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Tag: Chaperones

  • In situ analysis reveals the TRiC duty cycle and PDCD5 as an open-state cofactor

    In situ analysis reveals the TRiC duty cycle and PDCD5 as an open-state cofactor

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    Cell culture

    HEK (HEK Flp-In T-Rex 293, Invitrogen) cells were cultured in DMEM (Sigma-Aldrich) supplemented with 10% fetal bovine serum (Gibco) under standard tissue culture conditions (37 °C, 5% CO2). HEK293F (Thermo Fisher Scientific) cells were cultured in Freestyle medium (Thermo Fisher Scientific) at 37 °C, 8% CO2 and 120 rpm. Cells were negative for mycoplasma contamination.

    Native PAGE

    For immunoblotting, when HEK cells were at about 80% confluency, they were washed twice with ice-cold PBS and scraped in PBS, pelleted by centrifugation for 5 min at 1,000g, 4 °C and resuspended in modified native lysis buffer (50 mM HEPES pH 7.4, 50 mM KCl, 1.5 mM MgCl2, 10% glycerol, 0.1% NP-40, 1 mM PMSF, complete EDTA-free protease inhibitor cocktail and 1 mM DTT). Lysis buffer was also supplemented with 30 U ml−1 benzonase to remove DNA. Lysis was performed on ice for 20 min and the lysates were clarified by centrifugation for 10 min at 12,000g at 4 °C. The protein concentration was determined using a BCA assay (Thermo Fisher Scientific). 4× NativePAGE sample buffer (Thermo Fisher Scientific) was added to a final concentration of 1×. Then, 15 µg of each sample was resolved on 3–12% Bis-Tris NativePAGE gels (Thermo Fisher Scientific). NativePAGE was soaked in 0.1% SDS buffer for 15 min, then transferred to 0.45 µM PVDF membranes presoaked in methanol for 30 s. The membranes were blocked with 5% molecular biology grade BSA (Millipore Sigma) in Tris-buffered saline supplemented with 0.1% Tween-20 (TBST) for 1 h at room temperature, then probed with specific primary antibodies 4 °C for overnight. Primary antibodies was diluted in 1% BSA/TBST as follows: 1:10,000 rabbit anti-CCT5 (Abcam, ab129016). The secondary antibody was diluted 1:10,000 in TBST. Total protein was detected with Revert total protein stain. Fluorescence signal detection was performed using Li-Cor Odyssey infrared imager.

    PDCD5 knockdown

    HEK cells (5 × 105) were seeded into six-well plates. Then, 24 h after plating, 25 pmol siRNA (Thermo Fisher Scientific, s17467) were added with Lipofectamine RNAiMAX Transfection Reagent (Invitrogen). Cells were collected with ice-cold PBS after 48 h and then immunoblotting was run for further analysis.

    Expression and purification of recombinant PDCD5 and its mutants

    PDCD5 mutants were obtained using site-directed mutagenesis. A 6× His-tag was added to the C terminus of PDCD5. Plasmids containing WT and mutant PDCD5 were transformed into Escherichia coli Rosetta DE3 competent cells for expression. PDCD5 was expressed and purified as previously reported33. In brief, cell lysates were first passed through a nickel column, then PDCD5 bound to the nickel resin was eluted in high imidazole buffer, and pure PDCD5 was obtained by passing the elution twice through a Superdex 200 size-exclusion column. Proteins were concentrated by centrifugation and then quantified using the BCA colorimetric assay.

    TRiC ATPase activity

    The assay was performed as previously described48. In brief, stock solutions of 0.05% (w/v) quinaldine red, 2.32% (w/v) polyvinyl alcohol, 5.72% (w/v) ammonium heptamolybdate tetrahydrate in 6 M HCl and water were mixed in a 2:1:1:2 ratio to prepare the quinaldine red reagent fresh before each experiment. Then, 300 nM TRiC was diluted in ATPase buffer (50 mM Tris-HCl pH 7.4, 100 mM KCl, 5 mM MgCl2, 10% glycerol, 1 mM TCEP; 30 μl total reaction volume), preheated to 37 °C and added to 3 μl water or 10 mM ATP to start the reaction, then incubated for the indicated durations in the presence or absence of 3 µM PDCD5. The reactions were stopped by the addition of 5 μl of 60 mM EDTA in a Corning 96-well opaque non-sterile polystyrene plate (Sigma-Aldrich, CLS3992) on ice. After samples at all timepoints were collected, the reactions were developed by adding 80 μl quinaldine red reagent for 10 min, then quenched by adding 10 μl 32% (w/v) sodium citrate. The fluorescence intensity was measured (excitation, 430 nm; emission, 530 nm) using the CLARIOstar plate reader (BMG Labtech). Analysis was performed by fitting a phosphate standard curve with a one-phase decay function, and we derived the parameters for calculating the amount of phosphate released from CCT complexes.

    PDCD5 binding to TRiC

    To probe the binding affinity of PDCD5 for TRiC, increasing amounts of recombinant PDCD5 variants were incubated with a fixed concentration of TRiC (300 nM) for 20 min at 25 °C in ATPase buffer (50 mM Tris-HCl pH 7.4, 100 mM KCl, 5 mM MgCl2, 10% glycerol, 1 mM TCEP), in the absence of ATP. The reactions were run in native gels and immunoblotted using PDCD5 or CCT8 antibodies, as described above. To test whether PDCD5 binds to the TRiC open or closed conformations, 3 μM of WT or mutant PDCD5 was incubated with 300 nM TRiC for 20 min at 25 °C in ATPase buffer containing 1 mM of different nucleotides and ATP analogues. The reactions were run in native gels and immunoblotted using PDCD5 or CCT8 antibodies, as described above. To obtain insights about the binding kinetics of PDCD5 variants to TRiC, 3 μM of WT or mutant PDCD5 was incubated with 300 nM TRiC in ATPase buffer at 25 °C for 10, 15, 20 and 30 min. The reactions were run in native gels and immunoblotted using PDCD5 (Proteintech, 12456-1-AP, 1:1,000) and CCT8 (Santa Cruz Biotechnology, sc-377261, 1:250) antibodies, as described above.

    Co-IP

    For PDCD5–Flag co-IP, PDCD5-Flag constructs (GenScript) were transiently expressed in HEK293F for 48 h after transfection. Cells were washed with PBS before collection by centrifugation and frozen in liquid nitrogen. HEK293F cells were lysed in lysis buffer (PBS pH 7.4, 0.1% IGEPAL CA-630, 5 mM MgCl2, freshly added 0.6 mM phenylmethylsulphonyl fluoride and protease inhibitors), triturated through a 24-gauge needle ten times and incubated on ice for 5 min. After lysate clearing by centrifugation, 500 μg clarified protein extract was mixed with 20 µl packed anti-Flag M2 beads (Sigma-Aldrich) and incubated for 1 h at 4 °C. After three washes with lysis buffer, bound proteins were eluted by boiling in LDS sample buffer (Invitrogen). For western blotting, input and eluate (IP) samples were loaded onto 4–12% Bis-Tris gels (Invitrogen) and subsequently transferred to nitrocellulose membranes (Bio-Rad).

