Cloning

Sed5 (UniProt Q01590) (residues 1–319, truncating the transmembrane helix) was cloned from the S. cerevisiae S288c genome into a pETM-11 expression vector linearized at the XhoI and XbaI restriction sites using In-Fusion (Takara) technology. A flexible triple-glycine linker was added between the C-terminal residue (319) of Sed5 and a 6xHis-tag. The primers used were as follows:

Forward, 5′-GGTGTCCTCCTCCTCTATTACTCTTTATCCTGTCGAAG-3′

Reverse, 5′-GAGGAGGAGGACACCACCACCACCACCAC-3′

Sec23–Sec24, Sec13–Sec31 and Sar1 constructs previously described by Hutchings et al. (2021)15 were used here.

Protein expression and purification

The Sed5 pETM-11 vector was transformed into Escherichia coli (BL21) cells by heat shock. Cells were cultured at 37 °C with 220 rpm shaking in 2 L of LB medium supplemented with kanamycin. When cultures reached an optical density between 0.7 and 1, 0.2 mM IPTG was added and the incubation temperature was reduced to 16 °C. Culture pellets were harvested after approximately 16 h by centrifugation and flash-frozen in liquid nitrogen before storage at −80 °C.

Sed5 pellets from 2 L of culture were thawed and resuspended in 20 ml of Ni-A buffer (50 mM Tris (pH 8), 500 mM KCl, 0.1% Tween-20 (v/v), 10 mM imidazole and 1 mM DTT) supplemented with one complete protease inhibitor tablet (Roche). Then, 40 mg ml−1 lysozyme was added and cells were stirred on ice for 20 min. Cells were lysed using a cell disruptor. Unbroken cells were removed by ultracentrifugation at 20,200g for 25 min. The supernatant was loaded onto a Ni-NTA 5-ml His-trap column (GE Biosciences) equilibrated with Ni-A buffer and washed with five column volumes of Ni-A buffer. Sed5 was eluted from the column by applying a linear gradient of Ni-B buffer (50 mM Tris (pH 8), 500 mM KCl, 0.1% Tween-20 (v/v), 500 mM imidazole and 1 mM DTT). Fractions were analyzed by SDS–PAGE and those containing Sed5 were pooled before tenfold dilution in Q-A buffer (20 mM Tris (pH 8.0), 0.1% Tween-20 (v/v), 10% glycerol (v/v) and 1 mM DTT). Sed5 was loaded onto a 5-ml HiTrap Q column (GE Biosciences) equilibrated with Q-A buffer. The column was washed with two column volumes of Q-A buffer and two column volumes of a mixture of 90% Q-A buffer and 10% Q-B buffer (20 mM Tris (pH 8.0), 0.1% Tween-20 (v/v), 10% glycerol (v/v), 1 mM DTT and 1 M KCl). Sed5 was eluted with a linear gradient of Q-B buffer. Fractions were analyzed by SDS–PAGE and those containing Sed5 were pooled and concentrated using a protein concentrator with a 10-kDa molecular weight cutoff to a final concentration of 0.5 mg ml−1. Sed5 was separated into 100-μl aliquots and flash-frozen.

The final step of Sed5 purification was carried out on the day of use. One aliquot of Sed5 was thawed before injection onto a Superdex 200 Increase 3.2/300 column equilibrated with HKM buffer (20 mM HEPES, 50 mM potassium acetate and 1.2 mM MgCl2, pH 6.8). Fractions containing Sed5 were identified by SDS–PAGE and pooled together.

The purified protein was confirmed as Sed5 by analysis with SDS–PAGE combined with gel sequencing by MS at the MS and Proteomics Facility at the University of St. Andrews.

Sec23–Sec24, Sec13–Sec31 and Sar1 were expressed and purified as described previously from SF9 and E. coli cells, including the steps to cleave the 6xHis-tags in Sec23–Sec24 and Sec13–Sec31 (ref. 15).

Liposome flotation assays

Liposomes were generated as previously described34 using the ‘major–minor’ lipid mixture: 49 mol.% phosphatidylcholine, 20 mol.% phosphatidylethanolamine, 8 mol.% phosphatidylserine, 5 mol.% phosphatidic acid, 9 mol.% phosphatidylinositol, 2.2 mol.% phosphatidylinositol-4-phosphate, 0.8 mol.% phosphatidylinositol-4,5-bisphosphate, 2 mol.% cytidine diphosphate–diacylglycerol, supplemented with 2 mol.% Texas red–phosphatidylethanolamine, 2 mol.% Ni-NTA-tagged lipids (18:1 DGS–NTA(Ni)) and 20% (w/w) ergosterol.

