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Triggering conversion from protective antigen (PA) prepore to pore by in vitro acidification leads to rapid and irreversible aggregation. Attempts to prevent aggregation by screening detergents have largely failed15. By low-pH treatment of PA prepores directly on electron microscopy (EM) grids containing a thin layer of continuous carbon film, we obtained dispersed particles of PA pore without aggregation (Extended Data Fig. 1). We then acquired drift-corrected cryo-electron microscopy (cryoEM) images (Extended Data Fig. 1b–d) and reconstructed a map at an overall resolution of 2.9 Å using 60,455 particles (Fig. 1, Extended Data Fig. 2 and Supplementary Video 1). The resolution for most regions of the cryoEM map is ∼2.8 Å (Extended Data Fig. 2c). Our map reveals rich high-resolution structural features, including amino-acid side chains and 14 chelated Ca2+ ions (Extended Data Fig. 1e–h), and has allowed unambiguous de novo atomic modelling (Extended Data Table 1) and detailed structure and function analyses.

Figure 1: CryoEM reconstruction of the PA pore.
figure 1

a, b, Surface representations of the cryoEM map of the PA pore at 2.9-Å resolution as viewed from the top (a) and the side (b). Individual protomers of PA heptamer are colour-coded. Inset of b shows the unsharpened cryoEM map in which the flexible domains 4 (arrowheads) are visible.

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The overall structure of the PA pore has a ‘flower-on-a-stem’ architecture, including corolla, calyx, and stem from top to bottom (Figs 1b and 2a and Supplementary Video 1). Each PA protomer is divided into four domains in the PA prepore10, named 1′, 2, 3, and 4. In the PA pore, domains 1′, 3, and 4 form the corolla and domain 2 forms the calyx and the stem; therefore we designate the parts of domain 2 corresponding to the calyx and the stem as 2c (residues 259–274 and 354–487) and 2s (residues 275–353), respectively (Fig. 2b, c). Domains 1′ and 2c form a compact structure responsible for substrate protein binding and intake (Fig. 2). Domain 2s is an extended β-hairpin (2β2s and 2β3s), seven copies of which assemble to form a membrane-spanning 14-stranded β-barrel 105 Å in length and 27 Å (from Cα to Cα) in diameter (Fig. 2). Domain 3 is located peripherally and has close contact with domains 1′ and 2c (Fig. 2b). The cryoEM density of domain 4 is weak and has the lowest resolution among all domains (inset of Fig. 1b and Extended Data Fig. 2c), probably because of its flexibility from minimal contact with the other domains. Rigid-body fitting of domain 4 of the PA prepore crystal structure to the cryoEM map shows domain 4 shifts ∼4 Å towards the central axis in the PA pore (Extended Data Fig. 3).

Figure 2: Atomic model of the PA pore.
figure 2

a, Top and side views of the atomic model of the PA pore shown as ribbons. Protomers are colour-coded except for domain 4 (grey). b, Structural comparison of the protomers of the PA pore and prepore (PDB accession number 1TZO). The domains are coloured differently according to c. c, Domain organization of the PA protomer.

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The translocation channel of the PA pore has a funnel shape and can be divided into four parts based on diameters calculated with MOLE16: mouth, Φ-clamp, throat, and tube (Fig. 3a, b). Its surface is negatively charged and mainly hydrophilic, but hydrophobic patches are seen at the α-clamp14 of the mouth, near the Φ-clamp, and at the middle of the tube (Fig. 3a, c). As proposed on the basis of the PA prepore structure10, the negatively charged surface promotes passage of cations, and the hydrophilic surface facilitates passage of substrate proteins/polypeptides.

Figure 3: Translocation channel of the PA pore.
figure 3

a, Electrostatic surface (left) and channel radius profile (right) of the PA pore. An α-helix (green surface model of residues 555–574 of lethal factor; PDB accession number 1J7N) is modelled in the β-barrel. The green arrow depicts the direction of protein translocation. Colour scale, kcal (mol e)−1. b, The translocation channel (dots) running through the Φ-clamp. Two protomers of the PA pore are shown as ribbons. Residues Glu398, Asp425, Asp426, Phe427, and Ser429 (ball-and-stick) are exposed to the channel. c, d, Hydropathy surface of the PA pore (brown: hydrophobic; purple: hydrophilic; white: neutral). In c, the front half of the structure is removed to show the luminal surface. In d, two 1,2-dihexadecanoyl-sn-glycero-3-phosphocholine (DPPC) lipid molecules are modelled near the membrane insertion region. e, Bottom and side views of the segmented cryoEM map showing the β-barrel (yellow) bound with disordered detergent molecules (grey). f, The 14-stranded β-barrel (ribbons) and the hydrophobic residues (spheres) on its outer surface. The hydrophobic residues are depicted on different protomers for the ease of presentation.

