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

Wed Mar 12 15:05:10 PDT 2014

Hi Chuck, We did the FSC of the "gold-standard" calculations in the paper.
I was not sure about the PDB modeling in determining resolution in our
case, even though it was "better than nothing" as you said. Thanks for
sharing your thoughts. Qiu-Xing

On 3/12/14 4:45 PM, "Sindelar, Charles" <charles.sindelar at yale.edu> wrote:

>Hi Qiu-Xing, assuming the question you want to answer is "what is the
>highest resolution I can claim with certainty", then you have very few
>options.  Really the best one is the FSC of "gold-standard" calculations
>where the two halves of the data don't see each other, as was already
>mentioned. As you pointed out, FSC between your map and rigid-docked PDB
>can potentially under-estimate the resolution, but it's better than
>nothing, because it delivers a guaranteed lower limit.
>
>On the other hand, if your question is "how high could my resolution be"
>- then you are free to use any method you like, and pick the highest
>number. But obviously there are shortcomings with this approach.
>
>- Chuck
>
>On Mar 12, 2014, at 5:27 PM, Qiu-Xing Jiang
><Qiu-Xing.Jiang at utsouthwestern.edu>
> wrote:
>
>> 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
>>> 
>>> 
>>> 
>>> 
>>> _______________________________________________
>>> 3dem mailing list
>>> 3dem at ncmir.ucsd.edu
>>> https://mail.ncmir.ucsd.edu/mailman/listinfo/3dem
>> 
>> 
>> ________________________________
>> 
>> UT Southwestern Medical Center
>> The future of medicine, today.
>> 
>
>--
>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
>
>
>
>