I want to make a cryo-EM image for protein PDB ID: 1FLP

[b.beh...@gmail.com]

Hi,
I read the tutorial documentation and see all video tutorial in YouTube. But I'm confused and I don't know how I should start to make a 3D Cryo-EM images for a protein (for example: PDB ID: 1FLP).
Would you mind please say to me how I should start to do it?

[Steven Ludtke]

Hi. I think there must be some confusion here. The point of CryoEM is to solve the structure of unknown biological molecules and assemblies experimentally. CryoEM data is experimental data collected on purified molecules in a transmission electron microscope.  While some people may occasionally create synthetic data from PDB structures for purposes of testing the reconstruction software, it doesn't sound like that is what you are trying to do.  Could you explain what you're after?

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Steven Ludtke, Ph.D.

Professor, Dept of Biochemistry and Mol. Biol.         (www.bcm.edu/biochem)

Co-Director National Center For Macromolecular Imaging        (ncmi.bcm.edu)

Co-Director CIBR Center                          (www.bcm.edu/research/cibr)

Baylor College of Medicine                             

slu...@bcm.edu

[b.beh...@gmail.com]

Hi,
Thank you for comprehensive reply. I understand what you say.But for evaluating our work I need some protein which are modeled before by other method (e.g. crystallography) models by Cryo-EM too. I want to know how I can produce a Cryo-EM image for each protein? Is it possible or not that I select one protein randomly from PDB and produce Cryo-EM image by EMAN?

[Steven Ludtke]

I'm sorry, I'm having a very difficult time understanding what you are after. There seems to be some confusion about the specific definition of words, which is impeding the discussion. "modeled before by other method (e.g. cyrstallography)". To be clear, crystallography and cryoEM are experimental techniques, not modeling techniques. Modeling implies structures derived from theoretical not experimental methods. You say "for evaluating our work" but have not said what sort of work you are talking about? Have you collected CryoEM data, and you are hoping to simulate some date from PDB models to better understand what you are seeing?  Are you software developers trying to test an algorithm? Something else entirely?

My best guess from your statement is that you are trying to create synthetic CryoEM data for some purpose. If that is the case, then there are a range of different extents to which you may go in the simulation process, but even the most thoroughly simulated data still fails to capture all aspects of the experimental system. That is to say, when testing software, even if you use the most realistic simulated data, results are always far better than with real data, as there are simply some aspects of real data which are difficult to capture.

Is there a reason you don't wish to look at real experimental data? EMPIAR (https://www.ebi.ac.uk/pdbe/emdb/empiar/) contains an archive of raw image data associated with published maps, most at near-atomic resolution (so a PDB model also exists).

If you really want to do "simulation", then it depends on how thoroughly you wish to model the imaging process and specimen. The steps are:

1) convert the PDB to a density volume (e2pdb2mrc.py)

2) make projections of the volume (e2project3d.py  generally with a random orientation generator, e2help.py orientgen)

3) apply a CTF to the projections and add noise (e2proc2d.py ...  --process math.simulatectf:...   (see e2help.py processor simulatectf -v 2))

There are even more steps if you wish to add these into a simulated micrograph and/or simulate conformational variability, buffer molecules, impurities, radiation damage, etc.   A few people in the community have developed programs specifically designed to simulate data, but again, if you use such simulated data in software testing, simulated data is much too perfect to be realistic.

Is that closer to what you are trying to find out?

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[b.beh...@gmail.com]

Hi,
Thank you very much. I exactly understand what you say. I try to do it.

On Wednesday, May 25, 2016 at 12:01:55 PM UTC+4:30, b.beh...@gmail.com wrote: