Hi,
> What if instead of images we just collected a list of x-y coordinates of > photon hits vs time? This is how the Tristan detector works, it is based on timepix3 chips and the data is a read as time stamped x-y co-ordinates + counts and then diffraction images are reconstructed from this information downstream. This means you have complete freedom about how you want to bin your data in terms of time/dose. https://ieeexplore.ieee.org/document/9875340 We’ve used the detector to test Hadamard multiplexing of time/dose bins to boost the ‘weak images’ from small bins to help with indexing and integration prior to deconvolution back into time/dose resolved data. However, this is a work in process we are waiting for some software updates to help with the image reconstruction part of the process but the detector appears to work very well for this kind of application! Briony From: CCP4 bulletin board <CCP4BB@JISCMAIL.AC.UK> on behalf of Guillaume Gaullier <guillaume.gaull...@kemi.uu.se> Date: Friday, 17 May 2024 at 17:07 To: CCP4BB@JISCMAIL.AC.UK <CCP4BB@JISCMAIL.AC.UK> Subject: Re: [ccp4bb] Experimental phasing Selenomethionine data collection etc. tips CAUTION: External Message. Use caution opening links and attachments. Hello, > What if instead of images we just collected a list of x-y coordinates of > photon hits vs time? This already exists, but for electrons: https://doi.org/10.1107/S205225252000929X This is the technology in Falcon4 detectors. The advantage is that it allows you to decide how to slice the total dose into dose fractions *after* the data collection. So, when setting up the collection at the microscope, you only need to worry about how much total dose to use. With the previous generation of detectors, during set up you have to decide how much total dose but also how many dose fractions, and if it's not optimal you can't change it once the data is collected. I don't understand detector technology enough to tell whether this should be possible for X-rays. But out of curiosity, how would you use this detector if you had one? What would it enable that you can't do now? Cheers, Guillaume ________________________________ From: CCP4 bulletin board <CCP4BB@JISCMAIL.AC.UK> on behalf of James Holton <jmhol...@lbl.gov> Sent: Friday, May 17, 2024 5:27:02 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Experimental phasing Selenomethionine data collection etc. tips A few follow-up questions I got out-of-band: > how did you get to the 1:1 relationship between Bijvoet ratio and dose? > I got this from fitting a straight line to Table 1 of Banu's 2004 paper: 10.1107/S0907444904007917 Is this a rough estimate based on a singular result? Of course it is! This is how we roll in radiation damage research. > > Comment: with the more modern pixel array detectors (e.g. Eiger), you > can slice your dose even more finely than 0.1s, and not worry about > the readout time. yes. With a bit of a caveat on how many photons/pixel you need for stable background subtraction. XDS starts having issues around 1 photon/pixel or less, and DIALS claims to be able to get to 0.01 photons/pixel, but I have not personally pushed it that far. Not yet. I have a plan to try and push zero-dose extrapolation to the one-photon-per-image level, but that is on another thread. > is it better to collect 360 or 720º at half the dose Nothing wrong with going longer than 360, especially if you want to do zero-dose extrapolation, because it is only by repeating the same phi range (and everything else) exactly that you get a genuinely "same" increment in dose. However, once you go past 360 the "multiplicity" you gain starts turning into what you might call a "redundancy". What I mean by that is that in the first 360 each spot and its symmetry mates generally show up on different pixels. Each pixel has about 1% to 3% calibration error associated with it (depending on the detector). So, for the 2nd 360 you will re-measure all the same spots with the same pixels again, repeating a systematic error. You will also have the same sample self-absorption, etc. But, the pixel calibration error starts to really matter for anomalous at high "redundancy". To put it another way, if a particular pixel has 1% error, then counting more than 10,000 photons with it is a waste, because the systematic error of 1% will start to dominate the total error at higher photon counts. So, for anomalous especially, I recommend moving the detector between 