Dear Lindsey, I'm all with Nukri on this one. 12 Se atoms (whose incorporation you've shown) should be plenty to give you enough anomalous signal to phase the data and solve the structure. That's why I would do the simplest experiment first. Collect 360° of data at the peak energy, maybe a bit finer sliced than you have now (0.1° per image) and with fewer X-rays. Reduce exposure fivefold (just a guess, but most people tend to overexpose - see Winter et al., Acta D 75:242, 2019) and see what you can get from this one dataset after processing with FRIEDEL'S_LAW= FALSE.
Look at the table "SUBSET OF INTENSITY DATA WITH SIGNAL/NOISE >= -3.0 AS FUNCTION OF RESOLUTION" in CORRECT.LP and see how far the anomalous signal extends (Anomal Corr values above 15 to 20, highlighted by XDS with an asterisk) and how strong it is (SigAno values, above 3 gives you confidence, though I have solved structures with much less signal). If the anomalous signal isn't enough for structure solution, collect more data from the same crystal. This is one reason why you reduced the exposure in the first place. Move the detector in or out depending on the processing of the first 360° (How far does diffraction extend?) and collect another 360°. Or change chi/kappa. (See Basu et al., Acta D 75:262, 2019 for inspiration.) Or - and this is the best but unlikely to be necessary with your crystals - do two-wavelength MAD. This shouldn't be an afterthought to the SAD experiment, though, but properly planned as one comprehensive experiment. Using the inflection and remote energies might be a good idea because it maximizes the differences between the two datasets. Inverse beam can be powerful but also poses challenges. I use this technique only if I fail otherwise. All best. Andreas On Tue, Aug 27, 2019 at 3:55 AM 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 and here is an example of a frame: >> https://ibb.co/gR3ZR47. 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 >> > > ------------------------------ > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1 > ######################################################################## To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1