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The mouth has a 30-Å opening and inner diameters varying down to 20 Å (Fig. 3a); it can accommodate protein secondary structure elements, but not folded domains such as the amino (N)-terminal of lethal factor (LFN). The Φ-clamp below the mouth becomes the bottleneck of the entire channel, with a solvent-excluded inner diameter of only 6 Å (Fig. 3a, b), which is smaller than protein secondary structure elements and therefore may only allow passage of fully unfolded polypeptides. Underneath the Φ-clamp are the throat, which is an enlarged (∼18 Å) bulb-shaped chamber, and the tube formed by the 14-stranded β-barrel with inner diameters in the range 12–18 Å and rich in Ser and Thr residues in its middle region (Fig. 3a, b). The large diameter (>12 Å) of the throat and tube can accommodate an α-helix which may be formed by polypeptides after passing the Φ-clamp (Fig. 3a). The diameter and the hydrophilic property of this part of channel are similar to those of the exit tunnel of the ribosome, which is proposed to translocate α-helices17,18.

The mouth and the tube are the only two openings of the channel, and are accessible to the endosomal and cytosolic compartments, respectively (Fig. 3a). The rest of the channel is ‘water-tight’ and without holes permeable to small molecules. The substrate protein blocks the small hole of the Φ-clamp before or during translocation2. Thus the Φ-clamp may act as a gate separating the endosomal and cytosolic compartments. The differences of proton concentration (ΔpH) and electrical potential (Δψ) across the Φ-clamp (Fig. 3a) may serve as the primary driving force for substrate protein translocation3.

In contrast to the hydrophilic inner surface of the β-barrel, its outer surface is largely hydrophobic (Fig. 3c, d). The Phe residues (313, 314, and 324) form two aromatic belts on opposite sides of the lipid bilayer (Fig. 3d), which may stabilize membrane insertion19,20. The cryoEM map shows that the transmembrane region is surrounded by a cloud of disordered densities, which we interpret as bound detergent molecules that were added during sample preparation (Fig. 3e). Surprisingly, an additional hydrophobic surface in the middle of the β-barrel containing a cluster of hydrophobic residues (Ile289, Val332, Ile334, Leu338 and Leu340 from each protomer) was also bound with detergent molecules (Fig. 3d–f). However, the function of this additional hydrophobic region is unknown.

The overall architecture of the PA pore is similar to those of bacterial toxins α-haemolysin and Vibrio cholerae cytolysin in their membrane-inserted conformations (Extended Data Fig. 4) despite differences in functions21,22. The β-barrel of the PA pore is twice as long as those of the other two toxins and may facilitate LF and oedema factor translocation by entropically stabilizing an α-helix within the confinement of the cylindrical channel9,17. This longer barrel may also be necessary to accommodate its receptor situated between domain 4 and the host membrane11 (Extended Data Fig. 4). Except for this length difference, the 14-stranded β-barrels of the PA pore, α-haemolysin, and V. cholerae cytolysin share geometries, such as diameter, twist of β-strands, and pitch length (Extended Data Fig. 4).

In the PA pore, the seven 2β10–2β11 loops of the heptamer converge to form an iris with a 6-Å hole bounded by a symmetrical arrangement of the seven Phe427 residues (Fig. 4). By contrast, these loops in the PA prepore do not engage each other10,12, but circumscribe a 30-Å central hole with adjacent Phe427 residues spaced ∼14 Å apart (Extended Data Fig. 5a). In the PA pore, each 2β10–2β11 loop is stabilized by close interactions with the 2β7–2β8 loop of the same protomer and the 2β10–2β11 loops of its two neighbouring protomers. The hydrogen bonds between Asn399 and Ser428 and between Asn399 and Lys397′ of an adjacent protomer form a chain of interactions, which give rise to a ring of loops at the iris inside the PA pore (Extended Data Fig. 5b). Consistently, mutation of Ser428 abolished protein translocation, and mutation of Lys397 or Asn399 resulted in a dominant negative effect23,24,25. In the PA homologues found in Clostridium species, Lys397 and Asp426 are both replaced by uncharged Gln26, implying that this chain of interactions might be formed differently in these homologues.

