Article
affinity resin and size-exclusion chromatography using the same
protocol as for the EMC. In the binding assay, twice as much of the
purified TOM was pre-incubated with EMC for 1 h at 4 °C, and then ana-
lysed in a Superose 6 10/300 gel filtration column in buffer containing
0.01% GDN, 150 mM NaCl, 20 mM Tris-HCl, pH 7.4, 1 mM MgCl 2 and 1 mM
MnCl 2. As controls, the purified TOM and EMC proteins were analysed
separately using the same conditions.
Reporting summary
Further information on research design is available in the Nature
Research Reporting Summary linked to this paper.
Data availability
The cryo-EM 3D map of the S. cerevisiae EMC complex has been
deposited at the Electron Microscopy Data Bank (EMDB) database
with accession code EMD-21587. The corresponding atomic model
was deposited at the RCSB Protein Data Bank (PDB) with accession
code 6WB9. The TMT mass spectrometry data and the real-space cor-
relation coefficients of all residues with experimental densities data
are provided in Supplementary Tables 1–3. The uncropped SDS–PAGE
gels used in Figs. 1 a, 3g and Extended Data Fig. 6b can be found in
Supplementary Fig. 1.
- Funakoshi, M. & Hochstrasser, M. Small epitope-linker modules for PCR-based C-terminal
tagging in Saccharomyces cerevisiae. Yeast 26 , 185–192 (2009). - Kammers, K., Cole, R. N., Tiengwe, C. & Ruczinski, I. Detecting significant changes in
protein abundance. EuPA Open Proteom. 7 , 11–19 (2015).
33. Zheng, S. Q. et al. MotionCor2: anisotropic correction of beam-induced motion for
improved cryo-electron microscopy. Nat. Methods 14 , 331–332 (2017).
34. Mindell, J. A. & Grigorieff, N. Accurate determination of local defocus and specimen tilt in
electron microscopy. J. Struct. Biol. 142 , 334–347 (2003).
35. Zivanov, J. et al. New tools for automated high-resolution cryo-EM structure
determination in RELION-3. eLife 7 , e42166 (2018).
36. Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular
structure solution. Acta Crystallogr. D 66 , 213–221 (2010).
37. Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot.
Acta Crystallogr. D 66 , 486–501 (2010).
38. Pettersen, E. F. et al. UCSF Chimera—a visualization system for exploratory research and
analysis. J. Comput. Chem. 25 , 1605–1612 (2004).
39. Chen, V. B. et al. MolProbity: all-atom structure validation for macromolecular
crystallography. Acta Crystallogr. D 66 , 12–21 (2010).
40. Amunts, A. et al. Structure of the yeast mitochondrial large ribosomal subunit. Science
343 , 1485–1489 (2014).
Acknowledgements Cryo-EM images were collected in the David Van Andel Advanced
Cryo-Electron Microscopy Suite at Van Andel Research Institute. We thank G. Zhao and
X. Meng for assistance with data collection and D. Nadziejka for critical reading of the
manuscript. This work was supported by the US National Institutes of Health (NIH) (R01
CA231466 to H.L.) and Van Andel Institute (to H.L.).Author contributions L.B. and H.L. conceived and designed experiments. L.B. performed most
of the experiments. Q.Y., X.F. and A.K. helped with sample preparation and functional assays.
L.B. and H.L. analysed the data and wrote the manuscript.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-
2389-3.
Correspondence and requests for materials should be addressed to L.B. or H.L.
Peer review information Nature thanks Friedrich Förster and the other, anonymous, reviewer(s)
for their contribution to the peer review of this work.
Reprints and permissions information is available at http://www.nature.com/reprints.