    CCT3 co-IP was performed with non-transfected HEK293F cells subjected to in vivo cross-linking with 1.5 mM dithiobis(succinimidyl propionate) (DSP; Thermo Fisher Scientific) at 37 °C for 10 min. The cross-linking reaction was quenched by the addition of Tris (pH 8.0) to a final concentration of 160 mM and cells were collected and lysed as described above. Then, 2 mg of clarified protein extract was mixed with 10 μg rabbit anti-CCT3 antibody (Proteintech, 10571-1-AP) or rabbit control IgG (Proteintech, 30000-0-AP) as mock IP for 1 h at 4 °C, followed by addition of 50  μl equilibrated Protein G Magnetic Beads (Thermo Fisher Scientific) and incubation for 1 h at 4 °C. The samples were washed, eluted and evaluated using SDS–PAGE as described above.

    The percentage of IP efficiency was calculated by normalizing the measured intensities and the respective dilution factor of the loaded sample for western blotting (1% for the input sample and 5% for the IP sample), followed by IP/input. For the quantification, the mean ± s.d. values were as follows: PDCD5–flag (42.70 ± 16.16), CCT1 (86.66 ± 41.01), CCT2 (45.54 ± 15.25), CCT3 (45.57 ± 12.47), CCT4 (61.12 ± 15.08), CCT5 (98.98 ± 27.74), CCT6 (53.74 ± 21.34), CCT7 (65.99 ± 38.51), CCT8 (135.49 ± 64.48) and GAPDH (0.03 ± 0.06), with n representing the number of biologically independent experiments (n = 4). For the quantification of PDCD5 mutation experiments, the mean ± s.d. values were as follows: WT (100 ± 0), RKK (133.65 ± 59.63) and IL (11.04 ± 9.68), with n representing the number of biologically independent experiments (n = 4).

    To induce TRiC closure during co-IP, beads bound with TRiC–PDCD5–Flag (from co-IP, see above) were incubated in ATP/AlFx buffer (lysis buffer supplemented with 5 mM Al(NO3)3, 30 mM NaF and 1 mM ATP) for 1 h at 37 °C, followed by three washes with ATP/AlFx buffer. As a control, the beads bound with TRiC–PDCD5–Flag (from co-IP, see above) were incubated and washed in lysis buffer without the ATP/AlFx. For western blotting, 1% of input, 25% of released proteins after ATP/AlFx incubation and 25% of eluates (denoted as beads) were loaded.

    Without adding ATP in the TRiC sample before plunge freezing, around 100% TRiC particles are at open conformation based on the single-particle analysis13,14,19. With extra ATP/AlFx in TRiC solution before plunge freezing, a portion of TRiC particles were closed, although different papers show different closed/open ratios with ATP/AlFx at different conditions. Closed/open ratio: ~1.7 in buffer (1 mM ATP, 5 mM MgCl2, 5 mM Al (NO3)3 and 30 mM NaF) from ref. 13; ~5.1 in buffer (1 mM ATP, 1 mM Al3(NO3)3, 6 mM NaF, 10 mM MgCl2 50 mM KCl) from ref. 21; ~0.6 in buffer with ATP-AlFx from ref. 14; and ~2.2 in buffer (1 mM ATP, 5 mM MgCl2 and AlFx (5 mM Al(NO3)3 and 30 mM NaF) from ref. 16. In our experimental settings (Extended Data Fig. 7), we used the conditions from ref. 13 (1 mM ATP, 5 mM MgCl2, 5 mM Al (NO3)3 and 30 mM NaF).

    For the quantification in Extended Data Fig. 7, the mean ± s.d. values were as follows: PDCD5 (ATP/AlFx) (0.09 ± 0.05); PDCD5 (control) (0.10 ± 0.04); CCT1 (ATP/AlFx) (1.53 ± 0.51); and CCT1 (control) (0.38 ± 0.06); with n representing the number of biologically independent experiments (n = 4).

    Thermal protein profiling (heat-shock treatment of cells)

    WT (Abcam, ab255449) and PDCD5-knockout HEK293T cells (Abcam, ab266229) were used for the heart-shock assay and cultured in DMEM (Sigma-Aldrich) supplemented with 10% fetal bovine serum (Gibco) at 37 °C with 5% CO2. The experiment was conducted as described previously49,50. In brief, cells were collected and resuspended in PBS. Five aliquots were prepared and distributed into PCR tubes, each of the tubes containing 5 × 105 cells. Each tube was incubated for 3 min at various temperatures (37.0, 44.1, 49.9, 55.5 and 62.0 °C; or 56.8, 58.3, 59.5, 60.7 and 62.1 °C). The cells were then lysed in a buffer containing 1.5 Mm MgCl2, 0.8% NP-40, 0.4U μl−1 benzonase and protease inhibitor for 40 min at 4 °C. Protein aggregations were removed, and the soluble fraction was used for western blotting. For quantification of the western blotting of thermal protein profiling, the mean ± s.d. values of actin in WT cells at 37.0 °C to 62.0 °C were as follows: 100.0 ± 0.0, 85.3 ± 5.2, 73.8 ± 7.7, 46.3 ± 2.9 and 26.3 ± 9.4; the mean ± s.d. values of actin in PDCD5-knockout cells at 37.0 °C to 62.0 °C were as follows: 100.0 ± 0.0, 100.3 ± 7.0, 109.0 ± 9.7, 83.0 ± 2.0 and 57.6 ± 9.4; the mean ± s.d. values of tubulin in WT cells at 56.8 °C to 62.1 °C were as follows: 100.0 ± 0.0, 78.2 ± 4.2, 49.3 ± 5.5, 20.0 ± 4.9 and 5.4 ± 3.8; and the mean ± s.d. values of tubulin in PDCD5-knockout cells at 56.8 °C to 62.1 °C were as follows: 138.0 ± 22.3, 99.7 ± 6.4, 63.9 ± 15.9, 34.8 ± 0.4 and 8.3 ± 4.7.

