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. 2024 Jul 1;31(12):1848–1858. doi: 10.1038/s41594-024-01352-0

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© The Author(s) 2024

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 1 — a, Structural schematic of mtHsp60 domains showing V72I mutation (red circle), ATP and the apical–intermediate domain hinge (asterisk). b, SEC-MALS of mtHsp60 (black) and mtHsp60^V72I (purple) with (dashed lines) and without (solid lines) ATP. Normalized differential refractive index (left y axis) and average molecular mass (horizontal lines, right y axis) are shown versus elution volume (x axis). This experiment was repeated a total of three times with similar results. c, mtHsp60–10 refolding of chemically denatured human mtMDH, measured by the decrease in NADH absorbance at 340 nm (left, one biological replicate, data are presented as the mean ± s.d. of technical triplicates). Initial velocities are shown (right, data are presented as the mean ± s.e.m. of two or three biological replicates, overlaid with individual values). Significance testing was performed by one-way analysis of variance, with correction for multiple comparisons by Dunnett’s test. *P = 0.0305. Folded (green) and denatured mtMDH (mtMDH^denat) (orange) are shown for comparison. d, Top and side view 2D class averages of client-bound and client-unbound mtHsp60^V72I with client (arrow), and equatorial (+) and apical (^) domains indicated (Scale bar, 100 Å). e, Top and side views of the sharpened (opaque) and unsharpened (transparent) mtHsp60^apo consensus maps are shown, colored as in a. AD density is absent in the sharpened map contour, indicated in the top view (#). f, Map and model view of an mtHsp60^apo AD, with inward-facing helices H and I (dark purple) that are poorly resolved and flexible. g, Cryo-EM processing workflow to obtain maps with client and asymmetric AD conformations. h, Top view of the sharpened mtHsp60^apo focus map, showing improved AD density. i, Heptamer schematic (left) and unwrapped view of the ADs of the unsharpened mtHsp60^apo focus map colored to indicate AD rotation. Positive values (green) indicate an upward rotation (increased equatorial–apical distance) and negative values (red) indicate a downward rotation relative to the consensus map (dashed line). j, Slabbed side views of focused classifications showing unannotated client density (yellow) in different positions across the apical/equatorial regions.

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