[ccp4bb] Lousy diffraction at home but fantastic at the synchrotron?
Dear Klaus, With extensive travel in the meantime, we're sorry that it took us so long to get back to you on this. Well, the true answer is that any resolution might be expected on the PX Scanner. Thus, sometimes we have observed higher resolutions on the PX Scanner than observed from the 'same' protein crystals using a third generation synchrotron source. The point is that as well as determining the 'native' diffraction qualities (resolution limit, mosaicity and unit cell, etc.) of the chosen crystal, using the PX Scanner one can also establish the efficacy of the cryo-protection and the further 'handling' stages of the crystal workflow, etc. (So, in the cases above, perhaps the cryo-conditions were 'wrong', etc...). Moreover, using the PX Scanner, one can unambiguously identify the best-diffracting individual crystal in any droplet across the whole crystallisation plate: most importantly in situ without any kind of 'disturbance'... And even if the crystals do indeed diffract - on the PX Scanner, to lower resolution than they do on a more powerful source, of course you can still rank them and then pick up the clearly 'best' ones for further analysis. As experienced by many researchers: there can be very significant differences between crystals, even grown in the same drop. You, all, are most welcome to visit our labs, bringing your plates, in order to collect in situ crystal diffraction data: as an evaluation both of your crystals and the PX Scanner instrument. We look forward to this ! Yours, Tadeusz Skarzynski Marcus Winter (Oxford Diffraction Ltd. - now Agilent Technologies) -Original Message- From: Klaus Fütterer [mailto:k.futte...@bham.ac.uk] Sent: 30 September 2010 11:00 To: WINTER,MARCUS (A-Varian,ex1) Cc: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Lousy diffraction at home but fantastic at the synchrotron? Marcus, May I ask the following: assuming 8 A is obtained from a single crystal on the home source, what diffraction limit would one expect on the PX scanner? Best regards, Klaus === Klaus Fütterer, Ph.D. Reader in Structural Biology Undergraduate Admissions School of Biosciences P: +44-(0)-121-414 5895 University of Birmingham F: +44-(0)-121-414 5925 Edgbaston E: k.futte...@bham.ac.uk Birmingham, B15 2TT, UK W: www.biochemistry.bham.ac.uk/klaus/ === On 30 Sep 2010, at 10:44, Marcus Winter wrote: This recent discussion does tend towards the ideal scenario: of identifying ones best-diffracting crystals... before embarking on the synchrotron trip. The established Oxford Diffraction PX Scanner home laboratory instrument can therefore be most useful. This enables the direct X-ray screening of individual (putative) single crystal objects, in situ, in the (any SBS format) crystallisation plate. Yours sincerely, Marcus Winter (Oxford Diffraction Ltd. - now Agilent Technologies) -Original Message- From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Phil Jeffrey Sent: 28 September 2010 19:20 To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Lousy diffraction at home but fantastic at the synchrotron? Often this reflect crystal size - a small crystal in a big beam (or one with a long path in air) on a home source would see the small diffraction signal drop below the noise level quite quickly - often at the low resolution intensity dip that sits very approximately around 6 Angstrom. On a synchrotron source with a tight low-divergence beam that matches more closely the crystal dimensions that same crystal will appear to do rather better. Also one is more likely to expose the crystal longer (in terms of total photon numbers) at a synchrotron, which itself begets better signal/ noise. Alternatively: everyone tries harder before synchrotron trips Phil Jeffrey Princeton On 9/28/10 1:27 PM, 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
Re: [ccp4bb] 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 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
Re: [ccp4bb] Lousy diffraction at home but fantastic at the synchrotron?
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
[ccp4bb] Lousy diffraction at home but fantastic at the synchrotron?
This recent discussion does tend towards the ideal scenario: of identifying ones best-diffracting crystals... before embarking on the synchrotron trip. The established Oxford Diffraction PX Scanner home laboratory instrument can therefore be most useful. This enables the direct X-ray screening of individual (putative) single crystal objects, in situ, in the (any SBS format) crystallisation plate. Yours sincerely, Marcus Winter (Oxford Diffraction Ltd. - now Agilent Technologies) -Original Message- From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Phil Jeffrey Sent: 28 September 2010 19:20 To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Lousy diffraction at home but fantastic at the synchrotron? Often this reflect crystal size - a small crystal in a big beam (or one with a long path in air) on a home source would see the small diffraction signal drop below the noise level quite quickly - often at the low resolution intensity dip that sits very approximately around 6 Angstrom. On a synchrotron source with a tight low-divergence beam that matches more closely the crystal dimensions that same crystal will appear to do rather better. Also one is more likely to expose the crystal longer (in terms of total photon numbers) at a synchrotron, which itself begets better signal/noise. Alternatively: everyone tries harder before synchrotron trips Phil Jeffrey Princeton On 9/28/10 1:27 PM, 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
Re: [ccp4bb] Lousy diffraction at home but fantastic at the synchrotron?