    Antibodies

    Membranes from western blotting were incubated with primary antibodies (mouse anti-Flag M2 (Sigma-Aldrich, F1804, 1:2,000), rabbit anti-PDCD5 (Abcam, ab126213, 1:1,000), rabbit anti-CCT1 (Abcam, ab240903, 1:10,000), rabbit anti-CCT2 (Abcam, ab92746, 1:10,000), rabbit anti-CCT3 (Proteintech, 10571-1-AP, 1:30,000), rabbit anti-CCT4 (Proteintech, 21524-1-AP, 1:5,000), rabbit anti-CCT5 (Proteintech, 11603-1-AP, 1:3,000), rabbit anti-CCT6 (Proteintech, 19793-1-AP, 1:1,000), rabbit anti-CCT7 (Abcam, ab240566, 1:30,000), rabbit anti-CCT8 (Proteintech, 12263-1-AP, 1:2,000), rabbit anti-GAPDH (Proteintech, 10494-1-AP, 1:15,000), mouse anti-actin (Invitrogen, AM4302, 1:3,000), mouse anti-tubulin (Sigma-Aldrich, T5168, 1:3,000)), followed by incubation with HRP-conjugated secondary antibodies (anti-rabbit IgG (Cell Signaling, 7074, 1:10,000), anti-mouse IgG + IgM (Jackson ImmunoResearch, 115-035-044, 1:10,000)). Uncropped western blots are provided as Source Data.

    Grid preparation, data acquisition and tomogram reconstruction

    Cryo-ET sample preparation, data collection and tomogram reconstruction were performed essentially as described previously22. In brief, R2/2 gold grids with 200 mesh (Quantifoil) were glow discharged for 90 s and were positioned in 3.5 cm cell culture dishes (MatTek). Then, 2 ml HEK Flp-In T-Rex 293 cell suspension, with a concentration of 175,000 cells per ml, was added to the dish. For untreated samples, cells were cultured for 5 h before plunge-freezing. For HHT-treated samples, cells were cultured without HHT for 3 h and subsequently exposed to HHT (Santa Cruz Biotechnology) at a final concentration of 100 µM for 2 h before the plunge-freezing process. The grids were blotted from the backside for 6 s using the Leica EM GP2 plunger under 70% humidity and 37 °C. The grids were rapidly plunged into liquid ethane and stored in liquid nitrogen. Grids were FIB-milled using Aquilos FIB-SEM (Thermo Fisher Scientific). The samples were sputter-coated with an organometallic protective platinum layer using the gas injection system for 15 s. Lamella preparation was performed through a stepwise milling process with gallium ion-beam currents decreasing from 0.5 nA to 30 pA.

    The data acquisition area was focused on the cytoplasmic region within the cell. Tilt series were acquired on a Titan Krios G4 (Thermo Fisher Scientific) operated at 300 kV, and equipped with Selectris X imaging filter and Falcon 4 direct electron detector, at 4,000 × 4,000 pixel dimensions, pixel size of 1.188 Å, a total dose of 120 to 150 e Å−2 per tilt series, 2° tilt increment, tilt range of −60° to 60° and target defocus of −1.5 to −4.5 µm, using SerialEM software51. Tilt series were aligned automatically using the IMOD package52. The alignment files generated from IMOD were used for tomogram reconstruction in Warp53 v.1.0.9.

    Particle localization and refinement

    Template matching was performed similarly to previous studies22,54. For this work, the parameters were set as follows: 5° angular scanning step, low-pass filter radius=20, high-pass filter radius=1, apply_laplacian=0, noise_corelation=1 and calc_ctf=1. The cryo-EM map (EMD-32822)14 of TRiC downloaded from the Electron Microscopy Data Bank (EMDB) was used as the template covered by a sphere mask. The above optimized setting produced distinguished peaks visualized in napari55 (Extended Data Fig. 1b and Supplementary Video 1). To analyse all potential TRiC complexes within the datasets, we extracted the top 1,000 peaks per tomogram. The selection was based on the constrained cross-correlation (CCC) value from template matching, and these chosen coordinates were subsequently extracted as subtomograms in Warp. In total, 360,000 untreated and 352,000 treated subtomograms were extracted. 3D classifications (classes = 4, T = 0.5, iterations = 30, without mask) and refinements (C1 symmetry) were performed in RELION56 v.3.1. In total, 3,353 open TRiC particles and 4,054 closed TRiC particles in the untreated dataset, and 3,785 and 3,418 in the treated dataset were identified. Open TRiC particles from untreated and treated datasets were combined and refined to improve map resolution. Closed TRiC particles were merged from untreated and treated datasets and refined with C1 or D8 symmetry. Actin filaments were manually picked in ten tomograms. In total, 1,490 subtomograms were extracted and refined at bin4. Atomic models obtained from the PDB (7X3J, 7NVN, 7NVO, 7NVL, 7NVM and 8F8P)13,16,57 were fitted into our maps. ChimeraX58,59 was used to visualize EM maps and models.

    Subtomogram classification of TRiC states

    For 3,353 open TRiC particles in the untreated dataset, classification with a sphere mask covering the potential PFD region (classes = 3, T = 3, iterations = 50, C1 symmetry) of one ring (denoted ring1) was performed (Extended Data Fig. 2a), which generated 2,874 particles without PFD and 479 particles with PFD of ring1. Independently, the same classification was performed with a mask focused on the other ring (denoted ring2), which produced 2,791 particles without PFD and 562 particles with PFD of ring2. In total, 2,395 particles without PFD, 875 particles with 1 PFD and 83 particles with 2 PFD were identified by sorting particles based on the above two classifications. The same classification strategy was applied to 3,785 open TRiC particles in the treated dataset, resulting in 2,334 particles without PFD, 1,287 particles with 1 PFD and 164 particles with 2 PFD. The atomic model (PDB: 7WU7)14 was fitted into the maps with PFD. Different classification parameters were evaluated in attempts to resolve the density in the chamber of TRiC, but this did not result in meaningful insights. The densities inside the TRiC chamber were Gaussian filtered (sDev = 2 or 4) for visualization in Figs. 1b and 4d and Extended Data Figs. 3 and 10. For closed TRiC, 3D classification (classes = 4, T = 3, iterations = 35, C1 symmetry) was performed in untreated and treated datasets independently in RELION 3.1, which revealed several classes with different densities occupied in the chamber of the closed TRiC. Further classification with a mask focusing on the substrate position did not produce meaningful results (Supplementary Figs. 4 and 5). Fourier shell correlation (FSC) was calculated in RELION 3.1.

    AlphaFold-Multimer model of the CCT3–CCT1–CCT4–PDCD5 complex

    The structure of human PDCD5 in a complex with human CCT3, CCT1 and CCT4 was predicted using AlphaFold-Multimer31 (v.2.2.0). The prediction was executed using the default setting with AMBER relaxation, and 15 models were generated for each prediction. The same prediction setting was used for PDCD5 with the other CCT combinations. The full-length amino acid sequences of PDCD5 (UniProt: O14737)60 and the equatorial domain of CCT1–CCT8 (the sequences were the same as PDB 7NVO) were used for the above prediction. The monomeric model of PDCD5 (AF-O14737-F1) was downloaded from the AlphaFold Protein Structure Database30.

