Re: [ccp4bb] To bathe or not to bathe.
Oops, sorry. The x axis of the previous plot is actually not resolution, but Q. My bad. Richard On Nov 27, 2007, at 11:26 AM, Richard Gillilan wrote: Just a couple small images that may be of interest. The x scale is resolution in Angstroms, the y scale is intensity (arbitrary units). I don't recall if the plot is corrected for CCD pedestal values, so the difference is not quite as dramatic as x49, still very nice clean background if you are looking for a faint signal. The CCD images below are equal exposures shown with equal contrast settings: HeN2.tiff HeN2imgs.tiff
Re: [ccp4bb] To bathe or not to bathe.
Richard I think the sharp spot is just small angle scattering (e.g. from domain boundaries) resulting from the beam hitting one of the defining apertures in your collimation system. If you ray trace the beam from the last defining aperture, through the guard aperture then to the detector you should get an area of high background where the guard aperture does not cut this off and it is not covered by the beamstop. It then drops off as the beamstop comes in to play appearing to give a sharp spike. In fact, it is quite well set up with the beamstop not overly large. The appearance of a very diffuse ring for the scatter from nitrogen is due to the fact that the beamstop cuts off the air scatter, for the parts of the beam nearer the beamstop. Cheers Colin -Original Message- From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Richard Gillilan Sent: 27 November 2007 16:40 To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] To bathe or not to bathe. Oops, sorry. The x axis of the previous plot is actually not resolution, but Q. My bad. Richard On Nov 27, 2007, at 11:26 AM, Richard Gillilan wrote: Just a couple small images that may be of interest. The x scale is resolution in Angstroms, the y scale is intensity (arbitrary units). I don't recall if the plot is corrected for CCD pedestal values, so the difference is not quite as dramatic as x49, still very nice clean background if you are looking for a faint signal. The CCD images below are equal exposures shown with equal contrast settings: HeN2.tiff HeN2imgs.tiff DIVFONT size=1 color=grayThis e-mail and any attachments may contain confidential, copyright and or privileged material, and are for the use of the intended addressee only. If you are not the intended addressee or an authorised recipient of the addressee please notify us of receipt by returning the e-mail and do not use, copy, retain, distribute or disclose the information in or attached to the e-mail. Any opinions expressed within this e-mail are those of the individual and not necessarily of Diamond Light Source Ltd. Diamond Light Source Ltd. cannot guarantee that this e-mail or any attachments are free from viruses and we cannot accept liability for any damage which you may sustain as a result of software viruses which may be transmitted in or with the message. Diamond Light Source Limited (company no. 4375679). Registered in England and Wales with its registered office at Diamond House, Harwell Science and Innovation Campus, Didcot, Oxfordshire, OX11 0DE, United Kingdom /FONT/DIV
Re: [ccp4bb] To bathe or not to bathe.
To bathers and non bathers This is an interesting discussion with several relevant points. I agree that, if small beams can pick up the best bits of the crystal that is a very good reason for using them. The background arguments can be relevant and having the beam size at the detector matched to the detector resolution is a good idea to obtain the best signal above the background for weak spots. Several have raised the issue of radiation damage. The strategy which Bob mentions can make sense, ensuring fresh parts of the crystal are regularly brought in to the beam. I would have thought 5-10 micron beams were rather too small if your crystal is several times bigger than this. I think the microbeams can overcook the samples, particularly when collecting large rotation ranges. The centre part of the crystal (if centred!) is constantly in the beam in this case. With a crystal of uniform quality, one should try and make use of the entire volume if radiation damage is an issue. This could be by using either using the method outlined by Bob or by bathing the whole crystal in the beam. For large crystals, matching the beam at the crystal to the crystal size and that at the detector to the detector resolution can make a significant difference (see for example W. R. Wikoff, W. Schildkamp and J. E. Johnson Acta Cryst. (2000). D56, 890-893 , http://scripts.iucr.org/cgi-bin/paper?S090744495941). We occasionally hear reports (citations needed!) of better data from bending magnet beamlines and overcooking part of the crystal on an undulator may be one of the reasons. Undulator beamlines are great especially for small crystals. However, the parallel nature of the beam means that it is not always easy to get a big beam at the crystal and a smaller one at the detector, though it can be done if the set up is sufficiently flexible. Colin -Original Message- From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Robert Sweet Sent: 26 November 2007 00:28 To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] To bathe or not to bathe. Thanks, Ron, Regarding the bathing question, these days the major source of error we find in synchrotron-based data is crystal damage. Several groups, notably the two ID23s (one each of pairs of matched canted undulators at ESRF and APS) are producing small x-ray beams, on the order of 5-10 micron diameters, and are saying that the principal use to which they're put is to shoot a single larger crystal several times. There's a certain synergy here -- each new exposure is essentially a new and undamaged tiny crystal, and the orientations of each succesive spot on the crystal are essentially identical. The orientations abut nearly precisely and various data-reduction questions are simplified. These days, where x-tals tend to be smallish and synchrotron access is pretty available, this seems to us to be an excellent strategy. Regarding absorption corrections, here at the PXRR/NSLS a whole lot of data are taken with wavelengths on the order of one Angstroem. At these wavelengths the absorption of a typical crystal in its mount, even one that is, say 3M ammonium sulfate, is nearly negligible. On the other hand, if one tries to optimize S or I anomalous with 1.7 Angstroem radiation, all bets are off. Here I'm sure you would take excellent data. About empirical corrections: to employ Tony North's method in about 1969 I took what are formally psi scans (repeat mesurements on a diffractometer of one reflection at increments of rotation about one real-lattice vector) on the trypsin/soybean-trypsin-inhibitor complex. I was using Wyckoff scans* to do this. The crystal was an orthorhombic needle, and I was rotating about the needle axis. I observed that the rocking curve varied from narrow to wide, with the extremes being separated by about 180 deg. I'd jammed the needle into a tapered capillary and it was slightly bent(!), giving a focused beam in one direction (sharp) and an unfocused one in the opposite (broad). I'm told this happens these days with frozen needles sticking out of the gob of vitreous mother liquor. * Hal Wyckoff (the inventor of the true Inverse Beam Method**) reasoned that there were three ways to integrate a reflection: one could rotate the crystal through its rocking curve (the rotation method), one could use a broad bandpass of wavelengths (the Laue method), or one could use a widely convergent source (the Wyckoff scan). To do this one increased the take-off angle of the x-ray tube (Ron knows what this is, but those of you who don't know should ask the oldest crystallographer nearby) so that the angle subtended from the crystal would fill the rocking curve. In principal the single largest measurement would equal the integrated intensity. To hedge ones bets, one did a five-point scan and summed the top three. This was how I could see the sharpness of the scans. ** Wyckoff also invented a diffractometer that had two opposing x-ray generators
Re: [ccp4bb] To bathe or not to bathe.
