Hi,
You have to run CtfRefine with 4th order aberration refinement on.
Takanori
On 2020/11/27 16:00, Victor Banerjee wrote:
>
> Hi Takanori,
> Yeah, it indeed looks very weird and I basically discarded this and thought maybe I just need to play with the different twist and rise values in the
> refine step. Once I saw this email I thought it maybe a good place to see if this kind of curve is also fine or not. Did not that error in pixel size
> can cause this too. Thanks for the information. I have not checked the beam tilt or pixel size yet. Can you please tell me where can I see the
> "rlnEvenZernike"? It is not there at the data_optics table at the refine3D job.
> -Victor
>
>
>
> On Thu, Nov 26, 2020 at 3:37 PM Takanori Nakane <[log in to unmask] <mailto:[log in to unmask]>> wrote:
>
> Hi,
>
> Oh, sorry, I didn't look at Victor's attachment!
> I thought Victor was asking about the green curve in
> Marta's plot.
>
> Victor's curve is indeed bit strange.
> Victor, did you check beam tilt, pixel size, etc?
>
> Best regards,
>
> Takanori Nakane
>
> On 2020/11/26 21:33, Y. Mutum wrote:
> > Hi Takanori
> >
> > Sorry if I am redundant- I am quite new to EM. Could you please clarify?
> >
> > Why is this oscillating FSC regarded acceptable (Victor's one with the unmasked maps oscillating)?
> >
> > Thanks
> > Yaikhomba
> >
> ------------------------------------------------------------------------------------------------------------------------------------------------------
> > *From:* Collaborative Computational Project in Electron cryo-Microscopy <[log in to unmask] <mailto:[log in to unmask]>> on behalf of
> Victor Banerjee <[log in to unmask] <mailto:[log in to unmask]>>
> > *Sent:* 26 November 2020 21:12
> > *To:* [log in to unmask] <mailto:[log in to unmask]> <[log in to unmask] <mailto:[log in to unmask]>>
> > *Subject:* Re: [ccpem] [EXTERNAL] Re: [ccpem] Bump in FSC curves
> >
> > Thanks!
> > -Victor
> >
> >
> > On Thu, Nov 26, 2020 at 3:10 PM Takanori Nakane <[log in to unmask] <mailto:[log in to unmask]> <mailto:[log in to unmask]
> <mailto:[log in to unmask]>>> wrote:
> >
> > Hi,
> >
> > Yes, it is OK and FSC looks fine to me.
> >
> > Takanori
> >
> > On 2020/11/26 20:53, Victor Banerjee wrote:
> > > Does it mean OK to have such bumps at the 4.7A region? and the FSC is acceptable?
> > > -Victor
> > >
> > >
> > >
> > >
> > > On Thu, Nov 26, 2020 at 2:50 PM Takanori Nakane <[log in to unmask] <mailto:[log in to unmask]>
> <mailto:[log in to unmask] <mailto:[log in to unmask]>> <mailto:[log in to unmask] <mailto:[log in to unmask]>
> > <mailto:[log in to unmask] <mailto:[log in to unmask]>>>> wrote:
> > >
> > > Yes.
> > >
> > > Takanori
> > >
> > > On 2020/11/26 19:18, Victor Banerjee wrote:
> > > >
> > > > Hi,
> > > > Just checking if this kind of bump in the green line is also acceptable? The map is refined and postproceed.
> > > > -Victor
> > > >
> > > >
> > > > On Thu, Nov 26, 2020 at 12:24 PM Reza Khayat <[log in to unmask] <mailto:[log in to unmask]> <mailto:[log in to unmask]
> <mailto:[log in to unmask]>> <mailto:[log in to unmask] <mailto:[log in to unmask]>
> > <mailto:[log in to unmask] <mailto:[log in to unmask]>>> <mailto:[log in to unmask] <mailto:[log in to unmask]>
> <mailto:[log in to unmask] <mailto:[log in to unmask]>>
> > > <mailto:[log in to unmask] <mailto:[log in to unmask]> <mailto:[log in to unmask] <mailto:[log in to unmask]>>>>> wrote:
> > > >
> > > > Thanks!
