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- EMDB-16457: Drosophila melanogaster Rab7 GEF complex Mon1-Ccz1-Bulli -

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Basic information

Entry
Database: EMDB / ID: EMD-16457
TitleDrosophila melanogaster Rab7 GEF complex Mon1-Ccz1-Bulli
Map datadensity modified composite map
Sample
  • Complex: Trimeric metazoan guanine-nucleotide-exchange factor Mon1-Ccz1-Bulli
    • Protein or peptide: Mic1 domain-containing protein
    • Protein or peptide: Caffeine, calcium, zinc sensitivity 1
    • Protein or peptide: Vacuolar fusion protein MON1 homolog
KeywordsGuanain Nucleotide Exchange Factor / membrane traffic / lysosome / ENDOCYTOSIS
Function / homology
Function and homology information


guanyl nucleotide exchange factor inhibitor activity / Mon1-Ccz1 complex / RAB GEFs exchange GTP for GDP on RABs / guanyl-nucleotide exchange factor adaptor activity / protein targeting to vacuole / endosome to lysosome transport via multivesicular body sorting pathway / neuromuscular junction development / synaptic cleft / vesicle-mediated transport / guanyl-nucleotide exchange factor activity ...guanyl nucleotide exchange factor inhibitor activity / Mon1-Ccz1 complex / RAB GEFs exchange GTP for GDP on RABs / guanyl-nucleotide exchange factor adaptor activity / protein targeting to vacuole / endosome to lysosome transport via multivesicular body sorting pathway / neuromuscular junction development / synaptic cleft / vesicle-mediated transport / guanyl-nucleotide exchange factor activity / regulation of autophagy / autophagy / late endosome membrane / intracellular membrane-bounded organelle / lipid binding / cytosol
Similarity search - Function
Regulator of MON1-CCZ1 complex, C-terminal / Regulator of MON1-CCZ1 complex / : / Regulator of MON1-CCZ1 complex, C-terminal / Regulator of MON1-CCZ1 complex, N-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 ...Regulator of MON1-CCZ1 complex, C-terminal / Regulator of MON1-CCZ1 complex / : / Regulator of MON1-CCZ1 complex, C-terminal / Regulator of MON1-CCZ1 complex, N-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 / Regulator of MON1-CCZ1 complex / Vacuolar fusion protein CCZ1 homolog
Similarity search - Component
Biological speciesDrosophila melanogaster (fruit fly)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.2 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 15, 2023-
Header (metadata) releaseMay 17, 2023-
Map releaseMay 17, 2023-
UpdateJul 24, 2024-
Current statusJul 24, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_16457.png

Downloads & links

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Map

FileDownload / File: emd_16457.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationdensity modified composite map
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: 0.1
Minimum - Maximum-0.053569686 - 2.3558547
Average (Standard dev.)0.0010627309 (±0.023258902)
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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Additional map: composite map of L1 and L2

Fileemd_16457_additional_1.map
Annotationcomposite map of L1 and L2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: consensus map

Fileemd_16457_additional_2.map
Annotationconsensus map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: local mal MCB-bulli (L2)

Fileemd_16457_additional_3.map
Annotationlocal mal MCB-bulli (L2)
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: local map MCB-core (L1)

Fileemd_16457_additional_4.map
Annotationlocal map MCB-core (L1)
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
    • Protein or peptide: Mic1 domain-containing protein
    • Protein or peptide: Caffeine, calcium, zinc sensitivity 1
    • Protein or peptide: Vacuolar fusion protein MON1 homolog

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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 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Drosophila melanogaster (fruit fly)
Molecular weightTheoretical: 180 KDa

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Macromolecule #1: Mic1 domain-containing protein

MacromoleculeName: Mic1 domain-containing protein / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Drosophila melanogaster (fruit fly)
Molecular weightTheoretical: 72.058391 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MDNSNGIHYI ELTPNPIRFD AVSQLTNVFF DDSNKQIFAV RSGGATGVVV KGPGSPDDVV ISFCMSDRGG AIRSIKFSPD NQILAVQRK ENSVEFICFQ GDQPLLQDII THQVKTLIHG FVWVHNREVA LISNTGVEVY TVVPEKRQVR SVKSLSIGIK W FAWCCDAN ...String:
MDNSNGIHYI ELTPNPIRFD AVSQLTNVFF DDSNKQIFAV RSGGATGVVV KGPGSPDDVV ISFCMSDRGG AIRSIKFSPD NQILAVQRK ENSVEFICFQ GDQPLLQDII THQVKTLIHG FVWVHNREVA LISNTGVEVY TVVPEKRQVR SVKSLSIGIK W FAWCCDAN VALLCTSEGN SLIPVLVKQK VITKLPKVDL GNPSRDVQES KVTLGQVYGV LAVLILQSNS TTGLMEVEVH LL NGPGLAP RKCHVLRLSL LGRFAINTVD NLIVVHHQAS GTSLLFDISL PGEVINEITY HTPITPGRSI KPFGLKLPSL SPD GQILQC ELYSTHWVLF QPNIVIDAKL GCMWFLNLCI EPLCQLISDR IRLTEFLLQR SNGKQMLLKV IGQLVDDQYK GTLL PVLET IFSRINKIYA SWVQLELQNQ TAQPSNVKTT TLKQSTPPIV LIEQLDMVQI FQRIARRPYT ESILMLYLQS LNKFN IAAQ EELSKMIISE LISNRSFDTL RRLVSYSMLL ESKSVACFLL SHSNVDTAIS QVAIDMLGRI EAHEIIIEVM LGQGKV IDA LRLAKNSMGL EKVPARKFLE AAHKTKDDLI FHSVYRFFQM RNLKLYETLS FPKAEQCTEF IQHYNNTFPA DNPTRQP VS

