expression , purification , and reconstitution of fluc channels were as described in detail27,28 . in the final purification step ,
fluc protein was collected from a s200 size - exclusion column equilibrated in 100 mm naf ( or nacl for zero - f preps ) , 10 mm hepes ph 7.0 , 5 mm n - decylmaltoside ( dm ) .
bpe constructs carried two functionally neutral mutations to enhance expression , r29k / e94s or , for hg labeling , r29k / e94c .
hg labeling was achieved by incubating bpe with a 3-fold molar excess of hg(ii ) acetate for 30 minutes between the co - affinity and size exclusion columns .
ec2 constructs bore a single functionally neutral , expression - enhancing mutation , r25k , and , for selenomethionine incorporation , an additional methionine was introduced ( a51 m ) to enhance phasing power .
the c - terminal his6 tag was removed from bpe by treatment with lysine endoproteinase c ( roche)27 , but was left on ec2 .
fluc protein was typically reconstituted into liposomes at low density ( 0.1 - 0.2 g protein / mg lipid ) . for single - channel recording , liposomes were fused into planar lipid bilayers in symmetrical solutions of 300 mm naf , 15 mm mops , ph 7.0 , and channels were recorded at 200 mv holding voltage27,29 .
n - terminal his6 tags were removed while bound to talon beads by 16-hr treatment with tev protease also carrying a his tag ; monobodies with cleaved his tags were eluted from the affinity column with 150 mm nacl , 40 mm tris - hcl ph 7.5 . for the final purification step ,
the preparation was passed over a s75 size exclusion column in 100 mm naf ( or nacl ) , 10 mm hepes ph 7 .
monobodies were used immediately for crystallization or stored in frozen aliquots for channel - blocking experiments .
for crystallization from detergent micelles , fluc protein in solution containing 5 mm dm was concentrated to 10 mg / ml , a maneuver that concentrates the detergent 5 - 10-fold .
monobody solution ( 10 mg / ml ) was supplemented with 4 mm dm immediately before mixing with channel in a 1.2:1 molar ratio .
this protein solution was then mixed with an equal volume of crystallization solutions ( 0.5 l for sitting drops in 96-well plates or 1 l for hanging drops in 24-well plates ) .
bpe - s7 crystals grew in 3 - 5 days in crystallization solutions of 36 - 41% ( w / v ) polyethylene glycol 550 mme , 0.2 m mgcl2 or cacl2 , 0.1 m tris , ph 8.5 - 8.9 .
ec2-s9 crystals grew in 10 - 14 days in crystallization solutions containing 28 - 32% ( w / v ) polyethylene glycol 550 mme , 0.05 m lino3 , 0.1 m n-(2-acetamido)iminodiacetic acid , ph 6.0 - 6.7 .
crystals were frozen in liquid nitrogen for data collection . for lipidic cubic phase crystallization ,
fluc protein concentrated to 10 mg / ml as above was dialyzed overnight to reduce the dm concentration to 10 mm .
this was then mixed with monobody solution ( 10 mg / ml , with 4 mm dm ) in a 1:1.2 molar ratio .
the protein - laden mesophase was prepared by homogenizing 9.9 monoacylglycerol ( monoolein ) lipid with protein solution ( 10  mg / ml ) at a weight ratio of 1:1.5 ( protein : lipid ) using a coupled syringe mixing device at 20c30 .
crystallization trials were carried out at 19c in 96-well glass sandwich plates with 50  nl mesophase and 0.8  l precipitant solution using an in meso robot .
crystallization solutions consisted of 2226% ( v / v ) polyethylene glycol 500 dme , 0.1  m na - citrate ph 5.5 + /-
surfboard shaped crystals grew to a maximum size of 100    50   5  m in 5 - 10  days .
wells were opened using a tungsten  carbide glasscutter , and the crystals were harvested using 50 - 100  m micromounts ( mitegen ) .
crystals were snap - cooled directly in liquid nitrogen prior to data collection on the diamond light source beamlines i24 or i04 .
liposomes ( 10 mg lipid / ml , 0.2 - 1 g protein / mg lipid ) loaded with 300 mm kf or kcl solutions were freeze - thawed for 3 cycles and then extruded 21 times through a 400 nm filter .
immediately before the assay , a 100 l sample was centrifuged through a 1.5-ml sephadex column equilibrated with flux buffer ( fb , 300 mm k - isethionate , 1 mm kf or kcl , 25 mm hepes , ph 7 ) and was diluted 20-fold into a stirred chamber containing 3.8 ml fb .
halide concentration in the suspension was continuously monitored with a f or cl electrode amplified through a ph meter and digitized at 5 hz sampling frequency .
efflux was initiated by adding 1 m valinomycin , and after several minutes 30 mm octylglucoside was added to obtain the 100% efflux level .
