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- EMDB-18310: Helical reconstruction of yeast eisosome protein Pil1 bound to me... -

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Basic information

Entry
Database: EMDB / ID: EMD-18310
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)
Map dataSharpened helical map A&C, non-uniform refinement (D1, rise=5.408, twist=133.595)
Sample
  • Complex: Helical assembly of recombinant Pil1 protein tubulating +PIP2/+sterol lipid mixture (DOPC, DOPE, DOPS, cholesterol, PI(4,5)P2 35:20:20:15:10)
    • Protein or peptide: Sphingolipid long chain base-responsive protein PIL1
  • Ligand: D-MYO-INOSITOL-1,4,5-TRIPHOSPHATE
  • Ligand: O-[(R)-{[(2R)-2,3-bis(octadecanoyloxy)propyl]oxy}(hydroxy)phosphoryl]-L-serine
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: 3.61 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: 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, 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_18310.png

Downloads & links

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Map

FileDownload / File: emd_18310.map.gz / Format: CCP4 / Size: 1000 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSharpened helical map A&C, non-uniform refinement (D1, rise=5.408, twist=133.595)
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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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.83 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.055
Minimum - Maximum-0.18747392 - 0.35873693
Average (Standard dev.)0.0004193603 (±0.014356814)
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_18310_msk_1.map
Projections & Slices
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Additional map: Unsharpened helical map B (D3, rise=14.548, twist=276.716)

Fileemd_18310_additional_1.map
AnnotationUnsharpened helical map B (D3, rise=14.548, twist=276.716)
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Additional map: Unsharpened helical map K (D1, rise=5.740, twist=152.300)

Fileemd_18310_additional_2.map
AnnotationUnsharpened helical map K (D1, rise=5.740, twist=152.300)
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Additional map: Unsharpened helical map E (D1, rise=4.980, twist=-161.053)

Fileemd_18310_additional_3.map
AnnotationUnsharpened helical map E (D1, rise=4.980, twist=-161.053)
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Additional map: Unsharpened helical map D (D2, rise=11.142, twist=-137.653)

Fileemd_18310_additional_4.map
AnnotationUnsharpened helical map D (D2, rise=11.142, twist=-137.653)
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Additional map: Unsharpened helical map J (D1, rise=5.077, twist=-136.507)

Fileemd_18310_additional_5.map
AnnotationUnsharpened helical map J (D1, rise=5.077, twist=-136.507)
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Additional map: Unsharpened helical map F&G&H (D4, rise=20.968, twist=-140.956)

Fileemd_18310_additional_6.map
AnnotationUnsharpened helical map F&G&H (D4, rise=20.968, twist=-140.956)
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Additional map: Unsharpened helical map A&C, non-uniform refinement (D1, rise=5.408,...

Fileemd_18310_additional_7.map
AnnotationUnsharpened helical map A&C, non-uniform refinement (D1, rise=5.408, twist=133.595)
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Half map: Helical map A&C, non-uniform refinement - half map B

Fileemd_18310_half_map_1.map
AnnotationHelical map A&C, non-uniform refinement - half map B
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Half map: Helical map A&C, non-uniform refinement - half map A

Fileemd_18310_half_map_2.map
AnnotationHelical map A&C, non-uniform refinement - half map A
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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, cholesterol, PI(4,5)P2 35:20:20:15:10)
Components
  • Complex: Helical assembly of recombinant Pil1 protein tubulating +PIP2/+sterol lipid mixture (DOPC, DOPE, DOPS, cholesterol, PI(4,5)P2 35:20:20:15:10)
    • Protein or peptide: Sphingolipid long chain base-responsive protein PIL1
  • Ligand: D-MYO-INOSITOL-1,4,5-TRIPHOSPHATE
  • Ligand: O-[(R)-{[(2R)-2,3-bis(octadecanoyloxy)propyl]oxy}(hydroxy)phosphoryl]-L-serine

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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, cholesterol, PI(4,5)P2 35:20:20:15:10)
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)
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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Macromolecule #3: O-[(R)-{[(2R)-2,3-bis(octadecanoyloxy)propyl]oxy}(hydroxy)phospho...

MacromoleculeName: O-[(R)-{[(2R)-2,3-bis(octadecanoyloxy)propyl]oxy}(hydroxy)phosphoryl]-L-serine
type: ligand / ID: 3 / Number of copies: 14 / Formula: P5S
Molecular weightTheoretical: 792.075 Da
Chemical component information

ChemComp-P5S:
O-[(R)-{[(2R)-2,3-bis(octadecanoyloxy)propyl]oxy}(hydroxy)phosphoryl]-L-serine

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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: 20mM HEPES, pH 7.4, 150mM KoAc, 2mM 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: 5.408 Å
Applied symmetry - Helical parameters - Δ&Phi: 133.595 °
Applied symmetry - Helical parameters - Axial symmetry: D1 (2x1 fold dihedral)
Resolution.type: BY AUTHOR / Resolution: 3.61 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 77414
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-8qbd:
Helical 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)

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