ABSTRACT Chaperonins are large barrel-shaped complexes that mediate ATP-dependent protein folding1,2,3. The bacterial chaperonin GroEL forms juxtaposed rings that bind unfolded protein and

the lid-shaped cofactor GroES at their apertures. In vitro analyses of the chaperonin reaction have shown that substrate protein folds, unimpaired by aggregation, while transiently

encapsulated in the GroEL central cavity by GroES4,5,6. To determine the functional stoichiometry of GroEL, GroES and client protein in situ, here we visualized chaperonin complexes in their

natural cellular environment using cryo-electron tomography. We find that, under various growth conditions, around 55–70% of GroEL binds GroES asymmetrically on one ring, with the remainder

comprehensive quantification and conformational analysis of chaperonin complexes, we propose a GroEL–GroES reaction cycle that consists of linked asymmetrical and symmetrical subreactions

mediating protein folding. Our findings illuminate the native conformational and functional chaperonin cycle directly within cells. SIMILAR CONTENT BEING VIEWED BY OTHERS CRYOEM REVEALS THE

STOCHASTIC NATURE OF INDIVIDUAL ATP BINDING EVENTS IN A GROUP II CHAPERONIN Article Open access 06 August 2021 IN SITU ANALYSIS REVEALS THE TRIC DUTY CYCLE AND PDCD5 AS AN OPEN-STATE

COFACTOR Article Open access 11 December 2024 SNAPSHOTS OF ACTIN AND TUBULIN FOLDING INSIDE THE TRIC CHAPERONIN Article Open access 21 April 2022 MAIN The bacterial chaperonin GroEL

cooperates with its cofactor GroES in assisting the folding of roughly 10% of newly synthesized proteins, including proteins with α/β topology that fail to fold spontaneously1,2,7,8. GroEL

is a cylindrical complex of around 800 kDa containing two heptameric rings of 57 kDa subunits stacked back to back. The subunits consist of apical, intermediate and equatorial domains and a

flexible C-terminal tail protruding into the ring cavity9 (Fig. 1a, top left inset). The apical domains mediate substrate protein (SP) binding and the equatorial domains mediate ATP binding

and hydrolysis. Hydrophobic residues at the apical domains recruit unfolded SP. ATP-dependent binding of the lid-shaped GroES (a heptamer of 10 kDa subunits), capping the SP-containing ring

(the _cis_-ring), results in the burial of hydrophobic surfaces on GroEL and displaces the bound protein into an enclosed chamber. SP folds inside this chamber during ATP hydrolysis on the

GroEL _cis_-ring, and a second SP can bind to the _trans_-ring. The _cis_-chamber opens following ATP binding to the _trans_-ring, dissociating GroES through negative inter-ring allostery to

allow SP release1,2,10,11,12. Thus, the two rings of GroEL are sequentially folding active. However, in vitro studies1,2,13 showed that GroES not only binds asymmetrically with GroEL

(‘bullet’ complexes, EL–ES1), but can also associate symmetrically with both rings (‘football’ complexes, EL–ES2). Some reports have suggested that SP binding shifts GroEL entirely from an

asymmetrical cycle to a symmetrical mode14. The cell cytosol is characterized by a high degree of macromolecular crowding, which profoundly affects protein–protein interactions15. To

investigate how the available in vitro data apply to the situation in the intact cell, here we explored the chaperonin mechanism within its natural cellular context by cryo-electron

tomography (cryo-ET)—a technique enabling in situ visualization of macromolecular assemblies at subnanometre resolution16,17,18,19,20,21,22,23. We find that the native chaperonin cycle

consists of linked asymmetrical and symmetrical subreactions mediating protein folding. GROEL–GROES COMPLEXES IN SITU For visualization of GroEL by cryo-ET in situ, _Escherichia coli_

BL21(DE3) cells were vitrified on electron microscopy grids and thinned by cryogenic focused ion beam milling before imaging (Fig. 1a,b and Extended Data Fig. 1a). EL–ES1 and EL–ES2

complexes were readily observed in raw tomograms (Fig. 1a, right insets), whereas GroEL alone was undetectable. We used template matching with reference structures for systematic

identification and classification (Extended Data Fig. 1b), showing the relative proportions and cellular distribution of these complexes. In cells growing at 37 °C, EL–ES1 and EL–ES2

complexes occurred at an approximate ratio of 60:40% (Fig. 1c). To validate the accuracy of the template-matching results we compared the numbers of identified chaperonin complexes with

those of ribosomes, which can be readily identified in cryo-ET23,24. We localized essentially all cellular ribosomes (Extended Data Fig. 2a,b), and determined a median ratio of GroEL to

ribosomes of 1:23 during growth at 37 °C (Extended Data Fig. 2c). Quantification by mass spectrometry (MS) confirmed these results (Extended Data Fig. 2c, blue crosses), indicating that our

cryo-ET analysis had identified most GroEL complexes. However, owing to the inherent limitations of template matching, we cannot rule out a small fraction of false-positive or false-negative

particles. To load GroEL with chaperonin-dependent SP, we first increased the level of both GroEL and GroES by around sixfold (Extended Data Fig. 3a–c), to reduce occupancy with endogenous

