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Cross-linking mass spectrometry of purified human endosomes (DHSO, DMTMM)

ABSTRACT: EndoMAP aims to systematically dissect the human endosomal protein complexes. DHSO/DMTMM cross-linking experiment was performed on freshly prepared endosomal pellets from HEK293 cells to capture protein interactions and structures in purified organelles.

INSTRUMENT(S): Orbitrap Ascend

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Permanent Cell Line Cell, Cell Culture

SUBMITTER: Miguel A. Gonzalez-Lozano

LAB HEAD: J. Wade Harper

PROVIDER: PXD059547 | Pride | 2025-03-29

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	DHSO_Scout_ResiduePairs.csv	Csv
	DMTMM_pLink_cross-linked_sites.csv	Csv
	checksum.txt	Txt
	sa00263.raw	Raw
	sa00264.raw	Raw

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Cross-linking mass spectrometry of purified human endosomes

Project description:EndoMAP aims to systematically dissect the human endosomal protein complexes. Cross-linking experiments were performed on two biologically independent replicates of freshly prepared endosomal pellets from HEK293 cells to capture protein interactions and structures in purified organelles.

2025-03-29 | PXD054728 | Pride

Integrative Structural Characterization of the Human Canonical and Non-canonical Cop9 Signalosome Complexes

Project description:The COP9 signalosome (CSN) is an evolutionarily conserved protein complex that functions as a deneddylase to inactivate Cullin-RING E3 ligases for controlling protein ubiquitination. CSN possesses structural flexibility that is important for its activation upon binding to a diverse array of CRLs. The canonical and non-canonical CSN complexes consist of 8 (CSN1-8) and 9 (CSN1-9) subunits, respectively. Although CSN9 is not essential for CSN assembly and function, it appears to be important in non-catalytic regulation of CRLs by CSN. Here we employed a combinatory cross-linking mass spectrometry (XL-MS) approach to generate the largest PPI maps of human CSN complexes, which significantly enhanced the precision of integrative structural modeling. The resulting integrative structures allowed us not only to elucidate architectures of both complexes, but also to assess CSN structural dynamics that was not described in the crystal structure. In addition, we have determined CSN9 docking sites and its impact on the CSN structure. While CSN9 binding did not induce global conformational changes, it triggered subunit local reorientations that may be associated with CSN9 involvement in CSN-mediated steric regulation of CRLs.

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Reconstitution of mammalian Cleavage Factor II

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Biochemically validated structural model of the 15-subunit intraflagellar transport complex IFT-B

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Enabling Photoactivated Cross-linking Mass Spectrometric Analysis of Protein Complexes by Novel MS-cleavable Cross-linkers

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