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

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Basic information

Entry
Database: PDB / ID: 8qbd
TitleHelical reconstruction of yeast eisosome protein Pil1 bound to membrane composed of lipid mixture +PIP2/+sterol (DOPC, DOPE, DOPS, cholesterol, PI(4,5)P2 35:20:20:15:10)
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
D-MYO-INOSITOL-1,4,5-TRIPHOSPHATE / Chem-P5S / Sphingolipid long chain base-responsive protein PIL1
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 3.61 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
hetero molecules


Theoretical massNumber of molelcules
Total (without water)554,47342
Polymers537,50314
Non-polymers16,97028
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:

Ens-ID: ens_1

Dom-IDComponent-IDBeg auth comp-IDBeg label comp-IDEnd auth comp-IDEnd label comp-IDAuth asym-IDLabel asym-IDAuth seq-IDLabel seq-ID
d_11METMETTHRTHRFI1 - 2711 - 271
d_12I3PI3PI3PI3PFEA401
d_13P5SP5SP5SP5SFFA402
d_21METMETTHRTHRBB1 - 2711 - 271
d_22I3PI3PI3PI3PBQ401
d_23P5SP5SP5SP5SBR402
d_31METMETTHRTHRCC1 - 2711 - 271
d_32I3PI3PI3PI3PCS401
d_33P5SP5SP5SP5SCT402
d_41METMETTHRTHRDE1 - 2711 - 271
d_42I3PI3PI3PI3PDW401
d_43P5SP5SP5SP5SDX402
d_51METMETTHRTHREG1 - 2711 - 271
d_52I3PI3PI3PI3PEAA401
d_53P5SP5SP5SP5SEBA402
d_61METMETTHRTHRAA1 - 2711 - 271
d_62I3PI3PI3PI3PAO401
d_63P5SP5SP5SP5SAP402
d_71METMETTHRTHRGK1 - 2711 - 271
d_72I3PI3PI3PI3PGIA401
d_73P5SP5SP5SP5SGJA402
d_81METMETTHRTHRHM1 - 2711 - 271
d_82I3PI3PI3PI3PHMA401
d_83P5SP5SP5SP5SHNA402
d_91METMETTHRTHRID1 - 2711 - 271
d_92I3PI3PI3PI3PIU401
d_93P5SP5SP5SP5SIV402
d_101METMETTHRTHRJF1 - 2711 - 271
d_102I3PI3PI3PI3PJY401
d_103P5SP5SP5SP5SJZ402
d_111METMETTHRTHRKH1 - 2711 - 271
d_112I3PI3PI3PI3PKCA401
d_113P5SP5SP5SP5SKDA402
d_121METMETTHRTHRLJ1 - 2711 - 271
d_122I3PI3PI3PI3PLGA401
d_123P5SP5SP5SP5SLHA402
d_131METMETTHRTHRML1 - 2711 - 271
d_132I3PI3PI3PI3PMKA401
d_133P5SP5SP5SP5SMLA402
d_141METMETTHRTHRNN1 - 2711 - 271
d_142I3PI3PI3PI3PNOA401
d_143P5SP5SP5SP5SNPA402