I just noticed this thread. I should make a few comments. We regularly provide microbeam with and without Helium here at MacCHESS. Yes, there are cases in which microbeam can give you good diffraction on large crystals when a larger beam cannot. Just last week we had a user group collecting on column-shaped crystals about 40 microns wide by 200-300 microns long. When exposed using a 150 micron diameter beam, the diffraction patterns were a mess, producing multiple lattices. When they exposed the crystals using a 20 micron beam, they were able to find enough sweet spots with single lattice to give good results. I can only offer anecdotes at this time, but, based on user experiences so far, this is not uncommon at all. I don't usually think of microbeam alone as a significant reduction in background except that one avoids hitting solvent (which is a major source of scatter!). While the beam scatters less air due to its small size, microbeams also can have much increased flux which compensates for the smaller size ... so I don't think there will be less air scatter unless you are just using an aperture with no flux gain. Regarding helium: we regularly use helium at 95K combined with a helium enclosure. The setup is awkward for manual sample mounting, but quite convenient for automounting. The main advantage here is that direct beam scattering with air is nearly eliminated. How important is this effect? It depends upon how much free path there is between the end of the optic (slits, etc) and the beamstop. It also depends upon how small your sample is and how strongly it diffracts. In a carefully-controlled experiment using the identical crystal and orientation range with both nitrogen and helium, I saw a signal-to-noise improvement by over a factor of 3 in the 2.5 Angstrom range and lower. At higher resolution the benefit decreases, but still looks no worse than a factor of 2. This is with a 50 micron crystal illuminated with an 18 micron beam. I am currently working on guidelines for when helium and microbeam are necessary (based on both simulations and explicit measurements). At the present time, my feeling is that crystals below 50 micron can certainly make the extra hassle worthwhile. It really depends upon how badly you want that extra resolution. In the case above, it pushed the resolution from above to below the 2.0 Angstrom mark based on I/SIG. As for radiation damage, I do think it is important that a beam intensity profile be as flat at the top as possible (not a sharp hotspot) .. so defocusing a little may be useful. This is not usually a factor that regular users have any control over. I don't have any hard data on this. Richard Gillilan MacCHESS
Re: [ccp4bb] To bathe or not to bathe.
Richard Gillilan wrote: I am currently working on guidelines for when helium and microbeam are necessary (based on both simulations and explicit measurements). At the present time, my feeling is that crystals below 50 micron can certainly make the extra hassle worthwhile. It really depends upon how badly you want that extra resolution. In the case above, it pushed the resolution from above to below the 2.0 Angstrom mark based on I/SIG. Hi Richard, I/SigI based on which program ? Default modes or tweaked by expert ? I would give this particular dataset a chance to be processed by all available programs and then do the comparison, or actualy all the datasets you have with various setups. Should be a nice table comparing program X versus Y and Z with the given data. And if it's SeMet data that would even be better - runnig e.g. Shelx and demonstrating which setup leads to an interpretable electron density. Juergen -- Jürgen Bosch University of Washington Dept. of Biochemistry, K-426 1705 NE Pacific Street Seattle, WA 98195 Box 357742 Phone: +1-206-616-4510 FAX: +1-206-685-7002 Web: http://faculty.washington.edu/jbosch
Re: [ccp4bb] To bathe or not to bathe.
