Homologues of the human SMC head and hinge domains have been

Homologues of the human SMC head and hinge domains have been determined to atomic detail and served as templates for modelling the globular portions of SMC2 and SMC4, assembly of a draft structural model for SMC2/SMC4 was only made possible here by inclusion of constraints from the cross-linking analysis. This enabled us to pursue a template and fragment based approach and assemble the 13 fragments that were compatible with 117/120 high-confidence cross-links derived from the various condensin preparations into a low-resolution three-dimensional view of the entire SMC2/SMC4 dimer. The model reveals an intimate rod-like arrangement of the SMC2/SMC4 molecules dictated by the numerous, regularly arranged, intermolecular cross-links in the coiled-coil regions. Intriguingly, the remarkable consistency of the cross-link data with a single model seems to indicate that a single rod-like form [18] predominated in our samples (figure 9a), although alternative, V-shape conformations would not be detected with this protocol. Although it is not meaningful to talk of `resolution’ in a model structure such as ours, constraints owing to the presence of multiple cross-links and amino acid spacing in junctions between modelled fragments mean that the coiledcoil register of our model is likely to be correct within one heptad repeat (see Materials and methods). In addition to the precise domain boundaries and structural parameters derived from this analysis, threersob.order ARQ-092 royalsocietypublishing.org Open Biol. 5:(a)(b)(c)rsob.royalsocietypublishing.orgSMCSMC4 CAP-HCAP-GCAP-DOpen Biol. 5:Figure 9. Possible models of condensin complex structure based on cross-linking data. (a) Diagram of condensin as a rod-shaped complex suggested by the crosslinking data. (b) Alternative model suggesting that on chromosomes, cross-links between the SMC2 and SMC4 coiled-coils could arise owing to side-by-side association of condensin holocomplexes. (c) Cross-linking suggests that condensin can form multrimers ( possibly trimers in vitro) where CAP-H proteins interact.noteworthy observations arise from considerations relating to the model. First, the cross-linked SMC2/SMC4 complex appears to exhibit structural flexibility (and/or the ability for controlled movement) at the connection points between the long coiledcoil and globular domains with regard to the angle between coiled-coil and head and hinge domains. Second, two previously proposed disruptions to sequence periodicity within the long coiled-coil, referred to as `loop I’ and `loop III’ [43], line up ARQ-092MedChemExpress ARQ-092 opposite one another in our threedimensional model. That is, not only do loop I and loop III, which are at opposite ends of the coiled-coil in both SMC2 and SMC4, line up opposite one another in the anti-parallel coiled-coil within each molecule, the two loops from SMC2 also line up opposite their counterparts from SMC4 in the four-stranded coil. Our analysis additionally defines a proline-rich 33 residue insertion within SMC4 (`loop III’ residues 1035?067, not modelled). A recent alternative cross-linking approach has described the overall geometry of the bacterial condensin MukB [82] and revealed the presence of multiple interruptions of its coiled-coil. These interruptions, termed `knuckles’, have been suggested to impart flexibility to the coiled-coils in MukB and bacterial SMC [37]. Further experiments are required to determine the functional significance of the analogous structural features in condensin. Third, because SM.Homologues of the human SMC head and hinge domains have been determined to atomic detail and served as templates for modelling the globular portions of SMC2 and SMC4, assembly of a draft structural model for SMC2/SMC4 was only made possible here by inclusion of constraints from the cross-linking analysis. This enabled us to pursue a template and fragment based approach and assemble the 13 fragments that were compatible with 117/120 high-confidence cross-links derived from the various condensin preparations into a low-resolution three-dimensional view of the entire SMC2/SMC4 dimer. The model reveals an intimate rod-like arrangement of the SMC2/SMC4 molecules dictated by the numerous, regularly arranged, intermolecular cross-links in the coiled-coil regions. Intriguingly, the remarkable consistency of the cross-link data with a single model seems to indicate that a single rod-like form [18] predominated in our samples (figure 9a), although alternative, V-shape conformations would not be detected with this protocol. Although it is not meaningful to talk of `resolution’ in a model structure such as ours, constraints owing to the presence of multiple cross-links and amino acid spacing in junctions between modelled fragments mean that the coiledcoil register of our model is likely to be correct within one heptad repeat (see Materials and methods). In addition to the precise domain boundaries and structural parameters derived from this analysis, threersob.royalsocietypublishing.org Open Biol. 5:(a)(b)(c)rsob.royalsocietypublishing.orgSMCSMC4 CAP-HCAP-GCAP-DOpen Biol. 5:Figure 9. Possible models of condensin complex structure based on cross-linking data. (a) Diagram of condensin as a rod-shaped complex suggested by the crosslinking data. (b) Alternative model suggesting that on chromosomes, cross-links between the SMC2 and SMC4 coiled-coils could arise owing to side-by-side association of condensin holocomplexes. (c) Cross-linking suggests that condensin can form multrimers ( possibly trimers in vitro) where CAP-H proteins interact.noteworthy observations arise from considerations relating to the model. First, the cross-linked SMC2/SMC4 complex appears to exhibit structural flexibility (and/or the ability for controlled movement) at the connection points between the long coiledcoil and globular domains with regard to the angle between coiled-coil and head and hinge domains. Second, two previously proposed disruptions to sequence periodicity within the long coiled-coil, referred to as `loop I’ and `loop III’ [43], line up opposite one another in our threedimensional model. That is, not only do loop I and loop III, which are at opposite ends of the coiled-coil in both SMC2 and SMC4, line up opposite one another in the anti-parallel coiled-coil within each molecule, the two loops from SMC2 also line up opposite their counterparts from SMC4 in the four-stranded coil. Our analysis additionally defines a proline-rich 33 residue insertion within SMC4 (`loop III’ residues 1035?067, not modelled). A recent alternative cross-linking approach has described the overall geometry of the bacterial condensin MukB [82] and revealed the presence of multiple interruptions of its coiled-coil. These interruptions, termed `knuckles’, have been suggested to impart flexibility to the coiled-coils in MukB and bacterial SMC [37]. Further experiments are required to determine the functional significance of the analogous structural features in condensin. Third, because SM.