NCS oper:
IDCodeMatrixVector
1given(-0.981236439297, -0.19280495306, 0.00114029450587), (-0.192806326815, 0.981236035148, -0.00125046703497), (-0.000877801821861, -0.00144685981602, -0.999998568029)577.570511482, 56.5502218713, 488.282624634
2given(0.923551992778, 0.383472876803, -0.000519030335193), (-0.383472799828, 0.923548092734, -0.00274448537634), (-0.000573086226469, 0.0027337089543, 0.999996099196)-81.4433724109, 122.7684094, 86.2544729954
3given(0.870319330982, -0.492485919396, 0.00137161101084), (0.492487561291, 0.870318387953, -0.00138042156331), (-0.000513900101089, 0.00187690893322, 0.999998106558)164.919076902, -96.0598451526, 59.3403243933
4given(0.757645481251, -0.652666148027, 0.000473244437853), (0.652666272388, 0.757645503071, -0.000169005121736), (-0.000248247598393, 0.000436916649974, 0.999999873738)237.649903396, -108.885644716, 16.226289314
5given(0.980822047117, 0.194901804317, -0.00118260834606), (-0.194895883748, 0.980816077209, 0.00392647247705), (0.00192519784924, -0.00362068527416, 0.99999159209)-46.4543510022, 55.9951599239, 43.1569132953
6given(0.615164373024, 0.788398832345, -0.000274444154794), (-0.788398859929, 0.615164241236, -0.000440417429066), (-0.000178396356574, 0.000487300570374, 0.999999865356)-107.152175772, 311.7477446, 26.997748614
7given(0.449112907937, 0.893473916507, -0.00139873032331), (-0.893474778269, 0.449111109627, -0.00142541483435), (-0.000645385647124, 0.00188990246674, 0.999998005871)-90.6830884745, 383.952009117, 70.1762840213
8given(-0.999990900412, 0.00413683759957, 0.00104195378073), (0.00414314708197, 0.999972641383, 0.00612787221999), (-0.00101657526211, 0.00613213342665, -0.999980681571)530.385942767, -2.46489384227, 530.526871645
9given(-0.618190648428, -0.786019405487, -0.00371704102542), (-0.786027838262, 0.618178955496, 0.00387510756174), (-0.000748113203247, 0.00531725297826, -0.99998558347)639.6971242, 309.72615733, 503.547485154
10given(-0.453140305183, -0.891433233072, -0.0032641682565), (-0.891439099764, 0.45313539673, 0.00215491131586), (-0.000441849383381, 0.00388628438337, -0.999992350752)623.7307701, 382.060451412, 460.422070556
11given(-0.925133138064, -0.379641951053, -0.000816000497841), (-0.379642486772, 0.925127287694, 0.00332923428819), (-0.000509012673356, 0.00338977342259, -0.999994125154)612.825448711, 120.173420561, 444.296235997
12given(-0.868419826123, 0.495823817421, 0.00239743080662), (0.49582932979, 0.868414350031, 0.00312927789819), (-0.000530392802424, 0.0039062434783, -0.999992229942)364.459604334, -97.6713646483, 471.24379505
13given(-0.754688132759, 0.656071480803, 0.00400429143845), (0.656083399684, 0.754679576271, 0.00364826243922), (-0.000628436025177, 0.00538044950833, -0.999985327808)291.29511459, -110.254019251, 514.303408437

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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)
Gene: PIL1 / Plasmid: pCoofy6
Details (production host): a gift from Sabine Suppmann, Addgene plasmid #43990
Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
References: UniProt: P53252
#2: Chemical
ChemComp-I3P / D-MYO-INOSITOL-1,4,5-TRIPHOSPHATE


Mass: 420.096 Da / Num. of mol.: 14 / Source method: obtained synthetically / Formula: C6H15O15P3 / Feature type: SUBJECT OF INVESTIGATION
#3: Chemical
ChemComp-P5S / O-[(R)-{[(2R)-2,3-bis(octadecanoyloxy)propyl]oxy}(hydroxy)phosphoryl]-L-serine / phosphatidyl serine


Mass: 792.075 Da / Num. of mol.: 14 / Source method: obtained synthetically / Formula: C42H82NO10P / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY

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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, PI(4,5)P2 35:20:20:15:10)
Type: COMPLEX / Entity ID: #1 / 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 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 softwareName: PHENIX / Version: 1.21_5207 / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Helical symmertyAngular rotation/subunit: 133.595 ° / Axial rise/subunit: 5.408 Å / Axial symmetry: D1
3D reconstructionResolution: 3.61 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 77414 / 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: 97.6 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00231318
ELECTRON MICROSCOPYf_angle_d0.444842378
ELECTRON MICROSCOPYf_chiral_restr0.02994704
ELECTRON MICROSCOPYf_plane_restr0.00355474
ELECTRON MICROSCOPYf_dihedral_angle_d6.10264431
Refine LS restraints NCS
Ens-IDDom-IDAsym-IDAuth asym-IDRefine-IDTypeRms dev position (Å)
ens_1d_2IFELECTRON MICROSCOPYNCS constraints5.98324569112E-13
ens_1d_3IFELECTRON MICROSCOPYNCS constraints4.45772771535E-13
ens_1d_4IFELECTRON MICROSCOPYNCS constraints4.88855808386E-13
ens_1d_5IFELECTRON MICROSCOPYNCS constraints2.88870037815E-11
ens_1d_6IFELECTRON MICROSCOPYNCS constraints2.55351590574E-11
ens_1d_7IFELECTRON MICROSCOPYNCS constraints4.90565968215E-11
ens_1d_8IFELECTRON MICROSCOPYNCS constraints4.99397459462E-13
ens_1d_9IFELECTRON MICROSCOPYNCS constraints4.26791764378E-13
ens_1d_10IFELECTRON MICROSCOPYNCS constraints9.53576043406E-13
ens_1d_11IFELECTRON MICROSCOPYNCS constraints2.17646908084E-10
ens_1d_12IFELECTRON MICROSCOPYNCS constraints5.03549161415E-13
ens_1d_13IFELECTRON MICROSCOPYNCS constraints3.68862495403E-13
ens_1d_14IFELECTRON MICROSCOPYNCS constraints4.16004973109E-12

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