Note the density of air is approximately 1000 times less than a protein crystal. The total scatter for a beam going through a 50 micron thick crystal will be similar to that from 50mm air. Most beamlines will have a path length less than this but nevertheless the air scatter will be significant with small crystals. In principle, with smaller beams one can have smaller beamstops nearer the sample thus reducing the path length through the air. Colin -Original Message- From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Richard Gillilan Sent: 26 November 2007 16:24 To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] To bathe or not to bathe. I just noticed this thread. I should make a few comments. We regularly provide microbeam with and without Helium here at MacCHESS. Yes, there are cases in which microbeam can give you good diffraction on large crystals when a larger beam cannot. Just last week we had a user group collecting on column-shaped crystals about 40 microns wide by 200-300 microns long. When exposed using a 150 micron diameter beam, the diffraction patterns were a mess, producing multiple lattices. When they exposed the crystals using a 20 micron beam, they were able to find enough sweet spots with single lattice to give good results. I can only offer anecdotes at this time, but, based on user experiences so far, this is not uncommon at all. I don't usually think of microbeam alone as a significant reduction in background except that one avoids hitting solvent (which is a major source of scatter!). While the beam scatters less air due to its small size, microbeams also can have much increased flux which compensates for the smaller size ... so I don't think there will be less air scatter unless you are just using an aperture with no flux gain. Regarding helium: we regularly use helium at 95K combined with a helium enclosure. The setup is awkward for manual sample mounting, but quite convenient for automounting. The main advantage here is that direct beam scattering with air is nearly eliminated. How important is this effect? It depends upon how much free path there is between the end of the optic (slits, etc) and the beamstop. It also depends upon how small your sample is and how strongly it diffracts. In a carefully-controlled experiment using the identical crystal and orientation range with both nitrogen and helium, I saw a signal-to-noise improvement by over a factor of 3 in the 2.5 Angstrom range and lower. At higher resolution the benefit decreases, but still looks no worse than a factor of 2. This is with a 50 micron crystal illuminated with an 18 micron beam. I am currently working on guidelines for when helium and microbeam are necessary (based on both simulations and explicit measurements). At the present time, my feeling is that crystals below 50 micron can certainly make the extra hassle worthwhile. It really depends upon how badly you want that extra resolution. In the case above, it pushed the resolution from above to below the 2.0 Angstrom mark based on I/SIG. As for radiation damage, I do think it is important that a beam intensity profile be as flat at the top as possible (not a sharp hotspot) .. so defocusing a little may be useful. This is not usually a factor that regular users have any control over. I don't have any hard data on this. Richard Gillilan MacCHESS DIVFONT size=1 color=grayThis e-mail and any attachments may contain confidential, copyright and or privileged material, and are for the use of the intended addressee only. If you are not the intended addressee or an authorised recipient of the addressee please notify us of receipt by returning the e-mail and do not use, copy, retain, distribute or disclose the information in or attached to the e-mail. Any opinions expressed within this e-mail are those of the individual and not necessarily of Diamond Light Source Ltd. Diamond Light Source Ltd. cannot guarantee that this e-mail or any attachments are free from viruses and we cannot accept liability for any damage which you may sustain as a result of software viruses which may be transmitted in or with the message. Diamond Light Source Limited (company no. 4375679). Registered in England and Wales with its registered office at Diamond House, Harwell Science and Innovation Campus, Didcot, Oxfordshire, OX11 0DE, United Kingdom /FONT/DIV
Re: [ccp4bb] To bathe or not to bathe.
Hi Juergen, the original calculation was done with I/SIG's from scala. Yes, I am aware of the problems obtaining reliable and meaningful I/SIG with CCD data. I have gone through the exercise of trying to get agreement between scala and scalepack by optimizing error model parameters ... though not yet with this particular dataset. Keep in mind that these are nearly identical datasets (actually the same Bragg reflections), so it is a relative improvement figure and not absolute I/SIG that are relevant here. I had not thought to go as far as density comparison. That's a good idea. Unfortunately the datasets were kept incomplete on purpose to reduce possible radiation damage effects. Richard On Nov 26, 2007, at 11:38 AM, Juergen Bosch wrote: Richard Gillilan wrote: I am currently working on guidelines for when helium and microbeam are necessary (based on both simulations and explicit measurements). At the present time, my feeling is that crystals below 50 micron can certainly make the extra hassle worthwhile. It really depends upon how badly you want that extra resolution. In the case above, it pushed the resolution from above to below the 2.0 Angstrom mark based on I/SIG. Hi Richard, I/SigI based on which program ? Default modes or tweaked by expert ? I would give this particular dataset a chance to be processed by all available programs and then do the comparison, or actualy all the datasets you have with various setups. Should be a nice table comparing program X versus Y and Z with the given data. And if it's SeMet data that would even be better - runnig e.g. Shelx and demonstrating which setup leads to an interpretable electron density. Juergen -- Jürgen Bosch University of Washington Dept. of Biochemistry, K-426 1705 NE Pacific Street Seattle, WA 98195 Box 357742 Phone: +1-206-616-4510 FAX: +1-206-685-7002 Web: http://faculty.washington.edu/jbosch
Re: [ccp4bb] To bathe or not to bathe.
In our current helium box, there is a total of about 28 mm of beam exposed. 10 mm from the aperture of the optic and 18 mm from sample to beamstop. The 10 mm side working distance is very tight for hand mounting (little room for tongs) and falls just outside the shield stream for cryo. Could probably decrease the 28 mm some, but not below 10 mm I think. I think our standard setup places the collimator ion chamber about 20 mm from sample ... so 30-50 mm may not be far off the mark for typical stations. Usually, it is the virus crystallographers who are fussy about getting the smallest beamstop. Next time, I'll have to whip out a ruler and see what distance makes them happy. It would be interesting to know typical distances for other facilities. Anyone else made these measurements? Richard Gillilan MacCHESS Note the density of air is approximately 1000 times less than a protein crystal. The total scatter for a beam going through a 50 micron thick crystal will be similar to that from 50mm air. Most beamlines will have a path length less than this but nevertheless the air scatter will be significant with small crystals. In principle, with smaller beams one can have smaller beamstops nearer the sample thus reducing the path length through the air. Colin
Re: [ccp4bb] To bathe or not to bathe.
Concerning the bathing, I think there is at least this point, which I do not think has been mentioned, to wit: Considering that radiation damage is now present, I would guess, in virtually all synchrotron-collected datasets, and considering that probably this information could be used in virtually all cases to help phase the data or at least be corrected for, it really makes sense to bathe the crystal completely, as this way all of the crystal is exposed evenly (assuming radiation damage is not direction-specific, and assuming the beam flux is evenly distributed across the beam cross-section). This way, the damage should be a simple function of time, not confounded with various parts of the crystal entering or exiting the beam. Therefore, if one wants either to use or correct for the radiation damage, it seems obvious to me that, concerning this aspect, bathing is superior. Enjoying the debate, Jacob Keller *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program Dallos Laboratory F. Searle 1-240 2240 Campus Drive Evanston IL 60208 lab: 847.467.4049 cel: 773.608.9185 email: [EMAIL PROTECTED] *** - Original Message - From: Richard Gillilan [EMAIL PROTECTED] To: CCP4BB@JISCMAIL.AC.UK Sent: Monday, November 26, 2007 12:02 PM Subject: Re: [ccp4bb] To bathe or not to bathe. In our current helium box, there is a total of about 28 mm of beam exposed. 10 mm from the aperture of the optic and 18 mm from sample to beamstop. The 10 mm side working distance is very tight for hand mounting (little room for tongs) and falls just outside the shield stream for cryo. Could probably decrease the 28 mm some, but not below 10 mm I think. I think our standard setup places the collimator ion chamber about 20 mm from sample ... so 30-50 mm may not be far off the mark for typical stations. Usually, it is the virus crystallographers who are fussy about getting the smallest beamstop. Next time, I'll have to whip out a ruler and see what distance makes them happy. It would be interesting to know typical distances for other facilities. Anyone else made these measurements? Richard Gillilan MacCHESS Note the density of air is approximately 1000 times less than a protein crystal. The total scatter for a beam going through a 50 micron thick crystal will be similar to that from 50mm air. Most beamlines will have a path length less than this but nevertheless the air scatter will be significant with small crystals. In principle, with smaller beams one can have smaller beamstops nearer the sample thus reducing the path length through the air. Colin
Re: [ccp4bb] To bathe or not to bathe.
