the bssmc cdna was cloned from genomic dna by pcr , using pfu   polymerase ( lu and erickson , 1997 ) and adding ndei and bamhi sites to   allow subcloning into the expression vector pet11 ( studier et al . , 1990 ) .  
when induced in bl21 at 37c , the protein was abundantly expressed but   totally insoluble , and attempts to solubilize and renature it were unsuccessful . however , when the bacteria were maintained at 22c during induction , 1/3 of the protein remained soluble .
the bacterial supernatant   was partially purified by passing over a sephacryl hr-500 ( pharmacia   biotech , piscataway , nj ) column in 20 mm tris , 100 mm nacl , ph 8 ,   where the bssmc eluted ahead of most bacterial proteins .
the bssmc   from the leading and trailing sephacryl fractions sedimented identically at   6.3 s on glycerol gradients , indicating a homogeneity of the expressed   molecules .
the sephacryl fractions showed some contamination , and in   two cases were further purified by chromatography on mono q ( pharmacia biotech ) , where it eluted at 0.20.25 m nacl .
however , the best preparations for em were obtained by running the sephacryl - purified bssmc   directly on glycerol gradients .
mukbcoil is missing both the nh2- and cooh - terminal domains and includes only the coiled coils and hinge ( aa 3191125 ; our analysis of heptad repeats was slightly different from the coil prediction of   dnastar [ madison , wi ] , fig .
1 ) . mukbcoil tended to aggregate by sticking to small bits of bacterial debris ; however , some largely monomeric   fractions could be obtained from glycerol gradient sedimentation .
( we   also made a slightly larger coil construct , keeping the nh2 terminus at 319   and extending the cooh terminus to aa 1256 , which includes the last predicted segment of coiled coil [ fig . 1 ] .
this longer coil segment was highly   aggregated into rosettes , and there were no monomers .
we concluded   from this that the extra segment of predicted coiled coil is probably missing a partner to pair with , and therefore denatured , leading to aggregation . )
we then used the shorter mukbcoil as the basis for a chimera ,   fn - mukbcoil , in which the 40-kd cell adhesion domain of fibronectin ,   fn7 - 10 ( leahy et al . ,
we purified mukb as previously described using the expression plasmid pax814 kindly provided by dr .
sota hiraga , kumamoto university ,   kumamoto , japan ( niki et al . , 1992 ) .
we substituted a mono q column   for the deae sephacel and mukb eluted at 0.4 m nacl .
the best em results were obtained with material from the mono q column , subsequently   sedimented over glycerol gradients .
mukbcoil and fn - mukbcoil were purified by sephacryl chromatography followed by glycerol gradient sedimentation , omitting the mono q step .
sedimentation coefficients were estimated by zone sedimentation   through 1540% glycerol gradients in 0.2 m ammonium bicarbonate ( using the model sw50.1 or sw55.1 rotor ; beckman instruments , fullerton ,   ca ) ( erickson and briscoe , 1995 ) .
standard proteins catalase ( 11.3 s ) and   bsa ( 4.6 s ) were run in the same gradient as the samples , and the s value   was estimated by linear interpolation between these standards ( sedimentation of other proteins has shown our gradients to be linear in this region ) .
the stokes ' radius , rs in nm , was estimated by gel filtration on a superose 6 column using the following standards : thyroglobulin , rs = 8.5   nm ; catalase , rs = 5.2 nm ; and aldolase , rs = 4.8 nm .
s and rs were used   to calculate an experimental molecular weight as described by siegel and   monte ( 1966 ) ( see ohashi and erickson , 1997 , for an example and our assumed parameters ) .
samples from the glycerol gradients were rotary   shadowed ( fowler and erickson , 1979 ) and photographed at 50,000 magnification in an electron microscope ( model 301 ; philips electron optics ,   mahwah , nj ) .
2 shows the purification of bssmc after overexpression in e. coli using the pet system . because the protein is   so large and elongated
, the gel filtration chromatography   results in a substantial purification from the smaller bacterial proteins . a final step of glycerol gradient sedimentation resulted in fractions that showed only small contamination with other protein bands by sds - page and little   obvious contamination by em .  
the sedimentation coefficient of bssmc was 6.3 s ,   based on four determinations ranging from 6.2 to 6.4 s ,   and excluding three earlier results from 7 to 8 s. the sedimentation coefficient of mukb was 9.6 s , based on six   measurements ranging from 9.2 to 10.3 s , and excluding   two at 6.7 and 7.5 s. in several mukb preparations , some   of the protein was aggregated into rosettes , which sedimented ahead of the monomers .
the stokes ' radius was measured for bssmc , mukb , and fn - mukbcoils   by chromatography on a calibrated gel filtration column .  
the molecular masses calculated from these experimental   values of s and rs are close to those predicted for a homodimer of each protein ( table i ) .
the hydrodynamic   data for these full - length proteins and other constructs are   collected in table i and addressed further in the discussion .  
the fraction   of molecules in the different conformations varied considerably from one field to another and is probably affected   by the local conditions as the molecules are deposited on   the mica and dried .
both mukb and bssmc show the   same three or four characteristic conformations , which is   strong evidence that their basic structure is the same .  
the first conformation , which we call  folded - rod ,  is a   rod - shaped molecule with a large globular domain at one   end and a smaller one at the other ( fig .
the second   conformation , which we call  coils - spread ,  has similar   large and small globular domains , but the rod connecting   them is now split into two thinner rods ( fig .
a third conformation was seen frequently for bssmc but was rare for mukb ( fig .
the rods are together over most of   the molecule but veer apart near the pair of globular domains .
the fourth conformation , which we call  open - v ,    has both the rod and the large globular domain split into   two and splayed apart ( fig . 3 f ) .
