DMEM supplemented with penicillin–streptomycin, 1% non-essential
amino acids and 10% fetal bovine serum overnight. Thirty minutes after
sucrose stimulation by replacing the medium with 0.2 M sucrose, the
grids were blotted for 10 s using filter paper and vitrified by plunge-
freezing into a liquid ethane/propane mixture using a Vitrobot Mark
IV (FEI). Cryo-focused ion beam (FIB) microscopy cryo-electron tomo-
graphic data collection was performed as described in detail before^45.
In brief, electron microscopy grids were mounted onto modified
Autogrids sample carriers^46 , and then transferred into a dual-beam
(FIB/SEM) microscope (Quanta 3D FEG, FEI) using a cryo-transfer sys-
tem (PP3000T, Quorum). For the whole procedure, samples were kept
at a constant temperature of −180 °C. Thin lamellae with a thickness of
about 200 nm were prepared in the nuclear region of the cells using a
Ga2+ ion beam at 30 kV under a 20° stage tilt angle. The milling progress
was monitored by SEM imaging at 5 kV. The lamellas were transferred
to an FEI Titan Krios transmission electron microscope equipped with
a Gatan post-column energy filter and Gatan K2 Summit direct detector
for tomographic data collection. Tilt series were recorded from −50°
to +70° with an increment of 2° using SerialEM software^47 at a nominal
magnification of 42,000×, resulting in a pixel size of 3.42 Å at the speci-
men level. On average, six frames were collected for each image under
counting mode, resulting in a total dose of 110 e− Å−2 per tilt series. For
image processing, the MATLAB (Mathworks) TOM toolbox^48 was used
as a general platform for image processing. K2 frames were aligned
using in-house software (K2Align) based on previous work^49. K2Align
code is available at GitHub (https://github.com/dtegunov/k2align).
Using the IMOD software package, tilt series were first aligned using
fiducial-less tracking, and then tomograms were reconstructed by
weighted back-projection of the resulting aligned tilt series^50. To iden-
tify proteasomes in the tomograms, a mirrored single-capped protea-
some structure (EMD-3916) was filtered to 60 Å as an initial template
for template matching in the twice-binned tomograms (13.68 Å^3 per
voxel) using PyTom^51. The resulting subtomograms were cropped out,
CTF-corrected and classified using RELION^52. The resulting structure
clearly showed a 26S proteasome complex with correct handedness.
However, owing to limitations on the number of subtomograms, we
could not separate more conformational information.
Reporting summary
Further information on research design is available in the Nature
Research Reporting Summary linked to this paper.
Data availability
Raw cryo-ET data have been deposited to the Electron Microscopy
Data Bank under accession code EMD-10494. The mass spectrometry
proteomics data have been deposited to the ProteomeXchange Con-
sortium via the PRIDE partner repository with the data set identifier
PXD01637 and PXD016369. The uncropped blots and gels are provided
in Supplementary Fig. 1. Source Data for Figs. 1–4 and Extended Data
Figs. 1, 3–9 are provided with the paper.
Code availability
K2Align code is available at GitHub (https://github.com/dtegunov/
k2align).
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Acknowledgements We thank C.-G. Pack and M.-K. Jung for initial electron microscopy
analysis; S. Fukai, K. Iwai, T. Yamamoto and F. Zhang for reagents; N. Noda for helpful
discussion; and J. Horiuchi for critical reading of the manuscript. This research was supported
by AMED under grant numbers JP18gm1110003 (S.M. and Y.S.) and JP19gm1110010 (T.I.); MEXT/
JSPS KAKENHI grant numbers JP18K19352 (S.Y.), JP18K14913 (H.T.), JP26293018 (Y.S.),
JP18H03993 (Y.S.), JP18H05498 (F.O. and Y.S.), JP18H03977 (T.I.), JP 19H05281 (T.I.) and
JP21000012 (K.T.); the Takeda Science Foundation (Y.S., K.T. and T.I.); the Uehara Memorial
Foundation (Y.S.); the European Commission, FP7 GA ERC-2012-SyG_318987-ToPAG (Q.G., W.B.
and R.F.-B.); and the DFG, EXC 2067/1- 390729940 (R.F.-B.).Author contributions S.Y., K.T. and Y.S. designed most of the experiments; S.Y., A.K. and Y.S.
generated the KI/KO cell lines with assistance of S.M.; S.Y. and A.K. performed the fluorescent
microscopy analyses; S.Y. processed microscopy images; H.T., A.K., N.A. and A.E. performed
the immunoprecipitation and western blotting; H.T. performed the mass spectrometry
proteomics analysis with assistance of A.E. and F.O.; H.T. and N.A. prepared recombinant
proteins; S.Y. and Y.S. performed in vitro phase separation assay; Q.G., W.B. and R.F.-B.
performed and analysed cryo-ET experiments; K.I. and T.I. performed northern blotting; S.Y.,
K.T. and Y.S. wrote the manuscript with input from all co-authors.
Competing interests The authors declare no competing interests.Additional information
Supplementary information is available for this paper at https://doi.org/10.1038/s41586-020-
1982-9.
Correspondence and requests for materials should be addressed to K.T. or Y.S.
Reprints and permissions information is available at http://www.nature.com/reprints.