Folks, As expected, discussion about to bathe or not to bathe are starting to expand into other aspects of data collection and processing. Along the way I saw reference to how we use our small beams on GM/CA beamlines in sector 23 of APS. Without going into discussions of advantages and disadvantages of various beam sizes, I'd like to clarify our usage of small beams. Our mini-beams (5-10 micron) are not a primary tool and are being used when larger (100x25 um) beams have had problems. These problems include Small crystals (less than 20-30 um) Streaky spots (from any size crystal) Very high mosaicity Inconsistent quality (order) throughout the crystal When larger crystal is more or less homogeneous in its quality, larger beams are superior to be used and allow larger diffracting volume to work for you. This is especially true for avoiding the radiation damage as larger volumes allow equally lower flux to be used. We collect data from larger crystals and 5-10 um beams only when one or more of the above problems need to be resolved. For those who prefer bathing, the recommendation still is to keep the beam size closer to that of the sample. Air scatter, produced by the (part of the) beam which is not contributing to the Bragg spots, is still noticeable at most typical energies. Cheers, N. Ruslan Sanishvili (Nukri), Ph.D. GM/CA-CAT, Bld. 436, D007 Biosciences Division, ANL 9700 S. Cass Ave. Argonne, IL 60439 Tel: (630)252-0665 Fax: (630)252-0667 [EMAIL PROTECTED]
Re: [ccp4bb] To bathe or not to bathe.
- Original Message - From: Nave, C (Colin) [EMAIL PROTECTED] Several have raised the issue of radiation damage. The strategy which Bob mentions can make sense, ensuring fresh parts of the crystal are regularly brought in to the beam. I would have thought 5-10 micron beams were rather too small if your crystal is several times bigger than this. I think the microbeams can overcook the samples, particularly when collecting large rotation ranges. The centre part of the crystal (if centred!) is constantly in the beam in this case. On the other hand, why perfectly centre the crystal? A set-up where a narrow beam runs past rather than through the rotation axis will spare the central voxel entirely; apart from a somewhat more often exposed doughnut around the axis, only fresh parts of the crystal are illuminated that way. Sebastiaan Werten.
Re: [ccp4bb] To bathe or not to bathe.
Manjasetty, Babu wrote: Hi there, 1. See attached for life-time of crystals (How long will my crystal lost?) at various beamlines in the world. 2. The citations for the beauty and quality of the datasets from bending magnet beamlines. Please read most of the papers from Dauter group. Cheers, -Babu Well, it's certainly interesting to see how far my little document has gone! As one might infer from the URL in the header of the PDF Babu has just posted, I made this document for the radiation damage session at ACA this year. So, I thought I should take this moment to claim responsibility for it. If anyone out there feels like this document has errors or omissions, please do not flame Babu about it. Send all complaints and corrections to [EMAIL PROTECTED] Send them quickly too, I'm getting the impression that I should publish this document properly. As for the original question that launched this thread: I generally advise my users to use a beam that is the same size as their crystal. Bathing is important, but after you are clean, it is a waste. IMHO, The only time you want to use a beam smaller than your crystal is if there are parts of your crystal you want to avoid. An example given already is a needle crystal that has been bent. It is not hard to imagine a bent needle crystal as a pile of plate crystals stacked along a curved path. Thus the bend results in a wider mosaic spread. Using a smaller beam will make the mosaic spread smaller because plates nearer each other in the bent needle have more similar orientations. The only time you want to use a beam larger than your crystal is if you think you can live with the extra background. An over-sized beam obviously simplifies the crystal-centering problem as well as the flicker noise induced if the crystal is vibrating. However, in a properly done experiment (a centered crystal that doesn't shake), there is no real advantage to using an over-sized beam. Since the strength of diffraction spots is proportional to the crystal volume (roughly the third power of the linear size) as your shoot smaller and smaller crystals, the background will eventually kill you. I measured the background on ALS 8.3.1 on an absolute scale here: http://bl831.als.lbl.gov/~jamesh/pickup/background_scatter.png The rule of thumb (as Colin pointed out) is that 1 mm of air is about equal to 1 micron of sample. This is because the air and the sample are basically made of the same stuff (nitrogen) and air is ~1/1000 the density of a protein crystal. As you go to higher and higher angles, the form factor of water, protein or most any substance will approach that of a single atom of that substance. Hence, the rule of thumb applies in the domain of high-res spots. WRT helium paths: Scattering is proportional to the square of the number of electrons, so (at high res) He will produce (7/2)^2 ~1/12 the background from nitrogen (air). When it comes to forward scattering, N2 has 14 electrons in it, not 7. So, the difference in low-res scattering from N2 vs He is a factor of 49. My colleague Scott Classen has verified this experimentally: http://bl1231.als.lbl.gov/2006/05/helium_box.php So I would say a He box is certainly worth it, as long as your sample (crystal and surrounding material) is on the order of ~1/1000th the air path (or smaller). It is also important to bear in mind that not all of the background you see on a no-sample image is air scatter. A significant contribution to background can come from the pinhole/slits, or even from stuff upstream of them. The everything else line in the background_scatter.png graph above is generated by subtracting the calibrated air scatter from a no sample image. Note the sharp spike near the beamstop. I'm not sure what this is, but it is NOT air scatter. It is also not x-ray fluorescence from the pinhole or beamstop. I am still trying to figure out exactly what kind of physics produces these photons, but diffuse scatter from the pinhole (if there is such a thing) or SAXS from the mirrors may be culprits. If anyone has any ideas about what could give you a sharp spike just around the beamstop, I'd like to hear it. -James Holton MAD Scientist
Re: [ccp4bb] To bathe or not to bathe.