> > > >
> > > > Reza Khayat, PhD
> > > > Associate Professor
> > > > City College of New York
> > > > Department of Chemistry and Biochemistry
> > > > New York, NY 10031
> > > >
> > > > ________________________________________
> > > > From: Takanori Nakane <[log in to unmask] <mailto:[log in to unmask]> <mailto:[log in to unmask]
> <mailto:[log in to unmask]>> <mailto:[log in to unmask] <mailto:[log in to unmask]>
> > <mailto:[log in to unmask] <mailto:[log in to unmask]>>> <mailto:[log in to unmask] <mailto:[log in to unmask]>
> <mailto:[log in to unmask] <mailto:[log in to unmask]>>
> > > <mailto:[log in to unmask] <mailto:[log in to unmask]> <mailto:[log in to unmask]
> <mailto:[log in to unmask]>>>>>
> > > > Sent: Thursday, November 26, 2020 1:07 PM
> > > > To: Reza Khayat; [log in to unmask] <mailto:[log in to unmask]> <mailto:[log in to unmask]
> <mailto:[log in to unmask]>> <mailto:[log in to unmask] <mailto:[log in to unmask]> <mailto:[log in to unmask] <mailto:[log in to unmask]>>>
> > <mailto:[log in to unmask] <mailto:[log in to unmask]> <mailto:[log in to unmask] <mailto:[log in to unmask]>>
> <mailto:[log in to unmask] <mailto:[log in to unmask]> <mailto:[log in to unmask] <mailto:[log in to unmask]>>>>
> > > > Subject: [EXTERNAL] Re: [ccpem] Bump in FSC curves
> > > >
> > > > Hi,
> > > >
> > > > Because some CTF parameters (e.g. Cs) is defined assuming the right pixel size.
> > > > See
> > > >
> > >
> >
> https://urldefense.proofpoint.com/v2/url?u=https-3A__www3.mrc-2Dlmb.cam.ac.uk_relion_index.php_Pixel-5Fsize-5Fissues&d=DwIG-g&c=4NmamNZG3KTnUCoC6InoLJ6KV1tbVKrkZXHRwtIMGmo&r=1DzJFW0v6TgEhkW1gy_-ke-RbtvS1fzEbD5_hcb9Up0&m=Jyfz-YpMjUtIbokDFmZ3UMnIE5o6K4XUKZ0pYSn6l3Y&s=jQuIMopzoPDSd2-mfw-WIL_dlx2LXt4pagYeHGARyUw&e=
> <https://urldefense.proofpoint.com/v2/url?u=https-3A__www3.mrc-2Dlmb.cam.ac.uk_relion_index.php_Pixel-5Fsize-5Fissues&d=DwIG-g&c=4NmamNZG3KTnUCoC6InoLJ6KV1tbVKrkZXHRwtIMGmo&r=1DzJFW0v6TgEhkW1gy_-ke-RbtvS1fzEbD5_hcb9Up0&m=Jyfz-YpMjUtIbokDFmZ3UMnIE5o6K4XUKZ0pYSn6l3Y&s=jQuIMopzoPDSd2-mfw-WIL_dlx2LXt4pagYeHGARyUw&e=>
> >