SP, and then strongly overexpressed the obligate GroEL substrate _S_-adenosylmethionine synthase (MetK)25,26 (Extended Data Fig. 3d,e). Biochemical analysis by GroEL immunoprecipitation and

MS demonstrated that, on average, about 1.3 MetK molecules bound per GroEL complex, corresponding to over 50% of GroEL rings containing MetK (Extended Data Fig. 3f,g). The relative abundance

of EL–ES1 and EL–ES2 complexes in tomograms was about 55% and 45%, respectively, similar to growth without MetK overexpression (Fig. 1c). To explore changes in chaperonin function under

stress, we exposed cells to heat stress (HS) at 46 °C for 2 h. Note that _E. coli_ grows efficiently under HS in full medium (Extended Data Fig. 3d) although numerous proteins are

destabilized27, increasing the demand for chaperonin. HS induced a roughly threefold increase in GroEL and GroES abundance (Extended Data Fig. 3a,b), with a ratio of GroEL to ribosomes of

about 1:10 in MS and cryo-ET data (Extended Data Fig. 2c). Notably, the level of EL–ES1 complexes increased to 70% of total (Fig. 1c); GroEL alone remained undetectable. Thus, HS promotes

the formation of asymmetric chaperonin complexes. We next investigated whether EL–ES2 complexes form as a consequence of GroES:GroEL concentration ratio. Expression of the _groES_ and

_groEL_ genes (_groESL_), organized in an operon28, resulted in an approximate 1:1 GroES:GroEL ratio29, equivalent to around a twofold excess of GroES (7-mer) over GroEL (14-mer)30, with

both proteins being essential28. To reverse the physiological ratio of GroES and GroEL we selectively overexpressed GroEL (EL+ cells) at 37 °C, resulting in a roughly 4.5-fold increase in

GroEL (Extended Data Fig. 3h). EL+ cells grew essentially as wild-type (WT) (Extended Data Fig. 3d) but contained only free GroEL (EL complex) and EL–ES1 (around 90% and 10% of total GroEL,

respectively) and no EL–ES2 complexes (Fig. 1c). Notably, because EL–ES1 complexes were of similar abundance relative to ribosomes as in WT cells (Extended Data Fig. 3i), the absence of

EL–ES2 resulted in a reduction in the overall level of GroEL–GroES complexes. Nevertheless, overexpression of MetK did not impair the growth of EL+ cells (Extended Data Fig. 3e). In summary,

asymmetrical and symmetrical chaperonin complexes coexist in vivo, with EL–ES1 predominating under all growth conditions tested, including high SP load and HS. Cells grew efficiently when

EL–ES2 complexes were not populated, indicating that EL–ES1 complexes are sufficient for function. IN SITU STRUCTURES OF CHAPERONIN COMPLEXES Subtomogram averaging (STA) produced structural

models for EL–ES1, EL–ES2 and EL complexes at around 10–12 Å resolution following the application of symmetry (Fig. 2a–d, Extended Data Fig. 4a–d and Extended Data Table 1). Molecular models

were derived, starting from rigid-body fitting of high-resolution GroEL structures. EL–ES1 complexes were further classified based on the positioning of the apical domains of the GroEL

_trans_-ring, resulting in two conformations referred to as ‘narrow’ and ‘wide’ (Fig. 2a,b,e,f). In the narrow state the opening of the _trans_-ring has a diameter of around 45 Å (Fig. 2f),

similar to the EL–ADP7–ES1 crystal structure (PDB 1AON31) (Extended Data Fig. 4e). By contrast, the wide conformation shows a significant reorientation of the apical domains, extending the

ring opening to around 65 Å (Fig. 2f), which would facilitate the exit of larger SPs such as folded MetK (approximately 70 × 60 × 30 Å3 in size). Consistent with this interpretation, a

similar conformation was observed in a cryo-electron microscopy (cryo-EM) structure of EL–ES1 with bound ADP (PDB 7PBJ32) (Extended Data Fig. 4f). Under all conditions analysed, the wide

_trans_-ring conformation was more abundant than the narrow state, especially following HS (Extended Data Fig. 4g). The in situ structure of EL–ES2 at the given resolution showed no major

deviations from the crystal structure of the non-cycling symmetrical complex with bound ADP–BeFx (PDB 4PKO33) (Extended Data Fig. 4h). Interestingly, in the in situ structure of GroEL alone

at a resolution of about 9.8 Å (Extended Data Fig. 4d)—attained following GroEL overexpression (EL+)—one ring mirrored the wide _trans_-ring conformation of EL–ES1 whereas the other was in a

more narrow state (Fig. 2d) with continuous additional density at the apical domains (Extended Data Fig. 4i,j). This density probably resulted from symmetry-averaged, unfolded SP that had

accumulated on GroEL at substoichiometric GroES. Thus the GroEL complex shows intrinsic inter-ring asymmetry in vivo, reflecting the negative allosteric coupling between rings and leading to

preferential substrate binding to one ring. VISUALIZATION OF SUBSTRATE IN THE GROEL–GROES CYCLE Similar to GroEL alone, the _trans_-ring of EL–ES1 in the narrow state also contained central

density at the apical domains (indicated by arrowheads in Fig. 3a), presumably representing bound SP before encapsulation by GroES. No SP density was observed in the wide _trans_-ring, nor

was a narrow state without bound SP resolved (Fig. 3a). Indeed, the apical domains in the narrow state expose the functionally critical hydrophobic residues in helices αI and αH, forming a

continuous furrow for SP binding34, whereas in the wide state the coherent binding surface was disrupted (Fig. 2f). Thus, following GroES dissociation, the _trans_-ring in its wide

conformation would allow SP release whereas binding of new SP presumably occurs following conversion to the narrow conformation. Interestingly, the ratio of EL–ES1 with wide _trans_-ring to

