Dataset Information

3D topology of the SMC2/SMC4 sub-complex from chicken condensin I revealed by cross-linking and molecular modelling

ABSTRACT: SMC proteins are essential components of three protein complexes that are important for chromosome structure and function. The cohesin complex holds replicated sister chromatids together, whereas the condensin complex has an essential role in mitotic chromosome architecture. Both are involved in interphase genome organisation. SMC-containing complexes are large (>650 kDa for condensin) and contain long anti-parallel coiled-coils. They are thus difficult subjects for conventional crystallographic and electron cryomicroscopic studies. Here we have used amino acid-selective cross-linking and mass spectrometry (CLMS) combined with structure prediction to develop a full-length molecular draft 3D-structure of the SMC2/SMC4 dimeric backbone of chicken condensin. We assembled homology-based molecular models of the globular heads and hinges with the lengthy coiled-coils modelled in fragments, using numerous high-confidence cross-links and accounting for potential irregularity. Our experiments reveal that isolated condensin complexes can exist with their coiled-coil segments closely apposed to one another along their lengths and define the relative spatial alignment of the two anti-parallel coils. The centres of the coiled-coils can also approach one another closely in situ in mitotic chromosomes. In addition to revealing structural information, our cross-linking data suggest that both H2A and H4 may have roles in condensin interactions with chromatin.

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Gallus Gallus (chicken)

SUBMITTER: Zhuo Chen

LAB HEAD: Juri Rappsilber

PROVIDER: PXD001835 | Pride | 2015-07-13

REPOSITORIES: pride

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Dataset's files

Source:

			Action	DRS
	Rappsilber_Earnshaw_CLMS_SMC2-SMC4_PeakLists.zip	Other
	Rappsilber_Earnshaw_CLMS_SMC2-SMC4_SMC2-SMC4-subcomplex_1.RAW	Raw
	Rappsilber_Earnshaw_CLMS_SMC2-SMC4_SMC2-SMC4-subcomplex_2.RAW	Raw
	Rappsilber_Earnshaw_CLMS_SMC2-SMC4_SearchResults.csv	Csv
	Rappsilber_Earnshaw_CLMS_SMC2-SMC4_cohesin_1.raw	Raw

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Publications

Three-dimensional topology of the SMC2/SMC4 subcomplex from chicken condensin I revealed by cross-linking and molecular modelling.

Barysz Helena H Kim Ji Hun JH Chen Zhuo Angel ZA Hudson Damien F DF Rappsilber Juri J Gerloff Dietlind L DL Earnshaw William C WC

Open biology 20150201 2

SMC proteins are essential components of three protein complexes that are important for chromosome structure and function. The cohesin complex holds replicated sister chromatids together, whereas the condensin complex has an essential role in mitotic chromosome architecture. Both are involved in interphase genome organization. SMC-containing complexes are large (more than 650 kDa for condensin) and contain long anti-parallel coiled-coils. They are thus difficult subjects for conventional crystal ...[more]

PMID: 25716199

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Project description:The HOP2/MND1 heterodimer is essential for meiotic homologous recombination in plants and other eukaryotes, and promotes the repair of DNA double strand breaks. The HOP2/MND1 dimer forms via the central, split coiled coils (CC1 and CC2) present in both proteins, and the resulting complex contains several flexible regions such as the hinge between the coils. To investigate the conformational flexibility of the heterodimer, important for understanding mechanistic details of HOP2/MND1 function, we studied the spatial relation of the complex partners and their domains in solution. We performed chemical cross-linking in combination with mass spectrometry (XL-MS) to generate distance restraints, which were subsequently used for molecular modeling. The final XL-MS workflow encompassed the use of cross-linkers with varying spacer arm lengths, quenching, digestion, size exclusion enrichment and HCD based LC-MS/MS detection prior to data evaluation. We applied and systematically tested two different homobifunctional amine-reactive crosslinkers (DSS-11.4 Å and BS2G 7.7 Å) and one zero-length heterobifunctional crosslinker (EDC). Crosslinked peptides of four biological replicates were analyzed prior to 3D structure prediction by protein threading and protein-protein docking for crosslink guided molecular modeling. Our miniaturized SEC approach reduced the required starting material and led to a high amount of crosslinked peptides allowing the analysis of replicates. The majority of the identified crosslinks was found in the coiled coil domains, indicating a parallel orientation of the interaction partners. Furthermore, flexibility of the C-terminal head domains of HOP2 and MND1 was observed. The experimentally derived distance constraints combined with an iterative comparative modeling approach not only confirm the elongated, open conformation predicted by the crystal structure of the Giardia lamblia Hop2-Mnd1 heterodimer, but further suggest the coexistence of a closed complex conformation in solution.