Just a few comments on consider a crystal bathed in a uniform beam. I've not fully bought into the idea that it's OK to have the beam smaller than the crystal. I learned most of my crystallography in a lab dedicated to precise structure determinations, and somewhere along the line, I picked up the idea that it's better to remove systematic errors experimentally than to correct for them computationally. (Maybe that had something to do with the computing power and programs available at the time?) Anyway, I thought the reason people went to smaller beams was that it made it possible to resolve the spots on the film or detector. Isn't that the main reason for using small beams? In practice, I guess the change in crystal volume actually diffracting hasn't been a big issue and that frame-to-frame scaling deals with the problem adequately. I'm less convinced that frame-to-frame scaling can correct for absorption very well. Due to our irregular-shaped protein crystals, before the area detectors came along, we'd use an empirical correction (one due to North comes to mind) based on rotation about the phi axis of a four-circle goniostat. It was clearly an approximation to the more detailed calculations of path-lengths available for crystals with well-defined faces not surrounded by drops of mother liquor and glass capillaries. Has anyone checked to see how frame-to-frame scaling matches up with analytical determinations of absorption corrections? It'd be interesting to determine the validity of the assumption that absorption is simply a function of frame number. Ron
Re: [ccp4bb] To bathe or not to bathe.
It'd be interesting to determine the validity of the assumption that absorption is simply a function of frame number. ... and direction. See, eg: Acta Cryst. (1995). A51, 33-38[ doi:10.1107/S0108767394005726 ] An empirical correction for absorption anisotropy R. H. Blessing Best, Jon
Re: [ccp4bb] To bathe or not to bathe.
Thanks, Ron, Regarding the bathing question, these days the major source of error we find in synchrotron-based data is crystal damage. Several groups, notably the two ID23s (one each of pairs of matched canted undulators at ESRF and APS) are producing small x-ray beams, on the order of 5-10 micron diameters, and are saying that the principal use to which they're put is to shoot a single larger crystal several times. There's a certain synergy here -- each new exposure is essentially a new and undamaged tiny crystal, and the orientations of each succesive spot on the crystal are essentially identical. The orientations abut nearly precisely and various data-reduction questions are simplified. These days, where x-tals tend to be smallish and synchrotron access is pretty available, this seems to us to be an excellent strategy. Regarding absorption corrections, here at the PXRR/NSLS a whole lot of data are taken with wavelengths on the order of one Angstroem. At these wavelengths the absorption of a typical crystal in its mount, even one that is, say 3M ammonium sulfate, is nearly negligible. On the other hand, if one tries to optimize S or I anomalous with 1.7 Angstroem radiation, all bets are off. Here I'm sure you would take excellent data. About empirical corrections: to employ Tony North's method in about 1969 I took what are formally psi scans (repeat mesurements on a diffractometer of one reflection at increments of rotation about one real-lattice vector) on the trypsin/soybean-trypsin-inhibitor complex. I was using Wyckoff scans* to do this. The crystal was an orthorhombic needle, and I was rotating about the needle axis. I observed that the rocking curve varied from narrow to wide, with the extremes being separated by about 180 deg. I'd jammed the needle into a tapered capillary and it was slightly bent(!), giving a focused beam in one direction (sharp) and an unfocused one in the opposite (broad). I'm told this happens these days with frozen needles sticking out of the gob of vitreous mother liquor. * Hal Wyckoff (the inventor of the true Inverse Beam Method**) reasoned that there were three ways to integrate a reflection: one could rotate the crystal through its rocking curve (the rotation method), one could use a broad bandpass of wavelengths (the Laue method), or one could use a widely convergent source (the Wyckoff scan). To do this one increased the take-off angle of the x-ray tube (Ron knows what this is, but those of you who don't know should ask the oldest crystallographer nearby) so that the angle subtended from the crystal would fill the rocking curve. In principal the single largest measurement would equal the integrated intensity. To hedge ones bets, one did a five-point scan and summed the top three. This was how I could see the sharpness of the scans. ** Wyckoff also invented a diffractometer that had two opposing x-ray generators, literally shooting at the crystal from opposite directions, and then two opposing detectors, each on its own 2-theta arm. One detector would measure (h,k,l) at precisely the same time the other would measure (-h,-k,-l). What we do now, to flip the rotation axis by 180 degrees, seeing the (-h,-k,-l) reflections in a mirror image to the (h,k,l) ones, would better be termed the Friedel Flip. Keep those cards and letters coming, folks. Bob On Sun, 25 Nov 2007, Ronald E Stenkamp wrote: Just a few comments on consider a crystal bathed in a uniform beam. I've not fully bought into the idea that it's OK to have the beam smaller than the crystal. I learned most of my crystallography in a lab dedicated to precise structure determinations, and somewhere along the line, I picked up the idea that it's better to remove systematic errors experimentally than to correct for them computationally. (Maybe that had something to do with the computing power and programs available at the time?) Anyway, I thought the reason people went to smaller beams was that it made it possible to resolve the spots on the film or detector. Isn't that the main reason for using small beams? In practice, I guess the change in crystal volume actually diffracting hasn't been a big issue and that frame-to-frame scaling deals with the problem adequately. I'm less convinced that frame-to-frame scaling can correct for absorption very well. Due to our irregular-shaped protein crystals, before the area detectors came along, we'd use an empirical correction (one due to North comes to mind) based on rotation about the phi axis of a four-circle goniostat. It was clearly an approximation to the more detailed calculations of path-lengths available for crystals with well-defined faces not surrounded by drops of mother liquor and glass capillaries. Has anyone checked to see how frame-to-frame scaling matches up with analytical determinations of absorption corrections? It'd be interesting to determine the validity of the
Re: [ccp4bb] To bathe or not to bathe.
