zaire ebov ( strain mayinga-76 ) glycoprotein extracellular domain dna was synthesized ( uniprot entry kb - q05320 ) .
the expression construct gp contains two directly linked sections , amino acids 32 - 312 and 464 - 632 , with a t42a mutation to eliminate n40 glycosylation . at the n - terminus of the protein ,
4 amino acids etgr were added from the expression vector pneosec31 . at the c - terminus
, a foldon trimerization sequence from the bacteriophage t4 fibritin and a 6his tag were added with the sequence : gsgyipeaprdgqayvrkdgewvllstflgthhhhhh . the endotoxin free pneosec - gp plasmid was transiently transfected into the human embryonic kidney hek293 t ( atcc crl11268 ) cells with polyethylenmine ( pei , mw 25kd , sigma , uk ) . to inhibit the formation of complex glycosylation ,
the mannosidase inhibitor kifunensine ( cayman chemical , michigan , usa ) was added to a final concentration of 5 m .
after 5 days of transfection , the conditioned media was collected , dialysed against pbs and incubated with talon beads ( takara bio europe sas , france ) at 15c for 1 hour with gentle shaking .
the protein was eluted with 200 mm imidazole in pbs and further purified by size exclusion chromatography with a superdex 200 hiload 16/600 column ( ge healthcare , buckinghamshire , uk ) and a buffer of 10 mm mes , ph5.2 , 150 mm nacl .
10 l of solution containing 2 m glycosylated ebov gp protein , buffered by the addition of 10 l of 850 mm sodium malonate at the desired ph , was mixed with 10 l of 15 sypro orange dye ( thermo fisher scientific , uk ) , along with 10 l of 50 m compound in 10% dmso or just 10% dmso .
samples were placed in a semi skirted 96 well pcr plate ( 4 titude , surrey , uk ) , sealed and heated in an mx3005p qpcr machine ( stratagene , agilent technologies , usa ) from 24.5 to 98.5 c at a rate of 1 c min .
fluorescence changes were monitored with excitation and emission wavelengths at 492 and 610 nm respectively .
reference wells , i.e. solutions without chemical , but with same amount of dmso , were used to compare the melting temperature ( tm ) .
compounds tested included toremifene , tamoxifen , 4-hydroxyltamoxifen , anastrozole , benztropine , clomipramine , ibuprofen , diacylglycerol kinase inhibitor and benzodiazepine .
the serms endoxifen and clomiphene could not be tested since they bind to sypro orange directly .
hiv-1-derived pseudoviruses expressing the ebola virus envelope glycoproteins ( ebov pseudoparticle , ebovpp ) were produced as described previously32 .
hek-293 t cells in t175 flasks were transfected with 2 g pr8env , 2 g blam - vpr , 1 g pcrev and 3 g of plexm - ebov_gp plasmids ( containing zaire ebov gp residues 1 - 676 under control of -actin / cmv chimeric promoter ) . after 8 hrs transfection ,
viral supernatants were harvested and concentrated using the lenti - x concentrator ( clontech ) .
virus titres were determined by infecting tzm - bl cells ( pta-5659 , no mycoplasma contamination ) with a serial dilution of concentrated pseudovirus followed by a -gal assay .
since the tzm - bl cells contain a -gal expression cassette with an hiv-1-induced promoter infected cells can be identified through hydrolysis of x - gal33 .
24 hrs prior to the assay tzm - bl cells were plated at 410 cells / well in black clear - bottomed 96-well plates . on the day of assay
, cells were cooled on ice prior to the addition of ebovpp ( moi 0.5 ) , then centrifuged at 2100g for 30 mins at 4 c .
then , 100 l of dmem+10% fbs containing toremifene ( 15 m or 1.5 m ) , ibuprofen ( 150 m or 15 m ) , or the same amount of solvent for the above compounds ( 5% dmso ) was added to each well before placing in a 37 c , co2 incubator to initiate viral entry .
b / g loading kit ( life technologies ) and incubated at room temperature in the dark for 2 hrs .
following ccf2-am removal , cells were washed with pbs and fixed with 2% pfa prior to viewing .
cells were excited using a 405 nm continuous laser ( leica , manheim ) and the emission spectra between 430560 nm recorded pixel by pixel ( 512  512 ) using a leica sp8 x - smd microscope with a 20x objective .