3 f shows the structure of the molecule most clearly and explains the other   forms . the globular domain in the middle
is identified as   the hinge , and the thin rods extending on each side are   each a coiled coil . at the end of the coiled coils are the   globular domains , discussed below .
the coils - spread conformation is now interpreted as having the globular domains attached to each other but the two coiled coils separated and bowed out .
the folded - rod conformation , giving   the simple rod shape , is produced when the globular domains are attached to each other and the two coiled coils   lie close to each other .
measurements of several aspects of   the molecules are tabulated and explained in table ii and   will be addressed in the discussion .  
we initially anticipated that the coiled coils would be   parallel since all known long coiled coils are parallel .
this   arrangement would place the two nh2-terminal domains   at one end and the two cooh - terminal domains at the   other end of an open - v molecule .
we therefore expected   to see some difference in size or shape in the terminal   globular domains that would indicate this polarity .
we   also expected to see some difference in the lengths of the   two arms since they would be formed from different segments of coiled coils .
instead , we were surprised to find   that both bssmc and mukb showed a striking symmetry .  
the arms were indistinguishable in length , and most importantly the domains at the two ends always appeared   identical ( fig . 3 f ) .
some molecules showed a simple globular domain on each arm , but the best images of both   bssmc and mukb showed division of the terminal domain into a larger terminal globe and a smaller one somewhat closer to the hinge .
remarkably , whenever the two - part domain structure could be resolved on one arm , an   identical structure was seen on the other ( fig .
the   apparent twofold symmetry would only be possible with   an antiparallel arrangement of the coiled coils . to address the question of polarity , we prepared constructs of mukb with markers at the nh2 terminus .
we   had already prepared a truncated mukb , mukbcoil , in   which we had deleted both the nh2- and cooh - terminal   domains , leaving only the two coiled - coil segments and the   hinge .
this molecule frequently appeared as a rod where   the hinge was obvious as a single globular domain ; sometimes the two coiled - coil segments splayed apart in an   open - v or coils - spread conformation ( fig .
4 a ) . the   mukbcoil does not indicate the polarity because the nh2   and cooh termini are both truncated  helices .
to visualize the polarity , we made a new chimeric protein , fn - mukbcoil , in which we fused fn7 - 10 onto the nh2 terminus of mukbcoil .
fn7 - 10 , a 40-kd fragment of fibronectin ,   is well characterized by x - ray crystallography ( leahy et al .
,   1996 ) as a rod - shaped molecule about 3 nm in diameter   and 14 nm long , which should easily stand out from the   thinner coiled coil .
if the molecules were a parallel coiled   coil , we expected to see two fat fn rods projecting from   one end .
if they were antiparallel , we expected to see one   fat rod at each end .  
the fn - mukbcoil molecules visualized in the em were   nearly all in the open - v conformation and showed the fn   segment at each end ( fig . 4 b )
this provides the most   compelling evidence for the antiparallel arrangement of   the coiled coils .
there were several interesting contrasts   between the structures of mukbcoil and fn - mukbcoil .  
mukbcoil tended to aggregate , sedimenting as a smear   from 8 to 15 s , and the heavier fractions appeared as rosettes with the terminal segments of the coil aggregated at   the center and the hinge projecting out .
we believe that   the ends of these coils are sticky , probably because we misjudged the termini of the coils .
the sticky ends were also   evident in single molecules , since the two ends usually remained in contact giving the folded - rod or coils - spread   conformations ( fig .
, fn - mukbcoil sedimented as a sharp peak at 7 s , and the molecules were unaggregated and primarily in the open - v conformation in   the em .
thus , the fn segment at the terminus of the rods   seems to block the sticky sites and prevent association either within or between molecules .
in addition to demonstrating the antiparallel arrangement of the coiled coils , fn - mukbcoil provides definitive   proof that the hinge is flexible .
5 shows a histogram of   the angles of the two arms measured for different molecules .
we do not believe the apparent peaks and dips are   significant , and we conclude that the angles are probably   randomly distributed .
it is perhaps of interest to ask   whether the hinge can open to more than 180 , but these   measurements can not distinguish 270 from 90. we conclude that the hinge is quite flexible between 1 and 180   and perhaps can bend even further .  
the frequency of the different conformations seen by em   varied from field to field , but in most cases the folded - rod   was the predominant conformation .
the open - v conformation was the most important for demonstrating the fundamental structure of the molecule .
these open - v molecules also demonstrate the potential of the terminal   domains to separate and undergo a scissoring motion ,   which is likely to be important for biochemical functions .
the hydrodynamic data are consistent with the elongated shape seen by em and provide insight into the question of whether the molecule is in the folded - rod or open - v   conformation in solution .
the shape of a molecule is best   indicated by the frictional ratio f / fmin , where fmin is the frictional coefficient of an unhydrated sphere of the same   mass and density as the protein in question ( tanford ,   1961 ) .
f / fmin was calculated from the measured sedimentation coefficient , and the mass was predicted from the subunit aa sequence .
values are presented in table i for the   four molecules we purified and for the 8-s xcap - c/ xcap - e heterodimer ( hirano et al . , 1997 ) .
the fn - mukbcoil provides an important benchmark for comparison   because em indicated that this construct was entirely in   the open - v conformation .
we believe that its f / fmin = 2.3 is   characteristic of the open - v conformation .
the lower values of 1.92.0 for mukb and xcap are probably characteristic of the folded - rod conformation .
the f / fmin = 2.5 for bssmc is the largest of the group   and strongly suggests that this molecule is open - v in solution .
also , both bssmc and fn - mukbcoil have a larger   stokes ' radius than mukb , which is consistent with the interpretation that they are open - v , and mukb is folded - rod   in solution .
the only contradiction is that most of the   bssmc molecules appeared to be folded - rods in the em .  