On Sunday 25 November 2007 14:43, Ronald E Stenkamp wrote: Just a few comments on consider a crystal bathed in a uniform beam. Anyway, I thought the reason people went to smaller beams was that it made it possible to resolve the spots on the film or detector. Isn't that the main reason for using small beams? If you mean that the projected image of the crystal onto the detector is smaller because of a smaller beam, I think could only be relevant in the case of truly huge crystals. On the other hand, as mentioned earlier in this thread, there is a possibility that a small beam will illuminate a sweet spot on the crystal with lower mosaicity. In that case yes, the smaller beam may make it possible to resolve spots that would otherwise overlap due to high mosaicity. I think that is the strongest argument being advanced recently for the use of micro-beam apparatus. The other argument is that a smaller beam will generate lower background due to air-scatter. So for weakly diffracting crystals you want a beam that is no bigger than the crystal, as any part of the beam that doesn't hit the crystal contributes to the background but not to the signal. This is true, but if we really took the air-scatter argument seriously we would go back to the days of huge Helium-filled enclosures to get rid of the air scatter. Some beamlines currently do direct He outflow from the collimator toward the crystal, which reduces air scatter by the indident beam, but I have not seen many beamline helium box setups to reduce also the air scatter from the diffracted beams. I'm less convinced that frame-to-frame scaling can correct for absorption very well. Due to our irregular-shaped protein crystals, before the area detectors came along, we'd use an empirical correction (one due to North comes to mind) based on rotation about the phi axis of a four-circle goniostat. The current scaling algorithms for area detectors do more than generate a frame-to-frame scale. Separate correction factors are routinely calculated for different regions of the diffraction image. These map back onto a set of approximately equal X-ray paths through the crystal. Furthermore, the 3D profile fitting done by some processing programs is a logical extension of those same empirical corrections that we did back in the 70s. It'd be interesting to determine the validity of the assumption that absorption is simply a function of frame number. I don't think any of the current generation of programs make that assumption. But maybe I'm giving them too much credit? -- Ethan A Merritt Biomolecular Structure Center University of Washington, Seattle 98195-7742
Re: [ccp4bb] To bathe or not to bathe.
This is true, but if we really took the air-scatter argument seriously we would go back to the days of huge Helium-filled enclosures to get rid of the air scatter. Some beamlines currently do direct He outflow from the collimator toward the crystal, which reduces air scatter by the indident beam, but I have not seen many beamline helium box setups to reduce also the air scatter from the diffracted beams. Reducing air scatter between the collimator and beamstop makes the most significant reduction due to x-ray induced scattering background. Simply thinking, calculate the intensity times path-length before the beamstop and compare to the scattered beam intensity times the diffracted path length to estimate. This would suggest air-scatter from the diffracted beams (even totaled up) is small. Absorption/attenuation of the diffracted beam is an issue that can be addressed by the He-box, and this air scatter should be balanced against window(s) on the He-box that also absorb. My impression is that there are a few extreme cases where He box improves data quality, but that this is rarer than the number of cases where it is used.it would be nice to have someone perform a systematic study of this across a number of condition cases. IMHO mini-beams (at least those which retain a small divergence) are critical for small crystals and rods where otherwise multiple crystals would be needed, and seem to make a significant difference a number of cases, including mosiacity scanning as was earlier mentioned. - Thomas Thomas Earnest, Ph.D. Senior Scientist and Group Leader Structural Proteomics Development Group Physical Biosciences Division MS64R0121 Lawrence Berkeley National Laboratory Berkeley CA 94720 [EMAIL PROTECTED] 510 486 4603 Ethan A Merritt wrote: On Sunday 25 November 2007 14:43, Ronald E Stenkamp wrote: Just a few comments on consider a crystal bathed in a uniform beam. Anyway, I thought the reason people went to smaller beams was that it made it possible to resolve the spots on the film or detector. Isn't that the main reason for using small beams? If you mean that the projected image of the crystal onto the detector is smaller because of a smaller beam, I think could only be relevant in the case of truly huge crystals. On the other hand, as mentioned earlier in this thread, there is a possibility that a small beam will illuminate a sweet spot on the crystal with lower mosaicity. In that case yes, the smaller beam may make it possible to resolve spots that would otherwise overlap due to high mosaicity. I think that is the strongest argument being advanced recently for the use of micro-beam apparatus. The other argument is that a smaller beam will generate lower background due to air-scatter. So for weakly diffracting crystals you want a beam that is no bigger than the crystal, as any part of the beam that doesn't hit the crystal contributes to the background but not to the signal. This is true, but if we really took the air-scatter argument seriously we would go back to the days of huge Helium-filled enclosures to get rid of the air scatter. Some beamlines currently do direct He outflow from the collimator toward the crystal, which reduces air scatter by the indident beam, but I have not seen many beamline helium box setups to reduce also the air scatter from the diffracted beams. I'm less convinced that frame-to-frame scaling can correct for absorption very well. Due to our irregular-shaped protein crystals, before the area detectors came along, we'd use an empirical correction (one due to North comes to mind) based on rotation about the phi axis of a four-circle goniostat. The current scaling algorithms for area detectors do more than generate a frame-to-frame scale. Separate correction factors are routinely calculated for different regions of the diffraction image. These map back onto a set of approximately equal X-ray paths through the crystal. Furthermore, the 3D profile fitting done by some processing programs is a logical extension of those same empirical corrections that we did back in the 70s. It'd be interesting to determine the validity of the assumption that absorption is simply a function of frame number. I don't think any of the current generation of programs make that assumption. But maybe I'm giving them too much credit?
