Computational protocol: Reconstituted IMPDH polymers accommodate both catalytically active and inactive conformations

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Protocol publication

[…] Aliquots of purified protein, including WT IMPDH and the mutants Y12A, R356A, and S275L, were diluted to 2.5 µM in assembly buffer (50 mM Tris, 100 mM KCl, 1 mM DTT, pH 7.4) and incubated for 15 min at room temperature in the presence of various concentrations of ATP, GTP, IMP, and NAD+, as outlined in and . Following incubation, samples were applied to glow-discharged continuous carbon film EM grids and negatively stained using 1% uranyl formate. Grids were imaged by transmission electron microscopy using an FEI Tecnai G2 Spirit operating at 120 kV and a Gatan Ultrascan 4000 CCD via the Leginon software package (). Image processing, including CTF estimation, particle picking, and two-dimensional reference-free classification, was performed using the Appion and RELION software packages (; ). The image data collected from all ATP/GTP/IMP/NAD+ combinations were combined and processed en masse (77,525 helical repeats in total). Following two-dimensional classification, the resulting class averages were manually grouped according to the observed rise into one of three categories: “collapsed” (rise ∼94 Å), “expanded” (rise ∼114 Å), or “undetermined” (filament ends, distorted and bent octamers, and poorly aligned particles). The proportion of helical repeats from each experimental data set belonging to these three categories was then back-calculated.For three-dimensional reconstructions of IMPDH filaments, negative-stain images were acquired at a pixel size of 2.07 Å, and individual filaments were identified manually in Appion (). Reconstructions were performed by iterative helical real space refinement () in SPIDER (), imposing D4 symmetry on the helical asymmetric unit throughout. The structures of the collapsed and expanded IMPDH filament have been deposited in the EMDB () (EMD–8691, expanded; EMD-8690, collapsed). […]

Pipeline specifications

Software tools Leginon, Appion, RELION, SPIDER
Application cryo-EM
Organisms Homo sapiens
Chemicals Adenosine Triphosphate, Guanosine Triphosphate, Inosine Monophosphate, Nucleotides, Purine Nucleotides, Purines