it is essential for faithful partitioning of replicated chromosomes into nascent daughter cells during cell division . multisubunit complexes , termed condensins , are key mediators of this process ( hirano , 2012 ; thadani et  al . , 2012 ) . they commonly have a large core subunit ( > 1,100 amino acids ) that belongs to the family of structural maintenance of chromosome proteins ( smc ; in capital letters , used as a generic term for protein family ) . several types of condensins have been identified : three condensins in prokaryotes , smc - scpab , mukbef , and mksbef ; and two eukaryotic condensins , condensin i and condensin ii ( condensin i / ii ) . smc - scpab comprises a homodimer of smc and the two non - smc subunits scpa and scpb ( mascarenhas et  al . 2002 ) , whereas mukbef comprises a homodimer of the smc subunit mukb and the two non - smc subunits muke and mukf ( woo et  al . , 2009 ; smc - scpab is nearly ubiquitous in prokaryotes and more closely related to condensin in eukaryotes than mukbef , which is found in only some branches of -proteobacteria . i / ii are composed of the same heterodimer of smc2 and smc4 ( smc2 - 4 ) and a different set of three non - smc subunits ( onn et  al . , 2007 ) . the smc subunit exhibits a peculiar folding pattern : the extreme n- and c - terminal segments together form an abc - type nucleotide binding domain ( also called smc head ) , a middle segment folds into a so - called hinge domain , and the two intervening segments form an 50-nm - long antiparallel coiled coil connecting the two domains ( nolivos and sherratt , 2014 ) . the hinge domain is the interaction interface for the homo- or heterodimerization of smc subunits ( haering et  al . , 2002 ) . one of the non - smc subunits generally belongs to a superfamily of proteins called kleisins , which bind and bridge the head domains of the smc subunits ( schleiffer et  al . , 2003 ) . in smc - scpab condensin , the kleisin subunit scpa binds two distinct interfaces on and near the smc head domain to form a 1:1 asymmetric holocomplex between the smc dimer and the scpa1b2 subcomplex ( brmann et  al . , 2013 ) . likewise , in condensin i / ii , the head domains of the smc2 - 4 heterodimer are presumably bridged by the kleisin subunit cap - h / h2 that associates with two additional subunits , cap - g / g2 and cap - d2/d3 ( hirano , 2012 ) . the eukaryotic smc1-smc3 ( smc1 - 3 ) cohesin complex , which is evolutionarily related to condensin , is a chromosome concatenase that holds sister chromatid dna within its closed ring structure ( nasmyth , 2011 ) . striking architectural similarities between different smc complexes suggest that they all function using a fundamentally conserved mode of action ( brmann et  al . , 2013 ) . consistent with this notion , eukaryotic condensin , like cohesin , associates with minichromosomes by entrapment of dna within its ring ( cuylen et  al . the distant hinge and head domains are involved in the loading of smc - kleisin complexes onto dna . atp hydrolysis by smc1 and smc3 head domains is essential for stable binding of cohesin to chromosomes , whereas the smc hinge domains harbor affinity for dna in cohesin , condensin , and bacillus subtilis ( bs ) smc - scpab ( arumugam et  al . , 2003 ; chiu et  al . , 2004 ; griese et  al . , 2010 ; dna binding stimulates atp hydrolysis in smc - scpab and condensin in  vitro ( hirano and hirano , 2006 ; kimura and hirano , 1997 ) , and hinge opening appears to be required for loading dna into cohesin rings ( gruber et  al . , 2006 ) . whether ( and how ) atp binding and hydrolysis at the smc heads might be mechanistically coordinated with dna binding to the hinge and opening of the dna entry gate is largely unclear . conceivably , the coiled - coil arms could provide a mechanical link if they were somewhat stiff and rigidly connected to hinge and/or head domains . according to electron microscopy ( em ) and atomic force microscopy , the coiled coils in smc dimers and smc holocomplexes are not in random conformations but mostly v or o shaped or juxtaposed onto each other over their entire length ( anderson et  al . , 2002 ; fuentes - perez et  al . , 2012 ; haering et  al . , 2002 ; matoba et  al . , 2005 ; melby et  al . , 1998 ) . in the crystal structure of the thermotoga maritima ( tm ) smc hinge , two short coiled coils protrude from the hinge domain dimer in nearly opposite orientations ( haering et  al . , similar coiled - coil configurations were found in crystal structures of the mukb hinge domain ( ku et  al . , 2010 ; li et  al . , 2010 ; vos et  al . , 2013 ) . the observed variety in the conformations of smc coiled coils might be partly , or entirely , due to ( 1 ) intrinsic structural flexibility , ( 2 ) structural differences between classes of smc - kleisin complexes , or ( 3 ) experimental artifacts . thus , it is largely unclear what configurations smc - kleisin rings adopt on the chromosome , or in solution , and whether conformational changes are required during chromosomal loading and unloading cycles . using an integrative approach including crystallographic analyses of smc protein fragments with long stretches of coiled coil , we demonstrate that both prokaryotic and eukaryotic condensins form rod - shaped holocomplexes with rigid and juxtaposed coiled coils . we further reveal that binding of dna to smc dimers is incompatible with the reported coiled - coil arrangement at the hinge and uncover an interplay between dna and atp binding