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- PDB-8qb9: Helical reconstruction of yeast eisosome protein Pil1 bound to me... -

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Basic information

Entry
Database: PDB / ID: 8qb9
TitleHelical reconstruction of yeast eisosome protein Pil1 bound to membrane composed of lipid mixture -PIP2/+sterol (DOPC, DOPE, DOPS, cholesterol 30:20:20:30)
ComponentsSphingolipid long chain base-responsive protein PIL1
KeywordsLIPID BINDING PROTEIN / BAR domain / lipid reconstitution / membrane microdomain
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)
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 3.35 Å
AuthorsKefauver, J.M. / Zou, L. / Desfosses, A. / Loewith, R.J.
Funding supportEuropean Union, Switzerland, 4items
OrganizationGrant numberCountry
H2020 Marie Curie Actions of the European Commission101026765European Union
European Research Council (ERC)AdG TENDOEuropean Union
Swiss National Science FoundationCRSII5_189996 METEORIC 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, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jul 24, 2024Provider: repository / Type: Initial release
Revision 1.1Jul 31, 2024Group: Data collection / Database references / Category: citation / em_admin
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.title / _citation.year / _em_admin.last_update
Revision 1.2Aug 7, 2024Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.pdbx_database_id_PubMed / _citation.title / _em_admin.last_update
Revision 1.3Aug 28, 2024Group: Data collection / Database references / Category: citation / em_admin
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Sphingolipid long chain base-responsive protein PIL1
B: Sphingolipid long chain base-responsive protein PIL1
C: Sphingolipid long chain base-responsive protein PIL1
I: Sphingolipid long chain base-responsive protein PIL1
D: Sphingolipid long chain base-responsive protein PIL1
J: Sphingolipid long chain base-responsive protein PIL1
E: Sphingolipid long chain base-responsive protein PIL1
K: Sphingolipid long chain base-responsive protein PIL1
F: Sphingolipid long chain base-responsive protein PIL1
L: Sphingolipid long chain base-responsive protein PIL1
G: Sphingolipid long chain base-responsive protein PIL1
M: Sphingolipid long chain base-responsive protein PIL1
H: Sphingolipid long chain base-responsive protein PIL1
N: Sphingolipid long chain base-responsive protein PIL1


Theoretical massNumber of molelcules
Total (without water)537,50314
Polymers537,50314
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
d_1ens_1chain "F"
d_2ens_1chain "B"
d_3ens_1chain "C"
d_4ens_1chain "D"
d_5ens_1chain "E"
d_6ens_1chain "A"
d_7ens_1chain "G"
d_8ens_1chain "H"
d_9ens_1chain "I"
d_10ens_1chain "J"
d_11ens_1chain "K"
d_12ens_1chain "L"
d_13ens_1chain "M"
d_14ens_1chain "N"

NCS domain segments:

Component-ID: 1 / Ens-ID: ens_1 / Beg auth comp-ID: MET / Beg label comp-ID: MET / End auth comp-ID: THR / End label comp-ID: THR / Auth seq-ID: 1 - 271 / Label seq-ID: 1 - 271

Dom-IDAuth asym-IDLabel asym-ID
d_1FI
d_2BB
d_3CC
d_4DE
d_5EG
d_6AA
d_7GK
d_8HM
d_9ID
d_10JF
d_11KH
d_12LJ
d_13ML
d_14NN

NCS oper:
IDCodeMatrixVector
1given(-0.977662841136, -0.210175350563, -0.00130041401013), (-0.210170517131, 0.977659731, -0.00313115161514), (0.00192945329971, -0.00278790189909, -0.99999425239)581.42098347, 62.4604912656, 490.631595096
2given(0.911685077949, 0.410888977544, -0.0007528461852), (-0.410889282958, 0.911680189131, -0.00303807448503), (-0.000561956366348, 0.00307910360291, 0.999995101651)-85.6008354232, 133.294354286, 80.4517626144
3given(0.904179881653, -0.427148573476, 0.00168457454136), (0.427151377912, 0.904177893047, -0.00200949565638), (-0.000664801856462, 0.00253651388131, 0.999996562062)138.466372213, -87.5842400466, 65.4290223333
4given(0.794333039495, -0.60748211573, 0.00070812098267), (0.60748250624, 0.794332785978, -0.000655539563458), (-0.00016425515202, 0.000950887843224, 0.999999534416)215.807966635, -106.561735887, 25.1360339977
5given(0.977626084336, 0.210348157405, -0.000944406182362), (-0.210348713971, 0.977626275087, -0.000533657431435), (0.000811022440844, 0.000720372050997, 0.999999411653)-49.718832215, 61.9282734422, 39.9947720579
6given(0.648732018386, 0.761016877937, -0.000282517222415), (-0.761016919416, 0.64873190133, -0.00041056056278), (-0.000129165582835, 0.000481344168846, 0.999999875812)-108.763359645, 295.519737676, 15.1006734912
7given(0.474535845528, 0.880234600347, -0.00166723133623), (-0.880236078678, 0.474534034549, -0.00137689767597), (-0.000420834963013, 0.00212094447653, 0.999997662243)-93.9021626005, 373.742784893, 55.3236809884
8given(-0.999998785023, 0.00015829326328, -0.0015507728211), (0.000160247455305, 0.999999193237, -0.00126009755012), (0.00155057210504, -0.00126034452653, -0.999998003627)531.530921538, 0.230578265373, 530.889200017
9given(-0.648938171932, -0.760841139498, 9.72413437244E-5), (-0.760838853847, 0.648935898146, -0.00253743450656), (0.00186748116265, -0.00172062310259, -0.99999677598)640.061766254, 295.927243567, 515.732040223
10given(-0.47471574748, -0.880136925013, 0.00198804504246), (-0.880136755581, 0.474707942929, -0.00341473212215), (0.00206169105718, -0.00337077862543, -0.99999219361)625.08083534, 374.152626894, 475.537379199
11given(-0.9117510272, -0.410743172262, -0.00033292631515), (-0.410736842805, 0.911740925794, -0.00487136483943), (0.00230442239419, -0.00430472679264, -0.999988079411)617.050989922, 133.649088922, 450.37519538
12given(-0.904060684218, 0.427388629646, -0.00363847509317), (0.427398337298, 0.904059971645, -0.00249578535302), (0.00222272940764, -0.00381141961904, -0.99999026623)393.118996008, -87.5489235597, 465.460812187
13given(-0.794207365161, 0.607640467967, -0.00277899495786), (0.607644105833, 0.794209184441, -0.000641869607187), (0.00181707737056, -0.00219841747578, -0.999995932587)315.802444577, -106.606734047, 505.721612076

