I’m with Dale on this, the scientifically prudent thing is to set the rules and then play by them. Not to change the rules as you go. Of course, in a teaching environment where you know the correct answer, it is good to be educational and learn how to dig a bit more.
However, in a scientific setting this digging is not to come to a strong conclusion, but only to see if you should pursue the project and do additional experiments (e.g. longer soaks or using a higher ligand concentration). In this case the topic starter has poor density and fitting the ligand and refining gives negative difference density. Surely that is not enough evidence to reject the null hypothesis “the ligand is not bound”. In other words, there is no strong evidence that the ligand is bound. Perhaps you can look at the occupancy , but that is probably as far as you should go. The polder map is useful to get rid of the effect of the solvent mask blurring actual ligand density. But after fitting the ligand you shouldn’t need the polder map. Blurring and sharpening is something to make sense of the density shape to better fit your ligand, not to conclude whether or not you ligand is there. On a whole, for ligands we should try to stick to the so-called “bloody obvious” test: if the density is not bloody obvious, your ligand is not there. At least not all the time. Cheers, Robbie From: CCP4 bulletin board <CCP4BB@JISCMAIL.AC.UK> On Behalf Of Jon Cooper Sent: Wednesday, November 25, 2020 05:20 To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] phenix.refine with ligand with ambiguous electron density Hello Dale, the statistical rigour you describe is, of course, excellent, but in a learning environment, if someone gets a negative result, you have to go into overdrive to check that everything has been done correctly, since there is a fair chance that human error is the cause. It may be a terrible practice, but it would seem to be an important part of the process? Even as a relative newcomer to the field (well, since the mid-80's ;-) I have seen many people getting nothing in their initial difference maps, even if the ligand is there. Frequently it was just the contour level being too high and, depending on how far back you go, the solution varied from showing someone how to roll the mouse wheel in Coot to having the map recontoured at a computer centre 200 miles away and posted back on a magnetic tape, which took about 10 days - a timescale on which some people just gave up and did something else! I can't help thinking it would be a shame to robotically accept every negative result at face value, not least if you're doing something important like curing a pandemic. However, back to the original question which I think was whether polder map coefficients could be used as refinement targets and I think the answer to that one is probably 'no', at least in the X-ray field ;-) Best wishes, Jon Cooper -------- Original Message -------- On 24 Nov 2020, 16:02, Dale Tronrud < de...@daletronrud.com<mailto:de...@daletronrud.com>> wrote: Hi, To me, this sounds like a very dangerous way to use this tool decide if a ligand has bound. I would be very reluctant to modify my map with a range of arbitrary parameters until it looked like what I wanted to see. The sharpening and blurring of this tool is not guided or limited by theory or data. As you describe it, your choice of map is driven by its agreement with your ligand, and the proper way to make this decision is the other way around. The original poster has the problem that their density does not have the appearance they desire. They have chosen to run around trying to find some way to modify the map to get a variant that does. This is a terrible practice, since the final choice of map is being made in a fashion that is dominated by bias. I have no idea what sort of "structural characteristics" have convinced this poster of the presence of their ligand despite the absence of clear electron density. What other evidence does a diffraction pattern give? The map is your best and only source of information about your structure that you can get from the diffraction pattern. (Mass spec and other experimental techniques could, of course, be applied.) I think we, as a community, could learn a few things from the vaccine trial studies that are so much in the news now. In a modern clinical trial, to avoid bias in the interpretation of the results, all of the statistical procedures are decided upon BEFORE the study is even began. This protocol is written down and peer reviewed at the start. Then the study is performed and the protocol is followed exactly. If the results don't pass the test, the treatment is not supported. There is no hunting around, after the fact, for a "better" statistical measure until one is found that "works". This way of handling data analysis in clinical trials was adopted after the hard lesson was learned that many trails could be reproduced, their results were not. I would recommend that you decide what sort of map you think is the best at showing features of your active site, based on the resolution of your data set and other qualities of your project, before you calculate your first Fourier transform. If you think a Polder map is the bee's knees then calculate a Polder map and live with it. If you are convinced of the value of a FEM, or a Buster map, or a SA omit map, or whatever, calculate that map instead and live with it. If you have to calculate twenty different kinds of maps, with varying parameters in each, before you find the one that shows the density for your ligand; it probably didn't bind. Dale Tronrud On 11/24/2020 5:35 AM, John R Helliwell wrote: > Dear Nika, > A tool I am gaining experience with, but for a challenge like you > describe, may help:- > In Coot>Calculate you see “Blurring/Sharpening tool”. You are > presented with a choice of electron density map (here you would select > your Fo-Fc). There is then a slider tool, to the left and to the right, > and you can see the impact of negative or positive B factor on your map. > Blurring, slide right, may assist your density continuity versus > Sharpening, slide left, which may assist the detail of your map. The > logic of the tool is that your diffraction data, and of the Fo-Fc > differences, can be fine tuned, in or out. > Best wishes, > John > > Emeritus Professor John R Helliwell DSc > > > > >> On 24 Nov 2020, at 11:29, Nika Žibrat >> <nika.zib...@ki.si<mailto:nika.zib...@ki.si>> wrote: >> >> >> >> Hello, >> >> >> I have a question about protein-ligand, of which ligand displays an >> ambiguous electron density. I am solving a structure of protein with >> ligand which was obtained via soaking. Structural characteristics >> indicate the ligand is present however the electron density is quite >> vague and too small for the size of the whole ligand. I did a Polder >> map which showed much larger area of green density. After insertion of >> my ligand into the green density in Polder I ran phenix.refine and >> there is a lot of red on the spot where the ligand is which was to be >> expected. This leaves me wondering how, if even do I incorporate the >> polder map data into my refine input. >> >> >> My question is, how do I continue refining and validating the >> structure in this case? >> >> >> Thank you, >> >> >> Nika Žibrat >> >> >> >> ------------------------------------------------------------------------ >> >> To unsubscribe from the CCP4BB list, click the following link: >> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 >> <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 > <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<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 ######################################################################## 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/
