I don’t think there’s anything particularly difficult about scattering factors for cryoEM, but the patterns of dependences on atomic number differs from that in X-ray crystallography. For instance, in X-ray maps, oxygen atoms, common interactors of metal cations via carboxylic acids and/or water molecules, appear “stronger” due to this difference. Thus, at the same nominal resolution of ~2–3 A, the coordination sphere in cryoEM maps looks less well-defined than in X-ray crystallography maps (even though information content is the same). I find the default restraints insufficient, so I apply restraints manually in both Refmac/Servalcat and Phenix, e.g., I define Mg2+ octahedral geometry with proper distances in a mini script and include it in the refinement run. In other words, I vote for tighter geometry.
D. Dominika Borek, Ph.D. *** UT Southwestern Medical Center 5323 Harry Hines Blvd. *** Dallas, TX 75390-8816 214-645-9577 (phone) *** 214-645-6353 (fax) From: CCP4 bulletin board <[email protected]> On Behalf Of Frank von Delft Sent: Friday, July 25, 2025 10:10 AM To: [email protected] Subject: Re: [ccp4bb] Restraining metal sites in medium resolution cryo-EM Isn't there something about scattering factors being ... difficult... for CryoEM reconstructions? Which I imagine would cause issues in refinement. Frank On 25/07/2025 12:35, Matthew Snee wrote: Hi Everyone Id let to get the communities opinions on metal sites in Cryo-EM models. In X-ray structures the geometry of metal coordination (when restrained correctly) looks pretty good, even in low resolution structures, and at very high resolution you may wish to avoid applying restraints so that the precise coordination distances/angles can tell you something about the physical chemistry of the system (I.E reduction state of catalytic metals in chemical biology). I have found that EM structures between 2-3Å which are good enough to see individual features (un ambiguous sidechain rotamers and coordinated waters in the metal complex) refinement outputs don't look nearly as nice. My question is this,: Is this just "the way it is" because the optical resolution and true atomic resolvability is different between X-ray and EM. The constraints of the crystal might help to collapse the conformational landscape somewhat, and solvent flattening might repress (unidentifable) minor states that would distort the real-space fit, thus improving the convergence between ideal geometry and fit to the density. EM structures that have very high resolution FSC cutoffs can still be distorted by minor anisotropic flexibility in one or more particular direction, there are ways to isotropise maps, but I dont really like refining against these. The other option is that the restraints need to be up-weighted so that they have more parity with the protein bond restraints and other targets that one might use. Basically, are these structures where the coordination geometry is almost certainly not completely correct, likely to be better, more useful, or closer to the "true structure" or is it better to enforce the geometry more strictly than you would for an equivalent X-ray structure in order to achieve the most likely sensible cluster. I dont think either approach is "wrong" because you could make an argument for building what you see, only building the links that are directly suggested and restraining them as best you can, I'm not a metal cluster expert by any means, but I do know that the coordination number, and bond length/angles are quite well known. I know this is potentially more of a CCPEM/Phenix query, but X-ray people are generally more involved in the physical chemistry side (I know not always 🙂!), Best wishes Matthew Snee, PhD Post-doctoral Research Associate The Baldock Lab | Michael Smith Building C3.214 | Wellcome Trust Centre for Cell-Matrix Research |Division of Cell Matrix Biology & Regenerative Medicine| School of Biological Sciences| Faculty of Biology, Medicine and Health, University of Manchester, Oxford Road, Manchester M13 9PT Lab Tel: +44 (0)161 306 2869 _____ To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB <https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1> &A=1 _____ To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB <https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1> &A=1 ######################################################################## To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list hosted by www.jiscmail.ac.uk, terms & conditions are available at https://www.jiscmail.ac.uk/policyandsecurity/