the ratio of blue emission ( 440480 nm , cleaved ccf2-am ) to green ( 500540 nm , un - cleaved ccf2-am ) was then calculated pixel by pixel using a customized macro34 for imagej ( http://imagej.nih.gov/ij/ ) with ten different observation fields for each condition .
a blue / green threshold ( fusion threshold ) was set using hivnoenv virions containing vpr - blam as a background control to provide a fusion detection limit that corresponded to 0.75 + /-
the fusion threshold was calculated recovering the signal ( blue / green intensity ratio ) coming from individual cells plus 2x sd from ~200 cells in the observation field .
cells above the threshold where pseudocolored in red and cells below the threshold pseudocolored in blue .
" red " cells where then counted versus blue cells ( non - fusogenic ) as an accurate measure of fusion in different conditions . for crystallization ,
the gp was treated with endo--acetylglucosaminidase f1 at 37 c for 1 hour , further purified with size exclusion chromatography and concentrated to 10 - 12 mg / ml .
crystallization screen experiments were carried out using the nanolitre sitting - drop vapour diffusion method in 96-well plates as previously described35 .
crystals were initially obtained from hampton research pegrx 1 screen , condition 37 containing 10% ( w / v ) peg 6,000 and 0.1 m sodium citrate tribasic dihydrate ( ph 5.0 ) .
the best crystals were grown in optimized condition containing 9% ( w / v ) peg 6,000 and 0.1 m sodium citrate tribasic dihydrate at ph 5.2 . to obtain gp 
crystal soaking solutions were prepared by first dissolving the inhibitors in 100% dmso and then diluting with 15% ( w / v ) peg 6,000 and 0.1 m sodium citrate tribasic dihydrate ( ph 5.0 ) to a final dmso concentration of 10% .
crystals soaked for longer normally diffracted less well and the crystals from which the gp - toremifene and gp - ibuprofen complex structures were obtained were only soaked for 20 minutes .
crystals were cryo - protected using solutions containing 75% crystallization liquor ( or inhibitor soaking solution ) and 25% ( v / v ) glycerol and frozen in liquid nitrogen prior to data collection .
all data were collected from frozen crystals at 100 k. data were acquired as 0.1 images on pilatus 6 m detectors at diamond light source , uk , using beamline i03 for native data ( exposure time 0.1 s per frame , beam size 8020 m and 30% beam transmission ) , and i02 for inhibitor soaked crystals ( exposure time 0.05 s per frame , beam size 9025 m and 40% beam transmission ) .
diffraction images were indexed , integrated and scaled with the automated data processing program xia2 - 3dii36 .
the native data set was collected from four crystals to 2.23  resolution with 58-fold redundancy .
a total of 7 inhibitors were soaked , including toremifene , tamoxifen , 4-hydroxyltamoxifen , raloxifene , clomiphene , ibuprofen and benztropine , and diffraction data were collected with resolutions ranging from 3.5 to 2.3  .
the crystals belong to space group r32 with unit cell dimensions a = b = 114.0  and c = 307.0  approximately .
the apo structure was determined by molecular replacement with molrep37 using the gp structure of the gp - kz52 fab complex ( pdb i d , 3csy ) as a search model .
the apo structure was refined to 2.23  resolution with an rwork of 0.223 ( rfree , 0.251 ) and good stereochemistry .
close examination of the data from inhibitor soaked crystals showed that only toremifene and ibuprofen were fully bound with gp , and structures were refined to resolutions of 2.69  and 2.68  , respectively .
zaire ebov ( strain mayinga-76 ) glycoprotein extracellular domain dna was synthesized ( uniprot entry kb - q05320 ) .
the expression construct gp contains two directly linked sections , amino acids 32 - 312 and 464 - 632 , with a t42a mutation to eliminate n40 glycosylation . at the n - terminus of the protein ,
4 amino acids etgr were added from the expression vector pneosec31 . at the c - terminus
, a foldon trimerization sequence from the bacteriophage t4 fibritin and a 6his tag were added with the sequence : gsgyipeaprdgqayvrkdgewvllstflgthhhhhh . the endotoxin free pneosec - gp plasmid was transiently transfected into the human embryonic kidney hek293 t ( atcc crl11268 ) cells with polyethylenmine ( pei , mw 25kd , sigma , uk ) . to inhibit the formation of complex glycosylation ,
the mannosidase inhibitor kifunensine ( cayman chemical , michigan , usa ) was added to a final concentration of 5 m .
after 5 days of transfection , the conditioned media was collected , dialysed against pbs and incubated with talon beads ( takara bio europe sas , france ) at 15c for 1 hour with gentle shaking .
the protein was eluted with 200 mm imidazole in pbs and further purified by size exclusion chromatography with a superdex 200 hiload 16/600 column ( ge healthcare , buckinghamshire , uk ) and a buffer of 10 mm mes , ph5.2 , 150 mm nacl .
