Hello Robbie, Yes exactly, I agree. I thought that was what the poster faced: density with insufficient detail and not extending sufficiently for the whole ligand. To make the discussion thread more focussed a screenshot or two would assist us. Greetings, John
Emeritus Professor John R Helliwell DSc > On 25 Nov 2020, at 09:03, Robbie Joosten <robbie_joos...@hotmail.com> wrote: > > > 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> 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> 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, a mailing > list hosted by 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 ######################################################################## 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/