UniProtKB: Regulator of MON1-CCZ1 complex

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Macromolecule #2: Caffeine, calcium, zinc sensitivity 1

MacromoleculeName: Caffeine, calcium, zinc sensitivity 1 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Drosophila melanogaster (fruit fly)
Molecular weightTheoretical: 58.308566 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MAKLLQRVEI TLRSFYIFNS TFGQVEGEEH KKVLFYHPND IELNTKIKDV GLSEAIIRFT GTFTSEDDCQ ALHTQKTTQL FYQPEPGYW LVLVLNVPKE VRLKEGVEVA DYRGAEISDR IYRAILRQCY QMFRFQNGCF SSCGSEEPNP DKRRELLCQK L LQFYDQHL ...String:
MAKLLQRVEI TLRSFYIFNS TFGQVEGEEH KKVLFYHPND IELNTKIKDV GLSEAIIRFT GTFTSEDDCQ ALHTQKTTQL FYQPEPGYW LVLVLNVPKE VRLKEGVEVA DYRGAEISDR IYRAILRQCY QMFRFQNGCF SSCGSEEPNP DKRRELLCQK L LQFYDQHL TNLRDPAQCD IIDMLHSIQY LPLDKTLFLR AQNFGTLCET FPDIKESIML YQEQVLCGGK LSPEDLHCVH SY VVQHVLK VEASSSTIAV SPSLKRSISE CQVGGFVRSR QKVAGDEHDA VNEEDHPMKV YVTLDKEAKP YYLLIYRALH ITL CLFLNA DQVAPKQDLY DDLHAYMAPQ LTSLARDISS ELTKEAVGAA GQDNSSGNSE TAPKYLFINE QSLQHHTNFQ RHLP QGLPR NVLSIIADLA NGSGKAEMES APAEEVQVKT TNDYWIVKRR CNYRQYYVIL CNSKATLLDV TQEARRIFEQ ELTDD VFFD KDYKDHDGDY KDHDIDYKDD DDK

UniProtKB: Vacuolar fusion protein CCZ1 homolog

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Macromolecule #3: Vacuolar fusion protein MON1 homolog

MacromoleculeName: Vacuolar fusion protein MON1 homolog / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Drosophila melanogaster (fruit fly)
Molecular weightTheoretical: 59.904152 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MEVEQTSVRS DTNSTCEYLD AEGDPESPNL YQEADPDQEA EQQNHSIISE LRDGLGTMRD NSALSPEPGQ ENKGLAASVE SLALSTSTS AKTEDSIGGG LEEEYDYQHD SLWQGQKKHI FILSEAGKPI FSLHGNEDKL ATLFGVIQAL VSFVQMGQDA I TSIHAGGI ...String:
MEVEQTSVRS DTNSTCEYLD AEGDPESPNL YQEADPDQEA EQQNHSIISE LRDGLGTMRD NSALSPEPGQ ENKGLAASVE SLALSTSTS AKTEDSIGGG LEEEYDYQHD SLWQGQKKHI FILSEAGKPI FSLHGNEDKL ATLFGVIQAL VSFVQMGQDA I TSIHAGGI KFAFMQRSSL ILVAASRSNM SVQQLQLQLG DVYNQILSIL TYSHMTKIFE RRKNFDLRRL LSGSERLFYN LL ANDSSSA KVSNNIFTFL TNSIRVFPLP TTIRSQITSA IQSNCSKIKN LVFAVLIANN KLIALVRMKK YSIHPADLRL IFN LVECSE SFKSSENWSP ICLPKFDMNG YLHAHVSYLA DDCQACLLLL SVDRDAFFTL AEAKAKITEK LRKSHCLEAI NEEL QQPFN AKLYQQVVGI PELRHFLYKP KSTAQLLCPM LRHPYKSLTE LERLEAIYCD LLHRIHNSSR PLKLIYEMKE REVVL AWAT GTYELYAIFE PVVDKATVIK YVDKLIKWIE KEYDVYFIRN HATF

UniProtKB: Vacuolar fusion protein MON1 homolog

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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
GridModel: C-flat-1.2/1.3 / Material: COPPER / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 45 sec. / Pretreatment - Atmosphere: AIR / Details: 15 mA
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeTFS GLACIOS
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
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 130000

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Image processing

Startup modelType of model: INSILICO MODEL
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.2) / Number images used: 390520
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.2)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.2)
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: AB INITIO MODEL / Overall B value: 129
Output model

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

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