Since MFS transporters typically lack additional domains outside their transport unit, which is a major impediment for accurate particle alignment in single particle cryo-EM approaches, we assessed three fiducial marker strategies introducing additional density outside of detergent micelles containing DtpC, by analyzing the quality of 2D class averages (Figure 1). To obtain conformation specific binders against DtpC, we first immunized llamas with recombinant DtpC and selected nanobodies (Nbs) following standard procedures (Pardon et al., 2014). Three out of five selected binders (Nb17, Nb26, and Nb38) co-eluted with DtpC on gel filtration (Supplementary Figure S1) and increased the melting temperature of the respective DtpC-Nb complex by 20°C, 16°C, and 12°C. (Figures 2A,B). DtpC in complex with Nb17 and Nb26 yielded crystals in various conditions, but despite extensive optimization efforts, the crystals of the DtpC-Nb26 complex did not diffract X-rays better than 5 Å resolution. In a second step, we decided to increase the size of Nb26, which formed a tight complex with DtpC, by fusing one copy of the maltose binding protein (MBP) to its C-terminus as described previously (Botte et al., 2022). This resulted in a 52 kDa Pro-macrobody (short Mb26), and we expected it to bind to the periplasmic side of the transporter as seen in other MFS transporter-Nb complexes (Figure 1). In a third approach, we fused the two self-assembling parts of split-sfGFP; with β1-6 on the N-terminus of DtpC, and β7-11 on the C-terminus. We named this construct split sfGFP-DtpC_FL. In order to minimize the mobility between the membrane protein and the split sfGFP fiducial, we also generated two additional constructs where the last five (split sfGFP-DtpC_1-475), or ten residues (split sfGFP-DtpC_1-470) of the transporter were deleted. We then assessed proper folding and complementation by monitoring the fluorescence of the chromophore on an HPLC system (Figure 2C). All constructs eluted at similar retention times and the fluorescence was highest in the non-truncated construct (split sfGFP-DtpC_FL) and lowest in the most truncated version (split sfGFP-DtpC_1-470). In order to extend this observation to other MFS transporters, we repeated this experiment with the human POT homologue PepT1, and noticed a similar trend upon shortening of the termini. Yet, since the decrease of fluorescence was only minor in split sfGFP-DtpC_1-475 in comparison to split sfGFP-DtpC_FL, we proceeded to imaging with the shorter construct in the presence of Nb26.

The particle density and distribution in the vitrified solution was similar in the three imaged samples. However, DtpC-Nb26 produced the best 2D class averages considering the sharpness of secondary structure elements inside the micelle, as judged by visual inspection (Figure 1, Supplementary Figure S2). The Pro-macrobody Mb26 fiducial was clearly visible in 2D class averages, but it adopted various positions in relation to the transporter, therefore making accurate alignment of the particles more difficult than in its shorter but more rigid and stable nanobody version (Figure 1, Figure 3A, B, Supplementary Figure S2). The split sfGFP-DtpC_(1–475)-Nb26 sample allowed clear visualization of the transmembrane helices after clustering a small subset of particles, but the majority of particles clustered in classes with blurry density for the split sfGFP fiducial, or with the two complementary parts β1-6 and β7-11 not assembled (Supplementary Figure S2). AlphaFold2 predictions on the imaged construct, as well as on the full length construct later suggested a destabilization of the beta-barrel upon increasing termini restrains, resulting in partial unfolding of β7 and exposure of the chromophore to solvent quenching. Interestingly, this effect could partially be reverted by adding a linker of five glycine residues between the C-bundle and β7 based on in silico data. We conclude that termini restraining using the split-sfGFP approach is a promising fiducial strategy for structural studies of MFS transporters, in addition to the previous demonstrated showcases on small membrane proteins (2, 4 and 6TMs) (Liu et al., 2020; Liu et al., 2022). However, the amount of restraining in larger membrane proteins such as MFS transporters where both termini are placed far from each other need to be optimized experimentally or in silico, to produce a stable and rigid fiducial; two crucial aspects for high resolution structure determination of MFS transporters by single particle cryo-EM.