EL–ES1 with narrow _trans_-ring (Extended Data Fig. 4g) correlated closely with the overall ratio of EL–ES1 to EL–ES2 (Fig. 1c). This suggests that binding of SP to the _trans_-ring may

facilitate the formation of symmetrical complexes by lowering negative inter-ring allostery14. Furthermore, because EL–ES1 species with SP bound in _trans_ are populated, association of the

second GroES must be a relatively slow step. Next, for visualization of encapsulated SP we extracted and pooled GroEL–GroES chambers from all EL–ES1 and EL–ES2 complexes and analysed them by

averaging and three-dimensional classification of the chamber interior (Extended Data Fig. 4k). For each growth condition we identified two distinct classes of complex (Fig. 3b): the

GroEL–GroES chambers of class I contained a well-defined globular density close to the bottom of the cavity, consistent with structured SP. The chambers of class II showed only a weak and

fuzzy density, representing empty cavities and/or the presence of dynamic, non-native SP conformations that would be obscured by averaging. Sorting the EL–ES1 and EL–ES2 complexes in the in

situ datasets according to the presence of encapsulated and/or bound SP allowed us to quantify a total of seven different states of EL–ES1 and EL–ES2 (Fig. 3c). At 37 °C growth, the relative

proportions of these species were largely independent of MetK overexpression, with a subset of EL–ES2 complexes containing structured SP in both chambers. Interestingly, following HS,

EL–ES1 complexes with wide _trans_-ring conformation (no bound SP) were enriched (Fig. 3c(i,ii)) and EL–ES2 complexes reduced (Fig. 3c(v–vii)), perhaps due to changes in the ATP:ADP ratio

during HS35. This is consistent with SP binding to the _trans_-ring facilitating EL–ES2 formation. These results define the chaperonin species that are populated in vivo and demonstrate that

complexes EL–ES1 and EL–ES2 are both functionally active. STRUCTURE OF METK INSIDE CHAPERONIN To what extent does SP fold inside the chaperonin chamber during the functional GroEL–GroES

cycle in vivo? Previous in vitro cryo-EM analyses of encapsulated client protein under non-cycling conditions had shown a distinct density in the equatorial half of the chamber, representing

SP folding intermediates at low resolution36,37,38,39. We performed a similar in vitro analysis on encapsulated MetK, by both cryo-EM and cryo-ET, for comparison with the in situ cryo-ET

structures. We prepared SP-bound GroEL by heat denaturation of MetK in the presence of GroEL40. Encapsulation occurred following the addition of GroES and ATP-BeFx (Extended Data Fig. 5a,b).

BeFx favours the formation of stable (non-cycling) EL–ES2 complexes with bound ADP–BeFx (ref. 41). MS analysis indicated a stoichiometry of MetK to GroEL 14-mer of roughly 1.2 (Extended

Data Fig. 5c), similar to MetK overexpression (Extended Data Fig. 3f,g). Reference-free, two-dimensional classification demonstrated the presence of EL–ES2 as well as some EL–ES1 complexes

(Extended Data Figs. 5d and 6a–d). The latter exhibited subpopulations with wide and narrow _trans_-ring conformations resembling those observed in situ (Extended Data Fig. 6e,f), with

density for bound SP in the narrow state (Extended Data Fig. 6f). For visualization of encapsulated SP, GroEL–GroES chambers were processed for cryo-EM structure determination (Extended Data

Figs. 5d and 6c,d). Alignment and classification showed that around 40% of GroEL–GroES units contained density for an ordered MetK molecule close to the equatorial region of the chamber

(Fig. 4a–d, Extended Data Fig. 7 and Extended Data Table 1). The remainder contained only a faint, smeared-out density, representing empty chambers and chambers with incompletely folded or

misaligned MetK. The substructure of the ordered MetK molecules was solved at a resolution of approximately 3.7 Å, showing side-chain density in its hydrophobic core (Extended Data Fig.

7d–f). The encapsulated MetK was native-like, with a root mean squared deviation relative to the crystal structure (PDB 7LOO42) of 1.4 Å for 366 of the 379 Cα atoms (Fig. 4e). The main

difference was in the conformation of residues 97–111, the so-called core loop. This region packs against bound _S_-adenosylmethionine and an adjacent subunit in the MetK tetramer42,43

(Extended Data Fig. 8a), but in the encapsulated MetK subunit adopted a more extended conformation that was not well resolved (Fig. 4e). The core loop apparently remains unstructured until

tetramer assembly following release from chaperonin. The encapsulated MetK makes multiple contacts with the GroEL cavity wall, contacting two subunits at Phe44 in the equatorial GroEL domain

as well as five subunits at Phe281 and three at Tyr360, both protruding from the apical GroEL domains (Fig. 4b–d). These residues appear to interact with MetK via van der Waals contacts.