Liposomes were premixed with Sed5 and floatation assay experiments were performed without and with the addition of COPII components: 1 μM Sar1, 180 nM Sec23–Sec24, 173 μM Sec13–Sec31, 360 nM Sed5 with 1 mM GMP-PNP (Sigma-Aldrich) and 2.5 mM EDTA (pH 8.0). All flotation assays contained 0.27 mM liposomes in a total volume of 75 μl. Liposome flotation reactions were mixed with 250 μl of 1.2 M sucrose in HKM buffer in an ultracentrifuge tube. Next, 320 μl of 0.75 M sucrose in HKM was gently layered on top. A final layer of 20 μl of HKM was then layered on top of the sucrose solutions. Ultracentrifuge tubes were loaded into a SW-55 Ti ultracentrifuge rotor before spinning at 280,000g at 4 °C for at least 16 h. The top 20 μl of the sucrose gradient was carefully extracted before analysis by SDS–PAGE with silver staining.

Budding reactions

Purified microsomes from S. cerevisiae were prepared as described previously31. Then, 1.5 mg of microsomes were washed three times carrying out the following steps: resuspending the microsomes in 1 ml of B88 buffer (20 mM HEPES (pH 6.8), 150 mM potassium acetate, 250 mM sorbitol and 5 mM magnesium acetate), pelleting membranes by centrifugation on a chilled benchtop centrifuge at 20,000g for 2 min, removing the supernatant and resuspending the pellet in 50 μl of B88 buffer. After washing, the pellets were diluted a further eight times and chilled on ice before use in budding reactions.

Budding reactions in microsomes were prepared by incubating 1 μM Sar1, 180 nM Sec23–Sec24, 173 μM Sec13–Sec31 with 1 mM GMP-PNP (Sigma-Aldrich), 2.5 mM EDTA (pH 8.0) and 10% microsomes (v/v).

GUVs were prepared by electroformation35 from 10 mg ml−1 of a major–minor lipid mixture with 2 mol.% Ni-NTA tagged lipids (described above) in a 2:1 chloroform–methanol solvent mixture, as described previously14,36. The lipid mixture was spread over two indium tin oxide-coated glass slides. Then, 300 mM sucrose was suspended in a silicon O-ring between these glass slides and GUVs were generated using a NanIon Vesicle Prep Pro. GUVs in the sucrose solution were added to 500 μl of 300 mM glucose and left to sediment overnight at 4 °C. The supernatant was discarded, leaving a 50-μl pellet of GUVs.

Budding reactions in GUVs with Sed5 were prepared by incubating 1 μM Sar1, 180 nM Sec23–Sec24, 173 μM Sec13–Sec31, 360 nM Sed5 with 1 mM GMP-PNP (Sigma-Aldrich), 2.5 mM EDTA (pH 8.0) and 10% GUVs (v/v). GUVs were premixed with the Sed5 before addition to the COPII components. Budding reactions were incubated for at least 30 min before vitrification for cryo-ET.

Cryo-ET sample preparation

First, 5 nm BSA-blocked gold nanoparticles (BBI Solutions) were added to the budding reactions at a concentration of 10% (v/v). Then, 4 μl of budding reactions from GUVs or microsomes were added to glow-discharged Lacey carbon films on 300-mesh copper grids (Agar Scientific) and incubated for 60 s, before backblotting on a Leica-GP2 plunge-freezer in 95% humidity with a 4-s blotting time. Vitrified grids were stored in liquid nitrogen before data collection.

Cryo-ET data collection

Budding reactions with microsomes were imaged using cryo-ET at the European Molecular Biology Laboratory (EMBL) Imaging Center in Heidelberg on a Titan Krios microscope (Thermo Fisher Scientific) operated at 300 kV. The microscope was equipped with a SelectrisX energy filter (Thermo Fisher Scientific) and a Falcon 4 detector (Thermo Fisher Scientific) in counting mode. The pixel size was 1.526 Å and tilt series were taken with a defocus range of −3 μm to −5 μm. Tilt series were acquired using a dose-symmetric tilt scheme37 over a total exposure of 140 e− per Å2 with tilt angles ranging between −60° and +60° with 3° increments. Data collection was controlled using SerialEM38 and implementing PACE-tomo39. A total of 765 high-quality tilt series were collected, yielding the same number of tomograms.