    Sequence alignment

    Sequence alignment of CCT1–CCT8 (UniProt: P17987, P78371, P49368, P50991, P48643, P40227, Q99832 and P50990) was executed through Clustal Omega61. Sequence alignment of PDCD5 (UniProt: M. maripaludis, A9A8D7; S. pombe, O13929; C. elegans, Q93408; mouse, P56812; bovine, Q2HJH9; and human, O14737) and CCT1 (UniProt: H. volcanii, O30561; S. pombe, O94501; C. elegans, P41988; mouse, P11983; bovine, Q32L40; and human, P17987) were performed with ClustalO in Jalview62. The sequence conservation score of PDCD5 was calculated using the ConSurf server63.

    Spatial analysis of TRiC in situ

    The distance and angle examination of TRiC was performed similarly to as in previous studies22,64,65. For TRiC cluster tracing, the coordinates of TRiC determined by subtomogram averaging were used to localize the particles in the tomograms. The TRiC cluster (containing ≥2 TRiC particles) was defined by the distance between the coordinates of one TRiC and that of its nearest neighbour using a distance cut-off of 20 nm (centre-to-centre distance). As the coordinate represents the centre of the structure, the rotation of the particles would not affect the distance measurement. The particle closest to the previous particle in terms of Euclidean distance was selected as the trailing TRiC within the cluster, provided that it fell within the permitted distance threshold. Various distance thresholds ranging from 15 nm (the minimum centre-to-centre distance between two TRiC) to 40 nm were investigated (Fig. 4b,c). For each specific distance, the threshold was confined within a range of ±0.5 nm (for example, for 17 nm, the permissible distance ranged from 16.5 nm to 17.5 nm). A distance threshold of 20 nm was used to define whether TRiC belongs to the same cluster in this study.

    For the distance of TRiC pair analysis in Extended Data Fig. 9h,i, the number and the mean ± s.d. values were n2 (cluster length = 2) = 326 (17.35 ± 1.18); n3 = 218 (17.44 ± 1.27), n4 = 74 (17.01 ± 1.17), n5 = 35 (17.05 ± 1.16), n6 = 16 (16.87 ± 1.01) and n7 = 4 (17.33 ± 0.89), respectively, in the untreated dataset. The number and the mean ± s.d. were n2 = 195 (17.04 ± 1.28), n3 = 116 (17.42 ± 1.18), n4 = 27 (16.87 ± 0.96), n5 = 7 (17.09 ± 1.25) and n6 = 4 (16.65 ± 1.88), respectively, in the treated dataset. TRiC pairs with distances between 15 and 20 nm were analysed.

    The angle between TRiC and its closest neighbouring TRiC was investigated for particles within clusters in the untreated dataset (Extended Data Fig. 8d). The divided area of the hemisphere contains all points denoting cone rotation, described by Euler angles θ and ψ, of a vector (0, 0, 1). These rotations are projected onto the northern hemisphere (for vectors rotated with a z-coordinate greater than 0) and the southern hemisphere (for vectors rotated with a z-coordinate less than or equal to 0) using stereographic projection. The north pole corresponds to zero rotation, signifying a vector (0, 0, 1). The rotations of the neighbour TRiC were multiplied by the inverse rotations of the respective neighbour particles.

    To calculate the percentage of TRiC clusters with neighbouring actin filaments. The particles from the subtomogram averaging of TRiC and actin filaments were mapped back to tomograms for analysis. The threshold of the neighbouring distance (TRiC centre to the centre of actin dimer) was set to 20 nm.

    Spatial relation between ribosomes and TRiC in cells

    The spatial distribution of TRiC near the ribosome exit tunnel was investigated. The coordinates of ribosome, 60S and 40S determined by subtomogram averaging were used to localize the particles in the tomograms22. The ribosome was rotated to a reference position (zero rotation) through an inverse rotation, which means it was rotated by (−ψ, −θ, −φ)ribosome. Subsequently, TRiC underwent rotation by its respective angles (φ, θ, ψ)TRiC, followed by another rotation of (−ψ, −θ, −φ)ribosome, therefore aligning the ribosome–TRiC within a standard rotation frame (zero rotation of the ribosome), while maintaining their original angular relationship. The coordinates of the ribosome exit tunnel were subtracted from both the ribosome exit tunnel coordinates (setting it to zero) and TRiC coordinates. The new TRiC coordinates were rotated by (−ψ, −θ, −φ)ribosome to illustrate their positioning relative to the zero rotation of the ribosome. For the spatial analysis of ribosome and TRiC, ribosome particles were more abundant than TRiC particles. As a result, the same TRiC can be the nearest neighbour of several ribosomes. Our analysis focused on the ribosomes that acted as the nearest neighbours of TRiC. The mean ± s.d. in Extended Data Fig. 9c,k were as follows: untreated open TRiC in the ribosome ETS (55.1 ± 0.8%); untreated closed TRiC in the ETS (55.3 ± 0.3%); untreated open TRiC in the non-ETS (44.9 ± 0.8%); untreated closed TRiC in the non-ETS (44.7 ± 0.3%); treated open TRiC in the ETS (50.4 ± 0.4%); treated closed TRiC in the ETS (49.7 ± 1.0%); treated open TRiC in the non-ETS (49.6 ± 0.4%); and treated closed TRiC in the non-ETS (50.3 ± 1.0%). Data plotting and statistical analysis were performed using GraphPad Prism (v.10, GraphPad Software).

    Reporting summary

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

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  • Visualizing chaperonin function in situ by cryo-electron tomography

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    Plasmids and strains

    Escherichia coli BL21(DE3) Gold cells (Stratagene) were used for growth analysis, electron tomography and protein expression. For tomography and biochemical experiments, GroEL was expressed from a pBAD33 plasmid containing the groEL gene under the control of an araBAD promotor (EL+ cells)26. For overexpression of GroEL and GroES, a pBAD33 plasmid containing both groEL and groES genes under the control of an araBAD promotor was used48. MetK was expressed from a pET22b plasmid previously described26.

    Antibodies

    Polyclonal antisera used against GroEL, GroES, MetK and GAPDH were previously described26, and the rabbit antiserum against α-lactalbumin was a product of East Acres Biologicals immunization service.