in the dynamic control of smc arm conformation . these findings allow us to propose a mechanism by which the atpase head domains regulate dna binding to the hinge via engagement and disengagement of smc coiled coils . crystallographic studies supported by biochemical and genetic data have provided detailed insights into the structures of all globular parts of prokaryotic smc - scpab complexes . however , our understanding of the overall architecture of smc - scpab and , in particular , the arrangement of the smc coiled coils in the holocomplex of smc - scpab has remained rather limited so far . here , we have purified bs smc protein as well as smc - scpab holocomplexes produced in escherichia coli ( ec ) . as determined by size exclusion chromatography - multiangle light scattering ( sec - mals ) , our preparations comprise near homogenous solutions with molecular weights fitting well to isolated smc dimers and heteropentameric smc2-scpa1b2 complexes ( figure  s1 available online ) . the proteins were negatively stained and visualized by em ( figures 1a and 1b ) . smc dimers and smc - scpab holocomplexes were almost exclusively detected as straight objects comprising a single extended rod flanked by a small and a large globular density , which likely correspond to the smc hinge and head domains with or without scpab . in good agreement with data obtained for bs smc protein by rotary shadowing experiments , these images suggest that the two smc coiled coils are mostly aligned side by side ( melby et  al . , 1998 ) . we were concerned that the observed rod - like structure might arise during the harsh conditions used for em sample preparation . therefore , we probed the configuration of the smc coiled coils under more physiological conditions by estimating the distance between symmetry - related positions on the two coiled coils in solution using fluorescence resonance energy transfer ( fret ) . we produced a bs smc fragment comprising the smc hinge domain and a long stretch of coiled coil ( 100 residues ) , designated as bssmch - cc100 ( figure  1c ) . a single cysteine residue , cys437 , on the coiled coil was used for stochastic labeling with donor ( cy3 ) and acceptor ( cy5 ) dyes ( figure  1c ) . in order to discriminate the fret pair ( cy3-cy5 dimer ) from any non - fret pair ( cy3-cy3 and cy5-cy5 dimers ) in the sample , the single - molecule alternating - laser excitation fret ( alex - fret ) method was applied . the cy3-cy5 dimer species exhibited predominantly higher values of fret efficiency , e , indicating that the two coiled coils are close to each other in most or all dimers of bssmch - cc100 ( figure  1c ) . based on the observed fret efficiency , the distance between cy3 and cy5 was estimated to be around 44   ( e  = 0.86 ) or approximately twice the diameter of a coiled coil . similar experiments performed on a related fragment of the mukb protein , which exists in an open v conformation , demonstrated the validity of our fret approach ( figure  1d ) . furthermore , low real - time fluctuations in single - molecule total internal reflection fluorescence ( tirf)-fret suggest that the coiled coils of bs smc hinge fragments are mostly or always closely juxtaposed ( figure  s1 ) . to elucidate the molecular basis for the alignment of smc coiled coils , we determined the atomic structure of a fragment of pyrococcus furiosus ( pf ) smc containing its hinge and a significant part of its coiled coil . after we screened several constructs with different lengths of coiled coil , crystals of a pf smc hinge domain with a 60-residue stretch of coiled coil , referred to as pfsmch - cc60 , were obtained , and the phase problem was solved by molecular replacement using the structure of an isolated pf smc hinge ( table 1 ) ( griese and hopfner , 2011 ) . the asymmetric unit of the crystal contained two copies of the pfsmch - cc60 homodimer . as expected , the two hinge domains in the homodimer interact with each other at two identical interfaces to form a toroidal structure having a flat bottom and a central hole with the largest dimension of 16   ( figure  2a ; figure  s2a ) . the two coiled coils are aligned in parallel and are closely juxtaposed onto each other , thereby forming a rod - like overall structure . the coiled coils emanate from the smc hinge perpendicular to the bottom surface of the toroid , reminiscent of tentacles radiating from the body of a jellyfish . this shape is a result of a sharp 90 kink , governed by a single glycine residue ( gly507 ) , at the junction between the n - terminal helix of the coiled coil and the following  rooting helix 2 , which interacts with a hydrophobic groove at the bottom of the toroid ( figure  2b ) . the presented architecture of the pfsmch - cc60 homodimer is in sharp contrast with the v - shaped organization of the tm smc hinge with short coiled coils ( discussed later ) and of the ec mukb hinge with long coiled coils ( figure  s2b ) ( li et  al . , 2010 ) . right below the bottom surface of the pf smc hinge , the two n - terminal coiled - coil helices of the homodimer pack against each other and engage in hydrophobic contacts ( figure  2c ) . in addition , 80   ( or 50 residues ) below the toroid , the two c - terminal coiled - coil helices are in contact with each other in a similar fashion ( figure  2c ) . this two - site interaction is found in both dimers of the asymmetric unit and appears to be responsible for holding the coiled coils together . curiously , the