Marcus, May I ask the following: assuming 8 A is obtained from a single crystal on the home source, what diffraction limit would one expect on the PX scanner? Best regards, Klaus === Klaus Fütterer, Ph.D. Reader in Structural Biology Undergraduate Admissions School of Biosciences P: +44-(0)-121-414 5895 University of Birmingham F: +44-(0)-121-414 5925 Edgbaston E: k.futte...@bham.ac.uk Birmingham, B15 2TT, UK W: www.biochemistry.bham.ac.uk/klaus/ === On 30 Sep 2010, at 10:44, Marcus Winter wrote: This recent discussion does tend towards the ideal scenario: of identifying ones best-diffracting crystals... before embarking on the synchrotron trip. The established Oxford Diffraction PX Scanner home laboratory instrument can therefore be most useful. This enables the direct X-ray screening of individual (putative) single crystal objects, in situ, in the (any SBS format) crystallisation plate. Yours sincerely, Marcus Winter (Oxford Diffraction Ltd. – now Agilent Technologies) -Original Message- From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Phil Jeffrey Sent: 28 September 2010 19:20 To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Lousy diffraction at home but fantastic at the synchrotron? Often this reflect crystal size - a small crystal in a big beam (or one with a long path in air) on a home source would see the small diffraction signal drop below the noise level quite quickly - often at the low resolution intensity dip that sits very approximately around 6 Angstrom. On a synchrotron source with a tight low-divergence beam that matches more closely the crystal dimensions that same crystal will appear to do rather better. Also one is more likely to expose the crystal longer (in terms of total photon numbers) at a synchrotron, which itself begets better signal/ noise. Alternatively: everyone tries harder before synchrotron trips Phil Jeffrey Princeton On 9/28/10 1:27 PM, 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
[ccp4bb] Lousy diffraction at home but fantastic at the synchrotron?
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
Re: [ccp4bb] Lousy diffraction at home but fantastic at the synchrotron?
I find this interesting as well, mainly because I have never seen this myself and I have looked at plenty of badly diffracting crystals. In my hands, synchrotron data at most end up being ~1.5 angstrom better in terms of resolution than the same crystals on our home source. I'm wondering if this phenomenon is real, or whether it is just a matter of people screening more/better crystals at the synchrotron compared to at home? Or a lousy home source? Bert On 9/28/10 1:27 PM, Francis E Reyes francis.re...@colorado.edu 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
Re: [ccp4bb] Lousy diffraction at home but fantastic at the synchrotron?
On Tuesday 28 September 2010 10:27:17 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? Such a happy outcome is very rare. I normally expect the high intensity at a beamline to push the line separating weak but visible and too weak to see towards higher resolution usable data. But that isn't a change in the diffracting properties of the crystal, just a change in achievable signal-to-noise. Two possibilities come to mind. A) Unintentional annealing during transport/or and as a consequence of crystal handling after shipment. We've certainly seen this, but usually the result is bad ice rings rather than better diffraction. B) Home screening used a relatively large beam that saw the entirely of a highly imperfect crystal. The synchrotron beam used a much smaller aperture that happened to illuminate only a sweet spot. This is one argument advanced for the use of micro-focus beams. B') An extreme case of B. Multiple crystals in the loop. Home screening caught a bad one; beamline screening caught a good one. -- Ethan A Merritt Biomolecular Structure Center, K-428 Health Sciences Bldg University of Washington, Seattle 98195-7742
Re: [ccp4bb] Lousy diffraction at home but fantastic at the synchrotron?
Often this reflect crystal size - a small crystal in a big beam (or one with a long path in air) on a home source would see the small diffraction signal drop below the noise level quite quickly - often at the low resolution intensity dip that sits very approximately around 6 Angstrom. On a synchrotron source with a tight low-divergence beam that matches more closely the crystal dimensions that same crystal will appear to do rather better. Also one is more likely to expose the crystal longer (in terms of total photon numbers) at a synchrotron, which itself begets better signal/noise. Alternatively: everyone tries harder before synchrotron trips Phil Jeffrey Princeton On 9/28/10 1:27 PM, 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