However, the side chains of the interacting residues are poorly defined, indicating heterogeneity in these regions of the structure (Fig. 4f). The GroEL subunits contacting MetK show only

minor conformational rearrangements, with root mean squared deviation values of 0.5–1.0 Å compared with a new 2.5 Å cryo-EM structure of empty GroEL–(ADP–BeFx)7–ES chambers (Extended Data

Fig. 8b–g and Extended Data Table 1). Of note, the GroEL cavity wall does not contact the interface regions of the MetK subunit that become buried following assembly. These regions

apparently remain solvent exposed in the chamber (Extended Data Fig. 8a) but could be reached by flexible C-terminal Gly–Gly–Met repeat sequences (23 residues) of the GroEL subunits not

resolved in the cryo-EM structure. To further rationalize our in situ cryo-ET analysis of encapsulated SP (Fig. 3b), we next performed cryo-ET on isolated GroEL–GroES–MetK complexes using

the same imaging parameters as for in situ tomography (Extended Data Table 1). In agreement with the single-particle data, the classification of chambers within these complexes again yielded

two classes. Class I (around 40% of particles) contained a strong density in the chaperonin cavity, corresponding well with symmetry-averaged folded MetK (Fig. 4g, left), whereas class II

chambers (roughly 60% of particles) showed a weak, diffuse density (Fig. 4h, left). The location of the structured MetK near the equatorial region of the GroEL–GroES chamber and its density

relative to the GroEL wall (Fig. 4g, left) coincided with that of the folded SP in situ (Fig. 4g, right). Specifically, the position of the SP centre of mass following MetK overexpression,

in which MetK is highly enriched on GroEL (Extended Data Fig. 3f,g), was in almost perfect agreement with the position of the folded MetK in the in vitro tomograms (Extended Data Fig. 9).

Although other SPs besides MetK may be present within GroEL in situ, our data suggest that these proteins occupy a similar location within the chamber. Thus, encapsulation in vivo resulted

in SP folding to a native or native-like, compact state. CONCLUSIONS Our analysis of GroEL–GroES complexes in situ using cryo-ET allows us to define the intermediate steps of the bacterial

chaperonin cycle in vivo. We find that both asymmetric and symmetric chaperonin complexes operate in linked subreactions (Fig. 5). GroEL without bound GroES is below detectability and may

exist only transiently (Fig. 5(i)). By contrast, asymmetric EL–ES1 with and without bound SP on the _trans_-ring is abundant, defining the main SP acceptor state (Fig. 5(ii)). In the

asymmetric reaction the GroEL rings alternate between folding active and binding active. Following GroES dissociation, SP exits the folding chamber (Fig. 5(iii–i)), facilitated by a wide

conformation of the apical GroEL domains, possibly generating a short-lived GroEL-only intermediate (Fig. 5(i)). Alternatively, rather than completing the asymmetric cycle, GroES binding to

the _trans_-ring gives rise to EL–ES2 (Fig. 5(iii–iv)), in which both rings can be folding active. Because folding begins in the _cis_-chamber of EL–ES1 and can continue in the EL–ES2

complex, the symmetric cycle may benefit SPs with slow folding kinetics13. How is the partitioning between asymmetric and symmetric chaperonin reactions regulated? In the canonical

asymmetric cycle in vitro the GroEL rings are coupled by negative allostery, with ATP binding to the _trans_-ring causing ADP and GroES release from the _cis_-ring (Fig. 5 (species

ii/iii–i))1,44,45. Negative inter-ring allostery also operates in vivo, favouring EL–ES1 formation, because exclusively EL–ES1 complexes mediate protein folding at GroEL excess over GroES.

In WT cells, EL–ES2 complexes are also functional. Conversion of the EL–ES1 _trans_-ring from a wide conformation to the narrow, SP-binding state (Fig. 5(ii–iii)) appears to be limiting for

EL–ES2 formation (Fig. 5(iii–iv)), because the ratio of EL–ES1 wide to EL–ES1 narrow correlates closely with the overall EL–ES1 to EL–ES2 ratio. Our cryo-ET analysis also demonstrated that,

before release into bulk cytosol, SP reaches a folded state in the GroEL–GroES chamber. To validate this finding we solved as a reference the structure of stably encapsulated MetK, an

obligate GroEL substrate26, in vitro. The MetK subunit is natively folded and is located close to the equatorial region of the GroEL–GroES cavity36,38,39. Encapsulated MetK makes weak

contacts with specific GroEL residues (Fig. 4) and is in close proximity to flexible C-terminal GGM repeat sequences of the equatorial GroEL domains, which may promote efficient

folding46,47. The position and density of folded, encapsulated MetK closely resemble those of structured SP in the GroEL–GroES chamber in situ. In summary, our analysis provides a detailed

view of the chaperonin reaction cycle in vivo, in which asymmetric and symmetric GroEL–GroES complexes are functionally linked. SP accumulates inside the chaperonin chamber in a folded state

before release into cytosol. METHODS 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 8,000_g_ (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 16,000_g_ (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, 16,000_g_ 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 8,000_g_ 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 (_C_7 symmetry) and 11.9 Å for the EL–ES2 complex (_D_7 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 _C_2 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 _C_7

symmetry mismatch of the substrate and enclosing chamber, final averages were produced for all different biological conditions with _C_7 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 _C_2 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 _C_7 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. DATA AVAILABILITY