for experiments with the bpe n43d mutant , fb contained an additional 100 mm na - isethionate and 25 mm ches buffer .
single - channel block by monobodies was recorded in planar phospholipid bilayers exactly as described28 .
diffraction data for bpe - s7 were processed by the xia2 pipeline33 to xds34 and scaled using aimless35 .
the space group was determined to be p21212 with two bpe dimers and four s7 monobodies in the asymmetric unit ( extended data figure 1 ) .
a phasing strategy was devised that used pre - derivatisation of bpe mutated with a single cysteine residue ( e94c ) with hg ( ii ) acetate prior to crystallisation ( see above ) .
none of the native crystals were isomorphous with the hg derivatised crystals ( riso > 40 % ) , despite having similar cell dimensions .
indeed , hg derivatised crystals were observed to diffract x - rays to slightly higher resolution than native crystals , therefore effort was directed at these samples for phasing and refinement .
the four hg sites were located using the single wavelength anomalous dispersion ( sad ) method as implemented in shelx36 with the positions further refined and initial phases calculated using sharp37 with solvent flattening in solomon38 . to improve the phases a second and third dataset
were also collected at the se edge using both hg and seleno - l - methionine derivatized protein and another hg derivatised dataset respectively ( extended data table 1 ) .
all 16 se plus 4 hg sites were located using shelx and this dataset was combined with the initial 3.6 angstrom hg derivatized data .
we did not observe higher resolution diffraction in the native crystals , which typically gave diffraction between 3.6 - 3.8 angstroms .
our highest resolution dataset with optimal scaling statistics was one of the hg - derivatised crystals , we therefore used this dataset for subsequent refinement of the model built into the experimental electron density maps calculated from sharp ( see below ) .
for the bpe - l2 crystals , data were similarly processed and scaled in spacegroup p1 .
phases were calculated using molecular replacement as implemented in phaser39 using the experimentally determined bpe model and a homology model of the l2 monobody using a previously determined structure of a loop - library monobody ( pdb : 3rzw ) .
the electron density maps clearly showed major differences in the selected , variable regions of the monobody .
phases were calculated using se - sad with 8 se sites , and processed as above .
the experimental electron density maps were of exceptional quality following phase extension to the highest resolution shell of 2.58  .
data were collected at advanced light source beamlines 8.2.1 and 8.2.2 , and diamond light source , beamlines i24 and i04 . for the bpe - s7 complex structure , a model for the channel
was built into the experimental electron density maps calculated from sharp using o40 with sigma - a - weighted 2fo - fc and mfo - dfc electron density maps .
the s7 monobodies were initially built using a homology model based a previously determined structure of a side library monobody ( pdb : 4jeg ) .
the partial models were further cycled back into phase calculation in sharp to improve the initial solvent envelope used for the solvent flipping procedure .
the amino acid side chains were then built using the se and hg sites to determine the correct register .
refinement of the bpe - s7 model was carried out in refmac542,43 against the highest resolution dataset for these crystals , 3.6  , which came from one of the hg - derivatised crystals used for phasing ( extended data table 1 ) .
no prior phase information was used during the refinement , however refinement was improved following anisotropic truncation of the structure factors . to avoid biasing the model , ncs
the ec2-s9 model was built directly into the experimental maps , using se sites to ensure the correct register , and then monobodies were placed by molecular replacement using phaser with a homology model based on s7 .
the bpe - l2 model was built into the electron density maps calculated from phaser following iterative rounds of structure refinement in phenix45 and refmac5 .
expression , purification , and reconstitution of fluc channels were as described in detail27,28 . in the final purification step ,
fluc protein was collected from a s200 size - exclusion column equilibrated in 100 mm naf ( or nacl for zero - f preps ) , 10 mm hepes ph 7.0 , 5 mm n - decylmaltoside ( dm ) .
bpe constructs carried two functionally neutral mutations to enhance expression , r29k / e94s or , for hg labeling , r29k / e94c .
hg labeling was achieved by incubating bpe with a 3-fold molar excess of hg(ii ) acetate for 30 minutes between the co - affinity and size exclusion columns .
ec2 constructs bore a single functionally neutral , expression - enhancing mutation , r25k , and , for selenomethionine incorporation , an additional methionine was introduced ( a51 m ) to enhance phasing power .
the c - terminal his6 tag was removed from bpe by treatment with lysine endoproteinase c ( roche)27 , but was left on ec2 .
fluc protein was typically reconstituted into liposomes at low density ( 0.1 - 0.2 g protein / mg lipid ) . for single - channel recording , liposomes were fused into planar lipid bilayers in symmetrical solutions of 300 mm naf , 15 mm mops , ph 7.0 , and channels were recorded at 200 mv holding voltage27,29 .