it is possible that the folded - rod conformation of bssmc   was generated by the high glycerol and salt concentrations   when the specimens were dried on the mica .
there were two possibilities for the mechanics of the   hinge : it could be quite flexible , permitting free scissoring   motion of the two coiled coils , or it could be relatively   rigid , locking the coils into the parallel configuration seen   in the folded - rod .
a flexible hinge was implied by the   coils - spread conformation , which frequently showed an   angle of 60 or more for the coils at the hinge .
the flexibility of the hinge was demonstrated most convincingly by   the fn - mukbcoils construct , which appears by em to be   entirely in the open - v conformation .
since   the hinge is flexible , the folded - rod conformation must be   stabilized by an interaction of the terminal domains with   each other .
the measurements of the molecules in table ii show   that mukb is longer than bssmc ( 65 vs. 58 nm ) , which is   consistent with its larger mass and longer estimated coiled   coil ( fig .
1 ) . we particularly wanted to determine the   length of the coiled coil , as a guide to aligning the sequences .
as shown in table ii , the measurement of the   thin rod from the hinge to the outer globular domain ( subtracting the diameters of these domains from the length of   the whole molecule ) gives 51 and 41 nm for mukb and   bssmc .
these would actually estimate the minimum   length of the coiled coil since the coils could extend into   the hinge and globular domain . using the spacing of 0.15   nm per aa in an  helix
, these lengths would predict coiled   coils of 340 aa for mukb and 273 aa for bssmc .
this construct included aa 319 1125 , extending in both directions slightly beyond the   entire 330334-aa coiled - coil segments indicated in fig . 1 .  
however , the length of the coil measured from this construct was only 41 nm , corresponding to 273 aa .
it therefore seems likely that the short predicted coil from 1205 1243 , which is missing from mukbcoil , is a part of the   51-nm rod of the whole mukb .
it is not clear what this segment would be pairing with to make a coiled coil .
it is interesting that a mutation d1201n , just before this predicted coil segment , completely disrupts mukb function   ( yamanaka et al . , 1994 ) .
the precise alignment of the coiled coils is complicated   by the appearance of gaps in the predicted coils .
some of   these gaps may actually form coils and continue the coiled   coil , but others may bud out , shortening the length of the   rod .
most of these interruptions are small and might not   be visible even if they did fold into a globular domain .
we   note that the thin rods of the open - v molecules appear as   a uniform , thin diameter between the hinge and the two   globular domains at the ends .
the heterodimeric smcs of eukaryotes pose additional   ambiguity in alignment of the coils since there are now   four separate sequences that have to form the two coiled   coils . as shown in fig .
1 , reasonable alignments can be   made for cut3/cut14 of schizosaccharomyces pombe and   for xcap - c / xcap - e of xenopus laevis .
the following two sections diverge from the structural   analysis to discuss general features of the smc family of   proteins . from the databases ,
we have collected 18 eukaryotic   smc sequences , 6 bacterial sequences , and 4 archaeal sequences that appear to be bona fide smcs : mukb from e.   coli , which has the smc structure but limited sequence   identity ; and 8 sequences that show limited sequence identity at the nh2- and cooh - terminal domains and have   long coiled - coil segments between them .
we did a separate sequence alignment of the nh2-terminal domain of   230 aa and the cooh - terminal domain of 80 aa from   each of the 34 sequences .
the sequences were aligned by   the clustal algorithm of dnastar and then adjusted by   hand .
6 shows the two independently derived phylogenetic trees from the nh2- and cooh - terminal sequence   alignments ( trees were drawn with the drawtree algorithm of phylip , http://evolution.genetics.washington.edu/phylip.html ) .  
the trees from the nh2- and cooh - terminal domains   are remarkably similar , and a tree calculated from the   hinge region ( not shown ) also shows the same grouping .  
all three trees cluster the smc sequences into six groups   of smc proteins and a group of outliers .
the six - smc   groups comprise four eukaryotic smc groups , each containing one of the four s. cerevisiae smcs ; a group of bacterial smcs ; and a group of mostly archaeal smcs ( which ,   however , also includes the cyanobacterium synechocystis ) .  
we have labeled the eukaryotic groups 14 , corresponding   to the s. cerevisiae smc in the group .
( this is consistent   with the previously proposed nomenclature of koshland   and strunnikov   and a recent review by jessberger   et al .
) b is for the bacterial group , and a is for the   mostly archaeal group .
( 1998 ) have proposed that there are two   classes of heterodimers : smc2/4 and smc1/3 .
there are   three well - established examples of pairing a member of   the smc2 group with a member of the smc4 group :   xcap - e + xcap - c ( hirano and mitchison , 1994 ) ,   cut14 + cut3 ( saka et al . , 1994 ) , and mix-1 + dpy-27   ( lieb et al . , 1998 ) .
it is attractive to speculate that an smc   of group 2 always pairs with a group 4 partner .
mix-1 provides an interesting variation , since it is known to pair with   dpy-27 for its role in dosage compensation , but it probably has a different smc partner for its role in mitosis ( lieb   et al . , 1998 ) .
c. elegans has a second smc in group 4 , so far   identified only from genomic sequencing .
heterodimers of a group 3 and group 1 smc have been   identified for bovine smc ( jessberger et al . , 1998 ) and xenopus ( losada et al . , 1998 ) .
in s. cerevisiae , smc3 and   smc1 were each identified in a screen for mutants affecting sister chromatid cohesion ( michaelis et al . , 1997 ) , consistent with their forming a pair . a possible exception to   the generalization
is that smc2 from s. cerevisiae has been   shown to associate both with itself and with smc1 ( strunnikov et al . , 1995 ) .
it was not determined that either of   these was a specifically heterodimer association , but this   observation raises the possibility that heterodimer associations may be more complex than simply two classes .