two smc coiled coils within a dimer display slightly distinct angles of attachment to the hinge , thus creating an asymmetric overall architecture ( figure  2a , right panel ) . this asymmetry might allow a more stable interaction to be formed at the coils / coils interface . the coiled coils emanating from the smc hinge adopt juxtaposed configurations in fragments of bs and pf smc . however , multiple sequence alignments indicate that residues at the interfaces between the coiled coils in pf smc are not particularly well conserved ( figures 3a and 3b ) , raising the questions of whether this coils / coils interface is specific to archaeal smc proteins or a general feature conserved through coevolution of pairs of residues . to test this , we probed the conformation of the coiled coils in endogenous holocomplexes of prokaryotic condensin using cysteine - specific crosslinking in living bs cells . based on the structure of pfsmch - cc60 , we engineered single - cysteine residues into the predicted coils / coils interfaces of bs smc . as the coils / coils interface is located along the 2-fold symmetry axis , crosslinking of cysteines by the thiol reactive compound bmoe will occur between symmetry - related cys residues , which are in close proximity ( < 8   ) . to determine crosslinking efficiencies , we made use of a c - terminal halotag fusion to smc permitting in - gel fluorescence detection of smc species ( brmann et  al . , 2013 ) . based on sequence alignments and coiled - coil predictions , three residues in the n - terminal helix of the smc coiled coil were chosen to be mutated to cysteine ( d491c , m492c , and t495c ) ( figure  3a ) . all  three mutant smc proteins are functional , as judged by growth on rich medium ( data not shown ) ( gruber et  al . , 2014 ) . they displayed significant levels of smc - smc crosslinking after incubation with bmoe , whereas a wild - type control showed little or no crosslinking ( figure  3c ) . thus , the selected residues are located in close proximity of their symmetry mates , as predicted by the pfsmch - cc60 but not the tm smc hinge structure ( figure  3a ) . the alignment of sequences in the c - terminal helix of the smc coiled coil is more ambiguous ; therefore , several residues were mutated to cysteines in a region about 4560 residues from the smc hinge domain . three cysteine residues ( q708c , k712c , and d716c ) showed very little smc - smc crosslinking likely because their side chains are too far apart or facing opposite sides of the coils / coils structure . in stark contrast , residues a715c and e722c supported robust crosslinking of smc ( figure  3c ) . in summary , efficient crosslinking by specific cysteine residues demonstrates that the coiled coils emanating from the smc hinge are held together by a defined interface in endogenous smc - scpab complexes . given the fact that several smc hinge domains display dna binding affinity , we wondered whether the coiled - coil configuration would have any influence on the association of smc with dna . dna binding activity of smc hinges has been tentatively mapped to positively charged residues in the transition region between the hinge and the coiled coils in cohesin and smc - scpab ( chiu et  al . accordingly , bound dna might be located at the bottom surface of the hinge toroid ( figure  4a ) . intriguingly , this area is obstructed in our pf smc structure by the presence of the aligned coiled coils , thus highlighting the possibility that a conformational change at the smc hinge might control its association with dna . using fluorescence anisotropy measurements with short stretches of double - stranded dna ( dsdna ) , we first confirmed that bssmch - cc100 displays affinity for dna in the submicromolar range ( dissociation constant , kd , 0.1  m ) ( figure  4b ) . consistent with the notion that the transition region is involved in dna binding , we found that an isolated bs smc hinge ( bssmch ) lacking this region fails to bind to dna , whereas a slightly larger construct ( bssmch - cc8 ) binds dna ( kd , 0.2  m ) with an affinity similar to that of bssmch - cc100 . a smc protein fragment harboring almost the entire smc coiled coil attached to the hinge domain , designated as bssmch - cc300 , associated with dna only poorly , indicating that the long coiled coils interfere with efficient dna binding at the hinge , possibly because of stable occlusion of the dna binding site ( figure  4b ) . next , we wondered whether the coiled coils are still juxtaposed when bssmch - cc100 is bound to dna . to test this , we have purified bssmch - cc100 harboring t495c or a715c for crosslinking of smc coiled coils . intriguingly , formation of crosslinked dimers of bssmch - cc100 was strongly affected by the presence of dna ( figure  4c ) . in contrast , dna binding had no effect on the crosslinking of a pair of cysteines located at the hinge dimer interface ( r558c / n634c ) ( brmann et  al . , 2013 ) ( figure  4c ) . furthermore , a cysteine pair at the coils / hinge intersection ( q532c / s676c ) displayed a modest but reproducible increase in intramolecular crosslinking in the presence of dna ( increased from 50%   1% to 60%   1% ) , implying that this cysteine pair might preferentially capture the more open conformation of the coiled coils ( figures 4c and s3d ) . we next repeated the crosslinking of a715c using bis - maleimide compounds having long linkers between the reactive groups ( bm - peg3 , 30   ; and bm - peg11 , 55   ) , which