> <https://urldefense.proofpoint.com/v2/url?u=https-3A__www3.mrc-2Dlmb.cam.ac.uk_relion_index.php_Pixel-5Fsize-5Fissues&d=DwIG-g&c=4NmamNZG3KTnUCoC6InoLJ6KV1tbVKrkZXHRwtIMGmo&r=1DzJFW0v6TgEhkW1gy_-ke-RbtvS1fzEbD5_hcb9Up0&m=Jyfz-YpMjUtIbokDFmZ3UMnIE5o6K4XUKZ0pYSn6l3Y&s=jQuIMopzoPDSd2-mfw-WIL_dlx2LXt4pagYeHGARyUw&e= <https://urldefense.proofpoint.com/v2/url?u=https-3A__www3.mrc-2Dlmb.cam.ac.uk_relion_index.php_Pixel-5Fsize-5Fissues&d=DwIG-g&c=4NmamNZG3KTnUCoC6InoLJ6KV1tbVKrkZXHRwtIMGmo&r=1DzJFW0v6TgEhkW1gy_-ke-RbtvS1fzEbD5_hcb9Up0&m=Jyfz-YpMjUtIbokDFmZ3UMnIE5o6K4XUKZ0pYSn6l3Y&s=jQuIMopzoPDSd2-mfw-WIL_dlx2LXt4pagYeHGARyUw&e=>>
> > >
> >
> <https://urldefense.proofpoint.com/v2/url?u=https-3A__www3.mrc-2Dlmb.cam.ac.uk_relion_index.php_Pixel-5Fsize-5Fissues&d=DwIG-g&c=4NmamNZG3KTnUCoC6InoLJ6KV1tbVKrkZXHRwtIMGmo&r=1DzJFW0v6TgEhkW1gy_-ke-RbtvS1fzEbD5_hcb9Up0&m=Jyfz-YpMjUtIbokDFmZ3UMnIE5o6K4XUKZ0pYSn6l3Y&s=jQuIMopzoPDSd2-mfw-WIL_dlx2LXt4pagYeHGARyUw&e= <https://urldefense.proofpoint.com/v2/url?u=https-3A__www3.mrc-2Dlmb.cam.ac.uk_relion_index.php_Pixel-5Fsize-5Fissues&d=DwIG-g&c=4NmamNZG3KTnUCoC6InoLJ6KV1tbVKrkZXHRwtIMGmo&r=1DzJFW0v6TgEhkW1gy_-ke-RbtvS1fzEbD5_hcb9Up0&m=Jyfz-YpMjUtIbokDFmZ3UMnIE5o6K4XUKZ0pYSn6l3Y&s=jQuIMopzoPDSd2-mfw-WIL_dlx2LXt4pagYeHGARyUw&e=> <https://urldefense.proofpoint.com/v2/url?u=https-3A__www3.mrc-2Dlmb.cam.ac.uk_relion_index.php_Pixel-5Fsize-5Fissues&d=DwIG-g&c=4NmamNZG3KTnUCoC6InoLJ6KV1tbVKrkZXHRwtIMGmo&r=1DzJFW0v6TgEhkW1gy_-ke-RbtvS1fzEbD5_hcb9Up0&m=Jyfz-YpMjUtIbokDFmZ3UMnIE5o6K4XUKZ0pYSn6l3Y&s=jQuIMopzoPDSd2-mfw-WIL_dlx2LXt4pagYeHGARyUw&e= <https://urldefense.proofpoint.com/v2/url?u=https-3A__www3.mrc-2Dlmb.cam.ac.uk_relion_index.php_Pixel-5Fsize-5Fissues&d=DwIG-g&c=4NmamNZG3KTnUCoC6InoLJ6KV1tbVKrkZXHRwtIMGmo&r=1DzJFW0v6TgEhkW1gy_-ke-RbtvS1fzEbD5_hcb9Up0&m=Jyfz-YpMjUtIbokDFmZ3UMnIE5o6K4XUKZ0pYSn6l3Y&s=jQuIMopzoPDSd2-mfw-WIL_dlx2LXt4pagYeHGARyUw&e=>>>
> > > >
> > >
> >
> <https://urldefense.proofpoint.com/v2/url?u=https-3A__www3.mrc-2Dlmb.cam.ac.uk_relion_index.php_Pixel-5Fsize-5Fissues&d=DwIG-g&c=4NmamNZG3KTnUCoC6InoLJ6KV1tbVKrkZXHRwtIMGmo&r=1DzJFW0v6TgEhkW1gy_-ke-RbtvS1fzEbD5_hcb9Up0&m=Jyfz-YpMjUtIbokDFmZ3UMnIE5o6K4XUKZ0pYSn6l3Y&s=jQuIMopzoPDSd2-mfw-WIL_dlx2LXt4pagYeHGARyUw&e= <https://urldefense.proofpoint.com/v2/url