of 18 bacterial or archaeal genomes completely or almost completely sequenced , 13 have a clear smc , and 3   have no smc but a mukb ( which we now consider a divergent smc ; see also britton et al . , 1998 ) .
methanococcus   thermoautotrophican has only a distantly related protein   but no smc or mukb , and borelia burgdorferi has an   smc - like protein that is shorter than most and branches   ambiguously between bacterial smcs and outliers .
helicobacter pylori has nothing matching the conserved nh2 or   cooh termini of smc , mukb , or the distantly related   proteins ( erickson , h.p . , unpublished sequence analysis ;   sequence data for partially completed genomes were obtained from the institute for genomic research website   at http://www.tigr.org ) .
the most distinctive structural feature of the smc proteins is the presence of five domains : nh2-terminal , long   coiled coil , hinge , long coiled coil , and cooh - terminal .  
the coiled - coil segments of smc proteins are not highly   conserved ; however , the nh2-terminal , cooh - terminal ,   and hinge domains show a high level sequence match over   several motifs that seem characteristic of the smc proteins . for the present discussion , we will not show the full   alignment but will designate the motifs according to aa   numbers of the bssmc sequence ( swiss prot p51834 ) . in the nh2-terminal domain ,
the most striking motifs   are fks ( 1113 ) , gxngsgksn ( 3139 ) , which is the   walker a motif for ntp binding , and the dipeptide qg   ( 143144 ) .
the fks and gxngsgksn are almost totally   conserved in the bona fide smc proteins , and even the   outliers show mostly conservative substitutions .
a   most intriguing sequence motif , not previously noted , is   the qg , which is conserved in all 26 smc sequences examined .
it is also conserved in all three known mukb proteins ( from e. coli , h. influenzae , and vibrio cholerae [ sequence data for v. cholerae were obtained from the   institute for genomic research website at http://www .  
qg is conserved in methanococcus jannaschii   1322 and the sulfolobus protein , but not in methanobacter   thermoautotrophican nor in any of the rad proteins .
we   suggest that this qg is a diagnostic signature of the smc   proteins and is another argument for including mukb in   this group .
the hinge region shows no extended conserved motif ,   but the segment from aa 517 to 666 contains several isolated aa that are highly conserved .
it is interesting that   rad18 and rhc18 show a hinge region that matches that   of the smc proteins .
mukb has a 125-aa noncoil segment   that obviously functions as a hinge , but it shows no sequence similarity to the smc hinge sequence . the cooh - terminal domain as indicated in fig .
1 includes 200300 aa from the end of the coiled coil , but only   a short terminal segment of about 80 aa shows significant   sequence identity .
this segment begins with the highly   conserved lsgg ( 10911094 ) and then the pxphhhhdehdaald ( 11121126 ) , where  h  is a hydrophobic aa .
the   hhhhd may correspond to the walker b site , which is very   loosely defined as an aspartic acid preceded by four hydrophobic residues .
( 1995 ) for a more detailed analysis of this motif and its use in placing the smc   proteins in a larger family of transporters , helicases , and   repair enzymes .
a recent study of the e. coli dna - processing protein   sbcc has proposed including it as a member of the smc   family ( connelly et al . , 1998 ) .
this protein falls among the   outliers in our alignments of nh2- and cooh - terminal   domains ( fig .
6 ) , and moreover has no functional hinge   ( see below ) . although one could expand the definition of   the smc family to include the rad proteins and other outliers , we suggest for the present to limit the smc designation to the family members defined by the above characteristics , including specifically a functional hinge .
the antiparallel orientation   seems preferred for the short , interacting  helices found   in globular proteins , but coiled - coil proteins are predominantly parallel .
two exceptions are the antiparallel , 35-aa   coiled coil projecting as a helical arm of the bacterial seryl   trna synthetases ( oakley and kim , 1997 ) and a 45-aa   coiled coil of f1-atpase ( abrahams , 1994 ) .
the 300-aa   coiled coils of the smc proteins demonstrate for the first   time that the antiparallel arrangement can be used to form   very long coiled coils .
we predict that the eukaryotic smc heterodimers will   have the same antiparallel coiled coil structure ( fig .
1 ) ,   and this may also be true for the several proteins ( rad50 ,   rad18 , e. coli sbcc , m. jannaschii 1322 ) indicated as outliers in the phylogenetic tree .
a recent study of one of   these outliers , sbccd , included an electron micrograph   that provides some important insights ( connelly et al . ,   1998 ) .
the sbccd showed two large globular domains   separated by a single coiled coil 80 nm long .
although the   sequence indicates a short break in the middle of this   coiled coil , the images show no indication of a hinge in the   structure .
the coiled coil shows a gentle curvature that can   bring the terminal domains within 40 nm of each other ,   but this molecule seems incapable of achieving a folded - rod conformation .
the question of parallel or antiparallel   arrangement was not addressed in that study , and indeed   the structure is described as 
 however ,   there is no evidence of asymmetry in the structure ; the   globular domains at the two ends actually appear to be the   same size , consistent with an antiparallel arrangement of   the coiled coils in sbccd .
it is also interesting to note that the antiparallel structure provides a mechanism for bringing the proposed   walker a and b motifs together because the nh2- and   cooh - terminal domains are paired at each end of the   molecule .
alternatively , in the smc proteins they could   be brought together in the folded - rod conformation . however , since sbccd seems incapable of forming the folded - rod conformation , the antiparallel structure seems the   only possibility for bringing its nh2- and cooh - terminal   domains together .
we should caution that the resolution   of the analysis is not sufficient to actually demonstrate   contact of the domains , much less a functional association   of the walker a and b motifs .