Re: [ccp4bb] To bathe or not to bathe.
Hi I'm not convinced that the first sentence here has much to do with the second (although both might be true). The main reason was related to absorption. If you didn't completely bathe the crystal in the xray beam, then the diffracting volume of the crystal would be different during the data collection, and thus, scaling would be inaccurate, especially when there was radiation damage. Absorption can be a real problem when the path length through the crystal differs significantly, and is often not closely related to the diffracting volume - think of different paths through a flat plate or a needle. This is the main reason why old-fashioned crystallographers in days of yore used to grind their crystals into a sphere. Absorption problems are exacerbated when you have atoms present in your sample that absorb heavily (this should not be a surprise...). As a former small-molecule crystallographer, I always made sure the crystal was bathed in the beam (so that the diffracting volume was the same), and usually tried to make sure the the whole crystal was in the central part of the beam (to try to make sure I was using the more uniform part of the beam). When I moved to macromolecular work, I found that most people seemed to prefer to get the whole beam going through the crystal, and not worry too much about bits of crystal hanging off outside the beam. There are, of course, reasons for this, among which is that small molecule crystallographers are often spoilt for choice when it comes to picking out the right crystal, and protein crystallographers aren't (at least when I made the switch); they often needed all the crystals they could get in order to get a single dataset - this was in the days before cryo was standard, and room temperature data collection was de rigueur. While practising small molecule crystallography, I still needed to apply absorption corrections to the data, especially where I had loads of strongly absorbing atoms (e.g. in third-row transition metal clusters), in order to both solve and refine the structures. The absorption corrections I used were based on different methods (psi- scans, analytical corrections based on the making precise measurements of the crystal itself, and the (ahem) wonderful Walker Stuart DIFABS (which could turn a pig's ear into a silk purse, in spite of what the purists might say about what it was actually doing to the data!)). just my two ha'porth... This was especially true when you weren't sure that the crystal was well- centered in the xray beam (in a cryostat, and therefore not visible). We typically collected highly redundant data to help compensate for this. We also used to correct for absorption by assigning Bragg indices to the crystal and making precise measurements of crystal dimensions. Scaling programs are now more extensive, and include options to calculate a pseudo-absorption surface. In principle, if you have a beam that is ALWAYS smaller than the crystal, then the same crystal volume is illuminated by the xray beam, and will minimize scaling errors. Bernie Santarsiero On Fri, November 23, 2007 4:34 pm, Jim Pflugrath wrote: It probably goes back to the days of using a single-counter diffractometer where one didn't have multiple Bragg reflections on an image or film pack. That is, each reflection was collected by itself. Even in a small molecule crystal data collection nowadays, it would not hurt to have the crystal completely bathed in the beam. Also in the old days (let's say pre-cryo), there was plenty of radiation damage going on even with a sealed-tube source. We always corrected for radiation damage by extrapolating back to zero dose in those days. Jim -Original Message- From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Robert Sweet Sent: Friday, November 23, 2007 4:08 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] To bathe or not to bathe. Jorge, You said, I remember one former good (small molecule ?) crystallography book with words a kind of this the crystals should be completely bathed by the x-ray beam during the whole data collection ... The original motive for bathing the whole crystal was to assure that the relative intensity of the data on each successive film pack was very nearly constant. This was possible (one might say necessary) in the old days because the laboratory sources were very stable and the intensity was low enough that there wasn't a lot of x-ray damage to the crystals. There were a couple of other good reasons to pay attention to details like this. One was that methods for scaling images together were not quite as good as now, and another was that film data were relatively very much less accurate than what is achievable now with excellent detectors and brighter sources. To combat all of that, we tried to do everything possible to make things better
Re: [ccp4bb] To bathe or not to bathe.
One additional point to add not raised by Bob is that crystals are different. So you can shoot at one end of the crystal and say have a mosaicity of 0.2 degrees but somewhere else it might be 1.4 or even worse. In such cases e.g. rod like needles it pays off to have a smaller than crystal beam and walk over you crystal for the best spot to collect your dataset. Jürgen Robert Sweet wrote: Jorge, You said, I remember one former good (small molecule ?) crystallography book with words a kind of this the crystals should be completely bathed by the x-ray beam during the whole data collection and also some other concerns about beam homogeneity in its cross section. How serious is this nowadays ? Can processing programs easily overcome, in a certain mounting, the fact that not all crystal orientations have the same number of unit cells exposed to x-rays ? What about inhomogeneities at the beam ? I understand that technical difficulties may lead you to exposed your crystal partially to the beam, etc..., but how hard should we care about this (how much effort to avoid this) ? The original motive for bathing the whole crystal was to assure that the relative intensity of the data on each successive film pack was very nearly constant. This was possible (one might say necessary) in the old days because the laboratory sources were very stable and the intensity was low enough that there wasn't a lot of x-ray damage to the crystals. There were a couple of other good reasons to pay attention to details like this. One was that methods for scaling images together were not quite as good as now, and another was that film data were relatively very much less accurate than what is achievable now with excellent detectors and brighter sources. To combat all of that, we tried to do everything possible to make things better. These days scaling algorithms are good, the detectors are excellent, and very often it pays to employ a beam smaller than the x-tal. This, the non-uniformity of many synchrotron beams, and the systematic damage to crystals that we observe now with synchrotron sources cause serious systematic errors. We're forced to depend on good scaling and good detectors to get accurate measurements. Making the measurements in many different crystal orientations (redundancy) helps to smooth out these systematic errors. Nonetheless, it will always pay you to watch for EACH of these sources of error and to minimize them as best you can. Bob = Robert M. Sweet E-Dress: [EMAIL PROTECTED] Group Leader, PXRR: Macromolecular ^ (that's L Crystallography Research Resource at NSLSnot 1) http://px.nsls.bnl.gov/ Biology Dept Brookhaven Nat'l Lab. Phones: Upton, NY 11973631 344 3401 (Office) U.S.A. 631 344 2741 (Facsimile) = -- Jürgen Bosch University of Washington Dept. of Biochemistry, K-426 1705 NE Pacific Street Seattle, WA 98195 Box 357742 Phone: +1-206-616-4510 FAX: +1-206-685-7002 Web: http://faculty.washington.edu/jbosch
Re: [ccp4bb] To bathe or not to bathe.