10 l of solution containing 2 m glycosylated ebov gp protein , buffered by the addition of 10 l of 850 mm sodium malonate at the desired ph , was mixed with 10 l of 15 sypro orange dye ( thermo fisher scientific , uk ) , along with 10 l of 50 m compound in 10% dmso or just 10% dmso .
samples were placed in a semi skirted 96 well pcr plate ( 4 titude , surrey , uk ) , sealed and heated in an mx3005p qpcr machine ( stratagene , agilent technologies , usa ) from 24.5 to 98.5 c at a rate of 1 c min .
fluorescence changes were monitored with excitation and emission wavelengths at 492 and 610 nm respectively .
reference wells , i.e. solutions without chemical , but with same amount of dmso , were used to compare the melting temperature ( tm ) .
compounds tested included toremifene , tamoxifen , 4-hydroxyltamoxifen , anastrozole , benztropine , clomipramine , ibuprofen , diacylglycerol kinase inhibitor and benzodiazepine .
the serms endoxifen and clomiphene could not be tested since they bind to sypro orange directly .
hiv-1-derived pseudoviruses expressing the ebola virus envelope glycoproteins ( ebov pseudoparticle , ebovpp ) were produced as described previously32 .
hek-293 t cells in t175 flasks were transfected with 2 g pr8env , 2 g blam - vpr , 1 g pcrev and 3 g of plexm - ebov_gp plasmids ( containing zaire ebov gp residues 1 - 676 under control of -actin / cmv chimeric promoter ) . after 8 hrs transfection ,
viral supernatants were harvested and concentrated using the lenti - x concentrator ( clontech ) .
virus titres were determined by infecting tzm - bl cells ( pta-5659 , no mycoplasma contamination ) with a serial dilution of concentrated pseudovirus followed by a -gal assay .
since the tzm - bl cells contain a -gal expression cassette with an hiv-1-induced promoter infected cells can be identified through hydrolysis of x - gal33 .
24 hrs prior to the assay tzm - bl cells were plated at 410 cells / well in black clear - bottomed 96-well plates . on the day of assay ,
cells were cooled on ice prior to the addition of ebovpp ( moi 0.5 ) , then centrifuged at 2100g for 30 mins at 4 c .
then , 100 l of dmem+10% fbs containing toremifene ( 15 m or 1.5 m ) , ibuprofen ( 150 m or 15 m ) , or the same amount of solvent for the above compounds ( 5% dmso ) was added to each well before placing in a 37 c , co2 incubator to initiate viral entry .
b / g loading kit ( life technologies ) and incubated at room temperature in the dark for 2 hrs . following ccf2-am removal ,
cells were excited using a 405 nm continuous laser ( leica , manheim ) and the emission spectra between 430560 nm recorded pixel by pixel ( 512  512 ) using a leica sp8 x - smd microscope with a 20x objective .
the ratio of blue emission ( 440480 nm , cleaved ccf2-am ) to green ( 500540 nm , un - cleaved ccf2-am ) was then calculated pixel by pixel using a customized macro34 for imagej ( http://imagej.nih.gov/ij/ ) with ten different observation fields for each condition . a blue / green threshold ( fusion threshold )
was set using hivnoenv virions containing vpr - blam as a background control to provide a fusion detection limit that corresponded to 0.75 + /-
the fusion threshold was calculated recovering the signal ( blue / green intensity ratio ) coming from individual cells plus 2x sd from ~200 cells in the observation field .
cells above the threshold where pseudocolored in red and cells below the threshold pseudocolored in blue .
