Wrong size model?

[BG]

Hello,

We are testing EMAN2 on one of our proteins that has it structure
already determined. The model looks very close to our published model.
However, when we try to dock the solved structure into the model, the
model seems to be too small by at least a factor of 10. In addition,
when we compare the model with the EM images of the particles, there
appears to be a significant difference in size. Is there a specific
parameter that is being input on our part that has an error? Where are
we going wrong with our data?

Thank you,

BG

[Ludtke Steven]

Sounds like you are having problems with the A/pix value. Hopefully you have this correct within EMAN2, and are only having problems with it getting exported to Chimera, since if you have this number wrong in EMAN2, it means all of your CTF correction work is also incorrect. There was a bug in EMAN2 last fall that was preventing EMAN2 from copying the A/pix value into the output map correctly under some specific situations. That problem was fixed some time ago, so if you are using a recent EMAN2 snapshot, that, at least, shouldn't be the issue. If you have an older version, you may consider upgrading to a current snapshot.

As to solutions to the problem, the recommended file format for exporting data from EMAN2 -> Chimera is HDF, though it is also possible to use MRC or Spider. You can get the correct A/pix value in a couple of ways.
1) e2proc3d.py bdb:refine_xx#threed_filt_yy r_xx_yy.hdf --apix=<correct Apix value>
2) run chimera, and edit the size of the volume in the volume data interface (A/pix * box size in pixels)

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[Qian]

Hi

This is caused by incorrect a/pixel value stored in 3d volume file probably. If you use chimera you can check it in volume viewer windows by click tool-volume data- volume viewer. If voxel size is 1, that means you need to change it.

Qian

Department of molecular biology
The university of Sheffield
Uk

发自我的 iPod

[BG]

Hello,

We are curious to know the source we should be using for determining
the A/pix. We have been saving our EM images as DM3 files, but have
seen quite a large range of sizes of pix/nm even when the
magnification is the same. Since we are given the pix/nm (which varies
from file to file), should we be using this value to calculate the A/
pix?

Thank you,

BG

On Feb 16, 1:30 pm, Qian <x7d...@gmail.com> wrote:
> Hi
>
> This is caused by incorrect a/pixel value stored in 3d volume file probably. If you use chimera you can check it in volume viewer windows by click tool-volume data- volume viewer. If voxel size is 1, that means you need to change it.
>
> Qian
>
> Department of molecular biology
> The university of Sheffield
> Uk
>
> 发自我的 iPod
>

> 在 16 Feb 2012，15:19，BG <belindagale...@gmail.com> 写道：
>
>
>
> > Hello,
>
> > We are testing EMAN2 on one of our proteins that has it structure
> > already determined. The model looks very close to our published model.
> > However, when we try to dock the solved structure into the model, the
> > model seems to be too small by at least a factor of 10. In addition,
> > when we compare the model with the EM images of the particles, there
> > appears to be a significant difference in size. Is there a specific
> > parameter that is being input on our part that has an error? Where are
> > we going wrong with our data?
>
> > Thank you,
>
> > BG
>
> > --

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> > For more options, visit this group at

> >http://groups.google.com/group/eman2- Hide quoted text -
>
> - Show quoted text -

[Steven Ludtke]

This is a fairly fundamental experimental value. Do you not have much TEM experience ? What sort of microscope are you using ? 1 Å == 0.1 nm, so 10.0/<pix/nm> will give you A/pix IF your pix/nm value is accurate. This value must normally be carefully calibrated, and the typical microscope service technician may not do this with sufficient precision. The actual magnification may vary by up to +-2% or so with time as well.

----------------------------------------------------------------------------
Steven Ludtke, Ph.D.
Associate Professor, Dept. of Biochemistry and Mol. Biol. Those who do
Co-Director National Center For Macromolecular Imaging ARE
Baylor College of Medicine The converse
slu...@bcm.edu -or- ste...@alumni.caltech.edu also applies
http://ncmi.bcm.edu/~stevel

> --
> ----------------------------------------------------------------------------------------------

[Nicholas Gao]

Hi, 

Im running:

EMAN 2.11 (CVS 2015/06/05 19:02:06)

Your EMAN2 is running on: Mac OS 10.10.5 x86_64

Your Python version is:  2.7.10

I've got a similar problem.  I made a low-resolution volume by neg stain SPA, and i'm trying to dock a crystal structure into it.  But my volume is significantly smaller than the crystal structure.  I'm doing all of this in Chimera.  My volume is in .hdf format.  I will eventually use e2proc3d.py to change the Apix value to what I set it to during my data processing.  But before I do, I'd like to ask: how do I check what the Apix value currently is in my .hdf low-res volume file?

-Nick

[Steven Ludtke]

e2iminfo.py -H file.hdf

or

the 'info' button in the browser when the hdf file is selected

look at apix_x

You can adjust these values interactively in chimera with the volume viewer control panel, though, so this really shouldn't be a major problem...

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Professor, Dept. of Biochemistry and Mol. Biol.                Those who do

[Nicholas Gao]

Thanks.  I was able to view the Apix via e2imnfo.py.  And I also found the field to edit for voxel size in Chimera --> Volume Viewer --> Features --> Coordinates

The Apix at the time of data collection was 2.55 angstroms/pixel.

My volume's .hdf is 84 x 84 x 84 with apix_x, apix_y and apix_z all equal to 1.  

So is the correct voxel size = ?

-Nick

[Steven Ludtke]

Unless you resampled the data, the map should be 2.55 as well...

[suruchi singh]

Hi Steven,

I followed your reply as I was facing the same problem that the volume of my map in chimera was small. I used your second suggestion by increasing the voxel size in the volume viewer option of chimera. I saved the map with the same voxel parameters. Now, when I am estimating the resolution of the saved map using FSC validation or other tools, it is low resolution. Could you suggest anything?

Thanks

Suruchi

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[Steve Ludtke]

When you generate a map with EMAN2, it should be putting the A/pix value in the header, and Chimera should pick that up properly when reading the file (at least for .hdf and .mrc). If the file is showing the wrong A/pix value (e2iminfo.py -s map.hdf) then something funny is going on. If it resulted from an EMAN2 refinement this implies that you may not have had the A/pix value set properly during the refinement, which may produce inaccurate results. If you were just using EMAN2 for file format conversion, then specifying --apix at the e2proc3d.py command line is a good way to get the correct value into the header.

--------------------------------------------------------------------------------------
Steven Ludtke, Ph.D. <slu...@bcm.edu>                      Baylor College of Medicine 

Charles C. Bell Jr., Professor of Structural Biology
Dept. of Biochemistry and Molecular Biology                      (www.bcm.edu/biochem)

Academic Director, CryoEM Core                                        (cryoem.bcm.edu)
Co-Director CIBR Center                                    (www.bcm.edu/research/cibr)

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