Nature - USA (2019-07-18)

2

nature research | reporting summary

October 2018

Field-specific reporting

Please select the one below that is the best fit for your research. If you are not sure, read the appropriate sections before making your selection.

Life sciences Behavioural & social sciences Ecological, evolutionary & environmental sciences

For a reference copy of the document with all sections, see nature.com/documents/nr-reporting-summary-flat.pdf

Life sciences study design

All studies must disclose on these points even when the disclosure is negative.

Sample size No statistical methods were used to predetermine sample size. The determined sample size was adequate as the differences between

experimental groups was reproducible, as indicated. X-ray diffraction data were collected until completeness of the data set. For cryoEM,

sample sizes were determined by available electron microscopy time and density of particles on the electron microscopy grids. Sample sizes

were sufficient to obtain structures at the reported resolution.

Data exclusions Tomograms showing imaging problems were excluded from the data set. Otherwise, no data were excluded.

Replication The experimental findings were reproduced in multiple independent experiments. The number of independent experiments and biological

replicates in each data panel is indicated in the figure legends. CryoEM data sets were collected in different sessions, on different days from

repetitions of the same experimental setup (see methods part). For liposome binding and stimulated GTPase assays, at least two different

liposomes and protein batches were used for each reported data point.

Randomization Randomization was not formerly performed in this study as it did not involve animals and/or human research participants. When generating

yeast strains, random single clones were selected for further experiments.

Blinding Blinding is not relevant for protein structure determination and cryoET map interpretation since the results are not subjective. Investigators

were not blinded during functional assays. Importantly, data collection during yeast growth experiments (using a microplate reader) was

automated and non-subjective.

Reporting for specific materials, systems and methods

We require information from authors about some types of materials, experimental systems and methods used in many studies. Here, indicate whether each material,

system or method listed is relevant to your study. If you are not sure if a list item applies to your research, read the appropriate section before selecting a response.

Materials & experimental systems

n/a Involved in the study

Antibodies

Eukaryotic cell lines

Palaeontology

Animals and other organisms

Human research participants

Clinical data

Methods

n/a Involved in the study

ChIP-seq

Flow cytometry

MRI-based neuroimaging

Antibodies

Antibodies used If not otherwise mentioned, all used antibodies are custom-made polyclonal antisera. Antibodies against Mgm1 (GR796-5),

Mic10 (GR3367-7), Cox1 (GR1538-4), Ssc1 (GR119-3), described in Ieva et al., 2014, Malsburg et al. 2011 and Morgenstern et al.,

2017, were raised in rabbit using synthetic peptides and diluted 1:500 in TBST-5% (w/v) milk. HRP-conjugated secondary

antibodies were obtained from Merck/Millipore (catalogue number AP187P) and diluted 1:5000 in TBST-5% (w/v) milk.

Validation Antibodies were validated by Western blotting using yeast deletion mutants.

Eukaryotic cell lines

Policy information about cell lines

Cell line source(s) The YPH499 wild-type yeast strain has been described (Sikorski & Hieter, 1989) and was obtained from the laboratory of

Nikolaus Pfanner (University of Freiburg/Germany).

Authentication The PGAL1-MGM1 mutant yeast strain was generated as described in the methods section and validated by control PCR as

well as Western blotting after culturing on galactose-containing media.