pa63 prepores were prepared following the described procedures . for negative - stain em , 2 l 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 grids , therefore overcoming the problem of preferred orientation for the pa pore . immediately after removal of polylysine , 2 l of pa prepore ( ~50 g / ml ) 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 a day before use . the procedure for pore induction on cryoem grids was carried out following the same procedure for the negative - stain em except that the last step of staining was not used . about 1.5 l 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 . negative - stain em micrographs were acquired with leginon automation software 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  . 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  on the sample level ) and defocus values ranging from 1.8 to 5.1 m . the dose rate on the camera was set to ~8 e / pixel / s . 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 yifan cheng 's lab . the average images from all frames were used for defocus determination and particle picking , and those from the first 16 frames ( corresponding to ~20 e /  total dose on sample ) were used for 2d and 3d image classification . 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 . for negative - stain em single particle reconstruction , 140,775 particles were picked from 1,115 negative - stain em micrographs using the batchboxer program of eman . the defocus value of each micrograph was determined by ctffind and particles were corrected for contrast transfer function ( ctf ) by phase - flipping with the corresponding defocus and astigmatism values using bsoft . the refinement was then performed with 7-fold symmetry using eman . for cryoem single particle reconstruction , 21,200 particles ( 256256 pixels ) were initially picked by hand from 1,928 micrographs and subjected to auto - refinement by relion using the negative - stain em map obtained above as the initial model . the resulting cryoem map ( ~4  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 2d and 3d classifications using the recommended procedures of relion ( http://www2.mrc-lmb.cam.ac.uk/relion/index.php/recommended_procedures ) . 2d class averages and 3d class reconstructions were inspected and those without high - resolution and interpretable features were considered as " bad " classes . the c7 symmetry was applied throughout the 3d classification and 3d auto - refinement . to maximize usable signals from the frame images acquired with the k2 summit camera , we employed the resolution and dose - dependent model of radiation damage recently introduced in relion-1.3 in the following steps . first , the particle images averaged from all 32 frames with whole - image drift correction were used for a preliminary 3d 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 e /  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 3d auto - refinement in relion to generate the final cryoem map . relion post - processing with a soft auto - mask estimated a resolution of 2.9  by the gold - standard fsc at 0.143 criterion and a b - factor of 95  , and the post - processing without any mask reported a resolution of 3.3  . the accuracies of rotation and translation reported by relion 3d auto - refinement were 1.67 and 1.01  . 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 resmap using the two cryoem maps independently refined from halves of data . 3d classification and auto - refinement also identified a subset of 21,632 particles that led to a cryoem map at 3.6  resolution which is in the pore state but lacks the density corresponding to the 14-stranded  barrel , i.e. only has the corolla and calyx of the pa pore . de novo atomic model building of the pa pore except its domain 4 was carried out on the cryoem map at 2.9  resolution using coot . 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 cns . the amplitudes and phases of the structural factor the restraints of ramachandran , secondary structure , and non - crystallographic symmetry were used in the refinement . the available crystal structures of pa83 monomer ( e.g. pdb i d : 1acc and 3tew ) and pa63 heptameric prepore ( pdb i d : 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 pa pore in most situations and 1tzo was used when the inter - protomer interaction or domain movement were considered . for negative - stain em , 2 l 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 grids , therefore overcoming the problem of preferred orientation for the pa pore . immediately after removal of polylysine , 2 l of pa prepore ( ~50 g / ml ) 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 a day before use . the procedure for pore induction on cryoem grids was carried out following the same procedure for the negative - stain em except that the last step of staining was not used . about 1.5 l 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 . negative - stain em micrographs were acquired with leginon automation software 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  . 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  on the sample level ) and defocus values ranging from 1.8 to 5.1 m . the dose rate on the camera was set to ~8 e / pixel / s . 