[3dem] etomo question dual axis tomogram generation
Kaelber, Jason Toye
kaelber at bcm.edu
Thu Mar 13 13:33:35 PDT 2014
Hi Cristina,
The IMOD mailing list covers eTomo questions and is monitored by the developers. imod at lists.colorado.edu
I think to remove the bad fiducials from the second axis, during fiducial editing step, click the arrows next to "contour" until you have found the offending fiducial, and then use Shift+D to delete that fiducial from the model. Hit s to save the new model.
Jason
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Today's Topics:
1. etomo question dual axis tomogram generation
(cristina.suarez at bioquant.uni-heidelberg.de)
2. Re: 3dem Digest, Vol 79, Issue 11 (Qiu-Xing Jiang)
----------------------------------------------------------------------
Message: 1
Date: Thu, 13 Mar 2014 16:42:36 +0100
From: cristina.suarez at bioquant.uni-heidelberg.de
To: "3dem at ncmir.ucsd.edu" <3dem at ncmir.ucsd.edu>
Subject: [3dem] etomo question dual axis tomogram generation
Message-ID:
<20140313164236.mln5da23c4ccos8k at wwwmail.urz.uni-heidelberg.de>
Content-Type: text/plain; charset=ISO-8859-1; DelSp="Yes";
format="flowed"
Dear colleagues,
I am trying to get a tomogram combination from two single tomograms
using etomo, When I transfer the fiducials from other axis (tomogram
"a" to "b") if I check the seed model the fiducials are wrong and
etomo did not allow me to remove the wrong fiducials in order to put
them in the righ position in the tomogram.
Any of us have an idea of the solution for this problem?
Aditionaly information, when I took the image stacks they were
properly aligned after grid turning 90? and the single axis tomogram
were generated without any problem.
Best regards
Cristina
Cristina Su?rez Garc?a, PhD.
Electron Microscopy core facility & Dept. of Infectious diseases,
Heidelberg University,
Im Neuenheimer Feld 267, 69120
Heidelberg, Germany
------------------------------
Message: 2
Date: Thu, 13 Mar 2014 16:02:52 +0000
From: Qiu-Xing Jiang <Qiu-Xing.Jiang at UTSouthwestern.edu>
To: "rkhayat at ccny.cuny.edu" <rkhayat at ccny.cuny.edu>,
"3dem at ncmir.ucsd.edu" <3dem at ncmir.ucsd.edu>
Subject: Re: [3dem] 3dem Digest, Vol 79, Issue 11
Message-ID: <CF473CF1.13396%qiu-xing.jiang at utsouthwestern.edu>
Content-Type: text/plain; charset="us-ascii"
Hi,
Regarding the second point, at 15 angstroms we were enough to formulate a
pore forming mechanism of protein insertion and the charge segregation
mechanism for lipid-protein interaction. We could not probe the
interactions at the interfaces between monomers in the pore. Our filaments
tend to have local bending. We need to select enough straight filaments in
order to reach a resolution (say ~5A) suitable for recognizing the core
loops stabilized by C-C bonds. Qiu-Xing
On 3/13/14 6:02 AM, "rkhayat at ccny.cuny.edu" <rkhayat at ccny.cuny.edu> wrote:
>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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