Dataset Information

Dataset 4 from 'Atlas of the proteasome interactome by in situ structural biology'

ABSTRACT: In the hRpn1 pulldown(dataset1 and 2), we identified 2793 unique residue pairs (877 inter-protein) at a 2% residue-pair target-decoy false discovery rate (FDR), with an additional threshold limiting the protein-protein FDR to 5% (Lenz et al., 2021 PMID: 34117231). The hRpn11 pulldown (dataset 3) yielded 3371 unique residue pairs (1847 inter-protein) at the same FDR threshold. When combined, the two datasets included 5364 non-redundant crosslinked residue pairs. This includes 2266 hetero-protein links describing 278 PPIs, with a combined estimated PPI-FDR of 7.3%

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Kitaik Lee

LAB HEAD: Francis J.O'Reilly

PROVIDER: PXD066073 | Pride | 2026-07-16

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	E250309_01_KL_PSMB4_inSituXL_SDA_IDruns_inj1.pepXML	Pepxml
	E250309_01_KL_PSMB4_inSituXL_SDA_IDruns_inj1.pin	Other
	E250309_01_KL_PSMB4_inSituXL_SDA_IDruns_inj1.png	Other
	E250309_01_KL_PSMB4_inSituXL_SDA_IDruns_inj1.raw	Raw
	E250309_01_KL_PSMB4_inSituXL_SDA_IDruns_inj1_edited.pin	Other

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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.

Dataset Information

Dataset 4 from 'Atlas of the proteasome interactome by in situ structural biology'

Dataset's files

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