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 yifan cheng 's lab . the average images from all frames were used for defocus determination and particle picking , and those from the first 16 frames ( corresponding to ~20 e /  total dose on sample ) were used for 2d and 3d image classification . in total , 12,416 micrographs were taken in a continuous session . for negative - stain em single particle reconstruction , 140,775 particles were picked from 1,115 negative - stain em micrographs using the batchboxer program of eman . the defocus value of each micrograph was determined by ctffind and particles were corrected for contrast transfer function ( ctf ) by phase - flipping with the corresponding defocus and astigmatism values using bsoft . the refinement was then performed with 7-fold symmetry using eman . for cryoem single particle reconstruction , 21,200 particles ( 256256 pixels ) were initially picked by hand from 1,928 micrographs and subjected to auto - refinement by relion using the negative - stain em map obtained above as the initial model . the resulting cryoem map ( ~4  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 2d and 3d classifications using the recommended procedures of relion ( http://www2.mrc-lmb.cam.ac.uk/relion/index.php/recommended_procedures ) . 2d class averages and 3d class reconstructions were inspected and those without high - resolution and interpretable features were considered as " bad " classes . the c7 symmetry was applied throughout the 3d classification and 3d auto - refinement . to maximize usable signals from the frame images acquired with the k2 summit camera , we employed the resolution and dose - dependent model of radiation damage recently introduced in relion-1.3 in the following steps . first , the particle images averaged from all 32 frames with whole - image drift correction were used for a preliminary 3d 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 e /  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 3d auto - refinement in relion to generate the final cryoem map . relion post - processing with a soft auto - mask estimated a resolution of 2.9  by the gold - standard fsc at 0.143 criterion and a b - factor of 95  , and the post - processing without any mask reported a resolution of 3.3  . the accuracies of rotation and translation reported by relion 3d auto - refinement were 1.67 and 1.01  . 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 resmap using the two cryoem maps independently refined from halves of data . 3d classification and auto - refinement also identified a subset of 21,632 particles that led to a cryoem map at 3.6  resolution which is in the pore state but lacks the density corresponding to the 14-stranded  barrel , i.e. only has the corolla and calyx of the pa pore . de novo atomic model building of the pa pore except its domain 4 was carried out on the cryoem map at 2.9  resolution using coot . 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 cns . the amplitudes and phases of the structural factor the restraints of ramachandran , secondary structure , and non - crystallographic symmetry were used in the refinement . the available crystal structures of pa83 monomer ( e.g. pdb i d : 1acc and 3tew ) and pa63 heptameric prepore ( pdb i d : 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 pa pore in most situations and 1tzo was used when the inter - protomer interaction or domain movement were considered . some representative top - view and side - view particles are selected with circles and squares , respectively . b , a full - size drift - corrected cryoem micrograph of 3710  3710 pixels of pa pore particles acquired from a gatan k2 summit direct electron detection camera , and at 300 kv accelerating voltage , 2.7 m defocus , and a total dose of 39 e /  . h , superimposition of representative regions of the cryoem map ( mesh ) with the atomic model ( stick ) , including  helix ( e ) ,  strand ( f ) , loop ( g ) , and ca ions ( green spheres in h ) . a ,  gold - standard  fourier shell correlation ( fsc ) between two independently refined maps with an auto - mask that was corrected by phase randomization . b , fsc of the final atomic model versus the final cryoem map ( black ) ; of a model refined in the first of the two independent maps used for the gold - standard fsc versus the same map ( red ) and versus the second independent map ( blue ) . c , surface view and cut - through view of the unsharpened cryoem map colored by local resolution estimated by resmap . a , the crystal structure of domain 4 ( purple ribbons ) from pa prepore ( pdb i d : 1tzo ) fits the cryoem map ( grey surface ) of pa pore with a good agreement ( cross - correlation coefficient : 0.91 ) . domain 4 is shifted inwards as a rigid - body for 4  from the prepore conformation ( dark cyan ) to the pore conformation ( purple ) . the host cell receptor ( tem8 or cmg2 ) of pa that binds to domain 4 ( grey ribbons ) is schematically illustrated . a , comparison of the -clamp in the pa pore with its corresponding region in the pa prepore . the residues asp426 and phe427 missing in the crystal structure of the pa63 prepore ( pdb i d : 1tzo ) are modeled based the crystal structure of the pa83 monomer ( pdb i d : 1acc ) . b , close - up top view of the -clamp region in the pa pore . the hydrogen bonds between ser428 , asn399 , and lys397 , which form a chain tethering the 2728 and 210211 loops together , are depicted with dashed lines . a , superimposition of domains 2c of the pa pore and the crystal structure