are able to capture more distant pairs of cysteines . bm - peg3 and bm - peg11 exhibited robust crosslinking of a715c in the absence of dna but failed to do so in the presence of dna ( figure  s3 g ) , implying that the a715c residues are too distantly located to be bridged by either bm - peg3 or bm - peg11 when bssmch - cc100 is bound to dna . as positive control for long - distance crosslinking , we created a pair of cysteines ( r516c , s597c ) located about 30   apart from each other on the  smc hinge domain . as expected , this cysteine pair was efficiently crosslinked by bm - peg11 , but not by bm - peg3 or bmoe , regardless of the presence or absence of dna ( figure  s3 g ) . together , these findings strongly suggest that dna binding stabilizes an open conformation of the coiled coils at the smc hinge . intriguingly , this immediately implies a molecular model of regulated dna binding by smc : atp binding or hydrolysis at the smc heads might facilitate opening of smc arms and thus expose the dna binding site at the hinge . to test this , we purified full - length bs smc and cysless smc(a715c ) and performed dna binding and crosslinking studies in the absence and presence of atp . without atp , smc imposed only a weak effect on the fluorescence anisotropy of dna , indicating that the dna binding site at the hinge is at least partly occluded in full - length smc protein ( figure  4d ) . in the presence of atp , however , the anisotropy response was substantial , producing an affinity ( kd , 0.1  m ) similar to that of smc hinge fragments . exclusively under these conditions ( i.e. , with dna and atp ) , crosslinking of smc arms at a715c was strongly reduced ( figure  4e ) . a hydrolysis - defective smc mutant ( e1118q ) displayed normal smc arm opening , whereas a mutant blocked in smc head engagement ( s1090r ) was locked in the rod - like state , suggesting that atp - dependent head engagement drives dissolution of smc rods ( figure  4e ) . thus , smc arms undergo an extended structural transition , which is cooperatively promoted by binding of dna to the smc hinge and atp to the head domains . if the observed conformational change was physiologically relevant , then locking smc - scpab in the open or closed configuration should jeopardize its functionality . because of the extensive nature of the coils / coils interface and the rigid connection to the hinge , mutations in single residues are unlikely to have significant impact on the overall architecture . thus , we decided to replace parts of the bs smc protein with homologous protein fragments , which might intrinsically bear higher propensity for one or the other conformation . the tm hinge was chosen because it adopts an open , v - shaped organization in protein crystals . with the help of available structural information , a chimeric protein was constructed by splicing together n- and c - terminal sequences of bs smc with the central part of tm smc comprising its hinge domain and short stretches of the adjacent coiled coil ( figure  5a ) . it accumulated at normal levels in  vivo and efficiently formed smc dimers according to crosslinking analysis , implying that protein folding was mostly unperturbed ( data not shown ) . however , its functionality was severely compromised , as judged by colony formation assays ( figure  5a ) ( gruber et  al . , 2014 ) . to identify the underlying cause for this loss of function in bssmctmh , we mutagenized its tm hinge moiety and isolated suppressor mutations that enabled normal growth on rich medium ( figure  s4a ) . most suppressor mutations mapped in the vicinity of the connection between the tm smc hinge and the adjacent coils , suggesting that these mutations might indeed provide increased structural flexibility at the coils / hinge interface ( figure  5b ) . although the tm smc hinge bound dna in a more salt - sensitive manner than the bs smc hinge , the suppressor mutation s535n had no effect on the dna binding of a chimeric smc hinge fragment with long coiled coils ( figure  s4b ) . thus , defects in dna binding at the tm hinge are an unlikely explanation for the loss of functionality in bssmctmh . to measure the juxtapositioning of smc arms in chimeric smc proteins , we next fused a c - terminal halotag to bssmctmh and introduced the cysteine residue a715c for crosslinking . crucially , the arms of the nonfunctional bssmctmh protein were only poorly crosslinked by bmoe , suggesting that the tm hinge domain in bssmctmh promotes a more open coiled - coil arrangement , as suggested by its crystal structure ( figure  5c ) . notably , the suppressed version , bssmctmh(s535n ) , displayed wild - type levels of smc arm crosslinking . thus , the ability to efficiently adopt the rod - shaped conformation appears crucial for smc function . in combination with the observation that dna binding stabilizes the open form , this finding strongly supports the notion that both open and closed conformations are crucially important for condensin function . we propose that transitions from rod - like to ring - like states and vice versa are essential for the biochemical action of smc - scpab . eukaryotic condensin has been observed as a rod - like structure by em , suggesting that the architecture of the coils / hinge connection might be conserved between pro- and eukaryotic smc complexes . the structure of a mouse smc2 - 4 hinge heterodimer has recently been solved ( griese et  al . , 2010 ) . however , because of the lack of coiled coils , no insight into their arrangement with respect to the hinge was gained . to address this , we generated a number of yeast smc2 and smc4 constructs containing the hinge domain and long stretches of coiled coil . of these , smc2 ( residues 396792 ) and smc4 ( residues 555951 ) , referred to as scsmc2h - cc110 and scsmc4h - cc110 , respectively , were crystallized as a heterodimeric complex ( figure  6a ) . the coiled - coil stretches in these proteins correspond to about 150- - long  helices or approximately one third of the entire length of the smc2 and smc4 coiled coils . the hinge domains of scsmc2h - cc110 and scsmc4h - cc110 together form a toroid structure having a central hole similar to the counterparts in smc - scpab and cohesin ( figure  6b ) ( haering et  al . 