Hi Oli, Yes, this is a problem that has always plagued EM. Unfortunately it was never really dealt with because it's not much of an issue for lower resolution image reconstructions unless you're doing very detailed difference maps from the X-ray model. The problem is more pronounced now because atoms are finally being modelled into the reconstructions. This is something that most microscopists are not familiar with and thus do no go through the rigour of pixel correction. Most magnification/pixel size calibrations involve using diffraction from a 2D crystal with known cell parameters. This, in my opinion, is insufficient given that protein isomorphous x-tals are not all that common. Moreover, we know from our x-ray crystallography experiments that the final lattice constants of a x-tal are calculated during the scaling stage not just the indexing stage. I would imaging that the rmsd values for your bonds and angles are a bit messed up as well. Your fastest way of dealing with the situation is to write scripts that modify the pixel size and calculate CC-values or R/Rfree factors between your model and the image reconstruction. Reza Reza Khayat, PhD Assistant Professor City College of New York Department of Chemistry New York, NY 10031 ________________________________ From: Oliver Clarke Sent: Monday, February 8, 2016 7:36 AM To: Reza Khayat Cc: [email protected] Subject: Re: [phenixbb] Refine pixel size of map (for EM data)? Hi Reza, Second question first - not much (I can't detect any), assuming same scope and mag (which is relieving for multiple reasons). However, I don't know how close the pixel size I obtain by real space correlation with a crystal structure is to the "true" pixel size, and whether it is wrong in any systematic way. Re the other questions, I have not compared rigorously (I only refined against the map with the correct pixel size), but I undertook steps to correct it when I noticed during initial building in Coot that known (solved) domains were not quite fitting the density as well as expected. I would expect some consequences from this, particularly in terms of clash analysis and the use of reference restraints (not sure about electrostatics etc). Where it does make a big difference is in comparing structures of the same or related proteins solved by different groups - if the pixel size varies by even 1-2%, this really messes up structural superposition for large structures (if the structure is 300Å, this could be a 6 Å difference in size). In my experience many groups don't seem to do this - there are quite a lot of recent maps in the EMDB in which the nominal pixel size has been taken as correct, and calibration with a crystal structure shows that it is a ways off (this also affects the estimated resolution, sometimes substantially). Cheers, Oli On Mon, Feb 8, 2016 at 11:38 AM, Reza Khayat mailto:[email protected]> 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]mailto:[email protected] mailto:[email protected]> on behalf of Oliver Clarke mailto:[email protected]> Sent: Monday, February 8, 2016 4:28 AM To: [email protected]mailto:[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

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 ; Oliver Clarke ; [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]mailto:[email protected] mailto:[email protected] on behalf of Oliver Clarke mailto:[email protected] Sent: Monday, February 8, 2016 4:28 AM To: [email protected]mailto:[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 _______________________________________________ phenixbb mailing list [email protected]mailto:[email protected] http://phenix-online.org/mailman/listinfo/phenixbb Unsubscribe: [email protected]mailto:[email protected]

Hi Reza, It's a great idea to have a tool in Phenix to visualize NCS. We don't have one yet but I'll see what it would take to make one... We do have methods to identify symmetry axes from NCS operators that could be use in such a tool. All the best, Tom T ________________________________ From: [email protected] on behalf of Reza Khayat Sent: Tuesday, July 26, 2016 8:17 AM To: [email protected] Subject: [phenixbb] Visualizing phenix.autosol NCS axis Hi, Is it possible to visualize the NCS axis identified with phenix.autosol? Thanks. Best wishes, Reza Reza Khayat, PhD Assistant Professor City College of New York Department of Chemistry New York, NY 10031 ________________________________ From: Pavel Afonine Sent: Monday, February 8, 2016 11:07 AM To: Reza Khayat; Oliver Clarke; [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]mailto:[email protected] mailto:[email protected] on behalf of Oliver Clarke mailto:[email protected] Sent: Monday, February 8, 2016 4:28 AM To: [email protected]mailto:[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 _______________________________________________ phenixbb mailing list [email protected]mailto:[email protected] http://phenix-online.org/mailman/listinfo/phenixbb Unsubscribe: [email protected]mailto:[email protected]