Hello Dale, Well, warming to your theme, I start with a trust in Coot before a new project. Secondly, Coot’s blurring and sharpening tool is tethered directly to one’s measured diffraction data. Thirdly, scrutinising it at a sigma level above 5, Coot’s default, is certainly not the same as delving into the lower levels of a map’s sigma (and truly of noise). But, I have just rechecked the Coot manual and see no reference. There are several google hits but mainly to cryoEM maps. It does seem a free lunch though...... Greetings, John
Emeritus Professor John R Helliwell DSc > On 24 Nov 2020, at 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/