2011 ) . strikingly , however , the segments connected to the coiled coils are very different between the two subunits in their secondary structures and arrangement ( figures 6c and 6d ) . opposite orientations of the coiled coils with regard to their hinge domain make them run in parallel upon heterodimerization of smc2 and smc4 to produce a highly asymmetric , folded rod - like overall structure . the coiled coil of scsmc4h - cc110 is entirely visible and extends out by about 150   . in case of scsmc2h - cc110 , about half of its coiled coil is visible in the electron density map ( figure  6a ) . the end of the smc4 coiled coil is involved in crystal packing , whereas that of the smc2 coiled coil is not . does the crystal structure faithfully reflect a conformation adopted by condensin holocomplexes isolated from yeast ? to test this , we probed the complex by site - specific crosslinking with bmoe . we identified pairs of residues at the coils / coils interface of smc2 and smc4 that are in close proximity in the crystal structure and mutated them to cysteines ( figure  6e ) . for some cysteine combinations , the endogenous cys494 in smc2 was replaced by serine to prevent interference with the assay . cysteine mutations were combined with a halotag on smc2 and a pk6 tag on smc4 and introduced into the respective endogenous genetic loci of a haploid yeast strain . the modified genes were expressed as the sole source of smc2 and smc4 and supported viability , indicating that condensin remained functional . we then used antibodies against the pk epitopes on smc4 to immunoprecipitate holocomplexes from asynchronous cultures . immobilized complexes were treated with the crosslinker bmoe and conjugated to the halotag - tetramethylrhodamine ( tmr ) substrate . subsequently , crosslinked species of smc2-halotag were detected by in - gel fluorescence ( figure  6f ) . when wild - type complexes were treated with similarly , only insubstantial crosslinking was observed with cysteine pairs when their thiol group distance exceeded the 8   linker length of bmoe . in contrast , introduction of more closely positioned pairs of cysteines promoted robust crosslinking of smc2 and smc4 ( figure  6f ) . these data strongly suggest that the conformations of the coiled coils observed by x - ray crystallography are adopted by native condensin holocomplexes . quite extensive hydrophobic interactions are found at the coils / hinge interface in condensin , a feature that is much less pronounced or lacking in prokaryotic condensin . for description , we designate the n - terminal -helix of the smc2 coiled coil as n-h and the c - terminal -helix as c-h ; likewise , we designate those of the smc4 coiled coil as n-h and c-h . in the case of the smc4 coiled coil , n-h is longer than c-h , and the last part of n-h ( residues 665682 ) does not interact with c-h but with the smc4 hinge domain . this -helical segment of smc4which , in analogy to the pf structure , we call  rooting -helixhas at least three hydrophobic residues ( val672 , leu676 , and leu679 ) that interact with a hydrophobic groove on the smc4 hinge domain ( figure  6c ) . these interactions are quite extensive and appear to be responsible for fixing the position and the orientation of the smc4 coiled coil . notably , the key hydrophobic residues involved in the smc4 hinge / coiled coil interaction are conserved throughout eukaryotic condensins . consistent with this notion , we found that mutation of hydrophobic residues l676 or l731located at the smc4 hinge / coil interface  to aspartate rendered the protein nonfunctional in yeast ( figures s5a  s5f ) . the smc2 coiled coil associates with the hinge heterodimer at a hydrophobic interface involving leu676 , leu677 , and ile680 at the beginning of c-h and leu507 , phe531 , ile533 , leu552 , and phe553 on the smc2 hinge domain ( figure  6d ) . however , single mutations in the residues in c-h did not result in any obvious growth defects ( figures s5g  s5i ) . nevertheless , the high level of sequence conservation suggests that the observed conformations of the smc2 and smc4 coiled coils relative to the hinge domains are likely a general feature of condensin in eukaryotes . in addition to the coils / hinge interactions , also coils / coils interactions are found . the smc2 coiled coil is in contact with the smc4 coiled coil at one site via three exposed hydrophilic interactions and one ring - to - ring stacking interaction ( figure  s5k ) . the hydrophilic interactions are solvent exposed , and the contacting helices are not tightly packed against each other , unlike those observed in the pfsmch - cc60 structure ( compare figures 2c and s5k ) . therefore , these coils / coils