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- PDB-8c7g: Drosophila melanogaster Rab7 GEF complex Mon1-Ccz1-Bulli -

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Basic information

Entry
Database: PDB / ID: 8c7g
TitleDrosophila melanogaster Rab7 GEF complex Mon1-Ccz1-Bulli
Components
  • Caffeine, calcium, zinc sensitivity 1
  • Mic1 domain-containing protein
  • Vacuolar fusion protein MON1 homolog
KeywordsENDOCYTOSIS / Guanain Nucleotide Exchange Factor / membrane traffic / lysosome
Function / homology
Function and homology information


positive regulation of guanyl-nucleotide exchange factor activity / Mon1-Ccz1 complex / RAB GEFs exchange GTP for GDP on RABs / protein targeting to vacuole / endosome to lysosome transport via multivesicular body sorting pathway / neuromuscular junction development / synaptic cleft / vesicle-mediated transport / regulation of autophagy / positive regulation of GTPase activity ...positive regulation of guanyl-nucleotide exchange factor activity / Mon1-Ccz1 complex / RAB GEFs exchange GTP for GDP on RABs / protein targeting to vacuole / endosome to lysosome transport via multivesicular body sorting pathway / neuromuscular junction development / synaptic cleft / vesicle-mediated transport / regulation of autophagy / positive regulation of GTPase activity / autophagy / late endosome membrane / intracellular membrane-bounded organelle / cytosol
Similarity search - Function
Regulator of MON1-CCZ1 complex, C-terminal / Regulator of MON1-CCZ1 complex / Regulator of MON1-CCZ1 complex, C-terminal / Vacuolar fusion protein Ccz1 / Vacuolar fusion protein Mon1 / CCZ1/INTU, second Longin domain / CCZ1/INTU/HPS4, third Longin domain / Intu longin-like domain 2 / Intu longin-like domain 3 / CCZ1/INTU/HSP4, first Longin domain ...Regulator of MON1-CCZ1 complex, C-terminal / Regulator of MON1-CCZ1 complex / Regulator of MON1-CCZ1 complex, C-terminal / Vacuolar fusion protein Ccz1 / Vacuolar fusion protein Mon1 / CCZ1/INTU, second Longin domain / CCZ1/INTU/HPS4, third Longin domain / Intu longin-like domain 2 / Intu longin-like domain 3 / CCZ1/INTU/HSP4, first Longin domain / First Longin domain of INTU, CCZ1 and HPS4 / FUZ/MON1/HPS1, third Longin domain / FUZ/MON1/HPS1, second Longin domain / FUZ/MON1/HPS1, first Longin domain / First Longin domain of FUZ, MON1 and HPS1 / Second Longin domain of FUZ, MON1 and HPS1 / Third Longin domain of FUZ, MON1 and HPS1
Similarity search - Domain/homology
Vacuolar fusion protein MON1 homolog / Bulli / Caffeine, calcium, zinc sensitivity 1
Similarity search - Component
Biological speciesDrosophila melanogaster (fruit fly)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsSchaefer, J. / Herrmann, E. / Kuemmel, D. / Moeller, A.
Funding support Germany, 1items
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 15, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0May 17, 2023Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Mic1 domain-containing protein
B: Caffeine, calcium, zinc sensitivity 1
C: Vacuolar fusion protein MON1 homolog


Theoretical massNumber of molelcules
Total (without water)190,2713
Polymers190,2713
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area7650 Å2
ΔGint-35 kcal/mol
Surface area71480 Å2
MethodPISA

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Components

#1: Protein Mic1 domain-containing protein


Mass: 72058.391 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Drosophila melanogaster (fruit fly) / Gene: Bulli, Dmel\CG8270, CG8270, Dmel_CG8270 / Cell line (production host): Sf9 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q9VRX1
#2: Protein Caffeine, calcium, zinc sensitivity 1 / / RH02365p


Mass: 58308.566 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Drosophila melanogaster (fruit fly)
Gene: Ccz1, C7orf28B, Dccz1, Dmel\CG14980, NP_647835, CG14980, Dmel_CG14980
Cell line (production host): Sf9 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q9VZL5
#3: Protein Vacuolar fusion protein MON1 homolog


Mass: 59904.152 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Drosophila melanogaster (fruit fly)
Gene: Mon1, Dmel\CG11926, Dmon1, MON1, mon1, Mon1-RA, NP_608868, CG11926, Dmel_CG11926
Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q9VR38

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Trimeric metazoan guanine-nucleotide-exchange factor Mon1-Ccz1-Bulli
Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.18 MDa / Experimental value: NO
Source (natural)Organism: Drosophila melanogaster (fruit fly)
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm)
Buffer solutionpH: 7.3
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: 15 mA / Grid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: C-flat-1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 K

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Electron microscopy imaging

MicroscopyModel: TFS GLACIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 130000 X / Nominal defocus max: 2000 nm / Nominal defocus min: 800 nm
Image recordingElectron dose: 50 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k) / Num. of real images: 5931
EM imaging opticsEnergyfilter name: TFS Selectris / Energyfilter slit width: 10 eV

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Processing

SoftwareName: UCSF ChimeraX / Version: 1.6/v9 / Classification: model building / URL: https://www.rbvi.ucsf.edu/chimerax/ / Os: macOS / Type: package
EM software
IDNameVersionCategory
2EPU2.9image acquisition
4cryoSPARC3.2CTF correction
7Coot0.9model fitting
9cryoSPARC3.2initial Euler assignment
10cryoSPARC3.2final Euler assignment
113.2classification
12cryoSPARC3.23D reconstruction
19PHENIX1.19model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 390520 / Symmetry type: POINT
Atomic model buildingB value: 129 / Protocol: AB INITIO MODEL / Space: REAL
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00411805
ELECTRON MICROSCOPYf_angle_d0.56215987
ELECTRON MICROSCOPYf_dihedral_angle_d4.211553
ELECTRON MICROSCOPYf_chiral_restr0.0441837
ELECTRON MICROSCOPYf_plane_restr0.0052030

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