rsv in and its mutant forms were overexpressed in escherichia coli bl21 ( de3 ) and purified as previously described .
the proteins were stored in aliquots at 80c in 20 mm hepes - naoh ( ph 7.5 ) , 1.0 m nacl , 20m zncl2 , 5 mm 2-mercaptoethanol and 10% ( w / v ) glycerol .
the branched dna substrate mimicking the product of the concerted integration reaction was obtained by annealing three synthetic oligonucleotides ( integrated dna technologies ) .
a similar strategy had been used previously to prepare a pfv in - dna complex , which demonstrated that the in - dna complex assembled on the designed integration product has essentially an identical structure to the equivalent complex formed via the forward integration reaction .
the viral dna branches carrying the high - affinity gain - of - function mutant rsv u3 long terminal repeat ( ltr ) sequence ( gu3 )   were attached to the target dna duplex with a palindromic 6 bp spacer to generate a fully symmetrized structure . to prepare the rsv in - dna complexes for crystallization , 30 m half - site dna substrate was mixed with 120 m rsv in in 20 mm hepes - naoh ( ph 7.5 ) , 500 mm nacl , 20% ( w / v ) glycerol , and 1 mm tris-(2-carboxyethyl)phosphine ( tcep ) .
the mixture was dialyzed against low salt buffer ( 20 mm hepes - naoh ph 7.5 , 125 mm nacl , 20% ( w / v ) glycerol , 1 mm tcep ) at 25c for overnight . at the end of this first dialysis ,
the mixture was subsequently dialyzed against high salt buffer ( 20 mm mes - naoh ph 6.0 , 1.2 m nacl , 20% ( v / v ) dimethyl sulfoxide ( dmso ) , 5% ( w / v ) glycerol , 1 mm tcep ) at 25c for 24 hours . at the end of this second dialysis ,
the solubilized rsv in - dna complex ( intasome ) was purified through size - exclusion chromatography ( superdex 200 10/300 gl , ge healthcare ) running with 20 mm mes - naoh ph 6.0 , 1.2 m nacl , 20% ( v / v ) dmso , 5% ( w / v ) glycerol , and 1 mm tcep .
the isolated intasome remains stable in the high - salt condition containing 1.2 m nacl that precludes complex formation , suggesting that once the intasome forms , it is kinetically trapped .
the solubility - enhancing rsv in mutation f199k completely abolished intasome formation . for the sec - mals analysis
, a modified condition was used for the intasome re - solubilization and isolation for better baseline stability ( extended data fig .
the rsv intasome assembled through dialysis and purified by size - exclusion chromatography was concentrated to 46 mg / ml using centrifugal concentrator ( amicon ) .
various combinations of the lengths of the viral dna ( ranging from 16 bp to 25 bp ) and flanking target dna ( ranging from 14 bp to 22 bp , corresponding to the full target dna lengths of 34 bp to 50 bp ) were screened in crystallization trials .
the extensive screening yielded only one crystal form with a specific combination of 22-base ( length of the non - transferred strand ) viral dna branches and 16 base - pair ( bp ) target dna flanks on either side of the central six bp spacer ( fig .
dna substrates with two slightly different target sequences were used ( 5-aatgttgtcttatgcaatactc-3/5-gagtattgcataagacaacagtgcacgaatcttgaagacact-3/5-agtgtcttcaagattc-3 , or 5-aatgttgtcttatgcaatactc-3/5-gagtattgcataagacaacagtcgaccaaccttcaacttagc-3/5-gctaagttgaaggttg-3 ) , and they produced essentially the same crystals .
the rsv intasome crystals were grown through reverse vapor diffusion in hanging drops at 22c , by mixing 1.5 l in - dna complex solution with 1.5 l reservoir solution ( 3.2 m sodium formate ) .
crystals appeared within 35 days and reached a size of ~150300 m in 3~5 days . even though the rsv intasome crystals initially diffracted x - ray poorly ( ~ 10  ) , soaking the crystals with a metatungstate cluster compound dramatically improved the resolution ( extended data fig .
the tungsten cluster was later found to bind between in dimers from separate intasome complexes to mitigate crystal lattice disorder .