interactions appear to be a result , rather than a cause , of the juxtaposition of the coiled coils , although they might reinforce the parallel orientations of the coiled coils . the scsmc2h - cc110/scsmc4h - cc110 complex binds dsdna with high affinity ( kd 50  nm ; measured by fluorescence anisotropy ) . however , the crosslinking of two pairs of cysteines [ smc2(k487c)-smc4(e876c ) and smc2(k495c)-smc4(e866c ) ] at its coils / coils interface is unaffected by the presence of short dna molecules ( 40 base pairs [ bp ] ) ( data not shown ) . a surface potential map of the smc2 - 4 structure features a prominently positively charged area on top of the smc2 - 4 hinge ( figure  s6e ) , suggesting that initial dna contact might occur at the top hinge surface in condensin . possibly , additional elements such as atp binding to the smc2 - 4 heads , non - smc subunits , loading factors , or nucleosomes might control any binding of dna to the bottom hinge surface and/or opening of condensin smc arms . to address the first two possibilities , we purified endogenous yeast condensin from exponentially growing or mitotically arrested populations of cells harboring smc2(k495c ) and smc4(e866c ) mutations by immunoprecipitation or affinity tag purification . purified fractions of yeast condensin were then incubated with bmoe crosslinker in the presence or absence of atp and short dna . however , no significant differences in crosslinking were detected under the various conditions ( data not shown ) . thus , these attempts failed to provide evidence for the opening of smc arms in yeast condensin in  vitro . several em studies depict cohesin and mukbef in wide open conformations . in case of the latter , the open architecture is further supported by crystal structures of three isolated hinge fragments , all displaying diametrically opposed coiled coils ( ku et  al . , 2010 ; li et  al . , 2010 ; vos et  al . , 2013 ) conceivably , cohesin and mukbef might be fundamentally different in their structure from both condensin and smc - scpab . alternatively , all smc complexes might require closed and open conformations during the course of action but might have distinct intrinsic preferences for these arrangements . to our surprise , we found that the coils / hinge junction in cohesin bears a strong resemblance to condensin . a crystal structure of the cohesin smc1 - 3 hinge heterodimer was reported previously ( kurze et  al . , 2011 ) . it contains a very short smc3 coiled coil and a short -helix of smc1 that corresponds to the rooting -helix of smc4 . structural comparison with scsmc2h - cc110/scsmc4h - cc110 shows that the short smc3 coiled coil is oriented similarly as the smc2 coiled coil and that the short -helix of smc1 points in the same direction as the rooting -helix of smc4 ( figure  7a ) . furthermore , most hydrophobic residues important for the coils / hinge interaction in the smc2 - 4 heterodimer are conserved in smc1 and smc3 proteins . to address whether cohesin smc1 - 3 complexes may indeed be able to adopt a rod shape , we generated a scsmc1h - cc100/scsmc3h - cc50 dimer and analyzed its structure using small - angle x - ray scattering ( saxs ) , a robust technique for characterization of macromolecular conformations in solution ( hura et  al . , 2009 ; rambo and tainer , 2010 , 2013 ) . as proof of principle , we initially performed saxs analysis on pfsmch - cc60 and an ec mukb hinge fragment with long coiled coils , designated as ecmukbh - cc80 . for both proteins , molecular envelopes fitted accurately to the rod - shaped or open v - shaped structures obtained by x - ray crystallography ( figures 7b7d ; figure  s6c ; table s3 ) . these findings demonstrate that the coils / hinge junctions have a defined structure in solution as previously indicated by fret analysis ( figures 1c and 1d ) . in addition , these experiments confirm the validity and suitability of saxs for the study of smc coils / hinge organization . next , we analyzed the architecture of the scsmc1h - cc100/scsmc3h - cc50 dimer by saxs . remarkably , the saxs envelopes derived for the cohesin fragment were clearly too short to accommodate an open v - shaped dimer as seen for ecmukbh - cc80 but were similar in size and shape to the condensin structure ( figures 7b7d ; figure first , a comprehensive lysine proximity map of purified human cohesin  based on the identification of crosslinked peptides by mass spectrometry by the jan - michael peters laboratory  revealed 19 juxtaposed pairs of smc1 and smc3 coiled - coil residues ( out of a total of 51 on cohesin ) ( huis in almost all these chemical crosslinks occurred between residues with similar position ( plus or minus ten amino acids ) along the length of the smc1 and smc3 coiled coils , being consistent with a well - defined , physical association between the two coiled coils ( j .