Re: [ccp4bb] MR phasing using Negative Stain EM reconstruction

[Stefano Trapani]

 

Dear Pascal 

have a look at : 

https://doi.org/10.1107/S0907444910002763 

Acta Cryst. (2010). D66, 514-521
Macromolecular crystal data phased by negative-stained
electron-microscopy reconstructions
S. Trapani, G. Schoehn, J. Navaza and C. Abergel 

Best, 

---
Stefano Trapani

Maître de Conférences
http://www.cbs.cnrs.fr/index.php/fr/personnel?PERS=Stefano%20Trapani
-
Centre de Biochimie Structurale (CBS)
29 rue de Navacelles
34090 MONTPELLIER Cedex, France

Tel : +33 (0)4 67 41 77 29
Fax : +33 (0)4 67 41 79 13
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CNRS UMR 5048
INSERM UMR 1054
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Le 2016-10-25 03:49, Pascal Egea a écrit : 

> Dear All, 
> 
> I would like to know if it is possible to use a low resolution EM 
> reconstruction of a complex obtained in negative stain EM (not cryo EM) to 
> help molecular replacement in a 4.5A resolution X-ray diffraction data set of 
> the same complex 
> I am aware of the possibility of using low resolution cryoEM maps for MR as 
> described in the review from Jackson et al in Nature Protocols but I was 
> wondering if there is an intrinsically impossibility for negative stain 
> reconstructions. 
> 
> Any thoughts or advice will be greatly appreciated.
> 
> Best, 
> -- 
> 
> Pascal F. Egea, PhD
> Assistant Professor
> UCLA, David Geffen School of Medicine
> Department of Biological Chemistry
> Boyer Hall room 356
> 
> 611 Charles E Young Drive East 
> Los Angeles CA 90095
> office (310)-983-3515
> lab (310)-983-3516
> email pegea at mednet.ucla.edu [1] 
> -- 
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Re: [ccp4bb] MR phasing using Negative Stain EM reconstruction

[Savvas Savvides]

Dear Pascal,

the EM2DAM program from the ATSAS package for SAXS 
(https://www.embl-hamburg.de/biosaxs/manuals/em2dam.html 
) can convert any EM 
envelope to a dummy-atom model in pdb format.
Also, the recent SUPALM algorithm might provide additional options as well.
http://journals.iucr.org/j/issues/2016/03/00/ap5002/ap5002.pdf 



best wishes
Savvas


Savvas Savvides
Ghent University, L-ProBE
VIB Inflammation Research Center (IRC)
Technology Park 927, B-9052 Ghent (Zwijnaarde), Belgium
+32 (0)472 928 519 (mobile) ; +32 9 331 36 60 (office) ; Skype: 
savvas.savvides_skype
http://www.vib.be/en/research/scientists/Pages/Savvas-Savvides-Lab.aspx 




> On 25 Oct 2016, at 10:00, Randy Read  wrote:
> 
> Dear Pascal,
> 
> I'm assuming that you're talking about using the negative stain image as an 
> MR model.  I don't recall hearing of this having ever worked (though I would 
> be very interested of course if anyone has managed to do this!), but my 
> intuition is that it's not going to work.  Negative stain just gives you an 
> external shape, whereas a cryo-EM reconstruction has internal features as 
> well.
> 
> Presumably you don't have atomic models of the individual components of the 
> complex?  If you did, using those directly for MR would be my first choice, 
> but you could also consider making a pseudo-atomic model by docking them into 
> the shape of the negative stain image.  Such a model would add substantial 
> higher-resolution information.
> 
> Best wishes and good luck,
> 
> Randy Read
> 
>> On 25 Oct 2016, at 02:49, Pascal Egea > > wrote:
>> 
>> Dear All,
>> 
>> I would like to know if it is possible to use a low resolution EM 
>> reconstruction of a complex obtained in negative stain EM (not cryo EM) to 
>> help molecular replacement in a 4.5A resolution X-ray diffraction data set 
>> of the same complex
>> I am aware of the possibility of using low resolution cryoEM maps for MR as 
>> described in the review from Jackson et al in Nature Protocols but I was 
>> wondering if there is an intrinsically impossibility for negative stain 
>> reconstructions.
>> 
>> Any thoughts or advice will be greatly appreciated.
>> 
>> Best,
>> 
>> -- 
>> Pascal F. Egea, PhD
>> Assistant Professor
>> UCLA, David Geffen School of Medicine
>> Department of Biological Chemistry
>> Boyer Hall room 356
>> 611 Charles E Young Drive East
>> Los Angeles CA 90095
>> office (310)-983-3515
>> lab  (310)-983-3516
>> email pegea at mednet.ucla.edu 
> 
> --
> Randy J. Read
> Department of Haematology, University of Cambridge
> Cambridge Institute for Medical Research  Tel: + 44 1223 336500
> Wellcome Trust/MRC Building   Fax: + 44 1223 336827
> Hills RoadE-mail: rj...@cam.ac.uk 
> 
> Cambridge CB2 0XY, U.K.   www-structmed.cimr.cam.ac.uk 
> 

