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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 / protein localization / endocytosis / mitochondrial outer membrane / lipid binding ...protein localization to eisosome filament / eisosome filament / eisosome assembly / eisosome / lipid droplet / cell periphery / protein localization / endocytosis / 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: To Be Published
Title: CryoEM architecture of a native stretch-sensitive membrane microdomain
Authors: Kefauver, J.M. / Hakala, M. / Zou, L. / Alba, J. / Espadas Moreno, J. / Tettamanti, M.G. / Estrozi, L. / Vanni, S. / Roux, A. / Desfosses, A. / Loewith, R.
#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

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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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