    E. coli growth

    E. coli cells were grown in lysogeny broth (LB) medium that contained, depending on the plasmids used, the antibiotics ampicillin (200 μg ml−1, pET22b-MetK) and chloramphenicol (32 μg ml−1, pBAD33 variants). For overexpression of GroEL, GroES and MetK (MetK cells), transformed E. coli Bl21 (DE3) pBAD33-GroEL:ES pET22b-MetK cells were grown to early exponential phase at 37 °C, and GroEL–GroES expression using the pBAD33 promoter was induced for 90 min by supplementation of LB medium with arabinose to a final concentration of 0.2% (w/v). Cells were subsequently harvested by centrifugation at 8,000g (4 °C for 10 min) and resuspended to an optical density (600 nm, OD600) of 0.1–0.2 in fresh LB medium containing both antibiotics and 1 mM isopropyl β-d-thiogalactopyranoside (IPTG), to induce MetK expression under control of the T7 promoter for 40 min. GroEL expression (EL+) was induced in E. coli Bl21 (DE3) pBAD33-GroEL by supplementation of LB medium with arabinose to a final concentration of 0.1% (w/v) and growth of the culture at 37 °C. To expose E. coli Bl21 (DE3) cells to HS, cells were first cultured to early exponential phase at 37 °C and then incubated in a shaking water bath at 46 °C for 2 h.

    E. coli growth curves

    Cells were cultured as described above. Aliquots were removed at the time points indicated for optical density measurement at OD600. To ensure exponential growth conditions, growing cultures were diluted to an OD600 of 0.1 with prewarmed LB medium containing the necessary antibiotics and arabinose when OD600 just exceeded 0.4. Growth curves for MetK and EL+/MetK cells were measured following termination of GroEL induction by transfer of cells into arabinose-free medium containing 1 mM IPTG for MetK overexpression. The first sample was taken 5 min after changing the medium. Data were processed for fitting in R.

    Protein expression and purification

    GroEL, GroES and MetK proteins were expressed and purified as previously described26,49.

    Measurement of protein concentration

    Concentrations of purified proteins were determined by measurement of absorbance at 280 nm using absorbance coefficients calculated from the protein sequence with the program ProtParam50. Protein concentrations of cell lysates were determined with the Pierce Coomassie Plus (Bradford) Assay Kit (Thermo Fisher Scientific) as described by the manufacturer.

    Preparation of cell lysates

    Cultures were prepared as described above, harvested by centrifugation and the cell pellet flash-frozen in liquid nitrogen before further processing. Spheroplasts were prepared at 4 °C as previously described51. In brief, cells were resuspended in 100 mM Tris-HCl pH 8.0 and washed twice with 2 ml of buffer. The pellet was then resuspended in HMK buffer (50 mM HEPES-KOH pH 7.2, 20 mM Mg acetate, 50 mM K acetate) supplemented with 20% (w/v) sucrose and 0.25 mg ml−1 lysozyme. Cells were then incubated on ice for 7 min and transferred to 37 °C for 10 min. The resulting suspension was supplemented with Complete EDTA-free protease inhibitor cocktail (Roche), and spheroplasts were lysed by the addition of 0.1% (v/v) Triton X-100 and subsequent sonication.

    Mass spectrometry

    Cell lysates were reduced by the addition of dithiothreitol (DTT) to a final concentration of 10 mM and heated to 56 °C for 45 min. Acylation of thiol groups was performed by the addition of chloroacetamide to a final concentration of 55 mM and incubation for 45 min in the dark, followed by a first digestion step with Lys-C (Wako) at a w/w ratio of 1:20 for 2 h at 37 °C. This was followed by a second digestion step overnight with trypsin (Roche) at a 1:20 (w/w) ratio at 37 °C. The reaction was stopped by the addition of trifluoroacetic acid to a final volume of 1%. Peptides were desalted using OMIX C18 (100 μl) tips (Agilent Technologies, no. A57003100) according to the manufacturer’s instructions.

    Desalted peptides were dissolved in 12 µl of 5% formic acid, sonicated in an ultrasonic bath, centrifuged and transferred to autosampler vials (Waters). Samples were analysed on an Easy nLC-1200 nanoHPLC system (Thermo) coupled to a Q-Exactive Orbitrap HF mass spectrometer (Thermo). Peptides were separated on pulled-spray columns (ID 75 μm, length 30 cm, tip opening 8 μm, NewObjective) packed with 1.9 μm C18 particles (Reprosil-Pur C18-AQ, Dr Maisch) using either a stepwise 196 min gradient (comparison of 37 °C, HS and MetK) or a stepwise 67 min gradient (all other samples) between buffer A (0.2% formic acid in water) and buffer B (0.2% formic acid in 80% acetonitrile). Samples were loaded on the column by the nanoHPLC autosampler at a pressure of 900 bar. The high-performance liquid chromatography flow rate was set to 0.25 μl min−1 during analysis. No trap column was used. The following parameters were used for comparison of growth conditions 37 °C, HS and MetK: MS, resolution 60,000 (full-width at half-maximum (FWHM) setting); MS mass range 300–1,650 m/z; MS-AGC-setting 3 × 106; MS-MaxIT 50 ms; MS/MS fragmentation of the 15 most intense ions (charge state 2 or higher) from the MS scan; MS/MS resolution 15,000 (FWHM setting); MS/MS-AGC-setting 105; MS/MS-MaxIT 50 ms; MS/MS isolation width 1.8 m/z; collision-energy setting 29 (NCE). All other samples were analysed with the following parameters: MS resolution 120,000 (FWHM setting); MS mass range 300–1,650 m/z; MS-AGC-setting 3 × 106; MS-MaxIT 100 ms; MS/MS fragmentation of the ten most intense ions (charge state 2 or higher) from the MS scan; MS/MS resolution 15,000 (FWHM setting); MS/MS-AGC-setting 105; MS/MS-MaxIT 50 ms; MS/MS isolation width 1.2 m/z; collision-energy setting 29 (NCE).

    MS data analysis

    Protein identification was performed using MaxQuant with default settings. The E. coli K12 strain sequences of UNIPROT (v.2023-03-01) were used as the database for protein identification (Supplementary Information). MaxQuant uses a decoy version of the specified UNIPROT database to adjust false discovery rates for proteins and peptides below 1%.

    Quantification of MetK binding to GroEL

    To quantify the fraction of GroEL with bound MetK in MetK-overexpressing cells, we immunoprecipitated GroEL with GroEL antibody followed by GroEL and MetK immunoblotting and liquid chromatography–tandem mass spectrometry. Cells were prepared and lysed as described above, but with the addition of apyrase (25 U ml−1 final concentration) to rapidly deplete the ATP pool in the lysate and arrest the GroEL reaction cycle26. The lysate was clarified by centrifugation at 16,000g (4 °C for 10 min). Either 20 μl of a non-specific antibody (against α-lactalbumin) or a GroEL-specific antibody was coupled to 100 μl of recombinant protein A Sepharose 4B beads (Thermo Fisher Scientific) as described by the manufacturer. The beads were loaded with sample (180 μg of protein) and incubated in 650 μl of HMK buffer for 1 h. The beads were washed twice with 600 μl of HMK buffer and then twice more with HMK containing 0.1% Triton X-100. For immunoblotting, elution was performed with 50 μl of 2× lithium dodecyl sulfate (Pierce) containing β-mercaptoethanol 5% (v/v) as prescribed by the manufacturer. For liquid chromatography–tandem mass spectrometry analysis, elution and digestion were performed with the IST MS sample preparation kit (Preomics) using the manufacturer’s on-bead digestion protocol. Mass spectrometry was performed as described above.