As we obtained the best 2D class averages for DtpC with the fiducial marker Nb26, we proceeded to a large data collection (Table 1) and could cluster a subset of dimers within this data set (Figure 3). The presence of different oligomeric species was already expected based on the peak shape of the gel filtration run (Figure 4A). The large mass of the dimer, and the stable and rigid signal of the Nb26 fiducial, allowed us to reconstruct the DtpC-Nb26 dimer to 3.0 Å resolution and model this assembly (Figures 3, 4, Supplementary Figure S3). The quaternary structure consists of a non-symmetrical inverted dimer mediated by interactions through a large hydrophobic interface between the HA-HB helices of DtpC (Supplementary Figure S3). Although other inverted dimers were reported in homologous POT structures (Quistgaard et al., 2017), the source of such arrangements is likely to be artificial. We also investigated the oligomer heterogeneity in solution with small angle X-ray scattering and obtained a good fit at low angles (corresponding to the overall shape of particles in solution) for the cryo-EM volume of the dimer (Supplementary Figure S4). The fit to a monomeric cryo-EM volume was poor, indicating that in detergent solution a significant fraction of DtpC-Nb26 is dimeric. As for the interaction between the membrane protein and the fiducial marker, the CDR3 loop of Nb26 accounts for the strongest interactions with the periplasmic surface of the transporter with two salt bridges, while CDR1 and CDR2 contribute via hydrogen bonding (Figure 5). 3D variability analysis (Punjani and Fleet, 2021) revealed a small degree of flexibility between the two DtpC-Nb26 copies. Therefore, we performed a local refinement, focused on one copy of the membrane protein, which extended the resolution of the reconstruction to 2.7 Å and improved the accuracy of the atomic model for subsequent structural analysis (Figure 4).

The DtpC structure revealed the expected and well-known MFS transporter fold, with twelve transmembrane helices (TMs) organized in two helical bundles and additional two TMs specific for the POT family (known as HA and HB domains). It is highly similar compared to the previously determined DtpD structure (Zhao et al., 2014) with an overall RMSD value of 1.06 Å between the two (for 335 out of 436 C_α-atoms). The peptide binding site of DtpC is exposed to the cytoplasmic side (Figures 3, 4). Almost all bacterial POT structures described so far were determined by X-ray methods in a similar inward facing (IF) conformation. The extent to which the central cavity is open to the cytosol is regulated by a mechanism of occlusion mediated by TM4, TM5, TM10, and TM11, as supported by structures in IF occluded, partially occluded, and open states. In the case of the here described DtpC structure, the IF state is open (Figure 3).

Molecules from the periplasmic side, on the contrary, cannot enter the central cavity. Tight closure of both bundles above the binding site is mediated by a salt bridge between D43 (TM2, N-bundle) and R294 (TM7, C-bundle) and hydrogen bonds between H37 (TM1, N-bundle) and D293 (TM7) as well as R28 (TM1) and N421 (TM11, C-bundle) (Figure 6A). We also analyzed previously determined POT structures with clearly resolved side chain densities, to understand how the IF state is generally maintained in this transporter family. Except for human PepT2 and the POT transporter from Shewanella oneidensis (PepT_So), where the inter-bundle periplasmic salt bridge is formed between TM5 and TM7, the IF state is in all other analyzed structures stabilized by a salt bridge on the tip of TM2 and TM7 (Figure 6B). Additional hydrogen bonding networks as described in other studies, can occur, but vary greatly among different homologues. This analysis highlights that the alternate access mechanism in canonical and in so called ‘atypical’ POTs share similarities such as electrostatic clamping by formation and disruption of salt bridges. The differences in hydrogen bonding patterns however, could account for the various turnover rates seen among POT homologues.