the crystals were soaked overnight with 0.15 mm metatungstate cluster na6[h2w12o40 ] in a stabilization buffer consisting of 3.2 m sodium formate , 16 mm mes - naoh ph 6.0 , 0.8 m nacl , 16% ( v / v ) dmso , 4% ( w / v ) glycerol , and 1 mm tcep . after soaking ,
the crystals were cryo - protected in 3.2 m sodium formate , 16 mm mes ph 6.0 , 0.8 m nacl , 16% ( v / v ) dmso , 12% ( w / v ) glycerol , and 1 mm tcep , and were frozen by rapid immersion in liquid nitrogen .
the full - length wild - type rsv in(1286 ) , the c - terminally truncated rsv in(1270 ) , and its various mutant forms tested produced essentially the same crystals with indistinguishable x - ray diffraction properties .
x - ray diffraction data were collected at the advanced photon source northeastern collaborative access team beamlines ( 24-id - c / e ) and the advanced light source molecular biology consortium ( 4.2.2 ) beamline and processed using hkl2000   or xds .
the rsv intasome crystals showed varied degrees of pseudo - merohedral twinning with twin operator [ l , k , h ] , owing to the very similar a and c unit cell dimensions of the primitive monoclinic lattice .
thus , we screened a large number of crystals to identify ones that diffract to higher resolution and have smaller twin fractions .
the structure of rsv intasome was determined by molecular replacement with phaser , using the rsv in ccd , ctd ( pdb i d : 4fw1 ) , and a 16 bp b - form dna as search models .
8 copies of ccd , one copy of ctd and three copies of dna molecules were located .
refinement of the partial model revealed electron density for two copies of the metatungstate clusters .
subsequent iterative model building using coot   and refinement with phenix suite   allowed placement of the remaining 7 copies of ctd , 8 copies of ntd generated using modeller   based on hiv-1 ntd ( pdb i d : 1k6y ) , and building of the inter - domain linkers as well as the remaining parts of the dna molecule guided by the difference electron density maps .
a third metatungstate cluster , which is more weakly bound compared to the first two , was positioned manually into residual density .
the dna base - pairs and base - stacking restraints were used throughout the refinement .
the geometry restraints for protein included the reference - model restraints for ccd and ctd based on the higher resolution rsv in structure ( 4fw1 ) , and the secondary structure and zinc - coordination restraints for ntd .
atomic displacement parameters were refined with grouped b - factors per residue for protein and dna , and a total of 53 tls groups assigned by phenix .
the asymmetric unit of the crystal contains one complete rsv intasome , which includes eight in molecules and two viral dna branches emanating from a strongly bent 38 bp target dna .
the dataset used for the final refinement was from an rsv intasome crystal grown using selenomethionine - labeled rsv in ( 1270 ) with the following amino acid substitutions ; c23s , l112 m , l135 m , l162 m , l163 m , l188 m , and l189 m , which we confirmed to be active in concerted integration and inhibited by insti similarly to the wild - type rsv in , and a dna substrate carrying a nick at the middle of each target dna branch ( the 16-base dna strand shown in olive in fig .
the nick occasionally facilitated crystal growth , but it was not necessary for crystallization and did not change the space group or the unit cell parameters compared to crystals grown without the nick .
because this nick in the target dna is not biologically relevant , it is not shown in figs .
twin refinement protocol was not used as the dataset used for the final refinement had a low ( less than 10% ) twin fraction .
the summary of data collection and refinement statistics is shown in extended data table 1 .
the paired - refinement procedure   was performed in steps of 0.1  to determine the high - resolution limit ( extended data fig .
the register of amino acids in the final model was verified by the selenium anomalous difference fourier peaks ( extended data fig .
ramachandran analysis shows that 96.0 , 3.9 , and 0.1 % of the protein residues are in the most favored , allowed , and disallowed region , respectively .
the ntd - ccd linker in some of the non - catalytic in molecules and the last four bp at the distal end of one of the viral dna molecules were not built due to poor electron density .
rsv in and its mutant forms were overexpressed in escherichia coli bl21 ( de3 ) and purified as previously described .
the proteins were stored in aliquots at 80c in 20 mm hepes - naoh ( ph 7.5 ) , 1.0 m nacl , 20m zncl2 , 5 mm 2-mercaptoethanol and 10% ( w / v ) glycerol .
the branched dna substrate mimicking the product of the concerted integration reaction was obtained by annealing three synthetic oligonucleotides ( integrated dna technologies ) .
a similar strategy had been used previously to prepare a pfv in - dna complex , which demonstrated that the in - dna complex assembled on the designed integration product has essentially an identical structure to the equivalent complex formed via the forward integration reaction .