Budding reactions with GUVs and Sed5 were imaged using cryo-ET at the EMBL Imaging Center in Heidelberg over two sessions of data collection on a Titan Krios microscope operated at 300 kV. The microscope was equipped with a K3 (Gatan) detector and energy filter. The first session was collected in super-resolution mode and the second session was collected in counting mode. Pixel size was 1.33 Å and tilt series were taken with a defocus range of −1.5 μm to −3.5 μm. Tilt series were acquired using a dose-symmetric tilt scheme37 over a total exposure of 142 e− per Å2 with tilt angles ranging between −60 ° and +60 ° with 3° increments. Data collection was controlled using SerialEM. A total of 326 high-quality tilt series were collected, yielding the same number of tomograms.

Grids were screened and optimized at the Institute of Structural and Molecular Biology (ISMB) EM facility at Birkbeck College.

Cryo-ET data processing

The microsome dataset was processed using an alpha-phase development version of RELION 5.0 (4.1-alpha-1-commit-d2053c)40. Initially, .mdoc files were renamed as TS_[number]-style to ensure compatibility between RELION and Dynamo scripts used later in the processing workflow. Raw data were then imported into RELION 5.0. Individual tilt movies were motion-corrected and averaged using whole-frame alignment in the RELION implementation of MotionCor2 (refs. 41,42). Contrast transfer fraction (CTF) estimation was carried out using CTFFIND-4.1 (ref. 43) with a defocus range of −25,000 to −55,000 Å and a maximum CTF resolution of 20 Å. Tilt series were manually inspected and poor tilt images were removed using a Napari plug-in (https://github.com/napari/napari/blob/main/CITATION.cff) provided as part of the ‘exclude tilt images’ job type in RELION 5.0. Tilt series were automatically aligned using the IMOD wrapper for fiducial-based alignment in RELION with a fiducial diameter of 8 nm. Tomograms were reconstructed in RELION at a pixel size of 12.208 Å for visual inspection and particle picking. Tomograms were denoised and missing wedge-corrected using IsoNet for use in manual particle picking44. We also generated eight-binned CTF-corrected tomograms for use in PyTOM template matching45, using IMOD’s ‘etomo’ (ref. 46) function on the IMOD metadata generated by the ‘align tilt series’ job type in RELION.

The Sed5–GUV dataset was processed using the RELION4_Tomo_Robot (https://github.com/EuanPyle/relion4_tomo_robot/blob/master/CITATION.cff). Individual tilt videos were motion-corrected and averaged using whole-frame alignment with MotionCor2 (ref. 41). Videos collected in super-resolution mode were binned twice during motion correction. Tilt series were created from individual tilt images using IMOD’s ‘newstack’ function. Tilt series were manually inspected using IMOD’s ‘3dmod’ visualization function and bad tilts were removed using IMOD’s ‘excludeviews’ function. Tilt series were automatically aligned using Dynamo’s automated fiducial-based alignment in the RELION4_Tomo_Robot’s ‘fast_mode’ with a fiducial diameter of 5 nm (ref. 47). CTF estimation was carried out using CTFFIND-4.1 (ref. 43). The dataset was then imported into RELION 4.0 (ref. 16). Tomograms were reconstructed in RELION at a pixel size of 10.64 Å for visual inspection and particle picking. Tomograms were denoised and missing wedge-corrected using IsoNet for visual inspection44.

STAMicrosome dataset Inner coat

The surface of vesicles in IsoNet-denoised tomograms was defined and segmented using the ‘pick particle’ plug-in in Chimera as described previously48,49. The coordinates of the vesicle surface were used to mask the tomograms to enable manual particle picking in UCSF Chimera, which were assigned Euler angles normal to the membrane. A total of 3,579 particles were extracted in 48 voxel boxes from RELION-reconstructed (nondenoised) tomograms at a voxel size of 9.156 Å. Particles were assigned random in-plane rotation angles and were averaged to create a reference using Dynamo47 with 4,697 particles. Particles were then aligned and averaged in Dynamo with the following conditions: a cone range of 10° was applied while 360° in-plane rotation was allowed; particle translation was limited to one voxel in all directions because of the accuracy of the coordinates of the manually picked particles; a C2 symmetry was applied because of the pseudosymmetry of the inner coat at low resolution; a mask covering the area of one inner coat subunit was applied; alignment was carried out for 100 iterations. The resulting Dynamo table was converted to a .star file using ‘dynamo2relion’ (https://github.com/EuanPyle/dynamo2relion). Particles were imported into an alpha-phase development version of RELION 5.0 (4.1-alpha-1-commit-d2053c) and extracted as pseudosubtomograms at bin 4 (voxel size of 6.104 Å) in 64 voxel boxes. Pseudosubtomograms were generated from the raw tilt series and did not use denoised tomograms. A reference was reconstructed at the same box and voxel size using the ‘tomo reconstruct particle’ job type. Particles were refined using Refine3D with the reference low-pass-filtered to 30 Å, no mask applied, a particle diameter of 200 Å and all Euler angles limited to local refinements of approximately 9° using the additional argument ‘--sigma_ang 3’. Poorly aligned particles were removed by 3D classification without particle alignment, no mask applied, six classes and a regularization parameter (T value) of 0.2. A reference was reconstructed at bin 1 in a box size of 196 voxels before the tilt series alignment for each tomogram was refined using ‘tomo frame alignment’ without fitting per-particle motion or deformations. Particles were re-extracted as pseudosubtomograms at bin 4 and refined as before using a mask over one inner coat subunit.