Canonical POTs are characterized by i) the presence of the E₁XXE₂R motif on TM1 involved in proton coupling and ligand binding, and ii) the ability to accommodate dipeptides, tripeptides, and peptidomimetics, which relies on a set of conserved residues located in the central binding cavity. In DtpC, the E₁XXE₂R motif, has evolved to Q₁XXE₂Y (where Q₁ = N17, E₂ = E20, Y=Y21). In all high resolution X-ray structures of canonical POTs, R is in salt-bridge distance to E₂ and the C-terminus of substrate peptides. Mutation of either E₁ or E₂ in the conventional E₁XXE₂R motif to glutamine residues abolishes uptake (Aduri et al., 2015). A reverse mutation in DtpC, from Q₁XXE₂Y to E₁XXE₂Y or to E₁XXQ₂Y preserves high transport rates, while a Q₁XXQ₂Y motif significantly decreases it (Aduri et al., 2015). In addition, based on previous molecular dynamics experiments, a salt bridge switching mechanism from R-E₂ to R-E₁, upon protonation of E₂ in the E₁XXE₂R motif, was proposed (Aduri et al., 2015). This biochemical and in silico data strongly support a dual role of the E₁XXE₂R motif for both proton and peptide transport, where R can form a salt bridge interaction with the C-terminus of peptides or with E₁ when E₂ is protonated, and where the deprotonation event of the latter is required to disrupt the R-peptide interaction.

In DtpC, we now observe that the side chain pocket has a different architecture and characteristic in comparison with the one of canonical POTs. It displays an overall more acidic groove caused by the presence of the aspartate residue 392, conserved among atypical POTs. This residue has been predicted to be involved in substrate coordination and mutation of this residues in DtpC and homologues DtpD (corresponding residue is D395) abolished transport activity (Jensen et al., 2012b; Zhao et al., 2014). Canonical POTs have a conserved serine residue instead, yielding a slightly changed hydrophobicity pattern in the binding site (Figures 7A–D). A structural overlay of DtpC with a canonical POT structure bound to the dipeptide Ala-Phe allows us to position the peptide in the binding site. By replacing the phenyl group with a lysine side chain (generating the known DtpC dipeptide substrate Ala-Lys instead of Ala-Phe), we postulate a putative salt bridge between the carboxyl group of D392 and the ε-amino group of the lysine side chain. This observation, together with previous biochemical work (Jensen et al., 2012b; Aduri et al., 2015) allows us to hypothesize that the selectivity of DtpC for dipeptides with C-terminal lysine or arginine residues is caused by swapping a salt bridge between the recurrent carboxyl group of the peptide terminus and the transporter (R21Y mutation), to a side chain specific salt bridge with D392. Since the R-peptide interaction is lost in DtpC, there is no requirement for E1 to destabilize R-peptide for release, which would explain the presence of a Q₁XXE₂Y motif instead of E₁XXE₂R.

In summary, our work provides new insights into promiscuous versus selective substrate recognition in POTs and constitutes a step forward towards completing the family of E. coli POTs structures. Lastly, it displays some of the challenges related to high resolution cryo-EM structure determination of MFS transporters devoid of soluble domains, and manifests once again, the benefit of fiducial markers in overcoming those.

The full-length cDNA of DtpC wild type (WT) was amplified from the Escherichia coli genome, and cloned into a pNIC-CTHF vector by ligation-independent cloning (LIC). This vector contains a C-terminal His-Tag and a Tobacco Etch virus (TEV) cleavage site and a kanamycin resistance gene as selectable marker. The first 6 N-terminal beta strands of sfGFP were fused to the N-terminus of DtpC, and the beta strands 7 to 11 fused to the C-terminus. We named this construct split sfGFP-DtpC_FL. Two additional constructs were cloned with truncations of 5 (split sfGFP-DtpC_1-475), and 10 residues (split sfGFP-DtpC_1-470), on the C-terminal side of DtpC.

