Vesicle Picker: A tool for efficient identification of membrane protein complexes in vesicles DOI
Ryan Karimi, Claire E. Coupland, John L. Rubinstein

et al.

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2024, Volume and Issue: unknown

Published: July 16, 2024

Abstract Electron cryomicroscopy (cryo-EM) has recently allowed determination of near-atomic resolution structures membrane proteins and protein complexes embedded in lipid vesicles. However, particle selection from electron micrographs these vesicles can be challenging due to the strong signal contributed bilayer. This challenge often requires iterative laborious workflows generate a dataset high-quality images for subsequent analysis. Here we present Vesicle Picker, an open-source program built on Segment Anything model. Picker enables automatic identification cryo-EM with high recall precision. It then exhaustively selects all potential locations, either at perimeter or uniformly over surface projection vesicle. The is designed interface cryoSPARC, which performs both upstream micrograph processing downstream single image We demonstrate Picker’s utility by determining high-resolution map vacuolar-type ATPase native synaptic (SVs) identifying additional complex SV membrane.

Language: Английский

Structure of yeast RAVE bound to a partial V 1 complex DOI Creative Commons
Hanlin Wang,

Maureen Tarsio,

Patricia M. Kane

et al.

Proceedings of the National Academy of Sciences, Journal Year: 2024, Volume and Issue: 121(50)

Published: Dec. 3, 2024

Vacuolar-type ATPases (V-ATPases) are membrane-embedded proton pumps that acidify intracellular compartments in almost all eukaryotic cells. Homologous with ATP synthases, these multisubunit enzymes consist of a soluble catalytic V 1 subcomplex and proton-translocating O subcomplex. The subcomplexes can undergo reversible dissociation to regulate pumping, reassociation requiring the protein complex known as RAVE (regulator ATPase vacuoles endosomes). In yeast Saccharomyces cerevisiae , consists subunits Rav1p, Rav2p, Skp1p. We used electron cryomicroscopy (cryo-EM) determine structure bound . structure, is an L-shaped Rav2p pointing toward membrane Skp1p distant from both Only Rav1p interacts binding region subunit A not found corresponding synthase subunit. When RAVE, rotational state suitable for free complex, but it partially disrupted, missing five its 16 subunits. Other than conformation inhibitory H, appears poised reassembly

Language: Английский

Citations

1
NDT‐C11 as a Viable Novel Detergent for Single Particle Cryo‐EM DOI Creative Commons

Chimari Jiko,

Jiannan Li,

Youngsun Moon

et al.

ChemPlusChem, Journal Year: 2024, Volume and Issue: unknown

Published: June 17, 2024

Single particle cryo electron microscopy (cryo-EM) is now the major method for determination of integral membrane protein structure. For success a given project type mimetic used extraction from native cell membrane, purification to homogeneity and finally cryo-grid vitrification crucial. Although small molecule amphiphiles - detergents are most widely mimetic, specific tailoring detergent structure single cryo-EM rare demand effective not satisfied. Here, we compare popular lauryl maltose-neopentyl glycol (LMNG) with novel neopentyl glycol-derived triglucoside-C11 (NDT-C11) in its behavior as free when bound two types multisubunit complexes cyanobacterial photosystem I (PSI) mammalian F-ATP synthase. We conclude that NDT-C11 has high potential become very useful proteins.

Language: Английский

Citations

0
Control of Golgi- V-ATPase through Sac1-dependent co-regulation of PI(4)P and cholesterol DOI Creative Commons
Xin Zhou, Miesje M. van der Stoel, Shreyas Kaptan

et al.

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2024, Volume and Issue: unknown

Published: Nov. 5, 2024

Abstract Sac1 is a conserved phosphoinositide phosphatase, whose loss-of-function compromises cell and organism viability. Here, we employed acute auxin-inducible degradation to identify its immediate downstream effectors in human cells. Most of was degraded ∼1 h, paralleled by increased PI(4)P decreased cholesterol the trans-Golgi network (TGN) during following hour, superseded Golgi fragmentation, impaired glycosylation, selective TGN proteins ∼4 h. The disintegration resulted from deacidification caused disassembly V-ATPase. Mechanistically, mediated membrane composition maintained an assembly promoting conformation V 0 a2 subunit. Key phenotypes were recapitulated differentiated trophoblasts, causing processing defects chorionic gonadotropin, line with intolerance SACML1 gene. Collectively, our findings reveal that V-ATPase controlled via fuelled lipid exchange.

Language: Английский

Citations

0
TBC1D24 interacts with the v-ATPase and regulates intraorganellar pH in neurons DOI Creative Commons
Sara Pepe, Davide Aprile, Enrico Castroflorio

et al.

iScience, Journal Year: 2024, Volume and Issue: 28(1), P. 111515 - 111515

Published: Dec. 1, 2024

The vacuolar ATPase (v-ATPase) is essential for acidification of intracellular organelles, including synaptic vesicles. Its activity controlled by cycles association and dissociation the ATP hydrolysis (V

Language: Английский

Citations

0
Vesicle Picker: A tool for efficient identification of membrane protein complexes in vesicles DOI
Ryan Karimi, Claire E. Coupland, John L. Rubinstein

et al.

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2024, Volume and Issue: unknown

Published: July 16, 2024

Abstract Electron cryomicroscopy (cryo-EM) has recently allowed determination of near-atomic resolution structures membrane proteins and protein complexes embedded in lipid vesicles. However, particle selection from electron micrographs these vesicles can be challenging due to the strong signal contributed bilayer. This challenge often requires iterative laborious workflows generate a dataset high-quality images for subsequent analysis. Here we present Vesicle Picker, an open-source program built on Segment Anything model. Picker enables automatic identification cryo-EM with high recall precision. It then exhaustively selects all potential locations, either at perimeter or uniformly over surface projection vesicle. The is designed interface cryoSPARC, which performs both upstream micrograph processing downstream single image We demonstrate Picker’s utility by determining high-resolution map vacuolar-type ATPase native synaptic (SVs) identifying additional complex SV membrane.

Language: Английский

Citations

0