?u=https-3A__www3.mrc-2Dlmb.cam.ac.uk_relion_index.php_Pixel-5Fsize-5Fissues&d=DwIG-g&c=4NmamNZG3KTnUCoC6InoLJ6KV1tbVKrkZXHRwtIMGmo&r=1DzJFW0v6TgEhkW1gy_-ke-RbtvS1fzEbD5_hcb9Up0&m=Jyfz-YpMjUtIbokDFmZ3UMnIE5o6K4XUKZ0pYSn6l3Y&s=jQuIMopzoPDSd2-mfw-WIL_dlx2LXt4pagYeHGARyUw&e=> <https://urldefense.proofpoint.com/v2/url?u=https-3A__www3.mrc-2Dlmb.cam.ac.uk_relion_index.php_Pixel-5Fsize-5Fissues&d=DwIG-g&c=4NmamNZG3KTnUCoC6InoLJ6KV1tbVKrkZXHRwtIMGmo&r=1DzJFW0v6TgEhkW1gy_-ke-RbtvS1fzEbD5_hcb9Up0&m=Jyfz-YpMjUtIbokDFmZ3UMnIE5o6K4XUKZ0pYSn6l3Y&s=jQuIMopzoPDSd2-mfw-WIL_dlx2LXt4pagYeHGARyUw&e= <https://urldefense.proofpoint.com/v2/url?u=https-3A__www3.mrc-2Dlmb.cam.ac.uk_relion_index.php_Pixel-5Fsize-5Fissues&d=DwIG-g&c=4NmamNZG3KTnUCoC6InoLJ6KV1tbVKrkZXHRwtIMGmo&r=1DzJFW0v6TgEhkW1gy_-ke-RbtvS1fzEbD5_hcb9Up0&m=Jyfz-YpMjUtIbokDFmZ3UMnIE5o6K4XUKZ0pYSn6l3Y&s=jQuIMopzoPDSd2-mfw-WIL_dlx2LXt4pagYeHGARyUw&e=>> <https://urldefense.proofpoint.com/v2/url?u=https-3A__www3.mrc-2Dlmb.cam.ac.uk_relion_index.php_Pixel-5Fsize-5Fissues&d=DwIG-g&c=4NmamNZG3KTnUCoC6InoLJ6KV1tbVKrkZXHRwtIMGmo&r=1DzJFW0v6TgEhkW1gy_-ke-RbtvS1fzEbD5_hcb9Up0&m=Jyfz-YpMjUtIbokDFmZ3UMnIE5o6K4XUKZ0pYSn6l3Y&s=jQuIMopzoPDSd2-mfw-WIL_dlx2LXt4pagYeHGARyUw&e= <https://urldefense.proofpoint.com/v2/url?u=https-3A__www3.mrc-2Dlmb.cam.ac.uk_relion_index.php_Pixel-5Fsize-5Fissues&d=DwIG-g&c=4NmamNZG3KTnUCoC6InoLJ6KV1tbVKrkZXHRwtIMGmo&r=1DzJFW0v6TgEhkW1gy_-ke-RbtvS1fzEbD5_hcb9Up0&m=Jyfz-YpMjUtIbokDFmZ3UMnIE5o6K4XUKZ0pYSn6l3Y&s=jQuIMopzoPDSd2-mfw-WIL_dlx2LXt4pagYeHGARyUw&e=> <https://urldefense.proofpoint.com/v2/url?u=https-3A__www3.mrc-2Dlmb.cam.ac.uk_relion_index.php_Pixel-5Fsize-5Fissues&d=DwIG-g&c=4NmamNZG3KTnUCoC6InoLJ6KV1tbVKrkZXHRwtIMGmo&r=1DzJFW0v6TgEhkW1gy_-ke-RbtvS1fzEbD5_hcb9Up0&m=Jyfz-YpMjUtIbokDFmZ3UMnIE5o6K4XUKZ0pYSn6l3Y&s=jQuIMopzoPDSd2-mfw-WIL_dlx2LXt4pagYeHGARyUw&e= <https://urldefense.proofpoint.com/v2/url?u=https-3A__www3.mrc-2Dlmb.cam.ac.uk_relion_index.php_Pixel-5Fsize-5Fissues&d=DwIG-g&c=4NmamNZG3KTnUCoC6InoLJ6KV1tbVKrkZXHRwtIMGmo&r=1DzJFW0v6TgEhkW1gy_-ke-RbtvS1fzEbD5_hcb9Up0&m=Jyfz-YpMjUtIbokDFmZ3UMnIE5o6K4XUKZ0pYSn6l3Y&s=jQuIMopzoPDSd2-mfw-WIL_dlx2LXt4pagYeHGARyUw&e=>>>>
> > > > .