" red " cells where then counted versus blue cells ( non - fusogenic ) as an accurate measure of fusion in different conditions .
for crystallization , the gp was treated with endo--acetylglucosaminidase f1 at 37 c for 1 hour , further purified with size exclusion chromatography and concentrated to 10 - 12 mg / ml .
crystallization screen experiments were carried out using the nanolitre sitting - drop vapour diffusion method in 96-well plates as previously described35 .
crystals were initially obtained from hampton research pegrx 1 screen , condition 37 containing 10% ( w / v ) peg 6,000 and 0.1 m sodium citrate tribasic dihydrate ( ph 5.0 ) .
the best crystals were grown in optimized condition containing 9% ( w / v ) peg 6,000 and 0.1 m sodium citrate tribasic dihydrate at ph 5.2 . to obtain gp 
crystal soaking solutions were prepared by first dissolving the inhibitors in 100% dmso and then diluting with 15% ( w / v ) peg 6,000 and 0.1 m sodium citrate tribasic dihydrate ( ph 5.0 ) to a final dmso concentration of 10% .
crystals soaked for longer normally diffracted less well and the crystals from which the gp - toremifene and gp - ibuprofen complex structures were obtained were only soaked for 20 minutes .
crystals were cryo - protected using solutions containing 75% crystallization liquor ( or inhibitor soaking solution ) and 25% ( v / v ) glycerol and frozen in liquid nitrogen prior to data collection .
all data were collected from frozen crystals at 100 k. data were acquired as 0.1 images on pilatus 6 m detectors at diamond light source , uk , using beamline i03 for native data ( exposure time 0.1 s per frame , beam size 8020 m and 30% beam transmission ) , and i02 for inhibitor soaked crystals ( exposure time 0.05 s per frame , beam size 9025 m and 40% beam transmission ) .
diffraction images were indexed , integrated and scaled with the automated data processing program xia2 - 3dii36 .
the native data set was collected from four crystals to 2.23  resolution with 58-fold redundancy .
a total of 7 inhibitors were soaked , including toremifene , tamoxifen , 4-hydroxyltamoxifen , raloxifene , clomiphene , ibuprofen and benztropine , and diffraction data were collected with resolutions ranging from 3.5 to 2.3  .
the crystals belong to space group r32 with unit cell dimensions a = b = 114.0  and c = 307.0  approximately .
the apo structure was determined by molecular replacement with molrep37 using the gp structure of the gp - kz52 fab complex ( pdb i d , 3csy ) as a search model .
the apo structure was refined to 2.23  resolution with an rwork of 0.223 ( rfree , 0.251 ) and good stereochemistry .
close examination of the data from inhibitor soaked crystals showed that only toremifene and ibuprofen were fully bound with gp , and structures were refined to resolutions of 2.69  and 2.68  , respectively .
a - d , melt curves of ebov gp with toremifene , ibuprofen or protein alone at ph 5.0 , ph 6.0 , ph 7.0 and ph 8.0 respectively .
e , small effects of serm inhibitors tamoxifen , 4-hydroxytamoxifen and raloxifen on the melting temperature of ebov gp shown at ph 5.2 .
f , melt curves of ebov gp with diaglycerol kinase inhibitor , anastrozole and benztropine mesylate at ph5.2 .
g , h , shifts in melting temperature ( tm c in absolute value ) were plotted against different concentrations of toremifene ( g ) or ibuprofen ( h ) at ph 5.2 .
the affinity constant kd is calculated by a ligand binding 1:1 saturation fitting with the sigmaplot version 13 ( systat software inc ) .
sp , signal peptide ; fl , fusion loop ; nhr and chr , n- and c - terminal heptad repeats ; tm , trans - membrane helix .
the construct gp used for structure determination is made by deleting residues 313 - 463 of the gp mucin domain and residues 633 - 676 .
a foldon trimerization peptide and a 6his tag are added at the c - terminus .
b , the gp2 trimer in the prefusion state ( current structure ) . the trimer is shown as cartoon representation with the monomers coloured in red , green and blue , respectively .
a , the fusion loop that connects 19 and 20 of gp2 projects onto a shallow depression on the surface of a neighbouring monomer .
the fusion loop is shown as a red coil with side - chains drawn as grey sticks , the neighbouring monomer is shown in semi - transparent surface representation .
b , comparison of the fusion loop in the apo gp ( red and grey ) obtained at ph 5.2 with that in the kz52 fab complex ( cyan ) obtained at ph 8.3 .