The mass spectrometry data have been deposited to the ProteomeXchange Consortium83 via the PRIDE partner repository with the dataset identifier PXD042587. Model coordinates and electron

density maps have been deposited to the wwPDB database under PDB/EMDB accession code nos. 8P4M/EMD-17418 (empty GroEL–GroES chamber), 8P4N/EMD-17420 (GroEL–GroES chamber with no or

disordered MetK), 8P4O/EMD-17421/EMD-17422 (GroEL–GroES chamber with ordered MetK), 8QXS/EMD-18735 (EL–ES1–MetK wide), 8QXT/EMD-18736 (EL–ES1–MetK narrow), 8P4R/EMD-17426 (in situ EL–ES2),

8QXU/EMD-18737 (in situ EL–ES1 wide), 8QXV/EMD-18738 (in situ EL–ES1 narrow) and 8P4P/EMD-17425 (in situ EL). Primary electron density maps have been deposited to the wwPDB database under

EMDB accession code nos. EMD-17423 (in vitro GroEL–GroES chamber with no or disordered MetK), EMD-17424 (in vitro GroEL–GroES chamber with ordered MetK), EMD-17534 (empty EL–ES2), EMD-17535

(empty EL–ES1), EMD-17559 (GroEL–GroES chamber with no or disordered substrate), EMD-17560 (GroEL–GroES chamber with encapsulated, ordered substrate), EMD-17561 (70S ribosomes in 37 °C, HS

and MetK _E. coli_ cells), EMD-17562 (70S ribosomes in EL+ _E. coli_ cells), EMD-17563 (EL–ES1 with encapsulated ordered MetK), EMD-17564 (EL–ES1 with no or encapsulated disordered MetK),

EMD-17565 (EL–ES2 with two chambers with no or disordered MetK), EMD-17566 (EL–ES2 with ordered MetK in one chamber and no or disordered MetK substrate in the other) and

EMD-17567/EMD-17568/EMD-17569/EMD-17570/EMD-17571/EMD-17572/EMD-17573 (conformers 1–7 of EL–ES2 with two encapsulated, ordered MetK). Because of their large file size, original cryo-ET

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Google Scholar  Download references ACKNOWLEDGEMENTS We thank S. Gärtner, R. Lange and N. Wischnewski for expert technical assistance. We thank L. Zhang and C. Sitron for help in developing

the immunoprecipitation protocol and improving the text, respectively. This study used the infrastructure of the Department of Cell and Virus Structure at the MPI of Biochemistry. We thank

J. Plitzko for valuable technical advice. Funding was provided by the German Research Foundation (Deutsche Forschungsgemeinschaft) under Germany’s Excellence Strategy (EXC 2067/1-390729940)

and SFB 1035, as well as the European Research Council (ERC Advanced Grant no. 101052783-INSITUFOLD) and the Ministry of Science and Culture of the State of Lower Saxony (74ZN1949). FUNDING

Open access funding provided by Max Planck Society. AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Department of Cellular Biochemistry, Max Planck Institute of Biochemistry, Martinsried,

Germany Jonathan Wagner, Alonso I. Carvajal, Andreas Bracher, Roman Körner & F. Ulrich Hartl * Research Group Molecular Structural Biology, Max Planck Institute of Biochemistry,

Martinsried, Germany Jonathan Wagner & Wolfgang Baumeister * Institute of Neuropathology, University Medical Center Göttingen, Göttingen, Germany Jonathan Wagner & Ruben

Fernandez-Busnadiego * Cluster of Excellence “Multiscale Bioimaging: from Molecular Machines to Networks of Excitable Cells” (MBExC), University of Göttingen, Göttingen, Germany Jonathan

Wagner & Ruben Fernandez-Busnadiego * Research Group CryoEM Technology, Max Planck Institute of Biochemistry, Martinsried, Germany Florian Beck & Stefan Bohn * Vanderbilt University

Center for Structural Biology, Nashville, TN, USA William Wan * Institute of Structural Biology, Helmholtz Center Munich, Oberschleissheim, Germany Stefan Bohn * Faculty of Physics,

University of Göttingen, Göttingen, Germany Ruben Fernandez-Busnadiego Authors * Jonathan Wagner View author publications You can also search for this author inPubMed Google Scholar * Alonso

I. Carvajal View author publications You can also search for this author inPubMed Google Scholar * Andreas Bracher View author publications You can also search for this author inPubMed 