> > > >
> > > > Takanori
> > > >
> > > > On 2020/11/26 18:04, Reza Khayat wrote:
> > > > > Why would an incorrect pixel size cause a bump in the FSC? The wrong pixel size will move the bump (left or right), but I just
> > don't see
> > > why it
> > > > would create a bump. The pixel size is something we tell the computer to put the map on the laboratory point of reference.
> Perhaps
> > I'm missing
> > > > something important....
> > > > >
> > > > > Reza Khayat, PhD
> > > > > Associate Professor
> > > > > City College of New York
> > > > > Department of Chemistry and Biochemistry
> > > > > New York, NY 10031
> > > > >
> > > > > ________________________________________
> > > > > From: Collaborative Computational Project in Electron cryo-Microscopy <[log in to unmask] <mailto:[log in to unmask]>
> <mailto:[log in to unmask] <mailto:[log in to unmask]>>
> > <mailto:[log in to unmask] <mailto:[log in to unmask]> <mailto:[log in to unmask] <mailto:[log in to unmask]>>>
> > > <mailto:[log in to unmask] <mailto:[log in to unmask]> <mailto:[log in to unmask] <mailto:[log in to unmask]>>
> <mailto:[log in to unmask] <mailto:[log in to unmask]> <mailto:[log in to unmask] <mailto:[log in to unmask]>>>>> on behalf of Marino
> > > > Jacopo (PSI) <[log in to unmask] <mailto:[log in to unmask]> <mailto:[log in to unmask] <mailto:[log in to unmask]>>
> <mailto:[log in to unmask] <mailto:[log in to unmask]> <mailto:[log in to unmask] <mailto:[log in to unmask]>>>
> > <mailto:[log in to unmask] <mailto:[log in to unmask]> <mailto:[log in to unmask] <mailto:[log in to unmask]>>
> <mailto:[log in to unmask] <mailto:[log in to unmask]> <mailto:[log in to unmask] <mailto:[log in to unmask]>>>>>
> > > > > Sent: Thursday, November 26, 2020 10:27 AM
> > > > > To: [log in to unmask] <mailto:[log in to unmask]> <mailto:[log in to unmask] <mailto:[log in to unmask]>>
> <mailto:[log in to unmask] <mailto:[log in to unmask]> <mailto:[log in to unmask] <mailto:[log in to unmask]>>>
> > <mailto:[log in to unmask] <mailto:[log in to unmask]> <mailto:[log in to unmask] <mailto:[log in to unmask]>>
> <mailto:[log in to unmask] <mailto:[log in to unmask]> <mailto:[log in to unmask] <mailto:[log in to unmask]>>>>
> > > > > Subject: [EXTERNAL] Re: [ccpem] Bump in FSC curves
> > > > >
> > > > > I think it was already discussed somewhere it might be due to the detergent micelle. But I think Marta has a stronger
> feeling the
> > > initial pixel
> > > > size was off.
