Thank you James for a very interesting & informative discussion on the
subject.
- Gunnar
Gunnar Olovsson
gun...@byron.biochem.ubc.ca
University of British Columbia
Vancouver, Canada
On Tue, Oct 12, 2010 at 9:04 AM, James Holton <jmhol...@lbl.gov> wrote:
> 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
>>
>
