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The atomic coordinates derived from cryo-electron microscopy (cryo-EM) maps can be inaccurate when the voxel scaling factors are not properly calibrated. Here, we describe a method for correcting relative voxel scaling factors between pairs of cryo-EM maps for the same or similar structures that are expanded or contracted relative to each other. We find that the correction of scaling factors reduces the amplitude differences of Fourier-inverted structure factors from voxel-rescaled maps by up to 20-30%, as shown by two cryo-EM maps of the SARS-CoV-2 spike protein measured at pH 4.0 and pH 8.0. This allows for the calculation of the difference map after properly scaling, revealing differences between the two structures for individual amino acid residues. Unexpectedly, the analysis uncovers two previously overlooked differences of amino acid residues in structures and their local structural changes. Furthermore, we demonstrate the method as applied to two cryo-EM maps of monomeric apo-photosystem II from the cyanobacteria Synechocystis sp. PCC 6803 and Thermosynechococcus elongatus. The resulting difference maps reveal many changes in the peripheral transmembrane PsbX subunit between the two species., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

A minimal replication-transcription complex (RTC) of SARS-CoV-2 for synthesis of viral RNAs includes the nsp12 RNA-dependent RNA polymerase and two nsp8 RNA primase subunits for de novo primer synthesis, one nsp8 in complex with its accessory nsp7 subunit and the other without it. The RTC is responsible for faithfully copying the entire (+) sense viral genome from its first 5'-end to the last 3'-end nucleotides through a replication-intermediate (RI) template. The single-stranded (ss) RNA template for the RI is its 33-nucleotide 3'-poly(A) tail adjacent to a well-characterized secondary structure. The ssRNA template for viral transcription is a 5'-UUUAU-3' next to stem-loop (SL) 1'. We analyze the electrostatic potential distribution of the nsp8 subunit within the RTC around the template strand of the primer/template (P/T) RNA duplex in recently published cryo-EM structures to address the priming reaction using the viral poly(A) template. We carried out molecular dynamics (MD) simulations with a P/T RNA duplex, the viral poly(A) template, or a generic ssRNA template. We find evidence that the viral poly(A) template binds similarly to the template strand of the P/T RNA duplex within the RTC, mainly through electrostatic interactions, providing new insights into the priming reaction by the nsp8 subunit within the RTC, which differs significantly from the existing proposal of the nsp7/nsp8 oligomer formed outside the RTC. High-order oligomerization of nsp8 and nsp7 for SARS-CoV observed outside the RTC of SARS-CoV-2 is not found in the RTC and not likely to be relevant to the priming reaction.