Google Scholar * Florian Beck View author publications You can also search for this author inPubMed Google Scholar * William Wan View author publications You can also search for this author

inPubMed Google Scholar * Stefan Bohn View author publications You can also search for this author inPubMed Google Scholar * Roman Körner View author publications You can also search for

this author inPubMed Google Scholar * Wolfgang Baumeister View author publications You can also search for this author inPubMed Google Scholar * Ruben Fernandez-Busnadiego View author

publications You can also search for this author inPubMed Google Scholar * F. Ulrich Hartl View author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS

J.W. performed cryo-ET and cryo-EM single-particle analyses with help from W.W., F.B. and S.B. A.I.C. performed biochemical experiments and MS analysis together with J.W. and R.K. A.B. built

the structural models and helped with data interpretation. F.U.H., W.B. and R.F.-B. designed the project and wrote the manuscript together with the other coauthors. CORRESPONDING AUTHORS

Correspondence to Wolfgang Baumeister, Ruben Fernandez-Busnadiego or F. Ulrich Hartl. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing interests. PEER REVIEW PEER

REVIEW INFORMATION _Nature_ thanks Han Remaut and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available. ADDITIONAL

INFORMATION PUBLISHER’S NOTE Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. EXTENDED DATA FIGURES AND TABLES EXTENDED

DATA FIG. 1 CRYO-ET AND SUBTOMOGRAM AVERAGING. (A) Sample preparation for cryo-ET. _E. coli_ cells were vitrified, thinned by cryo focussed ion beam (FIB) milling and tomograms aquired in a

cryo-transmission electron microscope (TEM). Representative scanning electron micrograph of a sample before and after FIB milling is shown along with an overview of a lamella from a cryo-TEM

(a total of 166 tomograms were acquired for 37 °C, HS and MetK combined). (B) Processing flowchart used for EL–ES1 and EL–ES2 subtomogram averaging in situ. The color of the box indicates

whether the respective step was performed in STOPGAP (blue) or with the indicated program (white). See Methods for details. EXTENDED DATA FIG. 2 IN SITU STRUCTURAL ANALYSIS OF 70 S

RIBOSOMES. (A-B) Subtomogram averaging of ribosomes. Ribosomes from three datasets (37 °C, HS, MetK) were averaged and refined to a global resolution of 8.7 Å. The resulting subtomogram

structure with the superposed molecular model (PDB code 4V4A84) in ribbon representation (a) and the corresponding FSC curve (b) are shown. (C) Analysis of ribosome to GroEL 14-mer ratio in

tomograms (box plots; 37 °C n = 48, HS n = 58, MetK n = 60 tomograms) and by MS using intensity-based absolute quantification (iBAQ) (blue crosses; n = 3 independent experiments). Box plots

show median (center line), interquartile range (IQR) (box edges) and 1.5 × IQR (whiskers). The MS measurements fall mainly within the range of the first to third quartile of the tomography

data, indicating that most EL complexes were identified in situ. Source data EXTENDED DATA FIG. 3 BIOCHEMICAL ANALYSIS OF GROEL/GROES LEVELS AND METK BINDING. (A) Representative immunoblot

of GroEL and GroES for the different growth conditions analyzed (37 °C, HS, MetK and EL+). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as loading control. (B) Quantification of

GroEL and GroES levels by label-free mass spectrometry of cell lysates. The total amount of GroEL was quantified by label-free mass spectrometry using iBAQ. iBAQ values of GroEL and GroES

of cells grown at 37 °C cells were set to 1 and used for normalization (n = 3 independent experiments). The horizontal line in the boxplots indicates the median value; boxes indicate upper

and lower quartile and whisker caps the largest or smallest value within 1.5 times the interquartile range above the 75th percentile or below the 25th percentile, respectively. (C) Ratio of

GroEL 14-mer and GroES 7-mer, based on the iBAQ values from (b). The GroEL:GroES ratio in wild-type cells at 37 °C was set to 1 and used for normalization. The differences between the groups

were not statistically significant when compared with a 1-way ANOVA test. (D) Growth of _E. coli_ BL21(DE3) at 37 °C, upon exposure to HS at 46 °C or upon ~4,5-fold overexpression of GroEL

(EL+) at 37 °C. Data points are averages ± SD (n = 3, independent repeats). Growth curves were standardized to start at a log2(OD600) value of 0. (E) Growth of transformed _E. coli_

BL21(DE3) at 37 °C, upon sequential overexpression of GroEL/GroES and MetK (MetK cells) or upon ~4,5-fold overexpression of GroEL with subsequent overexpression of MetK (EL+/MetK) at 37 °C

(see Methods). For comparison, the growth of the latter strain without induction (n.i.) of MetK (EL+/MetK(n.i.)) is shown. Data points are averages ± SD (n = 3, independent repeats). Growth

curves were standardized to start at a log2(OD600) value of 0. (F) Quantification of MetK bound to GroEL complexes in 37 °C and MetK overexpressing cells. Apyrase treatment was performed

upon cell lysis to stop GroEL cycling. GroEL was immunoprecipitated (IP), followed by immunoblotting with antibodies against GroEL and MetK. Anti-lactalbumin antibodies were used as

non-specific control. (G) Quantification of MetK:GroEL stoichiometry by MS in GroEL IPs from (d). The fraction of MetK per GroEL 14-mer was calculated based on iBAQ values (n = 3 independent

experiments). Box plots show median (center line), interquartile range (IQR) (box edges) and 1.5 × IQR (whiskers). (H) Cellular abundance of GroEL in 37 °C and EL+ cells. The data was

normalized to a median of 1 for 37 °C (n = 3 independent experiments). Boxplots are defined as in (g). (I) Cellular abundance of EL–ES1 in 37 °C and EL+ cells. The abundance of EL–ES1

relative to ribosomes in tomograms was calculated as a proxy for its cytosolic concentration and normalized to a median of 1 for 37°C. Source data EXTENDED DATA FIG. 4 IN SITU STRUCTURAL