Re: [ccp4bb] MR phasing using Negative Stain EM reconstruction

[Alexandre OURJOUMTSEV]

Dear Pascal,

A while ago when I did  my tests on MR at a very low resolution, I did it both 
with “shaped” envelopes and with the “flat” ones. It did work for both (with 
simulated data, which is obviously not the same as the experimental), but MR 
with the “shaped” models was more robust.

Some traces of this are in the article in Acta Cryst D51 (1995) 888-895.

Best regards,

Sacha Urzhumtsev


De : CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] De la part de Pascal 
Egea
Envoyé : mardi 25 octobre 2016 03:49
À : CCP4BB@JISCMAIL.AC.UK
Objet : [ccp4bb] MR phasing using Negative Stain EM reconstruction

Dear All,

I would like to know if it is possible to use a low resolution EM 
reconstruction of a complex obtained in negative stain EM (not cryo EM) to help 
molecular replacement in a 4.5A resolution X-ray diffraction data set of the 
same complex
I am aware of the possibility of using low resolution cryoEM maps for MR as 
described in the review from Jackson et al in Nature Protocols but I was 
wondering if there is an intrinsically impossibility for negative stain 
reconstructions.

Any thoughts or advice will be greatly appreciated.

Best,

--
Pascal F. Egea, PhD
Assistant Professor
UCLA, David Geffen School of Medicine
Department of Biological Chemistry
Boyer Hall room 356
611 Charles E Young Drive East
Los Angeles CA 90095
office (310)-983-3515
lab  (310)-983-3516
email pegea at mednet.ucla.edu

Re: [ccp4bb] MR phasing using Negative Stain EM reconstruction

[Randy Read]

Dear Pascal,

I'm assuming that you're talking about using the negative stain image as an MR 
model.  I don't recall hearing of this having ever worked (though I would be 
very interested of course if anyone has managed to do this!), but my intuition 
is that it's not going to work.  Negative stain just gives you an external 
shape, whereas a cryo-EM reconstruction has internal features as well.

Presumably you don't have atomic models of the individual components of the 
complex?  If you did, using those directly for MR would be my first choice, but 
you could also consider making a pseudo-atomic model by docking them into the 
shape of the negative stain image.  Such a model would add substantial 
higher-resolution information.

Best wishes and good luck,

Randy Read

> On 25 Oct 2016, at 02:49, Pascal Egea  wrote:
> 
> Dear All,
> 
> I would like to know if it is possible to use a low resolution EM 
> reconstruction of a complex obtained in negative stain EM (not cryo EM) to 
> help molecular replacement in a 4.5A resolution X-ray diffraction data set of 
> the same complex
> I am aware of the possibility of using low resolution cryoEM maps for MR as 
> described in the review from Jackson et al in Nature Protocols but I was 
> wondering if there is an intrinsically impossibility for negative stain 
> reconstructions.
> 
> Any thoughts or advice will be greatly appreciated.
> 
> Best,
> 
> -- 
> Pascal F. Egea, PhD
> Assistant Professor
> UCLA, David Geffen School of Medicine
> Department of Biological Chemistry
> Boyer Hall room 356
> 611 Charles E Young Drive East
> Los Angeles CA 90095
> office (310)-983-3515
> lab  (310)-983-3516
> email pegea at mednet.ucla.edu 

--
Randy J. Read
Department of Haematology, University of Cambridge
Cambridge Institute for Medical Research  Tel: + 44 1223 336500
Wellcome Trust/MRC Building   Fax: + 44 1223 336827
Hills RoadE-mail: rj...@cam.ac.uk
Cambridge CB2 0XY, U.K.   www-structmed.cimr.cam.ac.uk