the viral dna branches carrying the high - affinity gain - of - function mutant rsv u3 long terminal repeat ( ltr ) sequence ( gu3 )   were attached to the target dna duplex with a palindromic 6 bp spacer to generate a fully symmetrized structure . to prepare the rsv in - dna complexes for crystallization , 30 m half - site dna substrate was mixed with 120 m rsv in in 20 mm hepes - naoh ( ph 7.5 ) , 500 mm nacl , 20% ( w / v ) glycerol , and 1 mm tris-(2-carboxyethyl)phosphine ( tcep ) .
the mixture was dialyzed against low salt buffer ( 20 mm hepes - naoh ph 7.5 , 125 mm nacl , 20% ( w / v ) glycerol , 1 mm tcep ) at 25c for overnight . at the end of this first dialysis ,
the mixture was subsequently dialyzed against high salt buffer ( 20 mm mes - naoh ph 6.0 , 1.2 m nacl , 20% ( v / v ) dimethyl sulfoxide ( dmso ) , 5% ( w / v ) glycerol , 1 mm tcep ) at 25c for 24 hours . at the end of this second dialysis ,
the solubilized rsv in - dna complex ( intasome ) was purified through size - exclusion chromatography ( superdex 200 10/300 gl , ge healthcare ) running with 20 mm mes - naoh ph 6.0 , 1.2 m nacl , 20% ( v / v ) dmso , 5% ( w / v ) glycerol , and 1 mm tcep .
the isolated intasome remains stable in the high - salt condition containing 1.2 m nacl that precludes complex formation , suggesting that once the intasome forms , it is kinetically trapped .
the solubility - enhancing rsv in mutation f199k completely abolished intasome formation . for the sec - mals analysis
, a modified condition was used for the intasome re - solubilization and isolation for better baseline stability ( extended data fig .
for crystallization , the rsv intasome assembled through dialysis and purified by size - exclusion chromatography was concentrated to 46 mg / ml using centrifugal concentrator ( amicon ) .
various combinations of the lengths of the viral dna ( ranging from 16 bp to 25 bp ) and flanking target dna ( ranging from 14 bp to 22 bp , corresponding to the full target dna lengths of 34 bp to 50 bp ) were screened in crystallization trials .
the extensive screening yielded only one crystal form with a specific combination of 22-base ( length of the non - transferred strand ) viral dna branches and 16 base - pair ( bp ) target dna flanks on either side of the central six bp spacer ( fig .
dna substrates with two slightly different target sequences were used ( 5-aatgttgtcttatgcaatactc-3/5-gagtattgcataagacaacagtgcacgaatcttgaagacact-3/5-agtgtcttcaagattc-3 , or 5-aatgttgtcttatgcaatactc-3/5-gagtattgcataagacaacagtcgaccaaccttcaacttagc-3/5-gctaagttgaaggttg-3 ) , and they produced essentially the same crystals .
the rsv intasome crystals were grown through reverse vapor diffusion in hanging drops at 22c , by mixing 1.5 l in - dna complex solution with 1.5 l reservoir solution ( 3.2 m sodium formate ) .
crystals appeared within 35 days and reached a size of ~150300 m in 3~5 days . even though the rsv intasome crystals initially diffracted x - ray poorly ( ~ 10  ) , soaking the crystals with a metatungstate cluster compound dramatically improved the resolution ( extended data fig .
the tungsten cluster was later found to bind between in dimers from separate intasome complexes to mitigate crystal lattice disorder .
the crystals were soaked overnight with 0.15 mm metatungstate cluster na6[h2w12o40 ] in a stabilization buffer consisting of 3.2 m sodium formate , 16 mm mes - naoh ph 6.0 , 0.8 m nacl , 16% ( v / v ) dmso , 4% ( w / v ) glycerol , and 1 mm tcep . after soaking ,
the crystals were cryo - protected in 3.2 m sodium formate , 16 mm mes ph 6.0 , 0.8 m nacl , 16% ( v / v ) dmso , 12% ( w / v ) glycerol , and 1 mm tcep , and were frozen by rapid immersion in liquid nitrogen .
the full - length wild - type rsv in(1286 ) , the c - terminally truncated rsv in(1270 ) , and its various mutant forms tested produced essentially the same crystals with indistinguishable x - ray diffraction properties .