    SDS–PAGE and immunoblotting

    Before SDS–polyacrylamide gel electrophoresis (SDS–PAGE) analysis, cells were resuspended in HMK buffer supplemented with 2 mM DTT, 1 mM EDTA and 5% glycerol and subsequently sonicated, followed by centrifugation (20 min, 16,000g at 4 °C). Protein samples were separated by electrophoresis on NuPAGE 10% Bis-Tris SDS gels (Invitrogen) using NuPAGE MES SDS running buffer (Invitrogen) at 150 V. Proteins were transferred to polyvinylidene difluoride membranes in blotting buffer (25 mM Tris, 192 mM glycine, 20% methanol) at 150 mA. Membranes were first incubated with primary antibodies in TBST buffer overnight at 4 °C and subsequently with horseradish peroxidase-conjugated secondary antibody for chemiluminescence detection. Uncropped immunoblots are provided in the Source Data file to Extended Data Fig. 3.

    In situ cryo-ET analysis

    Cell cultures were grown as described above. For cryo-ET analysis, cells in exponential growth (approximate OD600 0.4) were rapidly (for about 2 min) concentrated to an approximate OD600 of 10 by centrifugation at 8,000g and subsequently applied to R 2/1 100 Holey carbon film Cu 200 mesh grids (Quantifoil) that were previously plasma cleaned for 30 s. The sample was blotted for 9 s at force 10 and then plunge-frozen in a mixture of liquid ethane and propane cooled by liquid nitrogen using a Vitrobot Mark IV (Thermo Fisher Scientific) at 70% humidity and 22 °C. Frozen grids were transferred to a dual-beam, cryo-focused ion beam (FIB)/scanning electron microscope (Thermo Fisher Scientific; either Scios, Quanta, Aquilos or Aquilos 2). Cells were coated with a layer of inorganic platinum, if available in the system used, followed by the deposition of organometallic platinum using an in situ gas injection system (working distance, 10 mm; heating, 27 °C; time, 8 s). Removal of bulk material was done at a stage angle of 20–25° using gallium ions at 30 kV, 0.5 nA. Fine milling of lamellae was done at 11–13° stage tilt with successively lower currents between 0.3 nA and 30 pA, aiming for a final thickness of 100–200 nm (ref. 52). Lamellae for the selective GroEL overexpression dataset were prepared using Serial FIB53, and an additional layer of inorganic platinum was added following fine milling to avoid charging during image acquisition54. The resulting lamellae were transferred to a TEM (Titan Krios, field emission gun 300 kV, Thermo Fisher Scientific) equipped with an energy filter (Quantum K2, Gatan), a direct detection camera (K2 Summit, Gatan), and tomograms were acquired at a magnification of ×42,000 (pixel size 3.52 Å), defocus ranging from −5.0 to −3.0 μm and the energy filter slit set to 20 eV using SerialEM 3.9.0 (ref. 55). Tomograms were recorded in dose-fractionated super-resolution mode, with a total dose of roughly 120 e−/Å2 per tilt series. A dose-symmetric tilt scheme was used with an increment of 2–3° in a total range of ±60° from a starting angle of approximately 10° to compensate for lamellar pretilt (mostly around 11°)56. Frames were aligned using MotionCor2 (v.1.4.0, https://emcore.ucsf.edu/ucsf-software)57. The reconstruction was performed in IMOD using patch tracking (v.4.11.1, RRID:SCR_003297, https://bio3d.colorado.edu/imod/)58 using the TOMOgram MANager (TOMOMAN) wrapper scripts59. Tilt-series images were dose filtered using TomoMAN’s implementation of the Grant and Grigorieff exposure filter60. Defocus was estimated using CTFFIND4 (ref. 61).

    Tomograms of the EL+ dataset were acquired on a Krios G4 equipped with a Selectris X energy filter and Falcon 4 direct electron detector (Thermo Fisher Scientific). Tilt series were collected with a dose-symmetric tilt scheme using TEM Tomography 5 software (Thermo Fisher Scientific). A tilt span of ±60° was used with 2° steps, starting at ±10°, to compensate for lamellar pretilt. Target focus was changed for each tilt series in steps of 0.5 µm over a range of −2.5 µm to +5 µm. Data were acquired in EER mode of Falcon 4 with a calibrated physical pixel size of 3.02 Å and a total dose of 3e−/Å2 per tilt over ten frames. A 10 eV slit was used for the entire data collection. Data were preprocessed using TOMOMAN59. EER images were motion corrected using RELION’s implementation of MotionCor2 (ref. 62). Defocus was estimated using CTFFIND4 (ref. 61). Reconstruction was performed with IMOD using local deposits of the inorganic platinum that was applied by sputtering following milling as fiducials. All tomograms were reconstructed using NovaCTF63.

    E. coli membranes were segmented for visualization using TomoSegMemTV 1.0.

    Cryo-ET analysis of in vitro reconstituted GroEL–GroES complexes

    For generation of a GroEL–GroES reference for in situ tomographic analysis containing a defined substrate protein in a folded state and in a known topology, we imaged in vitro reconstituted GroEL–GroES–MetK complexes using the same data collection strategy and parameters as above for WT cells.

    Subtomogram averaging

    For subtomogram averaging, all datasets acquired on the same microscope (37 °C, HS, MetK) were combined and processed together; the EL+ dataset was processed separately. The overall processing workflow is depicted in Extended Data Fig. 1b.

    For template matching, PDB entry 1AON was used for EL–ES1, 4PKO for EL–ES2 and 5MDZ for 70S ribosomes to generate templates at a resolution of 40 Å using the molmap64 command in Chimera65. Initial positions for a subset of EL–ES1 and EL–ES2 complexes and ribosomes were determined using the noise correlation template-matching approach implemented in STOPGAP, by fourfold binning to a pixel size of 14.08 Å (ref. 66). This subset of the data was subsequently aligned and classified in STOPGAP to generate a reference from the tomographic data with a Fourier shell correlation (FSC) value close to 1 at 40 Å template-matching resolution. Template matching with various GroEL14 species was attempted, but never yielded an average of GroEL14 with a resolution better than the template resolution. The data-derived references of all three different structures were used for an additional round of template matching on the complete dataset. Cross-correlation cut-off was chosen separately for every tomogram by visual inspection of the generated hits and comparison with the tomogram. To reduce the level of false-positive detection, a mask for the cytosol of the cell was first created using AMIRA (Thermo Fisher Scientific) and subsequently used to filter out hits outside of the cytosol. Putative particles were deliberately overpicked with low-resolution templates in the initial stage to avoid false-negative assignments.