HsPepT1 was previously cloned into a pXLG vector containing an expression cassette composed of an N-terminal Twin-Streptavidin tag followed by the HRV-3C protease recognition sequence (Killer et al., 2021). Similarly, as for DtpC, the two self-assembling parts of split-sfGFP were first inserted into the N- and C-termini of the full-length version of HsPepT1, and on two other versions with i) a C-terminal truncation of 36 residues (split sfGFP-HsPepT11-672), and ii) a C-terminal truncation of 36 residues and a N-terminal truncation of 10 residues (split sfGFP-HsPepT110-672) were cloned.

Recombinant DtpC, and the three split sfGFP-DtpC constructs were expressed in E. coli C41(DE3) cells grown in terrific broth (TB) media supplemented with 30 μg/ml kanamycin according to established procedures (Löw et al., 2012; Löw et al., 2013). Cultures were grown at 37°C and protein expression was induced with 0.2 mM IPTG at an OD_{600 nm} of 0.6–0.8. After induction, culture growth continued at 18°C for 16-18 h. Cells were harvested by centrifugation (10,000 × g, 15 min, 4°C), and the pellet was stored at -20°C until further use. Cell pellets were resuspended in lysis buffer (20 mM NaPi at pH 7.5, 300 mM NaCl, 5% (v/ v) glycerol, 15 mM imidazole, with 3 ml of lysis buffer per Gram of wet weight pellet), supplemented with lysozyme, DNase and 0.5 mM tris(2-carboxyethyl)phosphine (TCEP). The cells were lysed by three cycles using an Avestin Emulsiflex homogenizer at 10,000–15,000 psi. Recovered material was centrifuged to remove non-lysed cells (10,000 × g, 15 min, 4°C) and the supernatant was subjected to ultracentrifugation to separate the membrane fraction (100,000 × g, 1 h, 4°C using an Optima XE-90, Beckman Coulter centrifuge). Membranes were resuspended in lysis buffer supplemented with cOmplete EDTA-free protease inhibitors (Roche), and solubilized by adding 1% n-Dodecyl-β-D-Maltoside (DDM) detergent (Anatrace). The sample was centrifuged for 50 min at 90,000 × g, and the supernatant was applied to Ni-NTA beads for immobilized-metal affinity chromatography (IMAC) on a gravity column. The beads were pre-equilibrated in lysis buffer and incubated with the solubilized membrane proteins for one hour at 4°C on a rotating wheel. Loaded beads were washed with buffer with increasing imidazole concentrations (20 mM NaPi at pH 7.5, 300 mM NaCl, 5% glycerol, 15-30 mM imidazole, 0.5 mM TCEP, 0.03% DDM). The proteins were eluted from the column with a buffer containing high imidazole concentration (20 mM NaPi at pH 7.5, 150 mM NaCl, 5% glycerol, 250 mM imidazole, 0.5 mM TCEP, 0.03% DDM) and combined with 1 mg of TEV protease to perform the His-tag cleavage during dialysis overnight at 4°C. The dialysis buffer contained 20 mM HEPES at pH 7.5, 150 mM NaCl, 5% glycerol, 0.5 mM TCEP, 0.03% DDM. The cleaved protein was recovered by negative IMAC, concentrated to 4 ml using a 50 kDa concentrator (Corning^® Spin-X^® UF concentrators) and run on an ÄKTA Pure system (GE Healthcare Life Sciences), using a HiLoad 16/ 600 Superdex 200 column for DtpC, and a Superdex 200 Increase 10/300 column for the split sfGFP-DtpC constructs. Fractions containing the protein were pooled, concentrated, flash frozen and stored at -80°C until further use.