Figure 4: Structure of the Φ-clamp.
figure 4

a, Top view of the Φ-clamp region of the PA pore showing the cryoEM map (mesh) superimposed with the atomic model (stick). b, Tilted view of the Φ-clamp with seven protomers coloured differently, showing the aromatic CH–π interaction. c, Cross-section side view of the translocation channel near the Φ-clamp region. The Φ-clamp (Phe427) and the conserved acidic residue Asp425 are coloured in orange and red, respectively.

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The aryl plane of Phe427 from each protomer is parallel to the central axis of the PA pore. Neighbouring Phe427 residues interact with each other by aromatic CH–π interaction in a tilted T-shaped configuration27 and possibly by hydrophobic interaction, thus forming the Φ-clamp (Fig. 4 and Extended Data Fig. 5). The integrity of the Φ-clamp is required for catalysis of protein translocation, as mutation of even a single Phe427 residue of the Φ-clamp severely reduced translocation efficiency and disrupted the seal against cation passage5. Because the PA pore translocates polypeptides with various side chains, the Φ-clamp may act like an elastic ‘O-ring’, changing its size and shape to allow an unhindered passage of different amino-acid residues while maintaining a good seal during translocation. In the recently reported structure of bacterial amyloid secretion channel CsgG, eight Phe residues form a Φ-clamp, which differs from that in the PA pore in having a 9.5-Å hole and facilitating protein translocation in an ungated manner28.

Evidence from electrophysiological studies indicates that the proton gradient across the endosomal membrane is the primary driving force for unidirectional translocation of proteins through the PA pore3. Proton-driven transporters usually involve protonation-dependent conformational changes of two or more alternating gates. In the PA pore, however, there is only one gate, namely the Φ-clamp, and neither the Φ-clamp nor its nearby residues probably undergo a protonation-dependent conformational change. Indeed, the PA pore structure supports the charge-state-dependent Brownian ratchet model2,3,4,5,6,7,8,9, proposed earlier. In this model, a negative electrostatic barrier within the pore hinders the passage, by Brownian motion, of deprotonated acidic residues3,4,29. The fact that acidic residues in the acidic environment of the endosome have a higher probability of being protonated, and are thus free to pass the barrier, than those in the neutral environment of the cytosol, leads necessarily to unidirectional movement of polypeptides across the barrier. Consistent with this model, the PA pore structure shows three acidic residues, Asp425, Asp426, and Glu398, to be near the Φ-clamp and proximal to the pore axis (Fig. 3b), generating a strong negative electrostatic barrier demarcating the endosomal and cytosolic compartments. In addition, the highly conserved acidic residue Asp425 located directly underneath the Φ-clamp is ideally positioned as a proton sink that may strip off protons from protonated acidic residues passing the Φ-clamp (Fig. 4c). Besides the charge barrier in the Φ-clamp, efficient protein translocation may require additional charged spots, such as the top region of the β-barrel8.

A key question about the low-pH-triggered conversion from prepore to pore is how PA senses pH. A notable conformational change during the conversion is that the 2β10–2β11 loop is flipped from one side to the other (Fig. 5a and Extended Data Fig. 6). Interestingly, this loop has different conformations in the two crystal structures of PA octameric prepores ([PA63]8, PDB accession number 3HVD; [PA63]8[LFN]4, PDB accession number 3KWV) whereas the remaining parts of the structures are largely unchanged9,13,14; that is, this loop is in the pore and prepore states in 3HVD (Extended Data Fig. 6b) and 3KWV, respectively. This comparison suggests that the 2β10–2β11 loop can switch between two conformations without affecting the overall structure. Mutation of the conserved Asn422 or Asp425 in this loop abolished the prepore to pore conversion at low pH in a dominant negative manner25. Therefore we interpret this loop as a pH sensor. Surprisingly, this loop does not possess any His, a residue whose pKa of approximately 6 falls in the pH range of 5–7 where the prepore to pore conversion occurs. The conformational change of this loop might result from destabilization and rearrangement of hydrogen bonds upon exposure to low pH, or alternatively from perturbed pKa of Asp residues (425 and 426) in this loop. We note that other parts of PA might impact pH sensing. Prior work has shown mutation of residues near or in the 2β2–2β3 loop, or at the domain 2/domain 4 interface, or deletion of the 2β2–2β3 loop changed the pH threshold for conversion but did not abolish the low-pH sensitivity25,30. These observations suggest that the involved residues might regulate pH sensing.

