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- PDB-8qbe: Compact state - Pil1 in native eisosome lattice bound to plasma m... -

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Basic information

Entry
Database: PDB / ID: 8qbe
TitleCompact state - Pil1 in native eisosome lattice bound to plasma membrane microdomain
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 / single particle reconstruction / cryo EM / Resolution: 3.67 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 Switzerland
Swiss National Science Foundation310030_207754 Switzerland
Citation
Journal: 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.
#1: Journal: Acta Crystallogr D Struct Biol / Year: 2018
Title: Real-space refinement in PHENIX for cryo-EM and crystallography.
Authors: Pavel V Afonine / Billy K Poon / Randy J Read / Oleg V Sobolev / Thomas C Terwilliger / Alexandre Urzhumtsev / Paul D Adams /
Abstract: This article describes the implementation of real-space refinement in the phenix.real_space_refine program from the PHENIX suite. The use of a simplified refinement target function enables very fast ...This article describes the implementation of real-space refinement in the phenix.real_space_refine program from the PHENIX suite. The use of a simplified refinement target function enables very fast calculation, which in turn makes it possible to identify optimal data-restraint weights as part of routine refinements with little runtime cost. Refinement of atomic models against low-resolution data benefits from the inclusion of as much additional information as is available. In addition to standard restraints on covalent geometry, phenix.real_space_refine makes use of extra information such as secondary-structure and rotamer-specific restraints, as well as restraints or constraints on internal molecular symmetry. The re-refinement of 385 cryo-EM-derived models available in the Protein Data Bank at resolutions of 6 Å or better shows significant improvement of the models and of the fit of these models to the target maps.
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 "A"
d_2ens_1chain "B"
d_3ens_1chain "C"
d_4ens_1chain "D"
d_5ens_1chain "E"
d_6ens_1chain "F"
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_1AA
d_2BB
d_3CC
d_4DE
d_5EG
d_6FI
d_7GK
d_8HM
d_9ID
d_10JF
d_11KH
d_12LJ
d_13ML
d_14NN

NCS oper:
IDCodeMatrixVector
1given(-0.999979563303, 0.00634171049912, 0.000809743381841), (-0.00633912039663, -0.99997490788, 0.00316214527957), (0.000829776473582, 0.00315694759498, 0.999994672562)338.612647096, 340.394550325, -0.656405680444
2given(0.973741290162, -0.00968800059077, 0.227451187021), (-0.0108434089113, 0.995986481291, 0.088844524695), (-0.227399033234, -0.0889779283286, 0.969728110325)-40.2438125553, -57.5731503977, 61.9370183058
3given(0.808473188352, -0.069989233219, -0.584356578597), (-0.0812792511027, 0.970111206966, -0.228643673559), (0.582893461154, 0.232348344844, 0.778620228091)212.864882187, 53.4183726063, -78.813958204
4given(0.657659182462, -0.138129150304, -0.740543541974), (-0.147838255054, 0.940263422066, -0.306673356305), (0.738666535092, 0.311167213828, 0.597952100902)277.756849886, 124.947996352, -69.8160818526
5given(0.97342283327, -0.00944678201301, -0.228820335588), (-0.01153295217, 0.995859066389, -0.0901759996117), (0.228724678781, 0.0904183510209, 0.969283004656)52.5297635266, 62.9592427178, -45.9383897161
6given(0.806704290457, -0.072389460353, 0.586504862545), (-0.0790857611664, 0.970317022117, 0.228539534809), (-0.585639505276, -0.230748006752, 0.777033929272)-123.580882124, -16.0849296319, 198.866798554
7given(0.655430249684, -0.145201527108, 0.741166448461), (-0.151011466942, 0.936333068407, 0.316979371348), (-0.74000454367, -0.319682501137, 0.591773921202)-112.950893108, -54.5244149533, 289.819626191
8given(-0.973402660213, 0.0139450680027, 0.228676181899), (0.00735864161162, -0.995727693081, 0.0920446175798), (0.228982775516, 0.0912792216782, 0.969141368535)286.428431611, 277.267728777, -46.0944534148
9given(-0.808973060035, 0.0710260160443, -0.583539110242), (0.0774747767539, -0.971132838071, -0.225607335401), (-0.582717982479, -0.227719818779, 0.780117578978)463.584967013, 355.807742406, 197.23625396
10given(-0.658118285257, 0.138120017453, -0.740137273341), (0.14404003976, -0.941775869499, -0.303826724591), (-0.739007976653, -0.306563325283, 0.599905107526)454.29279243, 394.68138876, 285.96920369
11given(-0.973679810495, 0.012234984001, -0.227591589917), (0.00781254886712, -0.996179712142, -0.0869766934175), (-0.227786282982, -0.0864655207822, 0.969864486927)379.49959652, 397.650699541, 61.6546714049
12given(-0.805674763635, 0.0771979307299, 0.587306269959), (0.0758780564146, -0.969854046598, 0.231572124513), (0.58747825134, 0.231135474966, 0.77552930081)124.891756947, 286.793028904, -78.6763896643
13given(-0.653934367656, 0.149961264487, 0.741539926067), (0.148039184936, -0.935846546987, 0.319805628806), (0.741926035749, 0.318908857898, 0.58978207656)59.3722814524, 212.354007664, -70.1587093029

