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- EMDB-16485: Cryo-EM structure of the yeast SPT-Orm1-Dimer complex, local refi... -

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Basic information

Entry
Database: EMDB / ID: EMD-16485
TitleCryo-EM structure of the yeast SPT-Orm1-Dimer complex, local refinement of a monomer
Map datamap-main
Sample
  • Complex: Heterotetrameric complex of Orm1 with Lcb1, Lcb2 and Tsc3
KeywordsSerine-Palmitoyl-Transferase / SPT / Orm-Protein / TRANSFERASE
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.0 Å
AuthorsSchaefer J / Koerner C / Parey K / Januliene D / Moeller A / Froehlich F
Funding support Germany, 2 items
OrganizationGrant numberCountry
German Research Foundation (DFG)SFB944 Germany
German Research Foundation (DFG)SFB1557 Germany
CitationJournal: Nat Commun / Year: 2023
Title: Structure of the ceramide-bound SPOTS complex.
Authors: Jan-Hannes Schäfer / Carolin Körner / Bianca M Esch / Sergej Limar / Kristian Parey / Stefan Walter / Dovile Januliene / Arne Moeller / Florian Fröhlich /
Abstract: Sphingolipids are structural membrane components that also function in cellular stress responses. The serine palmitoyltransferase (SPT) catalyzes the rate-limiting step in sphingolipid biogenesis. ...Sphingolipids are structural membrane components that also function in cellular stress responses. The serine palmitoyltransferase (SPT) catalyzes the rate-limiting step in sphingolipid biogenesis. Its activity is tightly regulated through multiple binding partners, including Tsc3, Orm proteins, ceramides, and the phosphatidylinositol-4-phosphate (PI4P) phosphatase Sac1. The structural organization and regulatory mechanisms of this complex are not yet understood. Here, we report the high-resolution cryo-EM structures of the yeast SPT in complex with Tsc3 and Orm1 (SPOT) as dimers and monomers and a monomeric complex further carrying Sac1 (SPOTS). In all complexes, the tight interaction of the downstream metabolite ceramide and Orm1 reveals the ceramide-dependent inhibition. Additionally, observation of ceramide and ergosterol binding suggests a co-regulation of sphingolipid biogenesis and sterol metabolism within the SPOTS complex.
History
DepositionJan 20, 2023-
Header (metadata) releaseOct 11, 2023-
Map releaseOct 11, 2023-
UpdateOct 11, 2023-
Current statusOct 11, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_16485.png

Downloads & links

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Map

FileDownload / File: emd_16485.map.gz / Format: CCP4 / Size: 216 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationmap-main
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.92 Å/pix.
x 384 pix.
= 354.816 Å		0.92 Å/pix.
x 384 pix.
= 354.816 Å		0.92 Å/pix.
x 384 pix.
= 354.816 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.924 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.25
Minimum - Maximum-1.547615 - 1.8007461
Average (Standard dev.)0.00021560177 (±0.04568118)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions384384384
Spacing384384384
CellA=B=C: 354.816 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_16485_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Dimer-Map-Masked-Half-A

Fileemd_16485_half_map_1.map
AnnotationDimer-Map-Masked-Half-A
Projections & Slices
AxesZYX

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Density Histograms

Histogram

Histogram (log scale)

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Half map: Dimer-Map-Masked-Half-B

Fileemd_16485_half_map_2.map
AnnotationDimer-Map-Masked-Half-B
Projections & Slices
AxesZYX

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Slices (1/2)
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Histogram

Histogram (log scale)

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Sample components

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Entire : Heterotetrameric complex of Orm1 with Lcb1, Lcb2 and Tsc3

EntireName: Heterotetrameric complex of Orm1 with Lcb1, Lcb2 and Tsc3
Components
  • Complex: Heterotetrameric complex of Orm1 with Lcb1, Lcb2 and Tsc3

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Supramolecule #1: Heterotetrameric complex of Orm1 with Lcb1, Lcb2 and Tsc3

SupramoleculeName: Heterotetrameric complex of Orm1 with Lcb1, Lcb2 and Tsc3
type: complex / ID: 1 / Parent: 0
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightTheoretical: 180 KDa

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration10.0 mg/mL
BufferpH: 6.8
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeTFS GLACIOS
Specialist opticsEnergy filter - Name: TFS Selectris / Energy filter - Slit width: 10 eV
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Number real images: 13604 / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 13000

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Image processing

Startup modelType of model: INSILICO MODEL
Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 3.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.2) / Number images used: 94884
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.2)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.2)
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: AB INITIO MODEL / Overall B value: 86

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