to shoot, so this includes the
"best part" of a bent, cracked or otherwise inhomogeneous "crystal".
The amount of background goes as the square of the beam size, so a 0.5
mm beam can produce up to 25 times more background than a 0.1 mm beam
(for a fixed spot intensity).
Also, if the beam has high "divergence" (the range of incidence angles
onto the crystal), then the spots on the detector will be more spread
out than if the beam had low divergence, and the more spread-out the
spots are the easier it is for them to fade into the background. Now,
even at home sources, one can cut down the beam to have very low
divergence and a very small size at the sample position, but this comes
at the expense of flux.
Another tenant of "collimation" (in my book) is the DEPTH of non-crystal
stuff in the primary x-ray beam that can be "seen" by the detector.
This includes the air space between the "collimator" and the beam stop.
One millimeter of air generates about as much background as 1 micron of
crystal, water, or plastic. Some home sources have ridiculously large
air paths (like putting the backstop on the detector surface), and that
can give you a lot of background. As a rule of thumb, you want you air
path in mm to be less than or equal to your crystal size in microns. In
this situation, the crystal itself is generating at least as much
background as the air, and so further reducing the air path has
diminishing returns. For example, going from 100 mm air and 100 um
crystal to completely eliminating air will only get you about a 40%
reduction in background noise (it goes as the square root).
Now, this rule of thumb also goes for the "support" material around your
crystal: one micron of cryoprotectant generates about as much background
as one micron of crystal. So, if you have a 10 micron crystal mounted
in a 1 mm thick drop, and manage to hit the crystal with a 10 micron
beam, you still have 100 times more background coming from the drop than
you do from the crystal. This is why in-situ diffraction is so
difficult: it is hard to come by a crystal tray that is the same
thickness as the crystals.
Absorption differences between home and beamline are generally because
beamlines operate at around 1 A, where a 200 um thick crystal or a 200
mm air path absorbs only about 4% of the x-rays, and home sources
generally operate at CuKa, where the same amount of crystal or air
absorbs ~20%. The "absorption correction" due to different paths taken
through the sample must always be less than the total absorption, so you
can imagine the relative difficulty of trying to measure a ~3% anomalous
difference.
Lower absorption also accentuates the benefits of putting the detector
further away. By the way, there IS a good reason why we spend so much
money on large-area detectors. Background falls off with the square of
distance, but the spots don't (assuming good collimation!).
However, the most common cause of drastically different results at
synchrotron vs at home is that people make the mistake of thinking that
all their crystals are the same, and that they prepared them in the
"same" way. This is seldom the case! Probably the largest source of
variability is the cooling rate, which depends on the "head space" of
cold N2 above the liquid nitrogen you are plunge-cooling in (Warkentin
et al. 2006).
-James Holton
MAD Scientist
On 9/28/2010 10:27 AM, Francis E Reyes wrote:
Hi all
I'm interested in the scenario where crystals were screened at home
and gave lousy (say < 8-10A) but when illuminated with synchrotron
radiation gave reasonable diffraction ( > 3A) ? Why the discrepancy?
Thanks
F
---------------------------------------------
Francis E. Reyes M.Sc.
215 UCB
University of Colorado at Boulder
gpg --keyserver pgp.mit.edu --recv-keys 67BA8D5D
8AE2 F2F4 90F7 9640 28BC 686F 78FD 6669 67BA 8D5D