Hi,
I had the same error while refining a membrane protein with a mask that was too tight. Run the refinement without mask, or try to check visually that the mask goes around your protein. With membrane proteins the mask that was being created was very strange and would end up giving Nyquist at the Refinement step.
Best,
Chiara
> On 16 Feb 2021, at 07:27, Dieter Blaas <[log in to unmask]> wrote:
>
> One more thing:
>
> I got some warnings during refinement:
>
> Are your groups large enough? Or is the reference on the correct greyscale?
> 3.42/4.17 min .................................................~~(,_,"> WARNING: norm_correction= 11.3176 for particle 4845 in group 3; Are your groups large enough? Or is the reference on the correct greyscale?
> 3.48/4.17 min ..................................................~~(,_,"> WARNING: norm_correction= 11.3181 for particle 4820 in group 3; Are your groups large enough? Or is the reference on the correct greyscale?
> 3.63/4.18 min ....................................................~~(,_,"> WARNING: norm_correction= 14.6129 for particle 4805 in group 3; Are your groups large enough? Or is the reference on the correct greyscale?
>
> can that be the reason for the distorted FSC curves? I now regrouped the particle images and, at present, refinement is running without warnings! I'll report the final outcome!
>
>
> best, Dieter
>
> ------------------------------------------------------------------------
> Dieter Blaas,
> Max Perutz Laboratories
> Medical University of Vienna,
> Inst. Med. Biochem., Vienna Biocenter (VBC),
> Dr. Bohr Gasse 9/3,
> A-1030 Vienna, Austria,
> Tel: 0043 1 4277 61630,
> Mobile: 0043 699 1942 1659
> e-mail: [log in to unmask]
> ------------------------------------------------------------------------
>
> On 16.02.2021 01:16, Basil Greber wrote:
>> Do your masks have any sharp edges? Are they large/soft enough? Are they zero all the way to the corners of the box, without artefacts near the box edges/corners?
>>
>> Basil
>>
>>
>>> Am 16.02.2021 um 00:02 schrieb Dieter Blaas <[log in to unmask]>:
>>>
>>> Hi,
>>>
>>> postprocess.star shows a reasonable resolution:
>>>
>>> _rlnFinalResolution 3.386336
>>>
>>> but the FSC curves are indeed screwed and show a bump. The curves start normally to go down but then go up again at about 1/A = 0.4. This reminds me a lot of the error I saw about a year ago....
>>>
>>> I am quite sure to not have duplicates....
>>>
>>> best, Dieter
>>>
>>> ------------------------------------------------------------------------
>>> Dieter Blaas,
>>> Max Perutz Laboratories
>>> Medical University of Vienna,
>>> Inst. Med. Biochem., Vienna Biocenter (VBC),
>>> Dr. Bohr Gasse 9/3,
>>> A-1030 Vienna, Austria,
>>> Tel: 0043 1 4277 61630,
>>> Mobile: 0043 699 1942 1659
>>> e-mail: [log in to unmask]
>>> ------------------------------------------------------------------------
>>>
>>> On 15.02.2021 22:12, Takanori Nakane wrote:
>>>> Hi,
>>>>
>>>> This is strange. You should trust the value from PostProcess.
>>>>
>>>> Refine3D reports the HIGHEST resolution where the FSC
>>>> crosses 0.143 (or 0.5 during iterations), while PostProcess
>>>> reports the LOWEST resolution. If your FSC curve has
>>>> oscillations or bumps, Refine3D can give too high resolution.
>>>>
>>>> Don't you have oscillations in the FSC?
>>>> Did you make sure you don't have duplicated particles?
>>>>
>>>> Best regards,
>>>>
>>>> Takanori Nakane
>>>>
>>>> On 2021/02/15 19:00, Dieter Blaas wrote:
>>>>> Sorry to come back to this issue but is it OK that this value is also output in run.out?
>>>>>
>>>>> .........................
