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- EMDB-14066: Structure of the Rab GEF complex Mon1-Ccz1 -

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Basic information

Entry
Database: EMDB / ID: EMD-14066
TitleStructure of the Rab GEF complex Mon1-Ccz1
Map dataCryo-EM structure of the Mon1-Ccz1 complex (CtMon1 aa141-665; CtCcz1 aa1-796delta361-460). Map sharpened by Phenix local anisotropic filtering.
Sample
  • Complex: Rab GEF complex Mon1-Ccz1
    • Protein or peptide: Vacuolar fusion protein MON1
    • Protein or peptide: Ccz1
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)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.85 Å
AuthorsKlink BU / Herrmann E / Antoni C / Langemeyer L / Kiontke S / Gatsogiannis C / Ungermann C / Raunser S / Kuemmel D
Funding support Germany, 2 items
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, 2021-
Header (metadata) releaseFeb 9, 2022-
Map releaseFeb 9, 2022-
UpdateFeb 16, 2022-
Current statusFeb 16, 2022Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.02
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.02
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7qla
  • Surface level: 0.02
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_14066.png

Downloads & links

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Map

FileDownload / File: emd_14066.map.gz / Format: CCP4 / Size: 22.2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryo-EM structure of the Mon1-Ccz1 complex (CtMon1 aa141-665; CtCcz1 aa1-796delta361-460). Map sharpened by Phenix local anisotropic filtering.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesX (Sec.)Y (Row.)Z (Col.)
1.09 Å/pix.
x 180 pix.
= 196.2 Å		1.09 Å/pix.
x 180 pix.
= 196.2 Å		1.09 Å/pix.
x 180 pix.
= 196.2 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.09 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.02 / Movie #1: 0.02
Minimum - Maximum-0.06390953 - 0.1044802
Average (Standard dev.)-1.1719719e-06 (±0.0031421822)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderZYX
Origin000
Dimensions180180180
Spacing180180180
CellA=B=C: 196.20001 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.091.091.09
M x/y/z180180180
origin x/y/z0.0000.0000.000
length x/y/z196.200196.200196.200
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ180180180
MAP C/R/S321
start NC/NR/NS000
NC/NR/NS180180180
D min/max/mean-0.0640.104-0.000

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Supplemental data

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Additional map: Cryo-EM structure of the Mon1-Ccz1 complex (CtMon1 aa141-665;...

Fileemd_14066_additional_1.map
AnnotationCryo-EM structure of the Mon1-Ccz1 complex (CtMon1 aa141-665; CtCcz1 aa1-796delta361-460). Map created by DeepEMhancer with high resolution training model.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Cryo-EM structure of the Mon1-Ccz1 complex (CtMon1 aa141-665;...

Fileemd_14066_additional_2.map
AnnotationCryo-EM structure of the Mon1-Ccz1 complex (CtMon1 aa141-665; CtCcz1 aa1-796delta361-460). Map created by Relion Postprocessing.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Cryo-EM structure of the Mon1-Ccz1 complex (CtMon1 aa141-665;...

Fileemd_14066_half_map_1.map
AnnotationCryo-EM structure of the Mon1-Ccz1 complex (CtMon1 aa141-665; CtCcz1 aa1-796delta361-460). Unfiltered half-map 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Cryo-EM structure of the Mon1-Ccz1 complex (CtMon1 aa141-665;...

Fileemd_14066_half_map_2.map
AnnotationCryo-EM structure of the Mon1-Ccz1 complex (CtMon1 aa141-665; CtCcz1 aa1-796delta361-460). Unfiltered half-map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Rab GEF complex Mon1-Ccz1

EntireName: Rab GEF complex Mon1-Ccz1
Components
  • Complex: Rab GEF complex Mon1-Ccz1
    • Protein or peptide: Vacuolar fusion protein MON1
    • Protein or peptide: Ccz1

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Supramolecule #1: Rab GEF complex Mon1-Ccz1

SupramoleculeName: Rab GEF complex Mon1-Ccz1 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all / Details: CtMon1 aa141-665; CtCcz1 aa1-796delta361-460
Source (natural)Organism: Chaetomium thermophilum (fungus)
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: BL21

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Macromolecule #1: Vacuolar fusion protein MON1