The main reason was related to absorption. If you didn't completely bathe the crystal in the xray beam, then the diffracting volume of the crystal would be different during the data collection, and thus, scaling would be inaccurate, especially when there was radiation damage. This was especially true when you weren't sure that the crystal was well-centered in the xray beam (in a cryostat, and therefore not visible). We typically collected highly redundant data to help compensate for this. We also used to correct for absorption by assigning Bragg indices to the crystal and making precise measurements of crystal dimensions. Scaling programs are now more extensive, and include options to calculate a pseudo-absorption surface. In principle, if you have a beam that is ALWAYS smaller than the crystal, then the same crystal volume is illuminated by the xray beam, and will minimize scaling errors. Bernie Santarsiero On Fri, November 23, 2007 4:34 pm, Jim Pflugrath wrote: It probably goes back to the days of using a single-counter diffractometer where one didn't have multiple Bragg reflections on an image or film pack. That is, each reflection was collected by itself. Even in a small molecule crystal data collection nowadays, it would not hurt to have the crystal completely bathed in the beam. Also in the old days (let's say pre-cryo), there was plenty of radiation damage going on even with a sealed-tube source. We always corrected for radiation damage by extrapolating back to zero dose in those days. Jim -Original Message- From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Robert Sweet Sent: Friday, November 23, 2007 4:08 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] To bathe or not to bathe. Jorge, You said, I remember one former good (small molecule ?) crystallography book with words a kind of this the crystals should be completely bathed by the x-ray beam during the whole data collection ... The original motive for bathing the whole crystal was to assure that the relative intensity of the data on each successive film pack was very nearly constant. This was possible (one might say necessary) in the old days because the laboratory sources were very stable and the intensity was low enough that there wasn't a lot of x-ray damage to the crystals. There were a couple of other good reasons to pay attention to details like this. One was that methods for scaling images together were not quite as good as now, and another was that film data were relatively very much less accurate than what is achievable now with excellent detectors and brighter sources. To combat all of that, we tried to do everything possible to make things better. These days scaling algorithms are good, the detectors are excellent, and very often it pays to employ a beam smaller than the x-tal. This, the non-uniformity of many synchrotron beams, and the systematic damage to crystals that we observe now with synchrotron sources cause serious systematic errors. We're forced to depend on good scaling and good detectors to get accurate measurements. Making the measurements in many different crystal orientations (redundancy) helps to smooth out these systematic errors. Nonetheless, it will always pay you to watch for EACH of these sources of error and to minimize them as best you can. Bob = Robert M. Sweet E-Dress: [EMAIL PROTECTED] Group Leader, PXRR: Macromolecular ^ (that's L Crystallography Research Resource at NSLSnot 1) http://px.nsls.bnl.gov/ Biology Dept Brookhaven Nat'l Lab. Phones: Upton, NY 11973631 344 3401 (Office) U.S.A. 631 344 2741 (Facsimile) =
Re: [ccp4bb] To bathe or not to bathe.
It probably goes back to the days of using a single-counter diffractometer where one didn't have multiple Bragg reflections on an image or film pack. That is, each reflection was collected by itself. Even in a small molecule crystal data collection nowadays, it would not hurt to have the crystal completely bathed in the beam. Also in the old days (let's say pre-cryo), there was plenty of radiation damage going on even with a sealed-tube source. We always corrected for radiation damage by extrapolating back to zero dose in those days. Jim -Original Message- From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Robert Sweet Sent: Friday, November 23, 2007 4:08 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] To bathe or not to bathe. Jorge, You said, I remember one former good (small molecule ?) crystallography book with words a kind of this the crystals should be completely bathed by the x-ray beam during the whole data collection ... The original motive for bathing the whole crystal was to assure that the relative intensity of the data on each successive film pack was very nearly constant. This was possible (one might say necessary) in the old days because the laboratory sources were very stable and the intensity was low enough that there wasn't a lot of x-ray damage to the crystals. There were a couple of other good reasons to pay attention to details like this. One was that methods for scaling images together were not quite as good as now, and another was that film data were relatively very much less accurate than what is achievable now with excellent detectors and brighter sources. To combat all of that, we tried to do everything possible to make things better. These days scaling algorithms are good, the detectors are excellent, and very often it pays to employ a beam smaller than the x-tal. This, the non-uniformity of many synchrotron beams, and the systematic damage to crystals that we observe now with synchrotron sources cause serious systematic errors. We're forced to depend on good scaling and good detectors to get accurate measurements. Making the measurements in many different crystal orientations (redundancy) helps to smooth out these systematic errors. Nonetheless, it will always pay you to watch for EACH of these sources of error and to minimize them as best you can. Bob = Robert M. Sweet E-Dress: [EMAIL PROTECTED] Group Leader, PXRR: Macromolecular ^ (that's L Crystallography Research Resource at NSLSnot 1) http://px.nsls.bnl.gov/ Biology Dept Brookhaven Nat'l Lab. Phones: Upton, NY 11973631 344 3401 (Office) U.S.A. 631 344 2741 (Facsimile) =