ANALYSIS OF GROEL COMPLEXES. (A-D) FSC curves for EL–ES1 narrow (a), EL–ES1 wide (b) and EL–ES2 complexes (c), as well as EL (d) from Fig. 2a–d. The resolution at the 0.143 FSC cut-off is

indicated. Note that free GroEL (EL) was only observed upon GroEL overexpression (EL+) and thus this structure was obtained from a separate data set. (E) Comparison of GroEL subunits in the

_trans_-ring of the in situ structure of EL–ES1 narrow (dark blue) and the crystal structure of GroEL·ADP7–GroES7 (yellow) (PDB 1AON31). Two orthogonal views of the superimposed models are

shown. The models are depicted in ribbon representation. (F) Comparison of GroEL subunits in the _trans_-ring of the in situ structure of EL–ES1 wide (light blue) and the cryoEM structure of

EL–ES1 in complex with 14 ADP molecules (orange) (PDB 7PBJ32), using the same representation as in (e). (G) Distribution of narrow and wide EL–ES1 complexes at 37 °C, upon overexpression of

GroEL, GroES and MetK at 37 °C (MetK cells) or upon exposure to HS at 46 °C (37 °C, n = 48; MetK, n = 60; HS, n = 58 tomograms). (H) Comparison of GroEL–GroES units of the in situ structure

of EL–ES2 (orange) and the crystal structure of EL–ES2 in complex with 14 ADP·BeFx ligands (teal) (PDB 4PKO33). The models are depicted in ribbon representation. (I) Overlay of the rings in

the wide conformation in the in situ structures of EL–ES1 (teal) and the EL complex (yellow). The models are depicted in ribbon representation. (J) Cross section through the EL complex

density. Additional density not accounted for by the molecular model is present in the more narrow ring at the SP binding sites, as shown in side and top view. There is no additional density

at the given contour level in the opposing ring in the wide conformation. (K) Processing workflow of tomograms for analysis of encapsulated SP. To discern the SP states in GroEL–GroES

chambers of EL–ES1 and EL–ES2 complexes, isolated chambers were aligned. After denoising the resulting subtomograms, initial structures for subsequent 3D classification were produced by

bootstrapping and k means clustering. The resulting averages were used as starting structures for 3D classification (see Methods). Source data EXTENDED DATA FIG. 5 PREPARATION AND CRYO-EM

ANALYSIS OF THE GROEL–GROES–METK COMPLEX. (A-B) Size exclusion chromatography of the stable GroEL–GroES–MetK complex prepared in the presence of ATP·BeFx (see Material and Methods). A

representative chromatogram is shown in (a). Eluate fractions F1−F5 were analyzed by SDS-PAGE and Coomassie staining (b). Purified GroEL, MetK and GroES were analyzed for comparison.

Fractions F1 and F2 contain the GroEL:ES-encapsulated MetK. (C) The complex was analyzed by MS and the ratio of MetK to the GroEL 14-mer calculated based on iBAQ values (n = 2 independent

samples, each 3 technical repeats). Box plots show median (center line), interquartile range (IQR) (box edges) and 1.5 × IQR (whiskers). (D) Data processing workflow for single particle

analysis of GroEL–GroES–MetK complexes. A flow diagram is shown. The color of the box borders indicate that the respective step was performed in CryoSPARC (green), RELION 3.1.3 (blue) or

with the indicated program (black). After data collection, particle picking and initial 2D classification (I), EL–ES1 and EL–ES2 complexes were processed separately (II). GroEL–GroES

chambers were extracted and combined for further processing. Subsequently, the GroEL–GroES density was subtracted and the chamber interiors separated by 3D classification without alignment

(III). The picture row shows central slices of the resulting 3D class averages, which were used to separate folded MetK from disordered MetK or empty chambers (IV). Red arrows mark the MetK

densities differing by 2π/7 rotation in the GroEL–GroES chambers. The other 3D class averages had no visible secondary structure elements. The GroEL–GroES–MetK chambers containing folded

MetK were aligned and refined to a resolution of 3.0 Å. Local refinement of MetK after signal subtraction resulted in a 3.7 Å resolution map. The final resolution for GroEL–GroES chambers

with disordered MetK or empty chambers was 2.9 Å. See Methods for details. Source data EXTENDED DATA FIG. 6 SINGLE PARTICLE ANALYSIS OF GROEL–GROES–METK COMPLEXES. (A) A representative

micrograph of the GroEL–GroES–MetK sample at a magnification of 22,500-fold. (8,945 micrographs were used after on-the-fly preselection) (B) Corresponding 2D classes of particles selected

for further refinement. (C, D) Surface representation of densities for MetK-containing EL–ES2 (c) and EL–ES1 (narrow conformation) complexes (d). The red boxes indicate the GroEL–GroES

chambers that were processed further to solve the GroEL–GroES–MetK complex structure. (E, F) Bottom views of the densities and molecular models for the apical domains of the _trans_-ring in