>>>>>
>>>>> Calculating solvent-corrected gold-standard FSC ...
>>>>> + randomize phases beyond: 4.34912 Angstroms
>>>>> Maximization ...
>>>>> 000/??? sec ~~(,_,"> [oo]^M3.08/3.08 mi$
>>>>> Auto-refine: Refinement has converged, stopping now...
>>>>> Auto-refine: + Final reconstruction from all particles is saved as: Refine3D/job030/run_cla$
>>>>> Auto-refine: + Final model parameters are stored in: Refine3D/job030/run_model.star
>>>>> *Auto-refine: + Final data parameters are stored in: Refine3D/job030/run_data.star**
>>>>> ** Auto-refine: + Final resolution (already with masking) is: 1.9804**
>>>>> *---------------------------
>>>>>
>>>>> Best, Dieter
>>>>>
>>>>> ------------------------------------------------------------------------
>>>>> Dieter Blaas,
>>>>> Max Perutz Laboratories
>>>>> Medical University of Vienna,
>>>>> Inst. Med. Biochem., Vienna Biocenter (VBC),
>>>>> Dr. Bohr Gasse 9/3,
>>>>> A-1030 Vienna, Austria,
>>>>> Tel: 0043 1 4277 61630,
>>>>> Mobile: 0043 699 1942 1659
>>>>> e-mail:[log in to unmask]
>>>>> ------------------------------------------------------------------------
>>>>>
>>>>> On 15.02.2021 16:57, Takanori Nakane wrote:
>>>>>> Hi,
>>>>>>
>>>>>> You are looking at wrong items.
>>>>>>
>>>>>> rlnCurrentResolution and rlnCurrentImageSize in model.star's
>>>>>> "model_general" table are those used for alignment and backprojection.
>>>>>> Since "the last iteration will use data to Nyquist frequency",
>>>>>> the value goes up.
>>>>>>
>>>>>> You should look at rlnEstimatedResolution in "model_classes" table.
>>>>>>
>>>>>> Best regards,
>>>>>>
>>>>>> Takanori Nakane
>>>>>>
>>>>>> On 2021/02/15 15:50, Dieter Blaas wrote:
>>>>>>> Hi,
>>>>>>>
>>>>>>> some time ago people reported that Postprocess reported erroneous resolution values and distorted FSC curves due to an incorrect b-factor determined for single particles (I hope I am right here). Is this still the case in the above version of relion? I used CtfRefine followed by Polishing and, on re-running the refinement with shiny.dat, I get the below output on "cat *mod*|grep Curr" reporting a erroneous resolution for the last step (i.e.combining the two half maps). Is this error related to the above one? Why is it occurring? Did I use wrong parameters? The map looks OK but does certainly not have 2 A resolution!
>>>>>>>
>>>>>>> I did not see this problem in another refinement following the same procedure....
>>>>>>>
>>>>>>> .................................
>>>>>>>
>>>>>>> _rlnCurrentResolution 3.759407
>>>>>>> _rlnCurrentImageSize 348
>>>>>>> _rlnCurrentResolution 3.759407
>>>>>>> _rlnCurrentImageSize 348
>>>>>>> _rlnCurrentResolution 1.980402
>>>>>>> _rlnCurrentImageSize 450
>>>>>>>
>>>>>>> Thanks, best, Dieter
>>>>>>>
>>>>>>> ------------------------------------------------------------------------
>>>>>>> Dieter Blaas,
>>>>>>> Max Perutz Laboratories
>>>>>>> Medical University of Vienna,
>>>>>>> Inst. Med. Biochem., Vienna Biocenter (VBC),
>>>>>>> Dr. Bohr Gasse 9/3,
>>>>>>> A-1030 Vienna, Austria,
>>>>>>> Tel: 0043 1 4277 61630,
>>>>>>> Mobile: 0043 699 1942 1659
>>>>>>> e-mail: [log in to unmask]
>>>>>>> ------------------------------------------------------------------------
>>>>>>>
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