Figure 5: Conversion from prepore to pore.
figure 5

a, Superimposition of domains 2c of the PA pore and prepore (monomer; PDB accession number 3TEW). Loops undergoing dramatic conformational changes during the prepore to pore conversion are labelled (blue). b, Overview of the three layers of conformational changes during the conversion. The interaction between the 2β9–2β10 loop and the β-sheet (blue box) is shown in e. c, Superimposition of the PA pore (pink, orange, and blue; coloured by domain) and prepore (light green; PDB accession number 1TZO). Domains 4 and 2s are removed for clarity. Between the two structures, domain 2c has a Cα root-mean-square deviation = 6.87 Å, whereas domains 1′ and 3 remain unchanged with a Cα root-mean-square deviation = 0.54 Å and 0.58 Å, respectively. d, Illustration of the steps of conformational changes during the prepore to pore conversion. For simplicity, only two protomers are illustrated. e, Interface between the 2β9–2β10 loop (green) and the bent β-sheet (gold) of 2β12, 2β6, and 2β7 of the adjacent protomer in the PA pore. Hydrogen bonds between the backbone of the loop and the side chains of the β-sheet are depicted with dashed lines.

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We find that the prepore to pore conversion involves three layers of conformational changes, all located within domain 2 (Fig. 5b). The first layer is the above-mentioned pH-sensing 2β10–2β11 loop. Low-pH-triggered conformational change of this loop is relayed to the following layers sequentially as detailed below.

The second layer involves the 2β7–2β8, 2β5–2β6, and 2β12–2β13 loops, which are positioned in an anti-parallel manner (Fig. 5a). In the PA pore, the N-terminal half of the 2β7–2β8 loop moves downwards to form a U-shaped turn tethered to the putative pH-sensing 2β10–2β11 loop via a hydrogen bond between Asn399 and Ser428 (Fig. 5a and Extended Data Figs 5b and 6e). This new inter-loop interaction may relay the low-pH-triggered conformational changes from the first layer to the second layer as supported by the dominant negative effect of Asn399 or Lys397 mutation24,25, which breaks the hydrogen bond network connecting these two loops (Extended Data Fig. 5b). As a result of the downward pull from the 2β7–2β8 loop, all three loops of the second layer become straightened in the PA pore, with parts of the 2β5–2β6 and 2β12–2β13 loops forming strands to augment 2β6 and 2β12, respectively (Fig. 5a and Extended Data Fig. 6c, f). This conformational change is supported by the observation that mutating any of Pro379, Val455, and Asn458 of these loops led to defects in protein translocation25. This conformational change leads to the convergence of domain 2c of each protomer and formation of the Φ-clamp by a 15° rotation of domain 2c around the hinge residues Ile261 and Tyr456, with its distal edge moving for 12 Å towards the central axis of the PA pore (Fig. 5c and Supplementary Video 2). Indeed, tethering domain 2 to domain 4 by receptor binding or stabilizing the interface between these two domains by a poly-γ-d-glutamate capsule impeded domain convergence and therefore decreased the pH threshold required for the prepore to pore conversion11,12,30. Conversely, destabilizing the domain 2/domain 4 interface by point mutations increased the pH threshold30.

The third layer, resulting from the second layer of conformational changes, involves release and refolding of the precursor of domain 2s (that is, 2β2, 2β3, 2α1, and the membrane insertion loop), and leads to the formation of the membrane-spanning β-barrel (Figs 2 and 5b and Extended Data Fig. 7). The rotation of domain 2c opens the pocket between domain 2c and domain 4 to release the precursors of domain 2s, which then come together to form the β-barrel (see details in Extended Data Fig. 7b). The β-barrel formation might be the result, rather than the cause, of the convergence of domain 2c, as supported by a cryoEM map obtained from a subset of particles that shows converged domain 2c in the pore state but lacks the β-barrel (Extended Data Fig. 8). We note that the stability of the domain 2/domain 4 interface was found to be a rate-limiting barrier to the conversion30 and the separation between domain 2c and domain 4 might also happen at an earlier stage of the conversion.

Taken together, these results suggest a multi-step mechanistic model of the low-pH-triggered conversion from prepore to pore (Fig. 5d). First, the 2β10–2β11 loop as a pH sensor changes its conformation upon acidification (layer 1). Second, consequently, the 2β7–2β8, 2β5–2β6, and 2β12–2β13 loops become ordered (layer 2), resulting in the convergence of domain 2c and the formation of the Φ-clamp. Last, as a result of the separation of domain 2c from domain 4, the precursors of domain 2s are released and refold into a uniform β-barrel (layer 3), which, as a final step, inserts into the endosomal membrane, ready for delivering the toxic enzymes into the cytosol.