the antiparallel coiled coil arrangement also suggests   the possibility of a twofold axis of symmetry passing   through the hinge . the twofold symmetry could be exact   for the homodimers and could be approximate for the heterodimers .
this would relate the nh2- and cooh - terminal domains on opposite arms by a 180 rotation , bringing   identical faces into contact in the folded - rod conformation .
perhaps the most important feature of the new model is   that the molecule is not a polar structure , with an atp - binding domain at one end and a dna - binding domain at   the other , but each terminus of the molecule contains a   complete and identical functional unit .
this means that the   two ends of the molecule can operate identically on two   strands of dna , separated by 100 nm for the fully open   molecule , or brought into contact in the folded - rod .
one   function well established for 13-s xcap condensin is the   generation of dna supercoils in vitro ( kimura and hirano , 1997 ) , and this may be the basis for dna condensation .
the symmetrical molecule with two complete functional units should suggest novel mechanisms for how the   smc complex might operate on dna to generate supercoils , and for how related complexes might manipulate   dna for cohesion or repair .
the frequency of the different conformations seen by em   varied from field to field , but in most cases the folded - rod   was the predominant conformation .
the open - v conformation was the most important for demonstrating the fundamental structure of the molecule .
these open - v molecules also demonstrate the potential of the terminal   domains to separate and undergo a scissoring motion ,   which is likely to be important for biochemical functions .
the hydrodynamic data are consistent with the elongated shape seen by em and provide insight into the question of whether the molecule is in the folded - rod or open - v   conformation in solution .
the shape of a molecule is best   indicated by the frictional ratio f / fmin , where fmin is the frictional coefficient of an unhydrated sphere of the same   mass and density as the protein in question ( tanford ,   1961 ) .
f / fmin was calculated from the measured sedimentation coefficient , and the mass was predicted from the subunit aa sequence .
values are presented in table i for the   four molecules we purified and for the 8-s xcap - c/ xcap - e heterodimer ( hirano et al . , 1997 ) .
the fn - mukbcoil provides an important benchmark for comparison   because em indicated that this construct was entirely in   the open - v conformation .
we believe that its f / fmin = 2.3 is   characteristic of the open - v conformation .
the lower values of 1.92.0 for mukb and xcap are probably characteristic of the folded - rod conformation .
the f / fmin = 2.5 for bssmc is the largest of the group   and strongly suggests that this molecule is open - v in solution .
also , both bssmc and fn - mukbcoil have a larger   stokes ' radius than mukb , which is consistent with the interpretation that they are open - v , and mukb is folded - rod   in solution .
the only contradiction is that most of the   bssmc molecules appeared to be folded - rods in the em .  
it is possible that the folded - rod conformation of bssmc   was generated by the high glycerol and salt concentrations   when the specimens were dried on the mica .
there were two possibilities for the mechanics of the   hinge : it could be quite flexible , permitting free scissoring   motion of the two coiled coils , or it could be relatively   rigid , locking the coils into the parallel configuration seen   in the folded - rod .
a flexible hinge was implied by the   coils - spread conformation , which frequently showed an   angle of 60 or more for the coils at the hinge .
the flexibility of the hinge was demonstrated most convincingly by   the fn - mukbcoils construct , which appears by em to be   entirely in the open - v conformation .
since   the hinge is flexible , the folded - rod conformation must be   stabilized by an interaction of the terminal domains with   each other .
the measurements of the molecules in table ii show   that mukb is longer than bssmc ( 65 vs. 58 nm ) , which is   consistent with its larger mass and longer estimated coiled   coil ( fig .
1 ) . we particularly wanted to determine the   length of the coiled coil , as a guide to aligning the sequences .
as shown in table ii , the measurement of the   thin rod from the hinge to the outer globular domain ( subtracting the diameters of these domains from the length of   the whole molecule ) gives 51 and 41 nm for mukb and   bssmc .
these would actually estimate the minimum   length of the coiled coil since the coils could extend into   the hinge and globular domain . using the spacing of 0.15   nm per aa in an  helix
, these lengths would predict coiled   coils of 340 aa for mukb and 273 aa for bssmc .
this construct included aa 319 1125 , extending in both directions slightly beyond the   entire 330334-aa coiled - coil segments indicated in fig . 1 .  
however , the length of the coil measured from this construct was only 41 nm , corresponding to 273 aa .
it therefore seems likely that the short predicted coil from 1205 1243 , which is missing from mukbcoil , is a part of the   51-nm rod of the whole mukb .
it is not clear what this segment would be pairing with to make a coiled coil .
it is interesting that a mutation d1201n , just before this predicted coil segment , completely disrupts mukb function   ( yamanaka et al . , 1994 ) .
the precise alignment of the coiled coils is complicated   by the appearance of gaps in the predicted coils .
some of   these gaps may actually form coils and continue the coiled   coil , but others may bud out , shortening the length of the   rod .
most of these interruptions are small and might not   be visible even if they did fold into a globular domain .
we   note that the thin rods of the open - v molecules appear as   a uniform , thin diameter between the hinge and the two   globular domains at the ends .
the heterodimeric smcs of eukaryotes pose additional   ambiguity in alignment of the coils since there are now   four separate sequences that have to form the two coiled   coils . as shown in fig .
1 , reasonable alignments can be   made for cut3/cut14 of schizosaccharomyces pombe and   for xcap - c / xcap - e of xenopus laevis .
the following two sections diverge from the structural   analysis to discuss general features of the smc family of   proteins . from the databases ,
we have collected 18 eukaryotic   smc sequences , 6 bacterial sequences , and 4 archaeal sequences that appear to be bona fide smcs : mukb from e.   coli , which has the smc structure but limited sequence   identity ; and 8 sequences that show limited sequence identity at the nh2- and cooh - terminal domains and have   long coiled - coil segments between them .
we did a separate sequence alignment of the nh2-terminal domain of   230 aa and the cooh - terminal domain of 80 aa from   each of the 34 sequences .
the sequences were aligned by   the clustal algorithm of dnastar and then adjusted by   hand .
