[3dem] 3dem Digest, Vol 79, Issue 11
rkhayat at ccny.cuny.edu
rkhayat at ccny.cuny.edu
Thu Mar 13 04:02:11 PDT 2014
Hi,
I would like to ask a much more naive question. Rather than asking how does
one determine resolution, I would like to ask how does resolution alter our
interpretation of an image reconstruction? For example, what more can we
learn of a complex if we generate a correct image reconstruction of it at
30Angstroms vs 25, 20, 16, 12, 9, 7, 5, 3 and 2Angstroms. How does this
interpretation change if we do not posses and do posses PDB(s) of the
components? What and where are the resolution landmarks that signify an
advance in interpreting an image reconstruction (e.g. subnanometer resolution
allows helices to be identified)?
Best wishes,
Reza
Reza Khayat, PhD
Assistant Professor
The City College of New York
Department of Chemistry, MR-1135
160 Convent Avenue
New York, NY 10031
Tel. (212) 650-6070
---- Original message ----
>Date: Wed, 12 Mar 2014 22:05:10 +0000
>From: 3dem-bounces at ncmir.ucsd.edu (on behalf of Qiu-Xing Jiang <Qiu-
Xing.Jiang at UTSouthwestern.edu>)
>Subject: Re: [3dem] 3dem Digest, Vol 79, Issue 11
>To: "3dem at ncmir.ucsd.edu" <3dem at ncmir.ucsd.edu>,"Sindelar, Charles"
<charles.sindelar at yale.edu>
>
>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
>>
>>
>>
>>
>
>
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