a , the several small pockets and three large tunnels in the gp trimer shown as grey surfaces .
protein backbones are drawn as ribbons and coloured as in fig . 2 of the main text .
a toremifene is bound at the entrance of each large tunnel and shown as yellow sticks .
a , the dff lid ( residues 192 - 194 , blue coil for main - chain and sticks for side - chains ) nestles at the entrance of the large tunnel in the apo structure .
b - c , toremifene ( yellow sticks in b ) and ibuprofen ( cyan sticks in c ) bind at same site by expelling the dff lid . in both panels ,
the inhibitor bound structure is shown in blue ( gp1 ) and red ( gp2 ) , the apo gp in grey .
the toremifene bound structure is shown in blue and red , the ibuprofen bound structure in grey .
the surfaces of 1 and 1 helices , which undergo large conformational changes upon receptor binding , are protected by the 287 - 293 loop from the glycan cap domain and the n563 glycan from gp2 in the apo gp .
a - b , the chemical structures of toremifene ( a ) and ibuprofen ( b ) .
c - d , |fo - fc| omit electron density maps for toremifene ( c ) and ibuprofen ( d ) contoured at 3. amino acid sequence alignment of 7 filovirus gps around the inhibitor binding site .
numbering corresponds to the full length zaire ebov gp , conserved residues are shown in a red background .
a , ccf2-loaded tzm - bl cells were exposed to ebov pseudoparticles ( ebovpp ) or control particles lacking envelope proteins ( noenv ) at 4 c to synchronise binding and receptor engagement before fusion was initiated by shifting cells to 37 c in the presence of toremefine ( 15 m and 1.5 m ) , ibuprofen- ( 150 m and 15 m ) , or just the compounds solvent ( 5 % dmso ) .
after 2h incubation , cells were loaded with the ccf2-am fret biosensor , fixed and the ratio of blue ( 440480 nm , cleaved ccf2-am ) to green ( 500540 nm , un - cleaved ccf2-am ) fluorescence measured .
cells are pseudocolored according to this ratio : blue represents no fusion , red represents fusion .
b , the percentage of fusogenic cells ( red versus blue ) was calculated taking the average max value coming from the negative control as a threshold for fusion , data are means  s.d .
analysis used an unpaired t test and compared to the ebov + dmso control , where ns ( not significant ) = p > 0.05 , * = p  0.05and * * * = p  0.001 .
 ebola viruses ( ebovs ) are responsible for repeated outbreaks of fatal infections , including the recent deadly epidemic in west africa . 
 there are currently no approved therapeutic drugs or vaccines for the disease . 
 ebov has a membrane envelope decorated by trimers of a glycoprotein ( gp , cleaved by furin to form gp1 and gp2 subunits ) which is solely responsible for host cell attachment , endosomal entry and membrane fusion17 . 
 gp is thus a primary target for the development of antiviral drugs . 
 here we report the first unliganded structure of ebov gp , and complexes with an anticancer drug toremifene and the painkiller ibuprofen . 
 the high - resolution apo structure gives a more complete and accurate picture of the molecule , and allows conformational changes introduced by antibody and receptor binding to be deciphered810 . 
 unexpectedly both toremifene and ibuprofen bind in a cavity between the attachment ( gp1 ) and fusion ( gp2 ) subunits at the entrance to a large tunnel that links with equivalent tunnels from the other monomers of the trimer at the 3-fold axis . 
 protein - drug interactions , with both gp1 and gp2 , are predominately hydrophobic . 
 residues lining the binding site are highly conserved amongst filoviruses except marburg virus ( marv ) , suggesting that marv may not bind these drugs . 
 thermal shift assays show up to a 14 c decrease in protein melting temperature upon toremifene binding , while ibuprofen has only a marginal effect and is a less potent inhibitor . 
 the results suggest that inhibitor binding destabilizes gp and triggers premature release of gp2 , therefore preventing fusion between the viral and endosome membranes . 
 thus these complex structures reveal the mechanism of inhibition and may guide the development of more powerful anti - ebov drugs . 
Methods
Protein cloning, expression and purification
Thermal shift assay
Ebola pseudovirus production and titration
BlaM assay and analysis
Crystallization and inhibitor soaking
Data collection and structure determination
Extended Data