In the PA pore, the convergence of domain 2c creates a large inter-protomer interface to stabilize the pore conformation (Extended Data Fig. 7a). Key to the formation of a functional PA pore is a newly identified interface between the 2β9–2β10 loop of one protomer and the triple-stranded β-sheet (2β12, 2β6, and 2β7) of its neighbour (Fig. 5e). The above-mentioned convergence of domain 2c brings them together to form a new binding interface primarily mediated by hydrogen bonds between the backbone of the loop and the side chains of Thr390, Thr393, and Asp451 of the β-sheet (Fig. 5e). These interactions explain the dominant negative effect of Ser382 or Thr393 mutant, the loss of PA activity of the Cys mutant of Thr390 or Asp451, and the conservation of Thr390 and Thr393 in PA homologues25. By contrast, these residues are accessible and free of interactions in the PA prepore, and thus might be potential drug targets for blocking the conversion to functional PA pores.

In summary, the 2.9-Å structure of the anthrax PA pore suggests the 2β10–2β11 loop as a pH sensor to trigger conformational changes for prepore to pore conversion, supports a charge-state-dependent Brownian ratchet model of proton-driven protein translocation, and can inform efforts both to engineer PA to target cancer cells and to design measures to block anthrax toxin entering cells14,21. A more detailed mechanism of protein translocation through the PA pore awaits additional experimental evidence, such as structures of the PA pore in the act of polypeptide translocation.

Methods

No statistical methods were used to predetermine sample size.

Preparation of PA prepores

PA63 prepores were prepared following the described procedures31.

EM sample preparation and data acquisition

For negative-stain EM, 2 µl of 0.1% polylysine solution (Polysciences) was first applied to a glow-discharged grid covered with carbon film and then removed by blotting with filter paper in 2 min. The polylysine treatment produced different orientations of particles on EM grids32, therefore overcoming the problem of preferred orientation for the PA pore. Immediately after removal of polylysine, 2 µl of PA prepore (∼50 µg ml−1) was applied to the grid and incubated for 1 min. The grid was then washed with the high-pH buffer (50 mM HEPES, 50 mM NaCl, pH 8.0) twice followed by two washes with the low-pH buffer (50 mM NaOAc, pH 5.0, 0.05% Igepal CA-630) to induce the conversion of prepore to pore. After removal of excess buffer, the grid was stained with 0.8% (w/v) uranyl formate.

For cryoEM, Quantifoil R1.2/1.3 holey grids were covered with a thin layer of continuous carbon film one day before use. The procedure for pore induction on cryoEM grids followed the same procedure for the negative-stain EM except that the last step of staining was not used. About 1.5 µl of low-pH buffer was left on the grids before they were transferred into an FEI Vitrobot Mark IV. The grids were then blotted and flash-frozen in liquid ethane in the Vitrobot at 100% humidity. The frozen grids were stored in liquid nitrogen before use.

Negative-stain EM micrographs were acquired with Leginon automation software33,34 and a TIETZ F415MP 16-megapixel CCD camera at ×68,027 magnification in an FEI Tecnai F20 electron microscope operated at 200 kV. The micrographs were saved by 2× binning to yield a pixel size of 4.4 Å.

Frozen-hydrated cryoEM grids were loaded into an FEI Titan Krios electron microscope operated at 300 kV for automated image acquisition with Leginon. CryoEM micrographs were recorded on a Gatan K2 Summit direct electron detection camera using the electron counting mode at ×22,500 nominal magnification (calibrated pixel size of 1.28 Å on the sample level) and defocus values ranging from −1.8 to −5.1 µm. The dose rate on the camera was set to about 8 electrons per pixel per second. The total exposure time was 8 s and fractionated into 32 frames of subimages with 0.25 s exposure time for each frame. Frame images were aligned and averaged for correction of beam-induced drift using the GPU-accelerated program from Y. Cheng’s laboratory35. The average images from all frames were used for defocus determination and particle picking, and those from the first 16 frames (corresponding to about 20 electrons per square ångström total dose on sample) were used for two- and three-dimensional image classifications. In total, 12,416 micrographs were taken in a continuous session. The best 7,062 micrographs were selected for the following in-depth data processing.

