Hi,

> it might affect the sharpening step

As I wrote in my previous mail, if the MTF is very off,
estimated B factors for "auto" sharpening can be off.

You can always override it by specifying a better
B factor in Postprocess. Indeed, this is often done
for modeling, because a single B factor does not necessarily
work well for the entire map. Some regions of the map
need blurring, while other regions need sharpening.
Indeed, CCPEM has a tool to calculate a series of maps with
different blurring and sharpening.

> make some microscope performance measurements

My recommendation is to run many Refine3D jobs with
various numbers of particle subsets and draw
the Rosenthal-Henderson plot (you can use RELION's
scripts/bfactor.py).

Since the FSC is not affected by the MTF used in PostProcess,
B factor estimated in this way is also not affected by the specified MTF.

Best regards,

Takanori Nakane

> Dear Gabe,
> Thanks for the clarification and for providing the curve!
>
> Dear all,
> Good to know it doesn't seem to affect your final maps so much.  (I
> thought
> it might affect the sharpening step but seems like not so much... thanks
> for the info, Takanori!). I just cared somewhat about the MTF because I
> was
> trying to make some microscope performance measurements.  (Not super
> critical though.)
>
> Thanks all!  I really appreciate the help.
> Best,
> Sara
>
>
>
> On Sun, Aug 11, 2019 at 10:55 PM Gabriel Lander <[log in to unmask]> wrote:
>
>> Hi Sara & all,
>> just want to clarify that the "Scripps" 200 keV is a fitted curve to
>> values that came from a "MTF_Summit-SRes_Plot.dm4" file that was
>> provided
>> to me by David Belnap in January 2017. I believe the data were collected
>> by
>> Gatan after their install, I have no details about exposure rate.
>> We've never really noticed much of a difference in the final densities
>> when we've tried out different MTF curves (even 300 keV ones).
>> -gabe
>>
>>
>> On Aug 9, 2019, at 8:30 PM, Sara L Campbell <[log in to unmask]> wrote:
>>
>> Thanks for making those plots!
>>
>> The Ruskin et al., JSB paper you linked to seems consistent with the MRC
>> data, but seems to disagree with the Gatan 200/300 keV data and Scripps
>> 200/300 keV data.
>>
>> That's a good thought about the dose rate.  In McMullan et al.,
>> Ultramicroscopy 2014 (
>> https://www.sciencedirect.com/science/article/pii/S030439911400151X?via%3Dihub),
>> they say 1.1 e-/pix/s for the MRC data, which is sufficiently low that I
>> don't think that dose rates can explain the discrepancy.
>>
>> Curiouser and curiouser... I was just trying to normalize some
>> resolution
>> measurements correctly :-)
>>
>> -Sara
>> <k2_mtf_300kev_compare.png>
>>
>>
>> On Fri, Aug 9, 2019 at 5:27 PM Daniel Asarnow <[log in to unmask]>
>> wrote:
>>
>>> Based on 10.1016/j.jsb.2013.10.016
>>> <http://www.sciencedirect.com/science/article/pii/S1047847713002815>,
>>> the difference could be explained by dose rates. Is anyone aware at
>>> what
>>> dose these were measured?
>>>
>>> Best,
>>> -da
>>>
>>> On Fri, Aug 9, 2019 at 5:11 PM Daniel Asarnow <[log in to unmask]>
>>> wrote:
>>>
>>>> For everyone's reference, here are plots of the available curves.
>>>> There
>>>> is indeed a bit of a discrepancy between the K2 @ 300kV measured by
>>>> the MRC
>>>> or from Niko's work and the ones measured by Gatan. At 200 kV though
>>>> there
>>>> is good agreement between Gatan's values and Gabe's. The second file
>>>> has
>>>> the K3 curves from Gatan as well.
>>>>
>>>> Best,
>>>> -da
>>>>
>>>> On Fri, Aug 9, 2019 at 3:40 PM Sara L Campbell <[log in to unmask]>
>>>> wrote:
>>>>
>>>>> Dear all,
>>>>>
>>>>> The K2 MTF data I found out there seems inconsistent?  Do folks have
>>>>> any thoughts or advice on this?  Forgive me if I've made a rookie
>>>>> mistake
>>>>> :-)
>>>>>
>>>>> This MTF data is all for:
>>>>> _rlnResolutionInversePixel
>>>>> _rlnMtfValue
>>>>>
>>>>> (first one is same as the one in the RELION tutorial download)
>>>>> https://www3.mrc-lmb.cam.ac.uk/relion/index.php/K2-summit_at_300kV
>>>>> http://www.gatan.com/sites/default/files/MTF_Curves/mtf_k2_300kV.star
>>>>>
>>>>> (tried to search CCPEM to see if the question had been asked already
>>>>> +
>>>>> found the first file)
>>>>> http://www.lander-lab.com/downloads/mtf-k2-200kev.star
>>>>> http://www.gatan.com/sites/default/files/MTF_Curves/mtf_k2_200kV.star
>>>>>
>>>>> If I square the MRC* 300 keV *values, they come remarkably close to
>>>>> the Gatan *200 keV *values.  Mysterious!
>>>>>
>>>>> Will the real MTF please stand up?
>>>>>
>>>>> Thanks for your help,
>>>>> -Sara
>>>>>
>>>>>
>>>>> ------------------------------
>>>>>
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>>>>>
>>>>
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>>
>>
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