360s. Sliding it horizontally is best. Or you can use 2theta. But, a small change in detector distance can usually do it and is almost always an available option. The only problem with all this "dose slicing" is the images get very very weak. And that brings us back to the "weak image limit". What if instead of images we just collected a list of x-y coordinates of photon hits vs time? Anyone have a suggestion for the name to give to the program that can process such data? -James Holton MAD Scientist On 5/15/2024 3:28 PM, James Holton wrote: > > Thank you to all who provided helpful suggestions so far. > > A few things I'd recommend for this particular beamline (which I have > been running for 20+ years) > > Do NOT collect one wavelength at a time. This was a good strategy on > old beamlines with noisy detectors and slow, drifty monochromators. > This is not the case at any of the ALS beamlines today. With modern > zero-read-noise detectors there is no penalty to spreading your > photons over a lot more images, and round-robin changes between at > least two wavelengths will double your phasing power for the same > dose. With 8.3.1's monochromator, wavelength changes take about 1 > second and are reproducible to well within the intrinsic width of the > Se peak. So you don't need to worry about missing or drifting off the > peak or inflection. The only thing you need to worry about is > over-cooking your crystal before you get all the data you need. > > No matter what beamline you use the number of photons your crystal > will give off before it dies is a fixed number. All you get to do is > decide how to spread them over the images. Doing two wavelengths > within this photon budget doesn't hurt. You can always scale and merge > them together. But keeping them separate gives you both kinds of > anomalous differences, which are 90 deg apart. So, when one zigs the > other zags. It is like having twice as many sites without the extra > damage you would get from them. Also, by taking shorter/weaker > exposures you maximize your chances of winning over radiation damage > "in-post" by cutting off images that degrade your signal. > > And before anybody says it: NO! Collecting fainter images does NOT > degrade your resolution. I don't know where this idea comes from, but > it never seems to die. It was true with film and image plates, but > with pixel arrays and modern CCDs there is no penalty to weak images. > Don't believe me? Read the manual for your detector. Modern PADs > actually sum a bunch of weak images internally before writing them to > disk. You can do the same "in post" if you want to. > > Yes, there are many cases where SAD is good enough, but my advice is > never to tempt fate. > > What I recommend is: > 1) collect two wavelengths: remote, and halfway between the peak and > inflection. > this will maximize both kinds of anomalous differences > 2) calculate your Bijvoet ratio here: > https://bl831.als.lbl.gov/xtalsize.html > 3) convert this into MGy. I.E. if your Bijvoet ratio is 3%, then 3 MGy > is the max dose to avoid. > 4) do a strategy and start at the recommended phi value > 5) set delta-phi to be 1/3 of your estimated mosaic spread, or 0.2 > deg, whichever is lower > this is all done automatically by the "index" program at 8.3.1 > 6) set your exposure time to be 0.1 s or more. > This is because the Pilatus M 6M has a 1 ms read-out and you want > that to be 1% of the exposure. > 7) attenuate the beam so that you will get complete data in less than > 1/2 your Bijvoet ratio in MGy. > This is handled by the exposure_time program at 8.3.1 > 8) collect data in inverse beam and round-robin for both wavelengths > (45 deg wedges) > In BLU-ICE, just enter the wavelengths into the list on the > Collect tab > 9) keep collecting until you get 360 deg for both wavelengths > 10) move the detector up by ~5 mm, this puts the next sphere of spots > onto new pixels > 11) multiply your exposure or de-attenuate by a factor of 4 > 12) goto 8 > > When the diffraction image is noticeably damaged, you are done with > this crystal. If it is bigger than the beam, move to a fresh spot and > do this again. When the crystal is all burnt up, mount the next one > and do this again. > > If you're lucky, the automatic processing will finish before you mount > your next crystal and you can try SHELXC/D/E on the 448-core > shared-memory computer we have for doing such things. I expect it > might be faster than the