> > > > >
> > > > >
> > > > > -----Original Message-----
> > > > > From: Collaborative Computational Project in Electron cryo-Microscopy <[log in to unmask] <mailto:[log in to unmask]>
> <mailto:[log in to unmask] <mailto:[log in to unmask]>>
> > <mailto:[log in to unmask] <mailto:[log in to unmask]> <mailto:[log in to unmask] <mailto:[log in to unmask]>>>
> > > <mailto:[log in to unmask] <mailto:[log in to unmask]> <mailto:[log in to unmask] <mailto:[log in to unmask]>>
> <mailto:[log in to unmask] <mailto:[log in to unmask]> <mailto:[log in to unmask] <mailto:[log in to unmask]>>>>> On Behalf Of Basil
> > > > Greber
> > > > > Sent: Thursday, 26 November 2020 16:25
> > > > > To: [log in to unmask] <mailto:[log in to unmask]> <mailto:[log in to unmask] <mailto:[log in to unmask]>>
> <mailto:[log in to unmask] <mailto:[log in to unmask]> <mailto:[log in to unmask] <mailto:[log in to unmask]>>>
> > <mailto:[log in to unmask] <mailto:[log in to unmask]> <mailto:[log in to unmask] <mailto:[log in to unmask]>>
> <mailto:[log in to unmask] <mailto:[log in to unmask]> <mailto:[log in to unmask] <mailto:[log in to unmask]>>>>
> > > > > Subject: Re: [ccpem] Bump in FSC curves
> > > > >
> > > > > Hi Marta,
> > > > >
> > > > > This bump (the valley, not the hill) is common and not an indication of processing issues. It may arise from the reduced
> > scattering power of
> > > > proteins in this frequency range.
> > > > >
> > > > > Best regards,
> > > > >
> > > > > Basil
> > > > >
> > > > >
> > > > >> Am 26.11.2020 um 15:16 schrieb Marta Sawicka <[log in to unmask] <mailto:[log in to unmask]>
> <mailto:[log in to unmask] <mailto:[log in to unmask]>> <mailto:[log in to unmask] <mailto:[log in to unmask]>
> > <mailto:[log in to unmask] <mailto:[log in to unmask]>>> <mailto:[log in to unmask] <mailto:[log in to unmask]>
> <mailto:[log in to unmask] <mailto:[log in to unmask]>>
> > > <mailto:[log in to unmask] <mailto:[log in to unmask]> <mailto:[log in to unmask] <mailto:[log in to unmask]>>>>>:
> > > > >>
> > > > >> Dear cryo-EM folks,
> > > > >>
> > > > >> I have observed a strange bump in FSC curves, which always occurs in a region between 5.5-4.5 A. I see the same pattern
> > regardless of a
> > > > protein I work with and data processing strategy. Having read what has been already discussed in relation to spikes in the
> FSC curves I
> > > suspect a
> > > > significant error in the pixel size to cause this bump. Also, having compared maps of the same protein coming from different
> > microscopes, I
> > > needed
> > > > to rescale the pixel size by approximately 5% to get a correct fit. Based on the RELION wiki entry on the pixel size issues,
> I have
> > redone
> > > > CtfRefine with estimation of the 4th order aberrations followed by another run to recalculate per-particle defocus. As far as I
> > understand this
> > > > should make things consistent with a wrong pixel size and after I feed a correct “absolute” pixel size in PostProcessing, I
> should
> > get FSC
> > > curves
> > > > without the bump. However, this is not the case and the FSCs look pretty much exactly the same. Is there something
> fundamental that I am
> > > missing
> > > > here? I would appreciate any ideas/suggestions.
> > > > >>
> > > > >> Many thanks,
> > > > >> Marta
> > > > >>
> > > > >> ########################################################################
> > > > >>
> > > > >> To unsubscribe from the CCPEM list, click the following link:
> > > > >>
> > > >
> > >
> >
> https://urldefense.proofpoint.com/v2/url?u=https-3A__www.jiscmail.ac.uk_cgi-2Dbin_WA-2DJISC.exe-3FSUBED1-3DCCPEM-26A-3D1&d=DwIGaQ&c=4NmamNZG3KTnUCoC6InoLJ6KV1tbVKrkZXHRwtIMGmo&r=1DzJFW0v6TgEhkW1gy_-ke-RbtvS1fzEbD5_hcb9Up0&m=4w70dDUPkTf3Atagtj-pfC2Mo8XeMifqPGrwhp0BQok&s=5J8vSpBsrl4r4H3fivRWK9iTlDVHhtqzKadpzbrtbx0&e=
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> >
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