n - terminal his6 tags were removed while bound to talon beads by 16-hr treatment with tev protease also carrying a his tag ; monobodies with cleaved his tags were eluted from the affinity column with 150 mm nacl , 40 mm tris - hcl ph 7.5 . for the final purification step ,
the preparation was passed over a s75 size exclusion column in 100 mm naf ( or nacl ) , 10 mm hepes ph 7 .
monobodies were used immediately for crystallization or stored in frozen aliquots for channel - blocking experiments .
for crystallization from detergent micelles , fluc protein in solution containing 5 mm dm was concentrated to 10 mg / ml , a maneuver that concentrates the detergent 5 - 10-fold .
monobody solution ( 10 mg / ml ) was supplemented with 4 mm dm immediately before mixing with channel in a 1.2:1 molar ratio .
this protein solution was then mixed with an equal volume of crystallization solutions ( 0.5 l for sitting drops in 96-well plates or 1 l for hanging drops in 24-well plates ) .
bpe - s7 crystals grew in 3 - 5 days in crystallization solutions of 36 - 41% ( w / v ) polyethylene glycol 550 mme , 0.2 m mgcl2 or cacl2 , 0.1 m tris , ph 8.5 - 8.9 .
ec2-s9 crystals grew in 10 - 14 days in crystallization solutions containing 28 - 32% ( w / v ) polyethylene glycol 550 mme , 0.05 m lino3 , 0.1 m n-(2-acetamido)iminodiacetic acid , ph 6.0 - 6.7 .
crystals were frozen in liquid nitrogen for data collection . for lipidic cubic phase crystallization ,
fluc protein concentrated to 10 mg / ml as above was dialyzed overnight to reduce the dm concentration to 10 mm .
this was then mixed with monobody solution ( 10 mg / ml , with 4 mm dm ) in a 1:1.2 molar ratio .
the protein - laden mesophase was prepared by homogenizing 9.9 monoacylglycerol ( monoolein ) lipid with protein solution ( 10  mg / ml ) at a weight ratio of 1:1.5 ( protein : lipid ) using a coupled syringe mixing device at 20c30 .
crystallization trials were carried out at 19c in 96-well glass sandwich plates with 50  nl mesophase and 0.8  l precipitant solution using an in meso robot .
crystallization solutions consisted of 2226% ( v / v ) polyethylene glycol 500 dme , 0.1  m na - citrate ph 5.5 + /-
surfboard shaped crystals grew to a maximum size of 100    50   5  m in 5 - 10  days .
wells were opened using a tungsten  carbide glasscutter , and the crystals were harvested using 50 - 100  m micromounts ( mitegen ) .
crystals were snap - cooled directly in liquid nitrogen prior to data collection on the diamond light source beamlines i24 or i04 .
liposomes ( 10 mg lipid / ml , 0.2 - 1 g protein / mg lipid ) loaded with 300 mm kf or kcl solutions were freeze - thawed for 3 cycles and then extruded 21 times through a 400 nm filter .
immediately before the assay , a 100 l sample was centrifuged through a 1.5-ml sephadex column equilibrated with flux buffer ( fb , 300 mm k - isethionate , 1 mm kf or kcl , 25 mm hepes , ph 7 ) and was diluted 20-fold into a stirred chamber containing 3.8 ml fb .
halide concentration in the suspension was continuously monitored with a f or cl electrode amplified through a ph meter and digitized at 5 hz sampling frequency .
efflux was initiated by adding 1 m valinomycin , and after several minutes 30 mm octylglucoside was added to obtain the 100% efflux level .
efflux rates were calculated after calibration with 25 m additions of naf or nacl . for experiments with the bpe n43d
mutant , fb contained an additional 100 mm na - isethionate and 25 mm ches buffer .
single - channel block by monobodies was recorded in planar phospholipid bilayers exactly as described28 .
diffraction data for bpe - s7 were processed by the xia2 pipeline33 to xds34 and scaled using aimless35 .
the space group was determined to be p21212 with two bpe dimers and four s7 monobodies in the asymmetric unit ( extended data figure 1 ) .
a phasing strategy was devised that used pre - derivatisation of bpe mutated with a single cysteine residue ( e94c ) with hg ( ii ) acetate prior to crystallisation ( see above ) .
none of the native crystals were isomorphous with the hg derivatised crystals ( riso > 40 % ) , despite having similar cell dimensions .
indeed , hg derivatised crystals were observed to diffract x - rays to slightly higher resolution than native crystals , therefore effort was directed at these samples for phasing and refinement .