cloud. > > Sorry if any of this sounds gruff, I don't mean to shout down on > anyone, but I want the message to be clear. This is something Gerard B > and I have struggled to communicate for decades: > Collecting one wavelength at a time is not MAD, but rather M-SAD. > Multiple, non-isomorphous SAD data sets. > > -James Holton > MAD Scientist > > On 5/13/2024 10:23 PM, dbellini wrote: >> Hi Marco, >> >> A few suggestions that I like to follow for MAD experiments: >> >> Before everything, check you have at least about 1 SeMet per 100 >> residues >> Then before crystallisation check by MassSpec that SeMet is properly >> incorporated in your protein >> After crystallisation collect first on the peak with (very) high >> redundancy and as little/gentle dose as possible >> Collecting the other wavelengths should give you better starting >> phases/maps, which might be very helpful at your resolution of 2.8 >> (especially if it is a very anisotropic 2.8...) >> >> Automated pipelines are so good nowadays, if you collect good data >> they should solve it without problems (as long as your crystal is not >> suffering from other pathologies like twinning or pseudosymmtries). >> >> Good luck! >> >> D >> >> >> On 2024-05-14 01:17, Marco Bravo wrote: >>> CAUTION: This email originated from outside of the LMB: >>> .-owner-ccp...@jiscmail.ac.uk-. >>> Do not click links or open attachments unless you recognize the sender >>> and know the content is safe. >>> If you think this is a phishing email, please forward it to >>> phish...@mrc-lmb.cam.ac.uk >>> -- >>> >>> Hello all, >>> I have a data collection trip next week and plan to collect data on >>> selenomethionine derivative crystals at the al831 beamline. Are there >>> any resources, tips, tutorials, literature etc. That you can recommend >>> to help me prepare for these experiments. Also is there a way to plug >>> in the experimental data into ccp4 cloud to do the automatic structure >>> solution? Do I need native and derivative data to solve the structure? >>> Last trip I collected a seemingly 2.8 angstrom resolution data on a >>> crystal of the native protein but could not get a solution depsite >>> extensive molecular replacement attempts. It seems that assigning a >>> space group for the crystals has been troublesome as well. here is my >>> last thread I posted about the issue for reference. >>> >>> https://www.jiscmail.ac.uk/cgi-bin/wa-jisc.exe?A2=ind2402&L=CCP4BB&O=D&X=CCE6DFA19FA3D40346&Y=mbrav005%40ucr.edu&P=112302 >>> >>> >>> ######################################################################## >>> >>> >>> To unsubscribe from the CCP4BB list, click the following link: >>> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 >>> >>> This message was issued to members of >>> www.jiscmail.ac.uk/CCP4BB<http://www.jiscmail.ac.uk/CCP4BB>, a >>> mailing list hosted by www.jiscmail.ac.uk<http://www.jiscmail.ac.uk/>, >>> terms & conditions are >>> available at https://www.jiscmail.ac.uk/policyandsecurity/ >> >> ######################################################################## >> >> To unsubscribe from the CCP4BB list, click the following link: >> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 >> >> This message was issued to members of >> www.jiscmail.ac.uk/CCP4BB<http://www.jiscmail.ac.uk/CCP4BB>, a >> mailing list hosted by www.jiscmail.ac.uk<http://www.jiscmail.ac.uk/>, terms >> & conditions are >> available at https://www.jiscmail.ac.uk/policyandsecurity/ > ######################################################################## To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 This message was issued to members of www.jiscmail.ac.uk/CCP4BB<http://www.jiscmail.ac.uk/CCP4BB>, a mailing list hosted by www.jiscmail.ac.uk<http://www.jiscmail.ac.uk/>, terms & conditions are available at https://www.jiscmail.ac.uk/policyandsecurity/ VARNING: Klicka inte på länkar och öppna inte bilagor om du inte känner igen avsändaren och vet att innehållet är säkert. 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For more information on how this is performed, please read here: http://www.uu.se/en/about-uu/data-protection-policy ________________________________ To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 ######################################################################## To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list hosted by www.jiscmail.ac.uk, terms & conditions are available at https://www.jiscmail.ac.uk/policyandsecurity/