- m . second , we found that the affinity of the human cohesin hinge for dna is reduced about 2-fold when 100 amino acids long coiled coils are attached to the smc1 and smc3 hinge domains , indicating that the dna binding site at the cohesin hinge might at least be partially occluded ( figure  s6d ) . although further studies are clearly necessary , these initial observations provide an indication that all smc complexes  with the possible exception of mukbef  might at least transiently adopt a rod - like structure with juxtaposed coiled coils . intriguingly , cohesin , condensin , and smc hinge domains , but not the mukb hinge ( ku et  al . , 2010 ) ( data not shown ) , display decent affinity for dna , highlighting the possibility of a conserved functional connection between hinge architecture and regulated association with dna . little information is available on the arrangement of the two long coiled coils in smc - kleisin complexes in  vivo . here , we start to fill this void by solving high - resolution x - ray structures of prokaryotic smc - scpab and eukaryotic condensin and by performing subsequent biochemical and genetic characterization . we identify close juxtapositioning of smc coiled coils at the hinge domain as a predominant architectural theme in smc complexes and establish a functional link between hinge structure and dna association . our work demonstrates how the long coiled coils in bs smc - scpab and yeast condensin are attached to their hinge domain dimers . the respective parts of these complexes share two striking structural features : the toroid - like hinge formed by homo- versus heterotypic interaction of two hinge domains and a four - helix bundle built by the intimate alignment of two smc coiled coils . the most pronounced difference between the two structures , however , is the orientation of the coiled coils with respect to the hinge - domain toroid . whereas the two coiled coils in the prokaryotic smc hinge are virtually symmetric and perpendicular to the hinge toroid , those in the eukaryotic hinge are highly asymmetric and roughly parallel to the plane of the bottom surface of the hinge toroid ( figure  s6a ) . the rooting -helix of the yeast smc4 hinge corresponds to 2 of the pf smc hinge . it can form a single straight -helix with the preceding n - terminal smc4 coiled - coil helix , as the smc2 coiled coil keeps the required space free by adopting a different orientation . in contrast , 2 and the n - terminal coiled coil helix in the pf smc hinge can not form a single continuous helix , because two such helices on the homodimer would inevitably clash with each other because of the molecular symmetry ( figure  s6b ) . the 90 bending at the junction between the two helices avoids this scenario and , instead , allows the coiled coils to stretch out in an i shape from the hinge toroid and juxtapose onto each other . apparently , as smc2 diverged from prokaryotic smc , it changed the orientation of its coiled coil drastically through a unique interaction between c-h and the hinge domain . it is important that , while eukaryotic smc proteins broke the symmetry at the hinge , they retained the parallel and juxtaposed organization of the coiled coils , thus underscoring its functional importance . the folded structure of prokaryotic smc - scpab and eukaryotic condensins may be a way to limit the total number of entrapped dna molecules within their circumference and/or to ensure that condensin would be occupied by selected dna fibers only at defined moments in its catalytic cycle . rather , smc - scpab complexes undergo marked transitions at the hinge between the folded rod and a more open ring - like configuration . the latter conformation is promoted by atp binding to smc heads and dna binding to the hinge , whereas the former seems to be an intrinsically more favorable resting state . em images suggest that smc arms are closely aligned along their entire length ( figure  1 ) . during the atp hydrolysis cycle , however , the head - proximal coiled coil might transiently become fixed in a conformation that is incompatible with coiled - coil juxtapositioning in this region ( haering et  al . , 2004 ) . this , in turn , might promote the progression of coiled - coil disengagement up to the hinge , as seen in our bmoe crosslinking experiments ( figure  4e ) . we propose that  the rod - like and ring - like configurations of smc - scpab resemble , in structural and functional terms , the inward- and outward - facing conformations of the related abc transmembrane transporters . in abc transporters , the transitions between these conformations are controlled by the occupancy of the substrate binding pocket , thus ensuring unidirectional transport of substrates across membranes via a defined series of conformational states ( oldham et  al . , 2008 ) . our results give an indication as to how binding of the substrate molecule , dna , might be restricted to the more open conformation of smc by exposure of an otherwise occluded interface for dna at the hinge . the hinge might thus serve as a sensor for dna that links smc arm architecture to the presence of dna . once dna is bound to the hinge , it might stimulate hydrolysis of atp by smc ( hirano and hirano , 2006 ) by simply promoting head engagement or through another long - range conformational change . the location of the dna binding site on the inner surface of the smc hinge also has strong implications on how dna might initially get in contact with smc - scpab rings . circular dna molecules , such as bacterial chromosomes , need to form loops within the circumference of a smc - kleisin ring so that dna can fully engage with the binding site at the inner face of the hinge ( figure  7e ) . it will be exciting to determine the fate of the hinge - bound stretch of dna  and the proposed dna loop reformation of smc rods will likely require the prior eviction of dna from between smc arms . we can think of two possible scenarios : ( 1 ) passage of dna toward the head domains or ( 2 ) exit of dna from the smc - scpa ring through a transiently opened hinge . the former could be related