of the pa heptameric prepore ( pdb i d : 1tzo ) . the dashed boxes highlight the conformational difference of the 210211 loop between these two structures . residues asp426 and phe427 are not solved in the crystal structure of pa prepore due to their flexibility . b , superimposition of domains 2c of the pa pore and the pa octameric prepore ( pdb i d : 3hvd ) . note the similarity of the 210211 loop ( dashed box ) between these two structures . c , superimposition of domains 2c of the pa pore and the pa prepore ( monomer ; pdb i d : 3tew ) . d  f , close - up views of the rearrangements of the 210211 loop ( d ) , the 2728 loop ( e ) , and the 2526 and 212213 loops ( f ) during the conversion from prepore to pore . val377 and leu378 of the 2526 loop and val455 and tyr456 of the 212213 loop are flipped upside down to obtain the  strand conformation in the pa pore , leading to an extension of 26 and 212 ( f ) . cryoem densities ( mesh ) corresponding to these loops are displayed to the right of d  f with atomic models superimposed , showing unambiguous atomic modeling . the superimpositions of the cryoem map and the atomic model are shown in views different from the left panels for clarity . the conformational changes of these loops result in a more compact domain 2c with a decrease of 567  in its surface area . a , surface views of two neighboring protomers ( the same domains in the two protomors are in different shades of the same colors ) of the pa pore and the pa prepore ( pdb i d : 1tzo ) visualized from inside pa heptamer . domains 1 ' , 2c , 2s , and 3 are colored differently and domain 4 is not shown . the inter - protomer interface in the pa prepore is largely formed by domains 1 ' and 3 and domain 2 only contributes to this interface by its membrane insertion loop and c - terminal region ( 213 , 214 , and 23 ) . in the pa pore , the convergence of domain 2c creates an inter - protomer interface without any gap , with an increase of interface area on domain 2c from 1,247  of the pa prepore to 2,106  of the pa pore as calculated using the pisa ( http://www.ebi.ac.uk/pdbe/pisa/ ) . additionally , formation of the  barrel also leads to extensive contacts , creating a new interface area of 1,195  between two protomers . b , schematic of the conversion of 22 , 23 , 21 , and the membrane insertion loop of the pa prepore to 22s and 23s of the pa pore . hydrogen bonds between 22 and 23 , which are depicted with dashed lines , are maintained during the conversion . by contrast , 21 and the connecting loops have to be fully unfolded and converted into  strands that collectively assemble the  barrel of the pa pore . although the detailed events of  barrel formation are not yet clear , it is likely that assembly starts from the top in a zipper - like manner . a favorable scenario is that the convergence of domains 2c would place the top ends of the  strands close to each other to form a short  barrel , which could extend by pulling more residues together via formation of ordered hydrogen bonds until it reaches the bottom end . it is less favorable that the assembly starts from other regions because disordered hydrogen bonds and hydrophobic interactions could generate enormous non - productive , possibly irreversible pairings between strands . b , cross - section side view of superimposition of the unsharpened cryoem map with the atomic model of the intact pa pore , showing the cryoem map has the same conformation as the pa pore except for the absence of the 14-stranded  barrel . c ,  gold - standard  fsc ( with an auto - mask that was corrected by phase randomization ) between two independently refined maps . d , top view of the -clamp region of the cryoem map ( mesh ) lacking the  barrel superimposed with the atomic model of the pa pore ( ribbons ) , showing correct assembling of the -clamp in the cryoem map .
summaryanthrax toxin , comprising protective antigen ( pa ) , lethal factor ( lf ) and edema factor ( ef ) , is the major virulence factor of bacillus anthracis , an agent that causes high mortality in human and animals . 
 pa forms oligomeric prepores that undergo conversion to membrane - spanning pores by endosomal acidification , and these pores translocate the enzymes lf and ef into the cytosol of target cells1 . 
 pa is not only a vaccine component and therapeutic target for anthrax infections but also an excellent model system for understanding the mechanism of protein translocation . 
 based on biochemical and electrophysiological results , researchers have proposed that a -clamp composed of phe427 residues of pa catalyzes protein translocation via a charge - state dependent brownian ratchet29 . 
 although atomic structures of pa prepores are available1014 , how pa senses low ph , converts to active pore and translocates lf and ef are not well defined without an atomic model of the pa pore . here , by cryo electron microscopy ( cryoem ) with direct electron counting 
 , we have determined the pa pore structure at 2.9- resolution . 
 the structure reveals the long - sought - after catalytic -clamp and the membrane - spanning translocation channel , and supports the brownian ratchet model for protein translocation . 
 comparisons of four structures reveal conformational changes in prepore to pore conversion that support a multi - step mechanism by which low - ph is sensed and the membrane - spanning channel is formed .