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- EMDB-26248: Yeast TRAPPII-Rab11/Ypt32 complex in the closed/open state (focus... -

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Basic information

Entry
Database: EMDB / ID: EMD-26248
TitleYeast TRAPPII-Rab11/Ypt32 complex in the closed/open state (focused refinement of Trs120 middle region in the closed monomer)
Map dataMap
Sample
  • Complex: TRAPPII complex bound to Rab11/Ypt32
    • Complex: Rab11/Ypt32
    • Complex: TRAPPII
Keywordscomplex / GTPase / Guanosine Exchange Factor / GEF / PROTEIN TRANSPORT
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.2 Å
AuthorsBagde SR / Fromme JC
Funding support United States, 2 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM136258 United States
National Science Foundation (NSF, United States)DMR-1719875 United States
CitationJournal: Sci Adv / Year: 2022
Title: Structure of a TRAPPII-Rab11 activation intermediate reveals GTPase substrate selection mechanisms.
Authors: Saket R Bagde / J Christopher Fromme /
Abstract: Rab1 and Rab11 are essential regulators of the eukaryotic secretory and endocytic recycling pathways. The transport protein particle (TRAPP) complexes activate these guanosine triphosphatases via ...Rab1 and Rab11 are essential regulators of the eukaryotic secretory and endocytic recycling pathways. The transport protein particle (TRAPP) complexes activate these guanosine triphosphatases via nucleotide exchange using a shared set of core subunits. The basal specificity of the TRAPP core is toward Rab1, yet the TRAPPII complex is specific for Rab11. A steric gating mechanism has been proposed to explain TRAPPII counterselection against Rab1. Here, we present cryo-electron microscopy structures of the 22-subunit TRAPPII complex from budding yeast, including a TRAPPII-Rab11 nucleotide exchange intermediate. The Trs130 subunit provides a "leg" that positions the active site distal to the membrane surface, and this leg is required for steric gating. The related TRAPPIII complex is unable to activate Rab11 because of a repulsive interaction, which TRAPPII surmounts using the Trs120 subunit as a "lid" to enclose the active site. TRAPPII also adopts an open conformation enabling Rab11 to access and exit from the active site chamber.
History
DepositionFeb 17, 2022-
Header (metadata) releaseMay 25, 2022-
Map releaseMay 25, 2022-
UpdateJan 17, 2024-
Current statusJan 17, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_26248.png

Downloads & links

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Map

FileDownload / File: emd_26248.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationMap
Voxel sizeX=Y=Z: 1.432 Å
Density
Contour LevelBy AUTHOR: 0.04
Minimum - Maximum-0.018606573 - 0.09697037
Average (Standard dev.)0.000002225767 (±0.0017509462)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions400400400
Spacing400400400
CellA=B=C: 572.8 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_26248_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
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Histogram

Histogram (log scale)

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Additional map: RESOLVE Density Modified Map

Fileemd_26248_additional_1.map
AnnotationRESOLVE Density Modified Map
Projections & Slices
AxesZYX

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Histogram

Histogram (log scale)

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Additional map: PostProcessed (sharpened) masked map

Fileemd_26248_additional_2.map
AnnotationPostProcessed (sharpened) masked map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map 1

Fileemd_26248_half_map_1.map
AnnotationHalf map 1
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map 2

Fileemd_26248_half_map_2.map
AnnotationHalf map 2
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 : TRAPPII complex bound to Rab11/Ypt32

EntireName: TRAPPII complex bound to Rab11/Ypt32
Components
  • Complex: TRAPPII complex bound to Rab11/Ypt32
    • Complex: Rab11/Ypt32
    • Complex: TRAPPII

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Supramolecule #1: TRAPPII complex bound to Rab11/Ypt32

SupramoleculeName: TRAPPII complex bound to Rab11/Ypt32 / type: complex / ID: 1 / Parent: 0
Molecular weightTheoretical: 1.1 MDa

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Supramolecule #2: Rab11/Ypt32

SupramoleculeName: Rab11/Ypt32 / type: complex / ID: 2 / Parent: 1
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)

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Supramolecule #3: TRAPPII

SupramoleculeName: TRAPPII / type: complex / ID: 3 / Parent: 1
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)

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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

Concentration5.6 mg/mL
BufferpH: 8
Component:
ConcentrationName
10.0 mMTris
150.0 mMSodium chloride
0.1 %CHAPS
1.0 mMMagnesium acetate
1.0 mMDTT
GridModel: UltrAuFoil R1.2/1.3 / Support film - Material: GOLD / Support film - topology: HOLEY / Support film - Film thickness: 50 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
Details: The sample was incubated on the grid for 10 seconds followed by blotting for 5 seconds before plunging in liquid ethane..

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 63000
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Digitization - Dimensions - Width: 5760 pixel / Digitization - Dimensions - Height: 4092 pixel / Number grids imaged: 3 / Number real images: 4998 / Average exposure time: 3.5 sec. / Average electron dose: 53.0 e/Å2 / Details: Images were collected as 50 frame movies.
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 979187
Startup modelType of model: INSILICO MODEL
In silico model: Used Ab-initio Reconstruction job in cryoSPARC to generate starting map.
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.2.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 4.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 74313
FSC plot (resolution estimation)

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