the four hg sites were located using the single wavelength anomalous dispersion ( sad ) method as implemented in shelx36 with the positions further refined and initial phases calculated using sharp37 with solvent flattening in solomon38 . to improve the phases a second and third dataset
were also collected at the se edge using both hg and seleno - l - methionine derivatized protein and another hg derivatised dataset respectively ( extended data table 1 ) .
all 16 se plus 4 hg sites were located using shelx and this dataset was combined with the initial 3.6 angstrom hg derivatized data .
we did not observe higher resolution diffraction in the native crystals , which typically gave diffraction between 3.6 - 3.8 angstroms .
our highest resolution dataset with optimal scaling statistics was one of the hg - derivatised crystals , we therefore used this dataset for subsequent refinement of the model built into the experimental electron density maps calculated from sharp ( see below ) . for the bpe - l2 crystals ,
phases were calculated using molecular replacement as implemented in phaser39 using the experimentally determined bpe model and a homology model of the l2 monobody using a previously determined structure of a loop - library monobody ( pdb : 3rzw ) .
the electron density maps clearly showed major differences in the selected , variable regions of the monobody .
phases were calculated using se - sad with 8 se sites , and processed as above .
the experimental electron density maps were of exceptional quality following phase extension to the highest resolution shell of 2.58  .
data were collected at advanced light source beamlines 8.2.1 and 8.2.2 , and diamond light source , beamlines i24 and i04 .
for the bpe - s7 complex structure , a model for the channel was built into the experimental electron density maps calculated from sharp using o40 with sigma - a - weighted 2fo - fc and mfo - dfc electron density maps .
the s7 monobodies were initially built using a homology model based a previously determined structure of a side library monobody ( pdb : 4jeg ) .
the partial models were further cycled back into phase calculation in sharp to improve the initial solvent envelope used for the solvent flipping procedure .
the amino acid side chains were then built using the se and hg sites to determine the correct register .
refinement of the bpe - s7 model was carried out in refmac542,43 against the highest resolution dataset for these crystals , 3.6  , which came from one of the hg - derivatised crystals used for phasing ( extended data table 1 ) .
no prior phase information was used during the refinement , however refinement was improved following anisotropic truncation of the structure factors . to avoid biasing the model , ncs
the ec2-s9 model was built directly into the experimental maps , using se sites to ensure the correct register , and then monobodies were placed by molecular replacement using phaser with a homology model based on s7 .
the bpe - l2 model was built into the electron density maps calculated from phaser following iterative rounds of structure refinement in phenix45 and refmac5 .
the asymmetric unit is shown in green and red ( channel and monobody , respectively ) , and symmetry mates are shown in black and blue . left panel :
right panel : single channel recording of bpe in the presence of 200 nm l2 .
channels were recorded in the presence of 300 mm n - methyl - glucamine - fluoride , from which all small cations were rigorously excluded .
left panel : cartoon schematic of ec2 with s9 monobodies bound , coloured as bpe in figure 1b .
right panel : cartoon view of tm4 from ec2 , with the solvent flattened electron density map calculated from sharp contoured at 1.8 sigma ( blue ) , and anomalous difference density from seleno - l - methionine contoured at 5 sigma ( magenta ) .
upper three panels : f- transport from liposomes by bpe mutants f82i , f85i , and n43d , in the presence and absence of 6 um blocking monobody .
f- efflux from proteoliposomes ( 0.2 g protein / mg lipid for phe mutants ; 1 g protein / mg lipid for n43d ) was monitored with a f electrode and normalized against total trapped f. lower panel : f dump by f85i measured at ph 7 and ph 9 .
highly conserved residues are shaded in grey . for the eukaryotic sequences , residues expected to line 
pore 2 ,  the pore mostly encompassed by the c - terminal domain , are colored red . for details on derivatisation
see supplementary information mn ( i ) half - set correlation as reported by aimless phasing power = rms ( |fh| / ( ( fh + fp)-(fph ) ) ) analogous experiments in which cl efflux was measured gave no detectable activity in any samples . estimated based on single channel currents .
 to contend with hazards posed by environmental fluoride , microorganisms export this anion through f -- specific ion channels of the fluc family14 . since the recent discovery of fluc channels , numerous idiosyncratic features of these proteins have been unearthed , including extreme selectivity for f- over cl- and dual - topology dimeric assembly56 . 
 to understand the chemical basis for f- permeation and how the antiparallel subunits convene to form a f -- selective pore , we solved crystal structures of two bacterial fluc homologues in complex with three different monobody inhibitors , with and without f- present , to a maximum resolution of 2.1  . 
 the structures reveal a surprising  
 double - barrelled  channel architecture in which two f- ion pathways span the membrane and the dual - topology arrangement includes a centrally coordinated cation , most likely na+ . 
 f- selectivity is proposed to arise from the very narrow pores and an unusual anion coordination that exploits the quadrupolar edges of conserved phenylalanine rings . 