Dataset Information

Cryo-EM structures of holo condensin reveal a subunit flip-flop mechanism

ABSTRACT: This data is from sulfo-SDA crosslinked condensin pentamer. Two datsets, one without atp aand one with ATP. How protein complexes of the SMC family fold DNA into the large loops that are fundamental for the 3D organization of genomes is a central unresolved question of chromosome biology. We used electron cryomicroscopy to investigate the reaction cycle of the SMC complex condensin, which is a key determinant of chromosome morphology and behavior during mitosis. Our structures of the Saccharomyces cerevisiae condensin holo complex at different functional stages suggest that ATP binding induces the transition from a folded-rod SMC conformation into an open architecture and triggers the exchange of the two HEAT-repeat subunits at the SMC ATPase head domains. We propose that these steps result in the interconversion of DNA binding sites in the catalytic core that form the basis of the DNA translocation and loop-extrusion activities of condensin.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Saccharomyces Cerevisiae (baker's Yeast)

SUBMITTER: Francis O'Reilly

LAB HEAD: Juri Rappsilber

PROVIDER: PXD019274 | Pride | 2020-07-28

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	1FDR_Condensin_pentamer_ATPbound_SDA	Other
	1FDR_Condensin_pentamer_ATPbound_SDA_Links_xiFDR1.4.1.csv	Csv
	1FDR_Condensin_pentamer_Apo_SDA	Other
	1FDR_Condensin_pentamer_Apo_SDA_Links_xiFDR1.4.1.csv	Csv
	B190124_11_Lumos_FO_CO_120_Condesin_SDA_SCX27.raw	Raw

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Publications

Cryo-EM structures of holo condensin reveal a subunit flip-flop mechanism.

Lee Byung-Gil BG Merkel Fabian F Allegretti Matteo M Hassler Markus M Cawood Christopher C Lecomte Léa L O'Reilly Francis J FJ Sinn Ludwig R LR Gutierrez-Escribano Pilar P Kschonsak Marc M Bravo Sol S Nakane Takanori T Rappsilber Juri J Aragon Luis L Beck Martin M Löwe Jan J Haering Christian H CH

Nature structural & molecular biology 20200713 8

Complexes containing a pair of structural maintenance of chromosomes (SMC) family proteins are fundamental for the three-dimensional (3D) organization of genomes in all domains of life. The eukaryotic SMC complexes cohesin and condensin are thought to fold interphase and mitotic chromosomes, respectively, into large loop domains, although the underlying molecular mechanisms have remained unknown. We used cryo-EM to investigate the nucleotide-driven reaction cycle of condensin from the budding ye ...[more]

PMID: 32661420

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Project description:[NiFe]-hydrogenases catalyse the reversible splitting of hydrogen. The catalytic metal centre (NiFe(CN)2CO) is unique in biology, and assembled by an intricate protein machinery, in a process that is still being explored. We hypothesised a structural, ATP-dependent, mechanistic explanation for the assembly of the Fe(CN)2CO fragment via the HypCD complex. We carried out a crosslinking mass spectrometry (crosslinking MS) analysis to study the structure of the HypCD complex, both with (holoprotein) and without (apoprotein) the metal cofactor, and both with and without the addition of ATP. We used the UV-photoactivatable crosslinking reagent sulfo-SDA, which has been shown to have excellent performance when studying dynamic and flexible protein complexes. For photoactivation we used a high-powered LED which enabled the use of exceptionally short reaction times (20 seconds) and gave strikingly clear results. From the resulting crosslinked residue pair patterns identified, we were able to unambiguously distinguish between holoprotein with and without ATP. Crosslinks found in holoprotein, in the absence of ATP, suggested a “closed” protein conformation. When the HypCD holoprotein was crosslinked in the presence of ATP, two distinct crosslink bands were almost entirely absent, indicating that the protein conformation had shifted to an “open” conformation. Interestingly, no shift in protein conformation was evident in the crosslinked apoprotein, which implied that the cofactor was central to protein conformational dynamics. Crosslinking MS data helped to explain, and was in agreement with, protein structures predicted by AlphaFold2 (which were subsequently refined by density functional theory (DFT) modeling for placing the cofactor). Considering all the experimental data from this study led to the conclusion that the binding of ATP alone, not its hydrolysis, is required for the transfer of the Fe(CN)2CO fragment to the apo-hydrogenase large subunit.