to processive extrusion of dna loops from the smc - scpab ring ( alipour and marko , 2012 ; nasmyth 2001 ) , whereas the latter could create a topological interaction between circular dna and smc - scpab , as seen with cohesin and condensin , starting from a dna loop . in this scenario , opening of the smc - scpab ring might occur in two steps : by initial disengagement of smc coiled coils and by subsequent hinge opening , possibly triggered by atp hydrolysis . intriguingly , causative mutations in genes for cohesin subunits in cornelia de lange syndrome ( cdls ) patients are mainly found in smc1 and smc3 coiled - coil sequences . in addition , several mutations that are located near the coils / hinge transition have been shown to increase dna binding by smc1 - 3 hinge heterodimers ( revenkova et  al . , 2009 ) . our data raise the exciting possibility that cdls cohesin might be defective in smc rod formation and , thus , display increased or misregulated association with dna . accordingly , cohesin in cdls patients might have lost tight coordination between dna binding at the hinge and the atpase activity located at the smc heads . smc proteins share their unusual architecture with rad50 , which uses a  zinc hook  rather than an smc hinge domain for dimerization . rad50 associates with nbs1 and the nuclease mre11 to form the mrn complex that is crucially important for efficient repair of dna double - strand breaks and other dna lesions ( williams et  al . , 2007 ) . simple binding of dna to isolated rad50 proteins has been suggested to convert ring - like dimers into straight rod - shaped structures ( moreno - herrero et  al . , 2005 ) . thus , transitions between rod - like and ring - like states might be a conserved , albeit differently regulated , feature of all smc - like proteins . the pfsmch - cc60 crystals grew from a precipitant solution containing 1  m sodium citrate , 0.1  m ches ( ph 9.5 ) , and 8% glycerol ; and the scsmc2h - cc110/scsmc4h - cc110 crystals grew from a solution of 16% polyethylene glycol 300 , 0.1  m na / k phosphate ( ph 6.0 ) , 8% glycerol , and 10  mm dithiothreitol ( dtt ) . the structure of pfsmch - cc60 was determined by molecular replacement using the structure of the coiled - coil - less pf smc hinge ( protein data bank [ pdb ] entry : 3nwc ) as a search model . the scsmc2h - cc110/scsmc4h - cc110 structure was solved by the single isormorphous replacement with anomalous scattering method ( table 1 ) . bl45xu of spring-8 ( hyogo , japan ) , 4c saxs ii beamline of pohang light source ii ( pohang , korea ) and a biosaxs-1000 system ( rigaku ) were used to collect saxs intensity data . the data were processed and analyzed using the software applications embedded in the atsas package . the labview software ( national instruments ) was used to select fluorescent bursts induced by single molecules . the distance between cy3 and cy5 was estimated by the equation of r  = r0(1/e  1 ) , with the r0 value of 6  nm for the cy3-cy5 pair . beads were washed , resuspended , and treated with bmoe ( 0.5  mm ) and incubated for 10  min on ice before quenching with 2-mercaptoethanol ( 2-me , 14  mm ) . fluorescence anisotropy titrations were performed at 25c using a biotek neo  plate reader in a buffer containing 50  mm tris - hcl at ph 7.5 , 50  mm nacl , and 3  mm mgcl2 ( plus 1  mm atp ) with 50  nm fluorescein - labeled dsdna ( 40  bp ) . bssmch - cc100 protein and double - stranded oligonucleotides ( 40  bp ) were mixed at 4  m and 20  m , respectively , in 50  mm tris , 50  mm nacl , 2  mm mgcl2 , 0.25  mm tcep ( ph 7.5)/23c ( final ) . after incubation at room temperature for 5  min , bmoe was added ( 0.5  mm final ) . reactions were incubated for 1  min at room temperature and quenched with 2-mercaptoethanol ( 14  mm ) . bmoe crosslinking of wild - type and mutant bs smc(a715c ) ( at 1  m ) was performed for 5  min at room temperature in the same buffer but with 3  mm mgcl2 , 10  m dna , with or without 1  mm atp , and quenched with dtt in sds loading buffer . y .- m.s . and h .- c.s . performed protein purification , structure determination , and biochemical experiments ; f.b . , , electron microscopy ; y .- g.k . , x - ray data collection ; c.p.t . , dna - binding measurements ; m .- s.k . and m .- l.d .- d . , protein purification ; and y .- m.s . , , s.g . , and b .- h.o . , conception of experiments and preparation of the manuscript .
summarysmc condensin complexes are central modulators of chromosome superstructure in all branches of life . 
 their smc subunits form a long intramolecular coiled coil , which connects a constitutive  hinge  dimerization domain with an atp - regulated  head  dimerization module . here 
 , we address the structural arrangement of the long coiled coils in smc complexes . 
 we unequivocally show that prokaryotic smc - scpab , eukaryotic condensin , and possibly also cohesin form rod - like structures , with their coiled coils being closely juxtaposed and accurately anchored to the hinge . upon atp - induced binding of dna to the hinge , however , smc switches to a more open configuration . 
 our data suggest that a long - distance structural transition is transmitted from the smc head domains to regulate smc - scpab s association with dna . 
 these findings uncover a conserved architectural theme in smc complexes , provide a mechanistic basis for smc s dynamic engagement with chromosomes , and offer a molecular explanation for defects in cornelia de lange syndrome .