x - ray diffraction data were collected at the advanced photon source northeastern collaborative access team beamlines ( 24-id - c / e ) and the advanced light source molecular biology consortium ( 4.2.2 ) beamline and processed using hkl2000   or xds .
the rsv intasome crystals showed varied degrees of pseudo - merohedral twinning with twin operator [ l , k , h ] , owing to the very similar a and c unit cell dimensions of the primitive monoclinic lattice .
thus , we screened a large number of crystals to identify ones that diffract to higher resolution and have smaller twin fractions .
the structure of rsv intasome was determined by molecular replacement with phaser , using the rsv in ccd , ctd ( pdb i d : 4fw1 ) , and a 16 bp b - form dna as search models .
8 copies of ccd , one copy of ctd and three copies of dna molecules were located .
refinement of the partial model revealed electron density for two copies of the metatungstate clusters .
subsequent iterative model building using coot   and refinement with phenix suite   allowed placement of the remaining 7 copies of ctd , 8 copies of ntd generated using modeller   based on hiv-1 ntd ( pdb i d : 1k6y ) , and building of the inter - domain linkers as well as the remaining parts of the dna molecule guided by the difference electron density maps .
a third metatungstate cluster , which is more weakly bound compared to the first two , was positioned manually into residual density .
the dna base - pairs and base - stacking restraints were used throughout the refinement .
the geometry restraints for protein included the reference - model restraints for ccd and ctd based on the higher resolution rsv in structure ( 4fw1 ) , and the secondary structure and zinc - coordination restraints for ntd .
atomic displacement parameters were refined with grouped b - factors per residue for protein and dna , and a total of 53 tls groups assigned by phenix .
the asymmetric unit of the crystal contains one complete rsv intasome , which includes eight in molecules and two viral dna branches emanating from a strongly bent 38 bp target dna .
the dataset used for the final refinement was from an rsv intasome crystal grown using selenomethionine - labeled rsv in ( 1270 ) with the following amino acid substitutions ; c23s , l112 m , l135 m , l162 m , l163 m , l188 m , and l189 m , which we confirmed to be active in concerted integration and inhibited by insti similarly to the wild - type rsv in , and a dna substrate carrying a nick at the middle of each target dna branch ( the 16-base dna strand shown in olive in fig .
the nick occasionally facilitated crystal growth , but it was not necessary for crystallization and did not change the space group or the unit cell parameters compared to crystals grown without the nick .
because this nick in the target dna is not biologically relevant , it is not shown in figs .
twin refinement protocol was not used as the dataset used for the final refinement had a low ( less than 10% ) twin fraction .
the summary of data collection and refinement statistics is shown in extended data table 1 .
the paired - refinement procedure   was performed in steps of 0.1  to determine the high - resolution limit ( extended data fig .
the register of amino acids in the final model was verified by the selenium anomalous difference fourier peaks ( extended data fig .
ramachandran analysis shows that 96.0 , 3.9 , and 0.1 % of the protein residues are in the most favored , allowed , and disallowed region , respectively .
the ntd - ccd linker in some of the non - catalytic in molecules and the last four bp at the distal end of one of the viral dna molecules were not built due to poor electron density .
the secondary structure elements for rsv in are color - coded based on the three in domains similarly to fig .
a , the half - site ( gaped duplex ) substrate prepared by annealing three oligonucleotides dimerizes via the self - complementary 6-base spacer sequence ( underlined ) to form a branched structure mimicking the product of the concerted integration reaction ( fig .
viral dna nucleotides are numbered , and some of the structural elements of rsv in involved in the viral dna interactions are shown .
c , x - ray diffraction pattern after the metatungstate - soaking ( see method for details ) .
is perpendicular to the two - fold screw ( b ) axis of the monoclinic lattice , which lies horizontally .
f , paired - refinement analysis   to assess the resolution limit of the rsv intasome diffraction data . for each pairwise comparison ,
model refinements were run at two different resolution limits and the r - factors calculated for a common ( lower ) resolution cutoff were compared .
a , a representative size - exclusion chromatography ( sec ) profile for rsv intasome , overlaid with that for a mixture of molecular weight markers .
b , sec profiles for rsv intasomes formed with in of varying c - termini .
c , sec - mals ( multi - angle light scattering ) analysis of rsv intasome .