6 shows the two independently derived phylogenetic trees from the nh2- and cooh - terminal sequence   alignments ( trees were drawn with the drawtree algorithm of phylip , http://evolution.genetics.washington.edu/phylip.html ) .  
the trees from the nh2- and cooh - terminal domains   are remarkably similar , and a tree calculated from the   hinge region ( not shown ) also shows the same grouping .  
all three trees cluster the smc sequences into six groups   of smc proteins and a group of outliers .
the six - smc   groups comprise four eukaryotic smc groups , each containing one of the four s. cerevisiae smcs ; a group of bacterial smcs ; and a group of mostly archaeal smcs ( which ,   however , also includes the cyanobacterium synechocystis ) .  
we have labeled the eukaryotic groups 14 , corresponding   to the s. cerevisiae smc in the group .
( this is consistent   with the previously proposed nomenclature of koshland   and strunnikov   and a recent review by jessberger   et al . , which did not include the bacterial and archaeal smcs . )
b is for the bacterial group , and a is for the   mostly archaeal group .
( 1998 ) have proposed that there are two   classes of heterodimers : smc2/4 and smc1/3 .
there are   three well - established examples of pairing a member of   the smc2 group with a member of the smc4 group :   xcap - e + xcap - c ( hirano and mitchison , 1994 ) ,   cut14 + cut3 ( saka et al . , 1994 ) , and mix-1 + dpy-27   ( lieb et al . , 1998 ) .
it is attractive to speculate that an smc   of group 2 always pairs with a group 4 partner .
mix-1 provides an interesting variation , since it is known to pair with   dpy-27 for its role in dosage compensation , but it probably has a different smc partner for its role in mitosis ( lieb   et al .
c. elegans has a second smc in group 4 , so far   identified only from genomic sequencing .
heterodimers of a group 3 and group 1 smc have been   identified for bovine smc ( jessberger et al . ,
1998 ) and xenopus ( losada et al . , 1998 ) . in s. cerevisiae , smc3 and   smc1
were each identified in a screen for mutants affecting sister chromatid cohesion ( michaelis et al . , 1997 ) , consistent with their forming a pair . a possible exception to   the generalization
is that smc2 from s. cerevisiae has been   shown to associate both with itself and with smc1 ( strunnikov et al . , 1995 ) .
it was not determined that either of   these was a specifically heterodimer association , but this   observation raises the possibility that heterodimer associations may be more complex than simply two classes .
of 18 bacterial or archaeal genomes completely or almost completely sequenced , 13 have a clear smc , and 3   have no smc but a mukb ( which we now consider a divergent smc ; see also britton et al .
methanococcus   thermoautotrophican has only a distantly related protein   but no smc or mukb , and borelia burgdorferi has an   smc - like protein that is shorter than most and branches   ambiguously between bacterial smcs and outliers .
helicobacter pylori has nothing matching the conserved nh2 or   cooh termini of smc , mukb , or the distantly related   proteins ( erickson , h.p . , unpublished sequence analysis ;   sequence data for partially completed genomes were obtained from the institute for genomic research website   at http://www.tigr.org ) .
the most distinctive structural feature of the smc proteins is the presence of five domains : nh2-terminal , long   coiled coil , hinge , long coiled coil , and cooh - terminal .  
the coiled - coil segments of smc proteins are not highly   conserved ; however , the nh2-terminal , cooh - terminal ,   and hinge domains show a high level sequence match over   several motifs that seem characteristic of the smc proteins . for the present discussion , we will not show the full   alignment but will designate the motifs according to aa   numbers of the bssmc sequence ( swiss prot p51834 ) . in the nh2-terminal domain ,
the most striking motifs   are fks ( 1113 ) , gxngsgksn ( 3139 ) , which is the   walker a motif for ntp binding , and the dipeptide qg   ( 143144 ) . the fks and gxngsgksn are almost totally   conserved in the bona fide smc proteins , and even the   outliers show mostly conservative substitutions .
a   most intriguing sequence motif , not previously noted , is   the qg , which is conserved in all 26 smc sequences examined .
it is also conserved in all three known mukb proteins ( from e. coli , h. influenzae , and vibrio cholerae [ sequence data for v. cholerae were obtained from the   institute for genomic research website at http://www .  
qg is conserved in methanococcus jannaschii   1322 and the sulfolobus protein , but not in methanobacter   thermoautotrophican nor in any of the rad proteins .
we   suggest that this qg is a diagnostic signature of the smc   proteins and is another argument for including mukb in   this group .
the hinge region shows no extended conserved motif ,   but the segment from aa 517 to 666 contains several isolated aa that are highly conserved .
it is interesting that   rad18 and rhc18 show a hinge region that matches that   of the smc proteins .
mukb has a 125-aa noncoil segment   that obviously functions as a hinge , but it shows no sequence similarity to the smc hinge sequence . the cooh - terminal domain as indicated in fig .
1 includes 200300 aa from the end of the coiled coil , but only   a short terminal segment of about 80 aa shows significant   sequence identity .
this segment begins with the highly   conserved lsgg ( 10911094 ) and then the pxphhhhdehdaald ( 11121126 ) , where  h  is a hydrophobic aa .
the   hhhhd may correspond to the walker b site , which is very   loosely defined as an aspartic acid preceded by four hydrophobic residues .
( 1995 ) for a more detailed analysis of this motif and its use in placing the smc   proteins in a larger family of transporters , helicases , and   repair enzymes .
a recent study of the e. coli dna - processing protein   sbcc has proposed including it as a member of the smc   family ( connelly et al . , 1998 ) .
this protein falls among the   outliers in our alignments of nh2- and cooh - terminal   domains ( fig .