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Components

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


Mass: 38393.043 Da / Num. of mol.: 14
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Strain: TB50 / Gene: PIL1 / Plasmid: pCoofy6
Details (production host): gift from Sabine Suppmann, Addgene plasmid #43990
Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
References: UniProt: P53252

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: HELICAL ARRAY / 3D reconstruction method: helical reconstruction

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Sample preparation

ComponentName: Helical assembly of recombinant Pil1 protein tubulating -PIP2/+sterol lipid mixture (DOPC,DOPE,DOPS,cholesterol 30:20:20:30)
Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / Strain: TB50
Source (recombinant)Organism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
Plasmid: pCoofy6
Buffer solutionpH: 7.4 / Details: 20mM HEPES, pH 7.4, 150mM KoAc, 2mM MgAc
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: EMS Lacey Carbon
VitrificationInstrument: LEICA EM GP / Cryogen name: ETHANE / Humidity: 90 % / Chamber temperature: 291 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 1800 nm / Nominal defocus min: 600 nm / Cs: 2.7 mm
Image recordingElectron dose: 50 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k)

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Processing

EM software
IDNameVersionCategoryDetails
1cryoSPARC4.1.2particle selectionFilament Tracer
4cryoSPARC4.1.2CTF correctionPatch CTF
7UCSF Chimera1.16model fitting
8Coot0.8.9.2model fitting
10PHENIX1.20-4459model refinement
11cryoSPARC4.1.2initial Euler assignment
12cryoSPARC4.1.2final Euler assignment
14cryoSPARC4.1.23D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Helical symmerty
IDImage processing-IDAngular rotation/subunit (°)Axial rise/subunit (Å)Axial symmetry
11-136.55.044D1
21-136.55.044D1
3D reconstructionResolution: 3.35 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 176005 / Symmetry type: HELICAL
Atomic model buildingSpace: REAL
Atomic model buildingAccession code: P53252 / Source name: AlphaFold / Type: in silico model
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 84.74 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.002528322
ELECTRON MICROSCOPYf_angle_d0.442838346
ELECTRON MICROSCOPYf_chiral_restr0.03084354
ELECTRON MICROSCOPYf_plane_restr0.00615026
ELECTRON MICROSCOPYf_dihedral_angle_d2.76283836
Refine LS restraints NCS
Ens-IDDom-IDAuth asym-IDRefine-IDTypeRms dev position (Å)
ens_1d_2IELECTRON MICROSCOPYNCS constraints1.01693336663E-12
ens_1d_3IELECTRON MICROSCOPYNCS constraints6.47489325711E-13
ens_1d_4IELECTRON MICROSCOPYNCS constraints9.00903061816E-11
ens_1d_5IELECTRON MICROSCOPYNCS constraints1.10943272885E-10
ens_1d_6IELECTRON MICROSCOPYNCS constraints1.28235134764E-12
ens_1d_7IELECTRON MICROSCOPYNCS constraints1.062690428E-11
ens_1d_8IELECTRON MICROSCOPYNCS constraints9.42800249269E-11
ens_1d_9IELECTRON MICROSCOPYNCS constraints8.20088129359E-13
ens_1d_10IELECTRON MICROSCOPYNCS constraints5.8725247828E-13
ens_1d_11IELECTRON MICROSCOPYNCS constraints1.78261561223E-11
ens_1d_12IELECTRON MICROSCOPYNCS constraints4.71662769523E-12
ens_1d_13IELECTRON MICROSCOPYNCS constraints9.90777876257E-10
ens_1d_14IELECTRON MICROSCOPYNCS constraints7.05416227993E-11

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