    This procedure yielded 176,408 initial subtomograms for the EL–ES1 reference and 125,860 for the EL–ES2 reference. These were then further aligned and classified separately in STOPGAP, each yielding classes containing both EL–ES1 and EL–ES2 particles. The combined number of particles contained in classes with emergent high-resolution features (Supplementary Fig. 1a) for the EL–ES1 reference was 19,239, and 17,614 for the EL–ES2 reference (Extended Data Fig. 1 and Supplementary Fig. 1b). Because both references pick up a subset of the other particles, the particles were then combined and duplicates removed. The resulting combined dataset was split by reference-free, three-dimensional classification in STOPGAP, resulting in a set of 17,598 EL–ES1 and 11,213 EL–ES2 complexes that were then independently refined. This resulted in a resolution at the FSC cut-off of 0.143 following the application of symmetry at 11.6 Å for the EL–ES1 complex (C7 symmetry) and 11.9 Å for the EL–ES2 complex (D7 symmetry). Classification was performed using simulated annealing stochastic hill-climbing multireference alignment as previously described67. All classifications were done repeatedly with different, random initial starting sets of 250–500 subtomograms to generate the initial references. Only particles that ended up in the same class for all independent rounds of classifications were retained67. Further refinements with the established WARP, RELION, M pipeline were attempted but did not yield any further improvements. EL–ES1 wide and narrow complexes were separated by classification with a focused, disk-shaped mask on the apical domains of the EL–ES1 trans-ring. This resulted in 6,681 narrow complexes that were refined to a resolution of 13.5 Å, and 10,130 wide EL–ES1 complexes refined to a resolution of 12.0 Å.

    The EL+ dataset was processed in the same way, but starting with the structures from the other datasets, low-pass filtered to 40 Å, as initial references for template matching. Template matching was then repeated once with structures generated by averaging a subset of particles from this dataset. To improve the resolution for model building, the dataset was exported to WARP68 and angles and positions refined using RELION v.3.0.8 (ref. 69). This yielded a GroEL14 structure at a global resolution of 13 Å. GroEL 14-mer particles were corefined for geometric distortions with ribosomes in M. The resulting GroEL 14-mer particles were exported for further alignment and classification in RELION. Classification was performed with a regularization parameter T of four and six classes for 25 iterations without angular search, resulting in a more homogeneous subset of 12,421 particles. These particles were again corefined in M for geometric distortions and per-particle defocus for contrast transfer function (CTF) estimation, resulting in a final structure with nominal resolution of 9.8 Å at 0.143 FSC cut-off.

    Owing to their high molecular weight and density, ribosome template matching achieves a higher precision and recall. During initial rounds of classification in STOPGAP, because no false-positive particles were detected, all ribosomal hits from template matching were aligned first in STOPGAP at progressively lower binnings (bin4, bin2, bin1). The resulting particles were then exported to WARP using TOMOMAN. Subtomograms were reconstructed for RELION v.3.0.8 using WARP at a pixel size of 3.52 Å per pixel. An iterative approach with subtomogram alignment in RELION and tilt-series refinement in M70 were performed until no further improvement in gold-standard FSC was obtained. This resulted in a final structure of the ribosome at a resolution of 8.6 Å for the combined 37 °C, HS and MetK datasets, and 6.3 Å for the EL+ dataset, which was processed separately.

    In vitro cryo-ET data for GroEL–GroES complexes were processed analogous to the in situ data, resulting in 39,518 initial hits for the EL–ES2 template and 46,093 for the EL–ES1 template, with both sets having a significant overlap. These were then further aligned and classified separately in STOPGAP, yielding 5,832 and 13,688 particles, respectively, following duplicate removal.

    Classification of SP occupancy of GroEL–GroES complexes in situ

    For the resolution of densities corresponding to substrate proteins in the GroEL–GroES chamber we first performed symmetry expansion around the C2 axis of the EL–ES2 complexes and aligned the new set of GroEL–GroES chambers with the cis-ring of the EL–ES1 complexes. The resulting subtomograms of the chambers were then denoised using TOPAZ’s three-dimensional pretrained denoising function71. Because initial attempts to classify the interior of the chamber using STOPGAP multireference-based alignment showed only separation by missing wedge, the subtomograms were combined into 5,000 random bootstraps containing 250 random subtomograms each. These averages were then used to perform k-means clustering with two classes. Bootstraps from the resulting clusters were averaged and used as initial start structures for multireference alignment in STOPGAP. For this, stochastic hill climbing was performed with a temperature factor of 10 for simulated annealing, followed by 40 iterations of multireference alignment with two classes and a mask around the interior of the chamber. This process was repeated five times. Only particles consistently assigned to the same classes were used for a final round of subtomogram averaging, resulting in one class showing weak diffuse density inside the chamber and a second showing strong density near the bottom. Attempts to further subdivide these two classes resulted only in separation based on missing wedge. Because it was not possible to resolve the C7 symmetry mismatch of the substrate and enclosing chamber, final averages were produced for all different biological conditions with C7 symmetry applied to increase the signal-to-noise ratio. The class showing a strong density near the bottom contained 12,255 subtomograms, the one showing only a weak diffuse density with 24,435 subtomograms for the combined 37 °C, HS and MetK datasets.

    In vitro data were processed analogously. The resulting classes were then again split into EL–ES1 and EL–ES2 complexes corresponding to their substrate state and exported to WARP68. An additional round of alignments was performed in RELION for all different classes and complexes. A prior was set for all angles. Local search was performed with a sigma of 0.5 and search angle of 0.9°. The resulting particles were separately refined in M, correcting for geometrical distortions. Particles were again exported from M70 and signal subtraction preformed in RELION of the trans-ring for EL–ES1 and the opposing chambers for EL–ES2. Based on their previous classification results in STOPGAP, the refined signal-subtracted, single-chamber complexes were combined in two groups resulting in 7,087 GroEL–GroES chambers containing an ordered SP and 14,371 that either contained a disordered SP or were empty. The resulting chambers were again locally refined in RELION using priors and a sigma on all angles, yielding a resolution of 9.4 Å for GroEL–GroES chambers containing ordered SP and 8.8 Å for the remaining chambers.

