>the top-hat profile is one of the reasons why inhouse machines produce better
>quality data than synchrotrons. However, the often much increased resolution
>you achieve at the synchrotron is generally worth more than the quality of the
>data at restricted resolution.
>
>Cheers,
>Tim
Several surprises to me:
-Data from in-house sources is better?
I have not heard of this--is there any systematic examination of this?
I saw nothing about this in a very brief Google foray.
-In-house beam profiles are top-hats?
Is there a place which shows such measurements? Does not pop out of
Google for me, but I would love to be shown that this is true.
-Resolution at the synchrotron is better?
This does not really seem right to me theoretically, although in
practice it does seem to happen. I think it is just a question of waiting for
enough exposure time, as the CCP4BB response quoted at bottom describes.
JPK
===========================
Date: Tue, 12 Oct 2010 09:04:05 -0700
From: James Holton <jmhol...@lbl.gov>
Re: Re: Lousy diffraction at home but fantastic at the synchrotron?
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