The structure generated by RELION was used to pick more particles in CTF-corrected (nondenoised) tomograms with PyTOM template matching45 with dose weighting and CTF correction applied. The template used was filtered to 25 Å.

Coordinates from PyTOM (29,496 particles from 475 tomograms) were imported into RELION 5.0. To remove junk particles, 3D classification was carried out with alignment using restricted tilt and psi Euler angles (‘--sigma_rot 3 --sigma_psi 3’) but leaving in-plane rotation free, a mask over one inner coat unit and over part of neighboring subunits, the map from the refined manually picked particle low-pass-filtered to 25 Å as a reference, four classes, a T value of 0.1 and a particle diameter of 330 Å. Particles clearly resembling the COPII inner coat were kept and refined under similar conditions to the preceding 3D classification. Particles were cleaned again using 3D classification without alignment with six classes and a T value of 0.2. The resulting particles .star file was merged with the manually picked particles generated earlier. Duplicate coordinates were deleted. Particles were exported to a Dynamo table using ‘relion2dynamo’ (https://github.com/EuanPyle/relion2dynamo) and were cleaned by neighbor analysis, as previously described48. Coordinates were converted back to a .star file using ‘dynamo2relion’ and reimported into RELION. Particles were refined as before but at bin 2 with all Euler angles limited to local refinements using ‘--sigma_ang 3’. One more round of tomo frame alignment, with per-particle motion, was carried out before tomo CTF refinement. A final refinement was carried out at bin 2 from 12,187 particles (from 352 tomograms), with limited Euler angles using ‘--sigma_ang 1.5’. The resolution according to Fourier shell correlation (FSC = 0.143) was 14.4 Å (Extended Data Fig. 6a).

A difference map, as described in Fig. 3a, between this structure and the inner coat from cargoless GUVs was generated. First, a model of the inner coat from cargoless GUVs (PDB 8BSH) was fitted into the inner coat map from microsomes. A volume representation of the fitted model was generated using ‘molmap’ in UCSF Chimera50 at high resolution (2 Å) before low-pass filtering to 14 Å in MATLAB. All maps were normalized to the same mean and s.d. before the map from the fitted model was subtracted from our map from microsomes. Another difference map, as described in Fig. 3b, was generated in the same way but using a 14-Å low-pass-filtered electron density map (EMD-15949) corresponding to the fitted PDB model (PDB 8BSH) instead of our map of the inner coat derived from microsomes.

Outer coat (vertex)

Outer coat vertices were manually picked in 30 tomograms. Particles were assigned Euler angles normal to their nearest membrane. Particles were extracted in 64 voxel boxes from RELION-reconstructed tomograms at a voxel size of 12.208 Å (bin 8). Particles were averaged as before for the inner coat to form an initial average. Particles were then aligned and averaged in Dynamo as for the inner coat but with a translational shift of four voxels allowed and with a mask covering the vertex. The resulting map was used as a template to pick more particles in CTF-corrected tomograms with PyTOM template matching on all tomograms45, as for the inner coat. Particles were cleaned on the basis of their proximity to the membrane of the vesicles. Particles were aligned in Dynamo again and the resulting Dynamo table was converted to a .star file using ‘dynamo2relion’.