For the split sfGFP-HsPepT1 constructs, expression was done in mammalian cells as described previously (Pieprzyk et al., 2018; Killer et al., 2021). Briefly, HEK293F cells were collected 48 h after transient transfection, and stored at −80°C until further use. Frozen cell pellets were resuspended in 300 mM NaCl, 20 mM NaPi (pH 7.5), 0.5 mM TCEP, and 5% glycerol, supplemented with cOmplete EDTA-free protease inhibitors, and were disrupted using an Avestin Emulsiflex homogenizer at 10,000–15,000 psi. The lysate was centrifuged for 10 min at 10,000 × g, 4°C, and the supernatant was centrifuged for 90 min at 100,000 × g, 4°C. The pellet containing the membrane fraction was solubilized in 1% N-Dodecyl-β-D-Maltopyranoside (DDM) and 0.1% cholesteryl hemisuccinate (CHS; Tris Salt, Anatrace) for 1 h at 4°C. The sample was centrifuged for 50 min at 90,000 × g, and the supernatant was applied to Strep-TactinXT beads (IBA). After 20 min of incubation on a rotating wheel, the suspension was transferred to a gravity column. Following two wash steps with 300 mM NaCl, 20 mM HEPES (pH 7.5), 0.03% DDM, and 0.003% CHS, split sfGFP-HsPepT1comnstructs were eluted with 0.03% DDM, 0.003% CHS, 150 mM NaCl, 20 mM HEPES (pH 7.5), and 10 mM desthiobiotin (Sigma-Aldrich).

To generate DtpC specific nanobodies, two non-inbred llamas were injected six times at weekly intervals with a mixture of 94 different proteins including DtpC purified in the detergent DDM (50 µg of each antigen weekly). After 6 weeks of immunization, two separate phage display libraries were constructed, one from each animal, in the pMESy2 vector, which is a derivative of pMESy4 that contain a C-terminal EPEA-tag for affinity purification. After pooling both libraries, nanobodies were selected against individual antigens in two rounds of parallel panning in 96-well plates containing one immobilized antigen in each well. After two selection rounds on DtpC, 60 clones were picked for sequence analysis, 13 clones encoded antigen-specific nanobodies as tested in ELISA, grouping them in 5 different sequence families. A nanobody family is defined as a group of nanobodies with a high similarity in their CDR3 sequence (identical length and >80% sequence identity). Nanobodies from the same family derive from the same B-cell lineage and likely bind to the same epitope on the target. Immunizations, library construction, selection by panning and nanobody characterization were performed according to standard procedures (Pardon et al., 2014). Five nanobodies were further characterized.

The nanobodies were expressed in E. coli WK6 cells and purified following standard procedures. Specifically, the cell pellet was resuspended in TES buffer (0.2 M TRIS, pH 8, 0.5 mM EDTA, 0.5 M sucrose) supplemented with one protease inhibitor tablet (Roche). Osmotic shock was performed by the addition of diluted TES buffer to release the periplasmic proteins. The solution was first centrifuged for 20 min at 10,000 × g and additionally for 30 min at 100,000 × g. The supernatant was applied to CaptureSelect beads (Thermo Fisher Scientific), which were equilibrated with wash buffer (20 mM NaPi, pH 7.5, 20 mM NaCl). After three column volumes of washing, the nanobody was eluted with 20 mM HEPES, pH 7.5, 1.5 M MgCl₂. The nanobodies were further purified on a HiLoad 16/600 Superdex 75 pg column in 20 mM HEPES, pH 7.5, 150 mM NaCl, 5% glycerol, concentrated with a 5 kDa cut-off concentrator, flash-frozen and stored at -80°C until further use.