6 ) , and moreover has no functional hinge   ( see below ) . although one could expand the definition of   the smc family to include the rad proteins and other outliers , we suggest for the present to limit the smc designation to the family members defined by the above characteristics , including specifically a functional hinge .
the antiparallel orientation   seems preferred for the short , interacting  helices found   in globular proteins , but coiled - coil proteins are predominantly parallel .
two exceptions are the antiparallel , 35-aa   coiled coil projecting as a helical arm of the bacterial seryl   trna synthetases ( oakley and kim , 1997 ) and a 45-aa   coiled coil of f1-atpase ( abrahams , 1994 ) .
the 300-aa   coiled coils of the smc proteins demonstrate for the first   time that the antiparallel arrangement can be used to form   very long coiled coils .
we predict that the eukaryotic smc heterodimers will   have the same antiparallel coiled coil structure ( fig .
1 ) ,   and this may also be true for the several proteins ( rad50 ,   rad18 , e. coli sbcc , m. jannaschii 1322 ) indicated as outliers in the phylogenetic tree .
a recent study of one of   these outliers , sbccd , included an electron micrograph   that provides some important insights ( connelly et al . ,   1998 ) .
the sbccd showed two large globular domains   separated by a single coiled coil 80 nm long .
although the   sequence indicates a short break in the middle of this   coiled coil , the images show no indication of a hinge in the   structure .
the coiled coil shows a gentle curvature that can   bring the terminal domains within 40 nm of each other ,   but this molecule seems incapable of achieving a folded - rod conformation .
the question of parallel or antiparallel   arrangement was not addressed in that study , and indeed   the structure is described as 
 however ,   there is no evidence of asymmetry in the structure ; the   globular domains at the two ends actually appear to be the   same size , consistent with an antiparallel arrangement of   the coiled coils in sbccd .
it is also interesting to note that the antiparallel structure provides a mechanism for bringing the proposed   walker a and b motifs together because the nh2- and   cooh - terminal domains are paired at each end of the   molecule .
alternatively , in the smc proteins they could   be brought together in the folded - rod conformation . however , since sbccd seems incapable of forming the folded - rod conformation , the antiparallel structure seems the   only possibility for bringing its nh2- and cooh - terminal   domains together .
we should caution that the resolution   of the analysis is not sufficient to actually demonstrate   contact of the domains , much less a functional association   of the walker a and b motifs .
the antiparallel coiled coil arrangement also suggests   the possibility of a twofold axis of symmetry passing   through the hinge . the twofold symmetry could be exact   for the homodimers and could be approximate for the heterodimers .
this would relate the nh2- and cooh - terminal domains on opposite arms by a 180 rotation , bringing   identical faces into contact in the folded - rod conformation .
perhaps the most important feature of the new model is   that the molecule is not a polar structure , with an atp - binding domain at one end and a dna - binding domain at   the other , but each terminus of the molecule contains a   complete and identical functional unit .
this means that the   two ends of the molecule can operate identically on two   strands of dna , separated by 100 nm for the fully open   molecule , or brought into contact in the folded - rod .
one   function well established for 13-s xcap condensin is the   generation of dna supercoils in vitro ( kimura and hirano , 1997 ) , and this may be the basis for dna condensation .
the symmetrical molecule with two complete functional units should suggest novel mechanisms for how the   smc complex might operate on dna to generate supercoils , and for how related complexes might manipulate   dna for cohesion or repair .
coiled - coil segments predicted by the program protean   ( dnastar ) are shown as black rectangles .
numbers above the   vertical lines indicate the aa number , and numbers in parentheses   between arrows indicates the total number of aa between the   lines .
the 275300-aa segment for bssmc and the 330335-aa   segment of ecmukb were initially selected as the coiled coil because they matched the 41- and 51-nm lengths measured by em .  
however , measurements of the truncated construct mukbcoil   ( see discussion ) indicate that the coiled coil of mukb probably   includes the short segment 12051243 .
the boundaries and alignment of the coiled - coil segments are therefore still ambiguous .  
heterodimeric smcs may also pair by the antiparallel coiled - coil   arrangement , as illustrated for cut3/cut14 and xcap - e / xcap - c .
about one - third of the bssmc is in the supernatant in these expressing bacteria grown at 22c .
the next seven lanes show fractions from the   sephacryl column , and the final three lanes show fractions from   the glycerol gradient ( of the peak sephacryl fraction ) .
m kd aa seq  is the mass calculated for the presumed dimer from the aa sequence .
m experim  was calculated   from these values ( siegel and monte , 1966 ) .
f / fmin is the ratio of the experimental frictional coefficient ( determined from s ) to that of an unhydrated sphere of the same   mass .   
selected fields are presented in a and b. ( c ) the most common conformation ,    folded - rod .  ( d ) the  coils - spread  conformation .
( e ) molecules with the coils together but with the terminal domains split , which   was seen reproducibly for bssmc .
( f ) the most informative  open - v  conformation . note the symmetry of the molecules in the open - v   conformation : whenever one arm shows two small globular domains , the other arm does also .
measurements of mukb and bssmc lengths and calculations   all dimensions listed here have been corrected for the presumed 1-nm shell of metal .   1 .
the most definitive measurement was the length of the whole molecule in the   folded - rod conformation and the lengths of single arms in the open - v ; these were the   same , and the measurements given here include both forms .   2 .
the smaller globular domain was identified as the hinge and had the same diameter   in all three conformations .  
