>at the beginning of my experience of S-SAD about 10 years ago, it was not too >difficult to do S-SAD phasing with inhouse data provided the resolution was >better than 2.0A, while it did not always work with synchrotron data. Purely >personal experience.
I assume that the synchrotron data were collected at similarly-low energy? >However, the inhouse machines I am familiar with have three circles, so that >you get much better real redundancy with equivalent reflections recorded at >different settings. This reduces systematic errors, I think. The most sophisticated synchrotron beamline I have been to offered a mini-kappa with 30degree range - that's not much compared to 10-20 different settings with varying phi- omega- and distance settings. Yes, I haven't seen much about people collecting multiple orientations of the same crystal, since I think people generally roast their crystals really fast to see higher-resolution spots. I am thinking recently that the best option might really be home sources with pixel-array detectors... >The top-hat comes from a quote I received from Bruker, and I have no reason to >believe the person acted purely with a salesperson's intent. Pretty interesting--wonder what's the best way to confirm this for our home source...? JPK Best, Tim On 01/12/2015 09:05 PM, Keller, Jacob wrote: >> 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 > -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A