Image processing

For negative-stain EM single particle reconstruction, 140,775 particles were picked from 1,115 negative-stain EM micrographs using the batchboxer program of EMAN36. Particles were windowed out in 96 pixels × 96 pixels. The defocus value of each micrograph was determined by CTFFIND37 and particles were corrected for contrast transfer function (CTF) by phase-flipping with the corresponding defocus and astigmatism values using Bsoft38. An initial model was generated using the startcsym program of EMAN. The refinement was then performed with sevenfold symmetry using EMAN.

For cryoEM single particle reconstruction, 21,200 particles (256 pixels × 256 pixels) were initially picked by hand from 1,928 micrographs and subjected to auto-refinement by RELION39,40 using the negative-stain EM map obtained above as the initial model. The resulting cryoEM map (approximately 4-Å resolution) was used to generate projections that were then served as templates to pick 259,719 particles from all of the 7,062 micrographs using the batchboxer of EMAN. In our procedure, the defocus values of the micrographs were determined by CTFFIND and particles were corrected for CTF by phase-flipping using Bsoft. The particles were processed with two- and three-dimensional classifications using the recommended procedures of RELION (http://www2.mrc-lmb.cam.ac.uk/relion/index.php/Recommended_procedures). Two-dimensional class averages and three-dimensional class reconstructions were inspected and those without high-resolution and interpretable features were considered as ‘bad’ classes. Particles contributing to the bad classes were discarded. The remaining 60,455 particles were selected for the final structure refinement. The C7 symmetry was applied throughout the three-dimensional classification and three-dimensional auto-refinement.

To maximize usable signals from the frame images acquired with the K2 Summit camera, we used the resolution and dose-dependent model of radiation damage recently introduced in RELION-1.3 in the following steps41. First, the particle images averaged from all 32 frames with whole-image drift correction were used for a preliminary three-dimensional auto-refinement. Second, particle images from individual frames were used to calculate translational alignments for the particle-based drift correction. A running average of seven frames, a standard deviation of one pixel, and fitting of linear tracks through the translations for all running averages were used for the optimal translational alignment following the suggested protocol of RELION. Last, particle images from frame 3 to frame 27 (∼30 electrons per square ångström total dose on sample) were translated using the above optimal alignment and weighted with different B-factors as estimated from the single-frame reconstructions to generate optimal ‘shiny’ average images. Application of this procedure to the above selected particles yielded 60,455 ‘shiny’ particles.

These ‘shiny’ particles were then subjected to three-dimensional auto-refinement in RELION to generate the final cryoEM map. RELION post-processing with a soft auto-mask42 estimated a resolution of 2.9 Å by the ‘gold standard’ Fourier shell correlation (FSC) at 0.143 criterion and a B-factor of −95 Å2, and the post-processing without any mask reported a resolution of 3.3 Å. The accuracies of rotation and translation reported by RELION three-dimensional auto-refinement were 1.67° and 1.01 Å. For visualization and atomic model building, the cryoEM map was sharpened and low-pass filtered by RELION post-processing using the above-mentioned B-factor and resolution. Local resolution was calculated by ResMap43 using the two cryoEM maps independently refined from halves of data.

Three-dimensional classification and auto-refinement also identified a subset of 21,632 particles that led to a cryoEM map at 3.6-Å resolution, which was in the pore state but lacked the density corresponding to the 14-stranded β-barrel: that is, it only had the corolla and calyx of the PA pore.

Atomic model building and refinement

De novo atomic model building of the PA pore except its domain 4 was performed on the cryoEM map at 2.9-Å resolution using Coot44. The coarse model was then refined using PHENIX in a pseudo-crystallographic manner45. Note this procedure only improved the atomic model and did not modify the cryoEM map. Briefly, the cryoEM map was put into an artificial crystal lattice to calculate its structure factor using the em_map_to_hkl.inp utility program in CNS46. The amplitudes and phases of the structure factor were used as pseudo-experimental diffraction data for model refinement in PHENIX. The restraints of Ramachandran, secondary structure, and non-crystallographic symmetry were used in the refinement.

The cryoEM maps and atomic models were visualized using UCSF Chimera47 or PyMOL48. The available crystal structures of PA83 monomer (for example, PDB accession numbers 1ACC and 3TEW) and PA63 heptameric prepore (PDB accession number 1TZO) are highly similar to each other; therefore 3TEW, which has the highest resolution among them, was used for structural comparison with our cryoEM structure of the PA pore in most situations and 1TZO was used when the inter-protomer interaction or domain movement were considered.