The nanobody 26 (Nb26) was first inserted into a pBXNPH3 vector containing a C-terminal penta-histidine tag preceded of a HRV-3C protease recognition sequence. The maltose binding protein (MBP) was then inserted in frame with the 3’ end of the nanobody, with two proline residues as a linker between the two genes similar as described in (Botte et al., 2021). The resulting Pro-macrobody 26 (Mb26) was expressed in E. coli WK6 cells as above. The cell pellet was resuspended in TES buffer (0.2 M TRIS, pH 8, 0.5 mM EDTA, 0.5 M sucrose) supplemented with one protease inhibitor tablet (Roche). Osmotic shock was performed by the addition of diluted TES buffer to release the periplasmic proteins. The solution was first centrifuged for 20 min at 10,000 × g and additionally for 30 min at 142,000 × g. The supernatant was further purified by immobilized-metal affinity chromatography (IMAC) on a gravity column. The beads were pre-equilibrated in 20 mM NaPi at pH 7.5, 300 mM NaCl, 5% glycerol, 15-30 mM imidazole, 0.5 mM TCEP and incubated. Loaded beads were washed with increasing imidazole concentrations (20 mM NaPi at pH 7.5, 300 mM NaCl, 5% glycerol, 15-30 mM imidazole, 0.5 mM TCEP, 0.03% DDM). The proteins were eluted from the column with a buffer containing high imidazole concentration (20 mM NaPi at pH 7.5, 150 mM NaCl, 5% glycerol, 250 mM imidazole, 0.5 mM TCEP, 0.03% DDM) and combined with 1 mg of 3C protease to perform the His-tag cleavage. The cleaved protein was recovered by negative IMAC, concentrated to 0.5 ml using a 30 kDa concentrator (Corning^® Spin-X^® UF concentrators) and run on an ÄKTA Pure system (GE Healthcare Life Sciences), using a Superdex 75 Increase 10/300 column. Fractions containing the protein were pooled, concentrated, flash frozen and stored at -80°C until further use.

The differential scanning fluorimetry method was used to follow the thermal unfolding event (Kotov et al., 2019) of Nb17, Nb26, Nb38, DtpC, DtpC-Nb17, DtpC-Nb26, DtpC-Nb38, Mb26, DtpC-Mb26, and split sfGFP-DtpC_1-475-Nb26 with a Prometheus NT.48 device (NanoTemper Technologies, Munich, Germany). The purified proteins were diluted to 16 µM, and the complexes were formed using a 1:1.5 M ratio of membrane protein: fiducial. The fluorescence at 330 and 350 nm was recorded over a temperature gradient scan from 15° to 95°C and processed in GraphPad Prism 9.0 (GraphPad Software).

Structures with the following sequences were used as input for AlphaFold2 structure prediction (Jumper et al., 2021), and AMBER relaxation. The best ranked models were used for visualization.

One hour before vitrification, the purified protein complexes were thawed on ice and run on a Superdex Increase 200 5/150 column in 0.015% DDM, 100 mM NaCl, 10 mM HEPES (pH 7.5), 0.5 mM TCEP in order to remove the excess of empty detergent micelles earlier generated upon sample concentration. The top fraction reached a concentration ranging between 3 and 6 mg/ml, and for each sample, 3.6 μl were applied to glow-discharged gold holey carbon 2/1 300-mesh grids (Quantifoil). Grids were blotted for 4 s at 0 force and 1-s wait time before being vitrified in liquid propane using a Mark IV Vitrobot (Thermo Fisher Scientific). The blotting chamber was maintained at 4°C and 100% humidity during freezing.

All movies were collected using a Titan Krios (Thermo Fisher Scientific) outfitted with a K3 camera and BioQuantum energy filter (Gatan) set to 10 eV. Automated data acquisitions were set using EPU (Thermo Fisher Scientific). The applied defocus ranged between -0.9 µm and -1.8 µm in all datasets.

For DtpC-Nb26 and DtpC-Mb26, movies were collected at a nominal magnification of ×105,000 and a physical pixel size of 0.85 Å, with a 70-μm C2 aperture and 100-μm objective aperture at a dose rate of 19.5 e−/pixel per second. A total dose of 75 e−/Ų was used with 2.8 s exposure time, fractionated in 50 frames. For split sfGFP-DtpC_1-475-Nb26, movies were collected at a nominal magnification of ×,130,000 and a physical pixel size of 0.67 Å, with a 50-μm C2 aperture and 100-μm objective aperture at a dose rate of 19.0 e−/pixel per second. A total dose of 57 e−/Ų was used with 3 s exposure time fractionated in 40 frames.

