Hi Lindsey, As I mentioned to you in the separate email, 180 degrees for each half is too little.
Here I'll try to explain some more about SAD vs. MAD: What I have observed at our beamlines is that the majority of those who collect MAD data, do it as as an afterthought of SAD. Priority in these experiments is given to SAD and after it is done, some folks decide to collect more data at a different wavelength "just in case". There are two big mistakes in this approach, also explaining why SAD so often "works better than MAD": 1. Unless one collects the second wavelength from a fresh part of the crystal, or from a different crystal, there is too much radiation damage in the second data set. Therefore, the differences in the intensities are mostly caused by radiation damage and not by anomalous or dispersive signal. This, of course, kills phasing. This is how SAD can be "better" than MAD. In a proper experiment, both wavelengths must be given equal priority. I.e. distribute the crystal life time equally between the two. 2. Another mistake also stems from the fact that the 2nd wavelength is treated as "addition" to SAD. Whether it is optimal or not is a different discussion but typically, the SAD data are collected at the absorption peak. Then, for 2-wavelength, one collects inflection point. What is lost in this approach is the whole purpose of a MAD experiment, which is to use the dispersive signal along with the anomalous one. Dispersive signal between the inflection point and the peak wavelengths is not so great. In a good experiment, one of the wavelengths is at the inflection point (as a must). One could argue that the other is not at the absorption peak but above the peak (in energy) in order to increase the dispersive signal. How much above, will depend on particular f' and f" plots. Further the better for the dispersive signal, but you also want to retain good anomalous one. So, some compromise needs to be made here. Bottom line is that SAD and MAD are two different experiments and one is not a simple expansion of the other. You need to make a decision which one you are doing and collect data accordingly. Cheers, Nukri On Tue, Aug 27, 2019 at 11:30 AM Doyle, Lindsey A <ldo...@fredhutch.org> wrote: > Hi Nukri, > > Thanks so much for your response. I appreciate the advice. > 1. Yes, I verified that the anomalous option is turned on during data > processing. Always a good question to ask > 2. I collected 180° for each half. I have not tried phasing just one half. > I’ll give a try but with my space group being P 21 the completeness and > redundancy might be pretty low. I have a couple inverse beam data sets with > wedges of 5° but they were about the same as the ones with 1° > 3. I’ve been collecting 0.5 sec exposures but without reducing the flux. > This seems to be one of the most recommended things and will be definitely > doing it on my next run. > 4. I’ve tried both SAD and MAD with peak 12661 (0.9793 Å) and inflection > 12658 (0.9795 Å) > > Thanks again, > Lindsey > > > On Aug 26, 2019, at 6:54 PM, Nukri Sanishvili <sannu...@gmail.com> wrote: > > Hi Lindsey, > Obviously, one would need a lot more information to properly diagnose the > problem and I am sure much smarter people them me will ask you for that. > But just to move the task by couple of steps, I want to point out couple of > things. > 1. Trivial question: did you have the anomalous option turned on during > data processing? (Just like from the IT help - is your computer turned on?) > 2. How much data did you collect for each half of the inverse beam > geometry? If you have enough, try phasing with only one half. When done > properly, inverse beam experiment is great but it can easily get tricky > introducing systematic errors and thus swamping anomalous signal. If you > redo the inverse beam, use little wider wedges, say, 5-10 degrees. > 3. I thought an example of diffraction image would not give any useful > information but... Judging by how smooth the background is on your Pilatus > image, I am guessing you have used a lot of exposure. Can you calculate how > much dose did you put in your crystal? If you are going to re-do the > experiment, I would suggest reducing the exposure level and collecting more > data. > 4. Because you are not showing f' and f" plots, I am guessing that you are > doing SAD. If it fails and you end up redoing your experiment and you have > crystals for it, you might want to try 2-wavelength MAD but for that you > would need to know exactly where is inflection point and collect one of the > datasets there. > > Good luck! > Nukri > > On Mon, Aug 26, 2019 at 5:45 PM L. Doyle <ldo...@fhcrc.org> wrote: > >> I have some Seleno-Methionine protein crystals (12 SeMet of 211 amino >> acids, incorporation verified by Mass Spec). I've already collected several >> datasets (ALS BL5.0.2) but I seem to be losing (rejecting?) a lot of >> anomalous signal during data processing. I'm most familiar with HKL2000, >> but I have tried XDS and DIALS auto-processing. Here is a scan: >> https://ibb.co/LZqm33p >> <https://urldefense.proofpoint.com/v2/url?u=https-3A__ibb.co_LZqm33p&d=DwMFaQ&c=eRAMFD45gAfqt84VtBcfhQ&r=2fgF7nXnZhu6kQ1ZLLvJeA&m=0-kIFi_aWaNeQ9Rl0Ca1A5Z5P41UhmeHF5atpPFQ1x8&s=AYn3f5GZQ2ZQv5oq-gYN2HEbikhDEnKryiUsbaQevVE&e=> >> and here is an example of a frame: https://ibb.co/gR3ZR47 >> <https://urldefense.proofpoint.com/v2/url?u=https-3A__ibb.co_gR3ZR47&d=DwMFaQ&c=eRAMFD45gAfqt84VtBcfhQ&r=2fgF7nXnZhu6kQ1ZLLvJeA&m=0-kIFi_aWaNeQ9Rl0Ca1A5Z5P41UhmeHF5atpPFQ1x8&s=MmgiUiUT2TmSx0xx3RskenKPWOJfYoTnLKXo2P8x2mw&e=>. >> Each frame is 0.25° and I'm using inverse beam with wedge size 1°. Maybe I >> need to adjust my collection strategy? All previous datasets have been in >> space group P 21 with dimensions of approx. 24.5Å, 85Å, 40Å, 90°, 96.5°, >> 90°. I'm sure there are additional things I can be doing in HKL but I've >> run out of ideas. Any advice or recommendations would be appreciated. >> Please let me know if you need additional information. >> >> Thank you, >> Lindsey >> >> ######################################################################## >> >> To unsubscribe from the CCP4BB list, click the following link: >> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1 >> <https://urldefense.proofpoint.com/v2/url?u=https-3A__www.jiscmail.ac.uk_cgi-2Dbin_webadmin-3FSUBED1-3DCCP4BB-26A-3D1&d=DwMFaQ&c=eRAMFD45gAfqt84VtBcfhQ&r=2fgF7nXnZhu6kQ1ZLLvJeA&m=0-kIFi_aWaNeQ9Rl0Ca1A5Z5P41UhmeHF5atpPFQ1x8&s=SBBWJJiu3emknBfFcxVTI545aZP1sEHSkBcmSVcwtqM&e=> >> > > ######################################################################## To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1
