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- EMDB-18309: Helical reconstruction of yeast eisosome protein Pil1 bound to me... -

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Entry
Database: EMDB / ID: EMD-18309
TitleHelical reconstruction of yeast eisosome protein Pil1 bound to membrane composed of lipid mixture +PIP2/-sterol (DOPC, DOPE, DOPS, PI(4,5)P2 50:20:20:10)
Map dataSharpened helical map Type c plusPIP2/-sterol (D3, rise=14.55, twist=-83.25)
Sample
  • Complex: Helical assembly of recombinant Pil1 protein tubulating +PIP2/-sterol lipid mixture (DOPC,DOPE,DOPS,PI(4,5)P2 50:20:20:10)
    • Protein or peptide: Sphingolipid long chain base-responsive protein PIL1
  • Ligand: D-MYO-INOSITOL-1,4,5-TRIPHOSPHATE
KeywordsBAR domain / lipid reconstitution / membrane microdomain / LIPID BINDING PROTEIN
Function / homology
Function and homology information


protein localization to eisosome filament / eisosome filament / eisosome assembly / eisosome / lipid droplet / cell periphery / endocytosis / protein localization / mitochondrial outer membrane / lipid binding ...protein localization to eisosome filament / eisosome filament / eisosome assembly / eisosome / lipid droplet / cell periphery / endocytosis / protein localization / mitochondrial outer membrane / lipid binding / mitochondrion / plasma membrane / cytoplasm
Similarity search - Function
Eisosome component PIL1/LSP1 / Eisosome component PIL1 / AH/BAR domain superfamily
Similarity search - Domain/homology
Sphingolipid long chain base-responsive protein PIL1
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodhelical reconstruction / cryo EM / Resolution: 3.86 Å
AuthorsKefauver JM / Zou L / Desfosses A / Loewith RJ
Funding supportEuropean Union, Switzerland, 4 items
OrganizationGrant numberCountry
H2020 Marie Curie Actions of the European Commission101026765European Union
European Research Council (ERC)AdG TENDOEuropean Union
Swiss National Science FoundationCRSII5_189996 Switzerland
Swiss National Science Foundation310030_207754 Switzerland
CitationJournal: Nature / Year: 2024
Title: Cryo-EM architecture of a near-native stretch-sensitive membrane microdomain.
Authors: Jennifer M Kefauver / Markku Hakala / Luoming Zou / Josephine Alba / Javier Espadas / Maria G Tettamanti / Jelena Gajić / Caroline Gabus / Pablo Campomanes / Leandro F Estrozi / Nesli E Sen ...Authors: Jennifer M Kefauver / Markku Hakala / Luoming Zou / Josephine Alba / Javier Espadas / Maria G Tettamanti / Jelena Gajić / Caroline Gabus / Pablo Campomanes / Leandro F Estrozi / Nesli E Sen / Stefano Vanni / Aurélien Roux / Ambroise Desfosses / Robbie Loewith /
Abstract: Biological membranes are partitioned into functional zones termed membrane microdomains, which contain specific lipids and proteins. The composition and organization of membrane microdomains remain ...Biological membranes are partitioned into functional zones termed membrane microdomains, which contain specific lipids and proteins. The composition and organization of membrane microdomains remain controversial because few techniques are available that allow the visualization of lipids in situ without disrupting their native behaviour. The yeast eisosome, composed of the BAR-domain proteins Pil1 and Lsp1 (hereafter, Pil1/Lsp1), scaffolds a membrane compartment that senses and responds to mechanical stress by flattening and releasing sequestered factors. Here we isolated near-native eisosomes as helical tubules made up of a lattice of Pil1/Lsp1 bound to plasma membrane lipids, and solved their structures by helical reconstruction. Our structures reveal a striking organization of membrane lipids, and, using in vitro reconstitutions and molecular dynamics simulations, we confirmed the positioning of individual PI(4,5)P, phosphatidylserine and sterol molecules sequestered beneath the Pil1/Lsp1 coat. Three-dimensional variability analysis of the native-source eisosomes revealed a dynamic stretching of the Pil1/Lsp1 lattice that affects the sequestration of these lipids. Collectively, our results support a mechanism in which stretching of the Pil1/Lsp1 lattice liberates lipids that would otherwise be anchored by the Pil1/Lsp1 coat, and thus provide mechanistic insight into how eisosome BAR-domain proteins create a mechanosensitive membrane microdomain.
History
DepositionAug 24, 2023-
Header (metadata) releaseJul 24, 2024-
Map releaseJul 24, 2024-
UpdateAug 28, 2024-
Current statusAug 28, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_18309.png

Downloads & links

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Map

FileDownload / File: emd_18309.map.gz / Format: CCP4 / Size: 1000 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSharpened helical map Type c plusPIP2/-sterol (D3, rise=14.55, twist=-83.25)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 640 pix.
= 531.2 Å		0.83 Å/pix.
x 640 pix.
= 531.2 Å		0.83 Å/pix.
x 640 pix.
= 531.2 Å

Surface

Projections

Slices (1/3)

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Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.83 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.05
Minimum - Maximum-0.13308172 - 0.2541855
Average (Standard dev.)0.0005284506 (±0.012177983)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions640640640
Spacing640640640
CellA=B=C: 531.2 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_18309_msk_1.map
Projections & Slices
AxesZYX

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Additional map: Unsharpened helical map Type d plusPIP2/-sterol (D1, rise=5.078,...

Fileemd_18309_additional_1.map
AnnotationUnsharpened helical map Type d plusPIP2/-sterol (D1, rise=5.078, twist=-136.523)
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Additional map: Unsharpened helical map Type b plusPIP2/-sterol (D1, rise=5.420,...

