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- EMDB-16462: MCB complex from drosophila melanogaster, local refinement map of... -

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Basic information

Entry
Database: EMDB / ID: EMD-16462
TitleMCB complex from drosophila melanogaster, local refinement map of the bottom part of the complex
Map datalocal-MCB-bulli
Sample
  • Complex: Trimeric metazoan guanine-nucleotide-exchange factor Mon1-Ccz1-Bulli
Biological speciesDrosophila melanogaster (fruit fly)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.7 Å
AuthorsSchaefer J / Herrmann E / Kuemmel D / Moeller A
Funding support Germany, 1 items
OrganizationGrant numberCountry
German Research Foundation (DFG) Germany
CitationJournal: Proc Natl Acad Sci U S A / Year: 2023
Title: Structure of the metazoan Rab7 GEF complex Mon1-Ccz1-Bulli.
Authors: Eric Herrmann / Jan-Hannes Schäfer / Stephan Wilmes / Christian Ungermann / Arne Moeller / Daniel Kümmel /
Abstract: The endosomal system of eukaryotic cells represents a central sorting and recycling compartment linked to metabolic signaling and the regulation of cell growth. Tightly controlled activation of Rab ...The endosomal system of eukaryotic cells represents a central sorting and recycling compartment linked to metabolic signaling and the regulation of cell growth. Tightly controlled activation of Rab GTPases is required to establish the different domains of endosomes and lysosomes. In metazoans, Rab7 controls endosomal maturation, autophagy, and lysosomal function. It is activated by the guanine nucleotide exchange factor (GEF) complex Mon1-Ccz1-Bulli (MCBulli) of the tri-longin domain (TLD) family. While the Mon1 and Ccz1 subunits have been shown to constitute the active site of the complex, the role of Bulli remains elusive. We here present the cryo-electron microscopy (cryo-EM) structure of MCBulli at 3.2 Å resolution. Bulli associates as a leg-like extension at the periphery of the Mon1 and Ccz1 heterodimers, consistent with earlier reports that Bulli does not impact the activity of the complex or the interactions with recruiter and substrate GTPases. While MCBulli shows structural homology to the related ciliogenesis and planar cell polarity effector (Fuzzy-Inturned-Wdpcp) complex, the interaction of the TLD core subunits Mon1-Ccz1 and Fuzzy-Inturned with Bulli and Wdpcp, respectively, is remarkably different. The variations in the overall architecture suggest divergent functions of the Bulli and Wdpcp subunits. Based on our structural analysis, Bulli likely serves as a recruitment platform for additional regulators of endolysosomal trafficking to sites of Rab7 activation.
History
DepositionJan 16, 2023-
Header (metadata) releaseMay 17, 2023-
Map releaseMay 17, 2023-
UpdateMay 17, 2023-
Current statusMay 17, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_16462.png

Downloads & links

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Map

FileDownload / File: emd_16462.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationlocal-MCB-bulli
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.92 Å/pix.
x 320 pix.
= 295.68 Å		0.92 Å/pix.
x 320 pix.
= 295.68 Å		0.92 Å/pix.
x 320 pix.
= 295.68 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: 0.16
Minimum - Maximum-1.2886788 - 1.8003194
Average (Standard dev.)0.00045745092 (±0.018197646)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 295.68 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_16462_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: local-MCB-bulli-half-A

Fileemd_16462_half_map_1.map
Annotationlocal-MCB-bulli-half-A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: local-MCB-bulli-half-B

Fileemd_16462_half_map_2.map
Annotationlocal-MCB-bulli-half-B
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Trimeric metazoan guanine-nucleotide-exchange factor Mon1-Ccz1-Bulli

EntireName: Trimeric metazoan guanine-nucleotide-exchange factor Mon1-Ccz1-Bulli
Components
  • Complex: Trimeric metazoan guanine-nucleotide-exchange factor Mon1-Ccz1-Bulli

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Supramolecule #1: Trimeric metazoan guanine-nucleotide-exchange factor Mon1-Ccz1-Bulli

SupramoleculeName: Trimeric metazoan guanine-nucleotide-exchange factor Mon1-Ccz1-Bulli
type: complex / ID: 1 / Chimera: Yes / Parent: 0 / Macromolecule list: #1-#3
Source (natural)Organism: Drosophila melanogaster (fruit fly)
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

Concentration1.0 mg/mL
BufferpH: 7.3
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeTFS GLACIOS
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 130000
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: 5931 / Average electron dose: 50.0 e/Å2

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Image processing

Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.2)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.2)
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.7 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.2) / Number images used: 390520
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: AB INITIO MODEL / Overall B value: 144

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