3 . molecules in the open - v conformation usually showed the terminal domain resolved into two globular domains .
the length of the coiled - coil was estimated by subtracting the diameters of the   hinge and outer globular domains from the length of the whole molecule .  
the length of the mukbcoil was measured from the outside of the globular hinge   domain to the termination of the thin coil .
this termination was sometimes ambiguous , hence the larger standard deviation of these measurements .  
the length of the coil was estimated by subtracting the diameter of the hinge ( 2 )   from the length of mukbcoil ( 5 ) .  
the larger ( nonhinge ) globular domain was approximately spherical in all conformations of bssmc , and its diameter is measure here .  
the coil of bssmc was estimated by subtracting the diameters of the hinge and   outer globular domain from the length of the whole molecule .     
( a ) mukbcoil , in which both the nh2- and cooh - terminal domains were deleted .
the   ends seem to be sticky and are frequently together ( right - hand   images ) , although some open - v molecules could be found .
( b )   fn - mukbcoil , in which a thick segment of fibronectin was attached to the nh2 terminus of the coiled coil .
this segment completely eliminated the stickiness of the ends , and all molecules are   in the open - v configuration .
the thick fn segment is seen projecting from each end , confirming the antiparallel coiled - coil arrangement .
molecules of fn - mukbcoil in the open - v conformation were measured for the angle of the two arms .
note   that angles larger than 180 are possible but could not be distinguished , so they would be grouped with the smaller angle .
separate trees are shown for the 220240-aa nh2-terminal domain ( flkrl ... lehve in bssmc ) , and the 80-aa cooh - terminal domain ( lsgge ... yssdt in bssmc ) .
the   circles indicate the six groups of smcs : 14 , eukaryal smcs ; b , bacterial ; a , archaeal .
the group under the circle  out  are outliers , distantly related to smcs .
each of the outlier sequences has an nh2-terminal domain with a related atp - binding motif , some limited sequence identity in the cooh - terminal domain , and long coiled coils separating the two , but usually no hinge .
group 1 smc : smc1 ( s. cer ) , p41003 ; h. sapiens , s78271 ; xsmc1 ( x. la ) , af051784 .
group 2 smc :   smc2 ( s. cer ) , p38989 ; cut14 ( s. pom ) , p41003 ; mix-1 ( c. el ) , u96387 ; xcap - e ( x. la ) , p50533 ; scii ( chick ) , q90988 .
group 3 smc :   smc3 ( s. cer ) , p47037 ; smc3 ( s. pom ) , al009197 ; a.(e . ) nidulans , s65799 ; d. melanog .
, u30492 ; bamacan ( rat ) , u82626 ; xsmc3 ( x. la ) ,   af051785 .
group 4 smc : smc4 ( s. cer ) , u53880 ; cut3 ( s. pom ) , p41004 ; dpy-27 ( c. el ) , p48996 ; smc4a ( c. el ) , z46242 ; xcap - c ( x. la ) ,   p50532 .
group b bacterial smcs : bacillus subtilis , p51834 ; mycobacterium tuberculosis , q10970 ; treponema palidum , orf00437 ; mycoplasma hyorhinis , p41508 ; mycoplasma pneumoniae , p75361 .
group a ( mostly ) archaeal smcs : methanococcus jannaschii , u67604 ;   aquifex aeolicus , ae000699 ; archeoglobus fulgidus , ae000995 ; synechocystis , d90905 ; pyrococcus horikoshii , d90905 . outliers 
proteins distantly related to smc : mukb ( e. coli ) , p22523 ; m. jannaschii 1322 , a64465 ; methanobacterium thermoautotrophican ,   ae000837 ; sulfolobus acidophilum , y10687 ; rad50 ( mouse ) , u66887 ; rad50 ( s. cer ) , p12753 ; rad18 ( s. pom ) , p53692 ; rhc18 ( s. cer ) ,   q12749 ; sbcc ( e. coli ) , p13458 . the model of the smc protein structure .
the nh2- and   cooh - terminal domains are shown schematically , without attempting to identify them with the two globular domains seen by   em in some open molecules .
the coiled - coil rods are rather rigid ,   but the hinge is quite flexible , permitting a scissoring movement   with the coils separated at angles from 0 to 180 or more .
the terminal domains associate with each other to lock the molecule   into the  folded - rod  conformation , but this association is reversible , permitting transition to the open - v conformation .
 structural maintenance of chromosomes   ( smc ) proteins function in chromosome condensation   and several other aspects of dna processing . 
 they are   large proteins characterized by an nh2-terminal nucleotide triphosphate ( ntp)-binding domain , two long   segments of coiled coil separated by a hinge , and a   cooh - terminal domain . here 
 , we have visualized by   em the smc protein from bacillus subtilis ( bssmc )   and mukb from escherichia coli , which we argue is a   divergent smc protein . 
 both bssmc and mukb show   two thin rods with globular domains at the ends emerging from the hinge . 
 the hinge appears to be quite flexible : the arms can open up to 180 , separating the terminal domains by 100 nm , or close to near 0 , bringing the   terminal globular domains together.a 
 surprising observation is that the 300amino   acid  long coiled coils are in an antiparallel arrangement . 
 known coiled coils are almost all parallel , and   the longest antiparallel coiled coils known previously   are 3545 amino acids long . 
 this antiparallel arrangement produces a symmetrical molecule with both an   nh2- and a cooh - terminal domain at each end . 
 the   smc molecule therefore has two complete and identical functional domains at the ends of the long arms .   the bifunctional symmetry and a possible scissoring action at the hinge should provide unique biomechanical   properties to the smc proteins . 
Materials and Methods
Results
Discussion
Conformations and Dimensions
Phylogenetic Tree of the SMC Family and  Consideration of Heterodimers
Conserved Sequences and the Defining Characteristics  of SMC Proteins
The Uniqueness of the Long, Antiparallel Coiled Coil
Figures and Tables