All movies were motion-corrected using Relion-3.1 (Scheres, 2012; Zivanov et al., 2018) own implementation of MotionCor2 (Zheng et al., 2017). Contrast transfer function parameters were calculated using CTFFIND4 (Rohou and Grigorieff, 2015) and putative particle coordinates were initially defined using CrYOLO (Wagner et al., 2019).

For DtpC-Mb26, 13,257 movies were collected, 3,062,337 coordinates were picked and used for 2D averaging and clustering. For split sfGFP-DtpC_1-475-Nb26, 7602 movies were collected, 1,049,399 coordinates were picked and used for 2D averaging and clustering. For DtpC-Nb26, 24,333 movies were collected, 6,464,070 coordinates were picked and used for 2D averaging and clustering, and 878,428 particles were used in the final 3D reconstruction. Briefly, DtpC-Nb26 dimeric population was clustered using 3D class averaging in Relion3.1 (Scheres, 2012). Particle trajectories and cumulative beam damage were further corrected by Bayesian polishing in Relion3.1 (Zivanov et al., 2019), and the resulting shiny particles were exported to cryoSPARCv3 (Punjani et al., 2017) for further 3D clustering via successive heterogeneous refinement cycles using “bad” and “good” volumes as references to denoise the dataset. Non uniform refinement (Punjani et al., 2020), followed by a local refinement using a soft mask around one transporter unit resulted in a 2.7 Å reconstruction of DtpC. The overall resolution was estimated in CryoSPARCv3 using the FSC = 0.143 cutoff. Local resolution estimations were also calculated in CryoSPARCv3 using the 0.5 FSC cutoff. The two half maps were used as inputs to assess various post-processing strategies such as the CryoSPARC’s sharpening tool, DeepEMhancer (Sanchez-Garcia et al., 2021), and Resolve_cryo-em (Terwilliger et al., 2020). The latter led to a slightly better defined contour of the atoms, and was subsequently used for the last atomic-model refinement of DtpC. The initial models of DtpC and Nb26 were generated using AlphaFold2, and refined against the experimental maps; first in Isolde (Croll, 2018), and last in Phenix (Afonine et al., 2018), principally to refine atomic displacement parameters (B-factors) and perform a slight energy minimization while keeping restrains from Isolde’s reference model. Half-maps, and postprocessed maps of the dimeric arrangement and of the focused refinement, as well as the atomic model of DtpC were deposited in the PDB and EMDB as deposition numbers 7ZC2, and EMD-14618. The atomic model of the dimeric DtpC-Nb26 is available upon request.

Synchrotron SAXS data from solutions of DtpC-Nb26 in β-DDM micelles (SEC-SAXS) were collected on the EMBL P12 (Blanchet et al., 2015) beamline at the PETRA III storage ring (Hamburg, Germany), in a buffer consisting of 0.015% DDM, 100 mM NaCl, 10 mM HEPES (pH 7.5), and 0.5 mM TCEP. Sample (10 mg/ml) was injected onto a Superdex Increase 200 10/300 column (Cytiva) and run at 0.5 ml/min at 20°C. 3000 successive 1 s frames were collected using a Pilatus 2M detector at a sample-detector distance of 3.1 m and at a wavelength of λ = 0.124 nm (I(s) vs. s, where s = 4πsinθ/λ, and 2θ is the scattering angle). The data were normalized to the intensity of the transmitted beam and radially averaged; the scattering of the solvent-blank was subtracted using CHROMIXS (Panjkovich and Svergun, 2018). Cryo-EM volume maps of DtpC-Nb26 were fit to the scattering data across the low-angle range (shape region only) using EM2DAM (Franke et al., 2017) at a density threshold of 0.1.

Graphs were generated using GraphPad Prism 9.0 (GraphPad Software). Molecular graphics and analyses performed with UCSF ChimeraX-1.2.5 (Pettersen et al., 2021). Figures were prepared in Adobe Illustrator 2021.