EL–ES1 complexes with wide (e) and narrow (f) conformation. Surface views of the density are shown. The models are depicted in ribbon representation, with the substrate binding helices αH

and αI highlighted in orange and yellow, respectively. Additional density in the narrow _trans_-ring not accounted for by the model – presumably from the substrate MetK – is high-lighted in

teal (f). The insert shows one apical domain of the narrow _trans_-ring in detail. EXTENDED DATA FIG. 7 RESOLUTION AND DENSITY FIT ANALYSIS OF GROEL–GROES CHAMBERS AND METK. (A-D) FSC curves

(top) and local resolution maps (bottom) of the empty GroEL–GroES chamber (a), the GroEL–GroES chamber with disordered MetK or without substrate (b), the GroEL–GroES chamber with ordered

MetK (c) and isolated MetK (d), respectively. The rainbow color gradient indicates the local resolution scale. (E, F) Cryo-EM density of MetK with superposed molecular model in ribbon

representation. Two views for the entire protein are shown (e). Exemplary portions of the structure are shown below with side chains in stick representation (f). The respective residue

ranges are indicated. (G) Angular sampling of MetK. Planar and spherical representation of the Fourier sampling of MetK depicted in (d). The color gradient from dark blue to pale yellow

corresponds to the number of images in each bin. The sampling compensation factor was calculated to be 0.987 with values over 0.81, generally indicating adequate sampling85,86. Image was

generated using the CryoSPARC Orientation Diagnostics job76. EXTENDED DATA FIG. 8 COMPARISON OF ISOLATED TETRAMERIC METK WITH GROEL–GROES-ENCAPSULATED METK AND THE EFFECT OF METK

ENCAPSULATION ON GROEL–GROES CHAMBERS. (A) Overview of the crystal structure of the MetK tetramer (left; PDB 7LOO42). One subunit is shown in ribbon representation in gold, the other three

as molecular surfaces in violet, blue and yellow, respectively. The insert highlights the location of the core loop, which is marked by a red asterisk. Bound ligands pyrophosphate and

S-adenosylmethionine are shown as stick models in green. Cut-away view of GroEL–GroES encapsulated, folded MetK in the GroEL–GroES chamber in the same orientation (right). MetK is shown in

ribbon representation (teal). The GroEL and GroES subunits are shown as molecular surfaces. The core loop of MetK is indicated by a red asterisk, and the last resolved residue Pro525 in the

GroEL subunits is indicated with the letter C. The disordered C-terminal GGM repeats, GroEL residues 536−548, could easily reach the exposed MetK interface regions. (B-D) Overlay of the

_C_7-symmetric model of the empty GroEL–GroES chamber (green) with the chamber containing either disordered MetK or no substrate (blue) at the level of the equatorial GroEL domains (b), the

intermediate domains and the hinge regions between equatorial and intermediate domains (c and d). (E-G) Overlay of the _C_7-symmetric model of the empty GroEL–GroES chamber (green) with the

chamber containing folded MetK (purple) at the level of the equatorial GroEL domains (e), the intermediate domains and the hinge regions between equatorial and intermediate domains (f and

g). EXTENDED DATA FIG. 9 ANALYSIS OF THE SP DENSITY IN GROEL–GROES CHAMBER SUBTOMOGRAMS. Central xy slices of the subtomogram averages of in vitro assembled GroEL–GroES chambers with ordered

MetK (left) and class I in situ chambers (containing structured substrate; see Fig. 3b) from cells overexpressing GroEL/GroES and MetK (right), with gray values normalized to 2 standard

deviations. The center of mass for the density within a spherical volume in the chamber indicated by a circle is depicted by a red asterisk. Within error, the centers of mass were identical

(x, y, z in voxels: 65, 65, 51). SUPPLEMENTARY INFORMATION SUPPLEMENTARY FIG. 1 Classification of GroEL–GroES complexes following template matching. REPORTING SUMMARY SUPPLEMENTARY DATA

Zipped folder containing Uniprot_Ecoli_20230301.fasta file and UniProt sequences used for mass spectrometry analysis. SUPPLEMENTARY DATA Validation reports for wwPDB and emDB deposition.

PEER REVIEW FILE SOURCE DATA SOURCE DATA FIG. 1 SOURCE DATA FIG. 3 SOURCE DATA EXTENDED DATA FIG. 2 SOURCE DATA EXTENDED DATA FIG. 3 SOURCE DATA EXTENDED DATA FIG. 4 SOURCE DATA EXTENDED

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ARTICLE Wagner, J., Carvajal, A.I., Bracher, A. _et al._ Visualizing chaperonin function in situ by cryo-electron tomography. _Nature_ 633, 459–464 (2024).

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