Vertex particles were imported into RELION 5.0 and extracted as pseudosubtomograms in a box size of 64 voxels and at a voxel size of 6.104 Å (bin 4). Particles were cleaned using 3D classification with refinement restricting the tilt and psi Euler angles (‘--sigma_rot 4 --sigma_psi 4’) but leaving in-plane rotation free. The 3D classification used three classes, a T value of 0.25 and a particle diameter of 600 Å. Particles containing the vertex were then refined under the same conditions used in 3D classification. Particles were extracted at bin 2 and further refined. Tomo frame alignment, tomo CTF refinement and subsequent refinement at bin 2 (voxel size of 3.052 Å) was iteratively repeated until resolution improvements stopped. The final map was reconstructed from 19,368 particles and had a resolution of 11.4 Å (FSC = 0.143) (Extended Data Fig. 6b).

Outer coat (rod)

Outer coat rods were manually picked in all tomograms. Particles were assigned Euler angles normal to their nearest membrane. Particles were extracted from RELION-reconstructed tomograms at bin 8 (voxel size of 12.208 Å), averaged to form a reference and aligned in Dynamo, as per the outer coat vertices. Rods of different length were selected and isolated using neighbor analysis. The resulting Dynamo table was converted to a .star file using ‘dynamo2relion’.

Rod particles were imported into RELION 5.0 and extracted as pseudosubtomograms in a box size of 64 voxels at bin 8. As for the outer coat vertices, particles were progressively unbinned from bin 8 to bin 2 (voxel size of 3.052 Å) and refined with restrictions to apply local Euler angle searches. The final map was reconstructed from 18,852 particles and had a resolution of 11.8 Å (FSC = 0.143) (Extended Data Fig. 6c).

To produce the maps in Fig. 5d, particles were separated into different groups depending on the position of the neighboring vertices as defined by masks on the neighbor plot. Classes contained between 1,500 and 2,500 particles each, were binned eight times and were not filtered beyond their Nyquist (at 24 Å).

Outer coat (vertex, five-way rods)

We manually picked vertices formed by the convergence of five rods (as judged from visual inspection; Fig. 4A). We extracted particles (n = 461) from IsoNet-corrected tomograms at bin 8 (voxel size of 12.208 Å) in 64 voxel boxes, assigned initial angles normal to the membrane and randomized the in-plane rotation, before averaging them to obtain a starting reference for alignments. We used Dynamo to align particles by restraining the angles normal to the membrane within a 20° cone and allowing full searches for in-plane rotation. After 50 iterations, alignments had converged. We imported the aligned coordinates in RELION 4.1 and ran a refinement at bin 8 using angular restraints (‘--sigma_ang 3’). The final map was reconstructed from 461 particles and had a resolution of 34 Å (FSC = 0.143).

Sed5–GUV dataset Inner coat

The surface of tubes in RELION-reconstructed tomograms was defined and segmented using the ‘pick particle’ plug-in in Chimera as described previously48,49. The surface of the tube was oversampled and coordinates were assigned Euler angles normal to the membrane. Particles were extracted in 32 voxel boxes from RELION-reconstructed tomograms at a voxel size of 10.8 Å. Particles were then aligned and averaged in Dynamo as before for the microsome inner coat dataset with several differences: in-plane rotation was restricted to 20° with azimuth flipping enabled; C1 symmetry was applied; particle translation was limited 15 voxels in all directions; alignment was carried out for one iteration. Duplicates defined as particles within four voxels of another particle and were deleted with Dynamo’s ‘separation in tomogram’ function during alignment. A previous inner coat structure (EMD-11199)16 was low-pass filtered and used as a reference. Particles were cleaned by neighbor analysis as before for the microsome inner coat dataset. The resulting Dynamo table was converted to a .star file using ‘dynamo2relion’.

Particles were imported into RELION 5.0 and extracted as pseudosubtomograms at bin 8. They were refined and progressively unbinned iteratively until bin 1 before tomo frame refinement and tomo CTF refinement as previously described16. The final map was reconstructed from 178,700 particles and had a resolution of 4.1 Å (FSC = 0.143) (Extended Data Fig. 6d). The map was sharpened using RELION’s LocalRes sharpening with a −50 B factor.

Outer coat (vertex)

To pick outer coat vertices, we used the refined coordinates for the inner coat lattice and radially shifted them away from the membrane by 12 pixels. This was done to randomly oversample outer coat subunits at the expected radial distance from the tubular membrane. We then extracted these particles in a 64 voxel box size from RELION-reconstructed tomograms using Dynamo before aligning to a low-pass filtered of a previous vertex structure (EMD-11194)15. Alignment parameters were the same as for the inner coat alignment from the Sed5–GUV dataset except C2 symmetry was applied. The resulting Dynamo table was converted to a .star file using ‘dynamo2relion’.

