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- PDB-7qla: Structure of the Rab GEF complex Mon1-Ccz1 -

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Basic information

Entry
Database: PDB / ID: 7qla
TitleStructure of the Rab GEF complex Mon1-Ccz1
Components
  • Ccz1
  • Vacuolar fusion protein MON1
KeywordsENDOCYTOSIS / guanine nucleotide exchange factor / TLD Rab GEF / longin domains / PIP binding
Function / homology
Function and homology information


protein targeting to vacuole / multivesicular body membrane / vacuolar membrane / vesicle-mediated transport / autophagy
Similarity search - Function
Vacuolar fusion protein Mon1 / 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
Similarity search - Component
Biological speciesChaetomium thermophilum (fungus)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.85 Å
AuthorsKlink, B.U. / Herrmann, E. / Antoni, C. / Langemeyer, L. / Kiontke, S. / Gatsogiannis, C. / Ungermann, C. / Raunser, S. / Kuemmel, D.
Funding support Germany, 2items
OrganizationGrant numberCountry
German Research Foundation (DFG)SFB944-P17 Germany
Max Planck Society Germany
CitationJournal: Proc Natl Acad Sci U S A / Year: 2022
Title: Structure of the Mon1-Ccz1 complex reveals molecular basis of membrane binding for Rab7 activation.
Authors: Björn U Klink / Eric Herrmann / Claudia Antoni / Lars Langemeyer / Stephan Kiontke / Christos Gatsogiannis / Christian Ungermann / Stefan Raunser / Daniel Kümmel /
Abstract: Activation of the GTPase Rab7/Ypt7 by its cognate guanine nucleotide exchange factor (GEF) Mon1-Ccz1 marks organelles such as endosomes and autophagosomes for fusion with lysosomes/vacuoles and ...Activation of the GTPase Rab7/Ypt7 by its cognate guanine nucleotide exchange factor (GEF) Mon1-Ccz1 marks organelles such as endosomes and autophagosomes for fusion with lysosomes/vacuoles and degradation of their content. Here, we present a high-resolution cryogenic electron microscopy structure of the Mon1-Ccz1 complex that reveals its architecture in atomic detail. Mon1 and Ccz1 are arranged side by side in a pseudo-twofold symmetrical heterodimer. The three Longin domains of each Mon1 and Ccz1 are triangularly arranged, providing a strong scaffold for the catalytic center of the GEF. At the opposite side of the Ypt7-binding site, a positively charged and relatively flat patch stretches the Longin domains 2/3 of Mon1 and functions as a phosphatidylinositol phosphate-binding site, explaining how the GEF is targeted to membranes. Our work provides molecular insight into the mechanisms of endosomal Rab activation and serves as a blueprint for understanding the function of members of the Tri Longin domain Rab-GEF family.
History
DepositionDec 20, 2021Deposition site: PDBE / Processing site: PDBE
Revision 1.0Feb 9, 2022Provider: repository / Type: Initial release
Revision 1.1Feb 16, 2022Group: Database references / Category: citation / Item: _citation.pdbx_database_id_PubMed / _citation.title

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Structure visualization

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  • Deposited structure unit
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  • EMDB-14066
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Assembly

Deposited unit
A: Vacuolar fusion protein MON1
C: Ccz1


Theoretical massNumber of molelcules
Total (without water)133,3522
Polymers133,3522
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Vacuolar fusion protein MON1


Mass: 57571.832 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Chaetomium thermophilum (fungus) / Strain: DSM 1495 / CBS 144.50 / IMI 039719 / Gene: CTHT_0067370 / Production host: Escherichia coli (E. coli) / References: UniProt: G0SGS3
#2: Protein Ccz1


Mass: 75780.461 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Chaetomium thermophilum (fungus) / Strain: DSM 1495 / CBS 144.50 / IMI 039719 / Gene: CTHT_0067370 / Production host: Escherichia coli (E. coli)

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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: Rab GEF complex Mon1-Ccz1 / Type: COMPLEX / Details: CtMon1 aa141-665; CtCcz1 aa1-796delta361-460 / Entity ID: all / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Chaetomium thermophilum (fungus)
Source (recombinant)Organism: Escherichia coli (E. coli) / Strain: BL21
Buffer solutionpH: 7.3
Buffer component
IDConc.NameFormulaBuffer-ID
125 mMHEPES1
2250 mMNaClSodium chloride1
31 mMMgCl21
40.5 TCEP1
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid type: Quantifoil R2/1
VitrificationInstrument: FEI VITROBOT MARK II / Cryogen name: ETHANE / Humidity: 100 %

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: SPOT SCAN
Electron lensMode: BRIGHT FIELDBright-field microscopy / Cs: 0.001 mm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 15 sec. / Electron dose: 73 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 11916
Details: Images were collected in movie-mode with 4 frames per second
EM imaging opticsEnergyfilter slit width: 20 eV / Phase plate: VOLTA PHASE PLATE
Image scansMovie frames/image: 60

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Processing

SoftwareName: PHENIX / Version: 1.19.1_4122: / Classification: refinement
EM software
IDNameCategory
1crYOLOparticle selection
2EPUimage acquisition
4MotionCorr2CTF correction
9SPHIREinitial Euler assignment
10RELIONfinal Euler assignment
11RELIONclassification
13Cootmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 7155250
3D reconstructionResolution: 3.85 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 911674 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0036983
ELECTRON MICROSCOPYf_angle_d0.5919467
ELECTRON MICROSCOPYf_dihedral_angle_d4.597948
ELECTRON MICROSCOPYf_chiral_restr0.0421076
ELECTRON MICROSCOPYf_plane_restr0.0041200

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