[3dem] 3dem Digest, Vol 79, Issue 11

[Qiu-Xing Jiang]

Hi Charles. I was asking whether a rigidly-docked PBD model is good enough
to determine resolution for the membrane-facilitated filament because I
did't have a crystal structure of the filament itself.

On 3/12/14 4:07 PM, "Sindelar, Charles" <charles.sindelar at yale.edu> wrote:

>Hi Qiu-Xing, the experiment that Ed refers to is one of the better ones
>you can do- the FSC between the PDB-generated map, and your experimental
>map.  Particularly if you do only rigid-body fitting (not flexible
>fitting), the 0.5 cut-off will give you a conservative estimate of the
>resolution- i.e. it is difficult or impossible to over-estimate the
>resolution in this way.  I was curious why your original email did not
>report the results of this type of experiment (forcing Ed to enlighten
>us).  The other methods you used can all significantly overestimate the
>resolution of your map - including, notably, the ResMap program.
>
>On Mar 12, 2014, at 4:33 PM, <3dem-request at ncmir.ucsd.edu>
> wrote:
>
>> ----------------------------------------------------------------------
>>
>> Message: 1
>> Date: Wed, 12 Mar 2014 15:59:30 -0400
>> From: Edward Egelman <egelman at virginia.edu>
>> To: 3dem at ncmir.ucsd.edu
>> Subject: Re: [3dem] Resolution estimate: FSC vs PDB modeling
>> Message-ID: <5320BCA2.3050703 at virginia.edu>
>> Content-Type: text/plain; charset="iso-8859-1"; Format="flowed"
>>
>> I feel compelled to respond to this, as I am the "senior colleague".
>>The
>> paper under discussion appeared recently in Nature (2014), Mukherjee et
>> al. Let me correct the record. If one compares the map (EMDB-5795) with
>> the atomic model (4MTH) all correlation disappears at better than about
>> 16 Angstroms, so the statement that there was strong correlation at 9.2
>> to 15 Angstroms is wrong. I urge others to make this comparison
>> themselves. This led me to raise questions about the 9.2 Angstrom
>> resolution claim for the map. As far as I can see, after more than 20
>> emails exchanged with Dr. Jiang before the submission of the paper,
>>this
>> resolution claim was simply fraudulent. That was why I asked for my
>>name
>> to be removed from this paper! I would welcome, however, for the
>>cryo-EM
>> field to move to a "reality-based" standard of resolution, and this
>> paper provides a good basis for discussing why such a standard is
>>needed.
>> Regards,
>> Ed Egelman
>>
>> On 3/12/14, 8:47 AM, Qiu-Xing Jiang wrote:
>>> Dear colleagues,
>>> This has been a topic discussed in a couple of map validation papers.
>>> I have a scenario encountered in a recent project and would like some
>>> input from those interested.
>>> We had a filament complex reconstruction, made of a small protein
>>> whose crystal structure contains mainly loops held together in the
>>> core by three pairs of disulfide bonds. At the time of resolution
>>> estimation, we used two independent maps calculated from either
>>> randomly selected halves or top/bottom halves to calculate FSC as
>>> usual in SPIDER. The FSC0.5 of the former gives 9.2 angstroms, which
>>> is more conservative than the FSC0.143 of the latter, and was used as
>>> a nominal estimate.
>>>
>>> The other opinion  from a senior colleague was PDB modeling, whose
>>> operations are to dock the X-ray structures of individual units into
>>> the map (assuming no change), filter the resulted PDB model to
>>> different resolutions, and visually determine at which resolution the
>>> map calculated from the PDB model and the experimental density map
>>> match the best(at certain thresholds), and could then be used for
>>> resolution estimate. When we were operating this procedure, we knew
>>> that our filaments had lipids associated and our map was not good
>>> enough to resolve the loops on the surface of the individual units in
>>> the filament. To make  the two maps match well to our eyes, we had to
>>> filter both to 12-15 angstroms, which would then say the resolution
>>> was 12-15 A. The calculated cross-correlation between the map from the
>>> pdb model and the experimental map was high at 9.2-15 range, but we
>>> were not sure whether it would be decisively meaningful.
>>>
>>> We had debates and disagreements on which to report. At the end we
>>> decided to use FSC0.5 as usual, and refrained from interpreting the
>>> map with mutations at the subunit interface due to the experience of
>>> PDB modeling. I  wonder if some of you had similar experience, and
>>> more generally whether PDB modeling is suitable to replace FSC.
>>>
>>> Thanks for sharing your experience.
>>>
>>> Best regards,
>>>
>>> Qiu-Xing
>>>
>
>Charles V. Sindelar, Ph.D.
>Dept. of Molecular Biophysics and Biochemistry
>Yale University
>SHMC-E25
>333 Cedar Street
>New Haven, CT 06520-8024
>
>Phone (203) 737-4752
>Lab (203) 737-4723
>Fax (203) 785-7979
>http://medicine.yale.edu/mbb/faculty/charles_sindelar.profile
>
>
>
>
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