Fileemd_18309_additional_2.map
AnnotationUnsharpened helical map Type b plusPIP2/-sterol (D1, rise=5.420, twist=133.600)
Projections & Slices
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Additional map: Unsharpened helical map Type a plusPIP2/-sterol (D4, rise=21.004,...

Fileemd_18309_additional_3.map
AnnotationUnsharpened helical map Type a plusPIP2/-sterol (D4, rise=21.004, twist=39.094)
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Additional map: Unsharpened helical map Type e plusPIP2/-sterol (D1, rise=4.714,...

Fileemd_18309_additional_4.map
AnnotationUnsharpened helical map Type e plusPIP2/-sterol (D1, rise=4.714, twist=-81.141)
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Additional map: Unsharpened helical map Type c plusPIP2/-sterol (D3, rise=14.55,...

Fileemd_18309_additional_5.map
AnnotationUnsharpened helical map Type c plusPIP2/-sterol (D3, rise=14.55, twist=-83.25)
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Half map: Helical map Type c plusPIP2/-sterol - half map A

Fileemd_18309_half_map_1.map
AnnotationHelical map Type c plusPIP2/-sterol - half map A
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Half map: Helical map Type c plusPIP2/-sterol - half map B

Fileemd_18309_half_map_2.map
AnnotationHelical map Type c plusPIP2/-sterol - half map B
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Sample components

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Entire : Helical assembly of recombinant Pil1 protein tubulating +PIP2/-st...

EntireName: Helical assembly of recombinant Pil1 protein tubulating +PIP2/-sterol lipid mixture (DOPC,DOPE,DOPS,PI(4,5)P2 50:20:20:10)
Components
  • Complex: Helical assembly of recombinant Pil1 protein tubulating +PIP2/-sterol lipid mixture (DOPC,DOPE,DOPS,PI(4,5)P2 50:20:20:10)
    • Protein or peptide: Sphingolipid long chain base-responsive protein PIL1
  • Ligand: D-MYO-INOSITOL-1,4,5-TRIPHOSPHATE

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Supramolecule #1: Helical assembly of recombinant Pil1 protein tubulating +PIP2/-st...

SupramoleculeName: Helical assembly of recombinant Pil1 protein tubulating +PIP2/-sterol lipid mixture (DOPC,DOPE,DOPS,PI(4,5)P2 50:20:20:10)
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / Strain: TB50

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Macromolecule #1: Sphingolipid long chain base-responsive protein PIL1

MacromoleculeName: Sphingolipid long chain base-responsive protein PIL1 / type: protein_or_peptide / ID: 1 / Number of copies: 14 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / Strain: TB50
Molecular weightTheoretical: 38.393043 KDa
Recombinant expressionOrganism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
SequenceString: MHRTYSLRNS RAPTASQLQN PPPPPSTTKG RFFGKGGLAY SFRRSAAGAF GPELSRKLSQ LVKIEKNVLR SMELTANERR DAAKQLSIW GLENDDDVSD ITDKLGVLIY EVSELDDQFI DRYDQYRLTL KSIRDIEGSV QPSRDRKDKI TDKIAYLKYK D PQSPKIEV ...String:
MHRTYSLRNS RAPTASQLQN PPPPPSTTKG RFFGKGGLAY SFRRSAAGAF GPELSRKLSQ LVKIEKNVLR SMELTANERR DAAKQLSIW GLENDDDVSD ITDKLGVLIY EVSELDDQFI DRYDQYRLTL KSIRDIEGSV QPSRDRKDKI TDKIAYLKYK D PQSPKIEV LEQELVRAEA ESLVAEAQLS NITRSKLRAA FNYQFDSIIE HSEKIALIAG YGKALLELLD DSPVTPGETR PA YDGYEAS KQIIIDAESA LNEWTLDSAQ VKPTLSFKQD YEDFEPEEGE EEEEEDGQGR WSEDEQEDGQ IEEPEQEEEG AVE EHEQVG HQQSESLPQQ TTA

UniProtKB: Sphingolipid long chain base-responsive protein PIL1

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Macromolecule #2: D-MYO-INOSITOL-1,4,5-TRIPHOSPHATE

MacromoleculeName: D-MYO-INOSITOL-1,4,5-TRIPHOSPHATE / type: ligand / ID: 2 / Number of copies: 14 / Formula: I3P
Molecular weightTheoretical: 420.096 Da
Chemical component information

ChemComp-I3P:
D-MYO-INOSITOL-1,4,5-TRIPHOSPHATE

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Experimental details

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Structure determination

Methodcryo EM
Processinghelical reconstruction
Aggregation statehelical array

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Sample preparation

BufferpH: 7.4 / Details: 20 mM HEPES, pH 7.4, 150 mM KoAc, 2 mM MgAc
GridModel: EMS Lacey Carbon / Support film - Material: CARBON / Support film - topology: LACEY
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 291 K / Instrument: LEICA EM GP

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 1.8 µm / Nominal defocus min: 0.6 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionApplied symmetry - Helical parameters - Δz: 14.547 Å
Applied symmetry - Helical parameters - Δ&Phi: -83.25 °
Applied symmetry - Helical parameters - Axial symmetry: D3 (2x3 fold dihedral)
Resolution.type: BY AUTHOR / Resolution: 3.86 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 85456
Startup modelType of model: NONE
Final angle assignmentType: NOT APPLICABLE

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Atomic model buiding 1

Initial modelPDB ID:

3252


Chain - Source name: AlphaFold / Chain - Initial model type: in silico model
RefinementSpace: REAL
Output model

PDB-8qbb:
Helical reconstruction of yeast eisosome protein Pil1 bound to membrane composed of lipid mixture +PIP2/-sterol (DOPC, DOPE, DOPS, PI(4,5)P2 50:20:20:10)

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