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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 natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / Strain: TB50 / 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: single particle reconstruction

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Sample preparation

ComponentName: Compact state - Helical lattice of native Pil1/Lsp1 protein bound to plasma membrane microdomain
Type: COMPLEX / Entity ID: all / Source: NATURAL
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 / Details: 50mM PIPES pH 7, 300mM NaCl, 1mM CHAPS, 0.5mM DTT
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
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: 800 nm / Cs: 2.7 mm
Image recordingElectron dose: 40 e/Å2 / Film or detector model: GATAN K2 QUANTUM (4k x 4k)

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Processing

EM software
IDNameVersionCategory
1RELION3.0.8particle selection
4Gctf1.06CTF correction
7UCSF Chimera1.16model fitting
8Coot0.8.9.2model fitting
10PHENIX1.20-4459model refinement
11RELION3.0.8initial Euler assignment
12cryoSPARC4.1.2final Euler assignment
14cryoSPARC4.1.23D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.67 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 63118 / Symmetry type: POINT
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: 104.67 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.003130576
ELECTRON MICROSCOPYf_angle_d0.654141328
ELECTRON MICROSCOPYf_chiral_restr0.03284592
ELECTRON MICROSCOPYf_plane_restr0.00615432
ELECTRON MICROSCOPYf_dihedral_angle_d3.86624200
Refine LS restraints NCS
Ens-IDDom-IDAsym-IDAuth asym-IDRefine-IDTypeRms dev position (Å)
ens_1d_2AAELECTRON MICROSCOPYNCS constraints2.22760671971E-10
ens_1d_3AAELECTRON MICROSCOPYNCS constraints5.01980452209E-13
ens_1d_4AAELECTRON MICROSCOPYNCS constraints6.88814950349E-13
ens_1d_5AAELECTRON MICROSCOPYNCS constraints2.09813699994E-11
ens_1d_6AAELECTRON MICROSCOPYNCS constraints8.9344041423E-13
ens_1d_7AAELECTRON MICROSCOPYNCS constraints5.15616320609E-13
ens_1d_8AAELECTRON MICROSCOPYNCS constraints3.28980056147E-13
ens_1d_9AAELECTRON MICROSCOPYNCS constraints5.03194487563E-10
ens_1d_10AAELECTRON MICROSCOPYNCS constraints1.56153539623E-13
ens_1d_11AAELECTRON MICROSCOPYNCS constraints1.6869914023E-10
ens_1d_12AAELECTRON MICROSCOPYNCS constraints1.37751792465E-10
ens_1d_13AAELECTRON MICROSCOPYNCS constraints4.063589992E-13
ens_1d_14AAELECTRON MICROSCOPYNCS constraints3.60077989345E-13

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