    Cryo-EM single-particle analysis of GroEL–GroES–MetK complexes

    For generation of substrate-bound GroEL–GroES complexes, 4 μM MetK was denatured in the presence of 1 μM GroEL (14-mer) in buffer A (20 mM MOPS-NaOH pH 7.4, 200 mM KCl, 10 mM MgCl2, 5 mM DTT) containing 30 mM NaF and 5 mM BeSO4 by first incubation of the mixture at 60 °C for 15 min and then cooling to 25 °C in a thermomixer (Eppendorf). The addition of 2 μM GroES (7-mer) and 1 mM ATP (pH 7.0) resulted in stable chaperonin complexes with encapsulated MetK40. Biochemical analysis of this preparation was performed by size exclusion chromatography on a Superdex 200 3.2/300 GL column. Fractions were analysed by SDS–PAGE electrophoresis (NuPAGE, Bis-Tris 4–12% gels), and MetK loading of GroEL–GroES complexes was estimated by mass spectrometry using intensity-based absolute quantification values72. For analysis by mass spectrometry, fractions F1 and F2 (Extended Data Fig. 5a) were analysed separately but intensities pooled for the determination of intensity-based absolute quantification ratios.

    GroEL–GroES–MetK samples were concentrated tenfold by ultrafiltration using a 100 kDa Amicon centrifugal concentrator (Millipore) at room temperature. As a control, GroEL and GroES were treated identically in the absence of MetK. Before freezing, 1 μl of a n-octyl-β-d-glucopyranoside stock solution (87.5 mg ml−1 in buffer A) was added per 50 μl of sample. For single-particle analysis and in vitro cryo-ET experiments, 4 μl of the sample was applied onto R 2/1 100 Holey carbon film Cu 200 mesh grids (Quantifoil) previously plasma cleaned for 30 s. This grid was blotted for 3.5 s at force 4 and plunge-frozen in a mixture of liquid ethane and propane cooled by liquid nitrogen using a Vitrobot Mark IV (Thermo Fisher Scientific) at 100% humidity and 4 °C.

    Cryo-EM data for the EL–ES–MetK dataset were acquired using a FEI Titan Krios transmission electron microscope and SerialEM software55. Video frames were recorded at a nominal magnification of ×22,500 using a K3 direct electron detector (Gatan), with a total electron dose of around 55 electrons per Å2 distributed over 30 frames at a calibrated physical pixel size of 1.09 Å. Micrographs were recorded within a defocus range of −0.5 to −3.0 μm.

    On-the-fly image processing and CTF refinement of cryo-EM micrographs were carried out using the Focus software package73. Only micrographs that met the selection criteria (ice thickness under 1.05, drift 0.4 Å < x < 70 Å, refined defocus 0.5 μm < x < 5.5 μm, estimated CTF resolution under 6 Å) were retained. Micrograph frames were aligned using MotionCor2 (ref. 57), and the CTF for aligned frames was determined using GCTF74.

    The control dataset of GroEL–GroES complexes without MetK was acquired similarly but with a nominal magnification of ×29,000, resulting in a calibrated pixel size of 0.84 Å.

    Image processing, classification and refinement for single-particle analysis

    From the resulting 8,945 micrographs of the GroEL–GroES–MetK dataset, 1,561,482 particles were picked using a trained crYOLO network75 and extracted with RELION v.3.1.3 (ref. 69). An initial round of two-dimensional classification was performed and the remaining particles were passed into CryoSPARC76 for further two-dimensional classification, ab initio model building, alignment and initial three-dimensional classification to separate EL–ES1 from EL–ES2 complexes. The remaining EL–ES2 (659,866 particles) and EL–ES1 (294,250 particles) complexes were then exported separately to RELION for additional alignment with imposed symmetry, CTF refinement and Bayesian polishing. For the EL–ES2 complexes, symmetry expansion around the C2 axis was performed and the opposing half removed using RELION’s signal subtraction.

    The resulting asymmetric EL–ES1 complexes were then classified further with CryoDRGN77, resulting in a clean subset of 242,276 particles. The trans-rings of the EL–ES1 complexes were classified in CryoSPARC using a focused mask on the apical domains of the trans-ring, resulting in 169,454 particles in the narrow conformation and 34,755 in the wide conformation. The resulting structures were refined in CryoSPARC under the application of C7 symmetry to a nominal resolution of 2.9 and 3.1 Å, respectively. For the analysis of the cis-chamber, all EL–ES1 particles were pooled and the trans-ring was removed by signal subtraction in RELION.

    The resulting GroES-bound, single-ring particles (1,562,002 particles) were then aligned to a common reference in RELION and exported to CryoSPARC for further alignment without imposed symmetry. The resulting mask and reference were reimported into RELION and used for an additional alignment step with the goal of aligning the asymmetric MetK substrate contained inside the chamber (Extended Data Fig. 5d). Subsequently a second round of signal subtraction was performed and the resulting particles, comprising only MetK density, were further subjected to three-dimensional classification without angular search in RELION. A subset of the resulting classes showed visible secondary structure elements in different orientations (Extended Data Fig. 5d). These classes were then combined and aligned into a single frame of reference in Matlab 2015b by manual rotation with the respective multiple of 360°/7 around the sevenfold symmetry axis. This was done by adding the corresponding increment to particle rotation angles in the particle table (.star file).

    These folded MetK (fMetK) particles were then further locally aligned in CryoSPARC. An additional round of three-dimensional classification was performed followed by a final round of local alignment (322,800 particles), resulting in density for MetK at a resolution of 3.7 Å.

    For the study of MetK contacts with the inner wall of GroEL–GroES chamber, we reverted the signal subtraction in RELION to generate single-ring GroEL–GroES–MetK particles for both the folded MetK and mixed population of chambers either containing disordered MetK or empty; both were refined and aligned in CryoSPARC. The subset containing a mixed population was additionally classified in CryoDRGN between the final alignment steps, resulting in a global resolution of 3.04 Å for the GroEL–GroES–MetK complex containing folded MetK and of 2.94 Å for the complex containing a mixed population of disordered MetK or empty chambers.

    GroEL–GroES complexes without MetK were processed analogously but without Bayesian polishing and CTF refinement in RELION. Signal subtraction was performed in CryoSPARC; using 293,974 particles, this resulted in a map with a global resolution of 2.5 Å following the application of C7 symmetry.

    Densities were visualized and rendered using ChimeraX78,79.

    Model building and refinement

    Model building was initiated by rigid-body fitting the GroEL subdomains, GroES and MetK from the crystal structures PDB 1SX3 (ref. 80), 5OPW12 and 7LOO42, respectively, into cryo-EM density, followed by manual editing using Coot81. The models were subsequently refined in real space with Phenix82. For the refinement of models against low-resolution data from STA, automatically generated restraints from reference structures such as PDB 8P4M (this study) were used. Residues with disordered sidechains were truncated at C-beta.

    Reporting summary

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

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