Vertex particles were imported into RELION 5.0 and extracted as pseudosubtomograms in a box size of 128 voxels at bin 4. As for the outer coat vertices from microsomes, particles were progressively unbinned from bin 8 to bin 2 and refined with restrictions to apply local Euler angle searches. The final map was reconstructed from 13,529 particles and had a resolution of 9.7 Å (FSC = 0.143) (Extended Data Fig. 6e). The map was sharpened using RELION’s LocalRes sharpening with a −175 B factor.

Outer coat (rod)

To pick outer coat rods, we used the refined coordinates of the outer coat vertices and used Dynamo’s subboxing function to create four new coordinates where the rods are placed relative to each vertex. As before for the outer coat vertices, particles were aligned in Dynamo to a low-pass-filtered reference (EMD-11193)15. Particles were cleaned by neighbor analysis and duplicates were deleted. The resulting Dynamo table was converted to a .star file using ‘dynamo2relion’.

Rod particles were imported into RELION 5.0 and extracted as pseudosubtomograms in a box size of 128 voxels at bin 4. As for the outer coat rods from microsomes, particles were progressively unbinned from bin 8 to bin 2 and refined with restrictions to apply local Euler angle searches. The final map was reconstructed from 39,757 particles and had a resolution of 9.5 Å (FSC = 0.143) (Extended Data Fig. 6f).

In all cases, relevant atomic model coordinates were rigid-body fitted into our maps using UCSF Chimera or ChimeraX. In all cases, fitting was unambiguous.

The number of particles for each dataset is summarized in Table 1. Local resolution was estimated using RELION 5.0 locres implementation.

Table 1 Cryo-EM data collection, refinement and validation statistics MS analysis

Total protein from the S. cerevisiae ER microsomes used in the reconstitution experiments (n = 1) was digested using the SP3 method51 with some adaptations. In brief, after reduction and alkylation of cysteines, total protein was precipitated onto magnetic beads (MagReSyn Hydroxyl, Resyn Biosciences) by adding ethanol to a final concentration of 80% (v/v). Digestion was carried out by incubating the washed magnetic beads and total protein aggregated material with 1 μg of trypsin (Promega) dissolved in 25 mM ammonium bicarbonate containing 0.1% RapiGest detergent (Waters). The sample was then acidified with trifluoroacetic acid to a final concentration of 0.5% (v/v) to stop digestion and induce RapiGest degradation. Magnetic beads and RapiGest-insoluble degradation products were pelleted by centrifugation at 11,000g for 15 min and the supernatant containing tryptic peptides was then taken for MS analysis.

Liquid chromatography (LC)–MS/MS was performed on an Ultimate U3000 high-performance LC system (Thermo Fisher Scientific) hyphenated to an Orbitrap QExactive Classic MS instrument (Thermo Fisher Scientific). Peptides were trapped on a C18 Acclaim PepMap 100 (5 µm, 300 µm × 5 mm) trap column (Thermo Fisher Scientific) and eluted onto a C18 Easy Spray Column (2 µm, 75 µm × 500 mm; Thermo Fisher Scientific) using 180-min gradient of acetonitrile (5–40%). For data-dependent acquisition, MS1 scans were acquired at a resolution of 70,000 (automatic gain control (AGC) target of 1 × 106 ions with a maximum injection time of 65 ms) followed by ten MS2 scans acquired at a resolution of 17,500 (AGC target of 2 × 105 ions with a maximum injection time of 100 ms) using a collision-induced dissociation energy of 25. Dynamic exclusion of fragmented m/z values was set to 40 s.

Raw data were imported and processed in Proteome Discoverer version 3.1 (Thermo Fisher Scientific). The raw files were submitted to a database search using Proteome Discoverer with Sequest HT against the UniProt reference proteome for S. cerevisiae. The processing step consisted of a double iterative search using the INFERIS rescoring algorithm on a first pass with methionine oxidation and cysteine carbamidomethylation set as variable and fixed modifications, respectively. For the second pass, all spectra with a confidence filter worse than ‘high’ were researched with Sequest HT including additional common protein variable modifications (deamidation (N,Q), Q to pyro-E (Q), N-terminal acetylation and methionine loss). The spectral identification was performed with the following parameters: MS accuracy, 10 ppm; MS/MS accuracy of 0.02 Da; up to two trypsin missed cleavage sites allowed. The percolator node was used for false discovery rate (FDR) estimation and only rank 1 peptide identifications of high confidence (FDR < 1%) were accepted.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.