the intasome formed with rsv in ( 1269 aa ) was separated by sec in a modified condition containing 20 mm hepes ph 7.5 , 1.0 m nacl , 5 % glycerol , and 1.0 mm tcep .
the absolute molecular mass was determined by light scattering using in - line detectors described previously .
a similar sec - mals analysis of the intasome formed with wt full - length rsv in ( 1286 aa ) yielded a molecular mass of 268 kda ( n=2 , data not shown ) .
the calculated mass of an intasome containing eight rsv in ( 1269 or 270 ) molecules is ~288 kda .
d , chemical cross - linking analysis of rsv intasome . the rsv intasome and free in ( 1269 aa ) were purified by size - exclusion chromatography in the running buffer : 20 mm hepes ( ph 7.5 ) , 1.0 m nacl , 5 % glycerol , and 1.0 mm tcep .
the peak fractions of the intasome and in were cross - linked with the indicated amount of ethylene glycol bis - succinimidylsuccinate ( egs ) as described previously   and analyzed by sds - page .
the majority of cross - linked species within the intasome were larger than a tetramer .
the nupage 412% gradient gel with a mes - based sds - page running buffer was used .
anomalous difference fourier maps calculated using the data collected on selemomethionine - labeled rsv intasome , contoured at 3.5 ( blue mesh ) or 5.0  ( orange mesh ) .
b , c , close - up view of a proximal ( b ) and distal ( c ) in dimer , respectively . a , b , protein arrangement in the octameric rsv intasome .
the inner and outer subunit of one proximal in dimer is colored in green and cyan , respectively , with the catalytic triad ( dde ) of the inner subunit shown in red .
c , a close - up view around the active site of the inner in subunit in the rsv intasome .
the dna strands are colored as in fig . 1 , and the catalytic triad residues ( dde )
the color scheme follows that used for the proximal in dimers of rsv in in ( a , b ) .
f , a close - up view around the active site of the inner in subunit in the pfv intasome ( pdb i d : 3os0 ) .
simulated annealing composite omit 2mfodfc density contoured at 1.0  , shown for area within 3.5  from any protein or dna atom in the final model . in a and b , electron densities around protein and dna are colored differently ( blue and green , respectively ) . a , b , dna structure in the rsv ( a ) or pfv ( b ) intasome , alternatively referred to as the strand - transfer complex ( stc ) .
c , a comparison of dna structures between the rsv and pfv intasomes ( stcs ) . the integration product dnas ( rsv in cyan , pfv in red ) superimposed at a viral dna terminus are shown in three different view angles . note the significant deviation in overall trajectory of the target dna , and difference in the orientation of the second viral dna molecule .
the region spanning the two integration sites on opposing target dna strands is 6 bp for rsv and 4 bp for pfv .
the sigma a - weighted 2mfodfc map contoured at 1.5  ( a ) or 2.5  ( b ) , overlaid with the final model for the central 6 bp region between the two integration sites .
data collection and refinement statistics statistics for the highest resolution shell are shown in parentheses .
a video showing the overall structure of the rsv intasome and positioning of the three structural domains of in within the intasome .
 integration of the reverse - transcribed viral dna into the host genome is an essential step in the lifecycle of retroviruses . 
 retrovirus integrase ( in ) catalyzes insertions of both ends of the linear viral dna into a host chromosome 1 . in from hiv-1 and closely related retroviruses 
 share the three - domain organization , consisting of a catalytic core domain flanked by n- and c - terminal domains essential for the concerted integration reaction . 
 although structures of the tetrameric in - dna complexes have been reported for in from prototype foamy virus ( pfv ) featuring an additional dna - binding domain and longer interdomain linkers 25 , the architecture of a canonical three - domain in bound to dna remained elusive . here 
 we report a crystal structure of the three - domain in from rous sarcoma virus ( rsv ) in complex with viral and target dnas . 
 the structure shows an octameric assembly of in , in which a pair of in dimers engage viral dna ends for catalysis while another pair of non - catalytic in dimers bridge between the two viral dna molecules and help capture target dna . 
 the individual domains of the eight in molecules play varying roles to hold the complex together , making an extensive network of protein - dna and protein - protein contacts that show both conserved and distinct features compared to those observed for pfv in . 
 our work highlights diversity of retrovirus intasome assembly and provides insights into the mechanisms of integration by hiv-1 and related retroviruses . 
METHODS
RSV intasome preparation
Crystallization
Structure determination
Extended Data
Supplementary Material