[3dem] Floating fsc
Steinar Halldorsson
steinar.halldorsson at crick.ac.uk
Sun Nov 4 14:27:41 PST 2018
Hi Ben
I have removed anything which is the same distance from the particle centre as the length of the protein itself. My tomograms are CTF corrected prior to processing so I hadn't considered the delocalised signa/noise, but that is an excellent suggestion, will try to reconstruct with a larger cut-off distance.
Cheers,
Steinar
________________________________
From: 3dem <3dem-bounces at ncmir.ucsd.edu> on behalf of Benjamin Himes <himes.benjamin at gmail.com>
Sent: 02 November 2018 22:06:19
To: 3dem at ncmir.ucsd.edu
Subject: [3dem] Floating fsc
Hi Steinar,
I will give a +1 to Vladan's suggestion. In emClarity, the option "fscSplitOnTomos" was added for just this reason.
I realize you have removed the overlapping particles, by editing your star files I suppose, but what distance do you use to ensure no overlap?
Keep in mind that there may be mixing of signal/noise some distance outside your particle envelope as well, especially if you are multiple micron off focus.
Ben H
On Fri, Nov 2, 2018, 3:00 PM <3dem-request at ncmir.ucsd.edu<mailto:3dem-request at ncmir.ucsd.edu>> wrote:
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Today's Topics:
1. Re: Add random noise to reconstruction (Vladan Lucic)
2. Re: Add random noise to reconstruction (Steinar Halldorsson)
----------------------------------------------------------------------
Message: 1
Date: Fri, 2 Nov 2018 18:21:04 +0100
From: Vladan Lucic <vladan at biochem.mpg.de<mailto:vladan at biochem.mpg.de>>
To: <3dem at ncmir.ucsd.edu<mailto:3dem at ncmir.ucsd.edu>>
Subject: Re: [3dem] Add random noise to reconstruction
Message-ID: <8b43672d-ec5e-c6ff-514d-bd06dc136257 at biochem.mpg.de<mailto:8b43672d-ec5e-c6ff-514d-bd06dc136257 at biochem.mpg.de>>
Content-Type: text/plain; charset="windows-1252"; Format="flowed"
Hi Stelnar,
Perhaps you have double (overlapping) particles, as a result of double
picking or translations during refinement for example. A quick way to
check for this is to make particle half-sets from different tomograms
(split tomograms in two groups and make each half particle set from one
group). In this way, double particles will always be in the same
particle half-set. If the FSC between these halves goes to 0, it's
double particles.
Just a guess, hope it helps
Best,
Vladan
On 11/2/18 5:44 PM, Steinar Halldorsson wrote:
>
> Hi all
>
>
> I have something which looks like an artefact in my FSC curves from
> tomography data calculated in Relion. Basically the curves hover
> around 0.1 instead of reaching 0... And this is not coming from my
> mask btw. There is quite a lot of information (protein) outside of the
> mask, you can see clear but slightly smeary density from neighbours in
> the final maps, and I wonder if this is?causing these correlations?
> One way I have thought to test this is to take my two half maps,
> somehow add random noise to parts outside of my region of interest and
> then recalculate my FSC.
>
>
> Firstly, does this sound like a reasonable thing to try?
> Secondly, does anyone have a suggestion of how to actually do the
> random noise addition outside of my region of interest?
>
> Cheers,
>
> Steinar
>
>
> ---
>
> Steinar Halldorsson
>
> Postdoctorate Fellow
>
> Rosenthal lab
>
> The Francis Crick Institute
> London, United Kingdom
>
> The Francis Crick Institute Limited is a registered charity in England
> and Wales no. 1140062 and a company registered in England and Wales
> no. 06885462, with its registered office at 1 Midland Road London NW1 1AT
>
>
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Message: 2
Date: Fri, 2 Nov 2018 18:19:11 +0000
From: Steinar Halldorsson <steinar.halldorsson at crick.ac.uk<mailto:steinar.halldorsson at crick.ac.uk>>
To: Vladan Lucic <vladan at biochem.mpg.de<mailto:vladan at biochem.mpg.de>>, "3dem at ncmir.ucsd.edu<mailto:3dem at ncmir.ucsd.edu>"
<3dem at ncmir.ucsd.edu<mailto:3dem at ncmir.ucsd.edu>>
Subject: Re: [3dem] Add random noise to reconstruction
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Hi Vladan
I have removed overlapping particles but it didn't change the FSC behaviour.
Steinar
________________________________
From: 3dem <3dem-bounces at ncmir.ucsd.edu<mailto:3dem-bounces at ncmir.ucsd.edu>> on behalf of Vladan Lucic <vladan at biochem.mpg.de<mailto:vladan at biochem.mpg.de>>
Sent: 02 November 2018 17:21:04
To: 3dem at ncmir.ucsd.edu<mailto:3dem at ncmir.ucsd.edu>
Subject: Re: [3dem] Add random noise to reconstruction
Hi Stelnar,
Perhaps you have double (overlapping) particles, as a result of double picking or translations during refinement for example. A quick way to check for this is to make particle half-sets from different tomograms (split tomograms in two groups and make each half particle set from one group). In this way, double particles will always be in the same particle half-set. If the FSC between these halves goes to 0, it's double particles.
Just a guess, hope it helps
Best,
Vladan
On 11/2/18 5:44 PM, Steinar Halldorsson wrote:
Hi all
I have something which looks like an artefact in my FSC curves from tomography data calculated in Relion. Basically the curves hover around 0.1 instead of reaching 0... And this is not coming from my mask btw. There is quite a lot of information (protein) outside of the mask, you can see clear but slightly smeary density from neighbours in the final maps, and I wonder if this is causing these correlations? One way I have thought to test this is to take my two half maps, somehow add random noise to parts outside of my region of interest and then recalculate my FSC.
Firstly, does this sound like a reasonable thing to try?
Secondly, does anyone have a suggestion of how to actually do the random noise addition outside of my region of interest?
Cheers,
Steinar
---
Steinar Halldorsson
Postdoctorate Fellow
Rosenthal lab
The Francis Crick Institute
London, United Kingdom
The Francis Crick Institute Limited is a registered charity in England and Wales no. 1140062 and a company registered in England and Wales no. 06885462, with its registered office at 1 Midland Road London NW1 1AT
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