MacromoleculeName: Vacuolar fusion protein MON1 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Chaetomium thermophilum (fungus) / Strain: DSM 1495 / CBS 144.50 / IMI 039719
Molecular weightTheoretical: 57.571832 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: TGTAGDLASL LAGDGLGRKS KAWRVLRAQQ QACGEGSGEE GEITEIEGLG LGEGMEGFER ELDNIPDTLP DDERLALWKG KLKHYLILS SAGKPIWSRH GDLSLVNSTM GVVQTIISFY EGARNPLLGF TAGKVRFVIL IKGPLYFVAI SRLRESDAQL R AQLEALYM ...String:
TGTAGDLASL LAGDGLGRKS KAWRVLRAQQ QACGEGSGEE GEITEIEGLG LGEGMEGFER ELDNIPDTLP DDERLALWKG KLKHYLILS SAGKPIWSRH GDLSLVNSTM GVVQTIISFY EGARNPLLGF TAGKVRFVIL IKGPLYFVAI SRLRESDAQL R AQLEALYM QILSTLTLPI LTNIFAHRPS TDLRGPLQGT ESLLASLADS FTKGSPSTLL SALECLRLRK SQRQAITNIF LK SRCEELL YGLLVAGGKL VSVIRPRKHS LHPSDLQLIF NMLFESGGIK GNGGENWIPL CLPAFNNTGY LYMYVSFLDD KAP DDQNQP PESSNLDASN KNSSNTPDDD LTALILISPS REAFYALQSM RTRLVSQLLS TGYLSLIRST ALSGRPSITS ILPK TPLLH FLYKSRPNVQ WCMSSLSSLT PPGATATETL LARRKLMSVY EELHAALHAR HAHLRVVYST ADEKEGEGLA CLGWS TPAF EVYCVAPGCV GRAGMAREVN RVVQWARREE ERLFILGGGV F

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Macromolecule #2: Ccz1

MacromoleculeName: Ccz1 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Chaetomium thermophilum (fungus) / Strain: DSM 1495 / CBS 144.50 / IMI 039719
Molecular weightTheoretical: 75.780461 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MTTPVSPSPS GIIPAQLGFL AIYNPALGTT DETLEDQIVY YATASTLSQA RRRHRRPRRR DRQRAQSVVK DSRPNAAGAT GDSEAVAED KDPVSKEERH ERLRQIGLAQ GMVEFAKSFS DGEPVDTIDT EKARVILVEV EEGWWILASI DLTRLPLPQI K TPTSSSAP ...String:
MTTPVSPSPS GIIPAQLGFL AIYNPALGTT DETLEDQIVY YATASTLSQA RRRHRRPRRR DRQRAQSVVK DSRPNAAGAT GDSEAVAED KDPVSKEERH ERLRQIGLAQ GMVEFAKSFS DGEPVDTIDT EKARVILVEV EEGWWILASI DLTRLPLPQI K TPTSSSAP PPAPNLNPLP PEPAYEYSSR EVKPPSLLRA DLLRAYDLFL LHHGSSLSSL LASQGRAQLV ASLTRFWDHF LA TWNVLLH GNPACDVFGG IKLAASGELG IGVGEEERGS GEREVLEGLV ERVEGLVDVV VGRYGGPPSE KGPEEEQWLG LGG EVGEED GAVFLGVGAL DRKSLRGVVQ WMEEVYVWGE NAFGKPRRDL STGHFLLGLS ECSEEELTSS QANPKAIFVE LKPS YQHPS RKIPPEDPQP LGKVGPELPR DHTARLRPVI YVSQPFIYIL LFSEITPSPS TWPTLAESLH AQLSPLQKPL LHSTS YRPE RPVVETTSSS GTTTQHQIFD LVYDTETLTL QSTIPNIPDP FPYSATTPTG HSTGQQHHQQ SIWTRVEALQ THAQIL AIL SSGRAIPTDP SSFTHLPWEE GERTCKTARG WWIVWTRVVE HSPPDAVSLH HARDDDDNDD DASCSVLGHL RSVSSSH AA GSTSSSSGSG FGLGAIPGLG GLGGWAADGA TRLAQGIGID TRRYVEGLLT SLGR

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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

BufferpH: 7.3
Component:
ConcentrationNameFormula
25.0 mMHEPES
250.0 mMNaClSodium chloride
1.0 mMMgCl2
TCEP
GridModel: Quantifoil R2/1 / Material: COPPER / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Instrument: FEI VITROBOT MARK II

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 0.001 mm
Specialist opticsPhase plate: VOLTA PHASE PLATE / 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 K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Number grids imaged: 1 / Number real images: 11916 / Average exposure time: 15.0 sec. / Average electron dose: 73.0 e/Å2
Details: Images were collected in movie-mode with 4 frames per second
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 7155250
CTF correctionSoftware - Name: MotionCorr2
Startup modelType of model: NONE
Details: The initial model was generated from the ISAC 2D class averages using RVIPER from SPHIRE
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: SPHIRE
Final 3D classificationNumber classes: 4 / Software - Name: RELION
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
Final reconstructionNumber classes used: 1 / Resolution.type: BY AUTHOR / Resolution: 3.85 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 911674
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementProtocol: AB INITIO MODEL
Output model

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

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