Hi Pavel,

Thanks, it’s as I anticipated.

Best wishes,
Reza

Reza Khayat, PhD

Assistant Professor

Department of Chemistry

City College of New York

85 Saint Nicholas Terrace, CDI 2.318

New York, NY 10031

http://www.khayatlab.org/

212-650-6070

From: Pavel Afonine [mailto:[email protected]]
Sent: Monday, February 08, 2016 11:08 AM
To: Reza Khayat <[email protected]>; Oliver Clarke <[email protected]>; [email protected]
Subject: Re: [phenixbb] Refine pixel size of map (for EM data)?

Hi Reza,

geometry of refined model may be severely distorted if your target map on a wrong scale (magnification).

Some relevant reading:

1)
Automatic estimation and correction of anisotropic magnification,distortion in electron microscopes,Timothy Grant, Nikolaus Grigorieff
Journal of Structural Biology 192 (2015) 204–208

2)
Electron cryomicroscopy observation of rotational states in a eukaryotic V-ATPase,
Jianhua Zhao, Samir Benlekbir & John L. Rubinstein
Nature 521, 241–245 (14 May 2015)

3)
Description and comparison of algorithms for correcting anisotropic magnification in cryo-EM images.
Jianhua Zhao, Marcus A. Brubaker, Samir Benlekbir, John L. Rubinstein

Pavel

On 2/8/16 02:38, Reza Khayat wrote:

Hi Oliver,

Out of curiosity:

    1. what kind of R-factors and CC-values do you get when refining against the two different pixel size?

    2. how different are your refined pixel sizes from one reconstruction to another?

    3. how much of an affect does the wrong pixel size have on your downstream structure analysis (e.g. BDA, ASA, electrostatic...)?

Best wishes,
Reza

Reza Khayat, PhD

Assistant Professor

City College of New York

Department of Chemistry

New York, NY 10031

From: [email protected] <[email protected]> on behalf of Oliver Clarke <[email protected]>
Sent: Monday, February 8, 2016 4:28 AM
To: [email protected]
Subject: [phenixbb] Refine pixel size of map (for EM data)?

Hello,

I wonder whether it would be possible to add an option for phenix.real_space_refine to allow refinement of the pixel size of the map (or the unit cell dimensions - just an overall size scale factor), and write out the altered map at the end of refinement.

Although we try to calibrate this as best as we are able at the time of data collection, it is never perfect - for example, in one case I have dealt with, our nominal pixel size out of the scope is 1.19 Å, but the pixel size calibrated based on a crystal structure of a fragment of the protein is 1.25 Å. This is not a huge difference, but it is sufficient I think to have a substantial impact on refinement, particularly as regards clash assessment and H-bond/sec struc restraints.

In cases where one does not have a solved crystal structure to use for calibration, perhaps refining the pixel size in conjunction with the geometry might be of some use?

Cheers,

Oli
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