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- PDB-6lt0: cryo-EM structure of C9ORF72-SMCR8-WDR41 -

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Basic information

Entry
Database: PDB / ID: 6lt0
Titlecryo-EM structure of C9ORF72-SMCR8-WDR41
Components
  • Guanine nucleotide exchange C9orf72
  • Guanine nucleotide exchange protein SMCR8
  • WD repeat-containing protein 41
KeywordsPROTEIN BINDING / ALS / FTD / GTPase / C9ORF72 / SMCR8 / WDR41 / GAP / GEF
Function / homology
Function and homology information


Atg1/ULK1 kinase complex / late endosome to lysosome transport / regulation of TORC1 signaling / negative regulation of autophagosome assembly / regulation of actin filament organization / guanyl-nucleotide exchange factor complex / negative regulation of immune response / regulation of autophagosome assembly / Flemming body / axon extension ...Atg1/ULK1 kinase complex / late endosome to lysosome transport / regulation of TORC1 signaling / negative regulation of autophagosome assembly / regulation of actin filament organization / guanyl-nucleotide exchange factor complex / negative regulation of immune response / regulation of autophagosome assembly / Flemming body / axon extension / presynaptic cytosol / negative regulation of exocytosis / positive regulation of autophagosome maturation / main axon / negative regulation of macroautophagy / protein kinase inhibitor activity / positive regulation of macroautophagy / positive regulation of TOR signaling / axonal growth cone / stress granule assembly / autophagosome / GTPase activator activity / negative regulation of protein phosphorylation / positive regulation of GTPase activity / guanyl-nucleotide exchange factor activity / cell projection / regulation of autophagy / P-body / autophagy / small GTPase binding / cytoplasmic stress granule / endocytosis / regulation of protein localization / presynapse / nuclear membrane / perikaryon / postsynapse / lysosome / endosome / lysosomal membrane / negative regulation of gene expression / intracellular membrane-bounded organelle / dendrite / chromatin / protein kinase binding / extracellular space / nucleoplasm / nucleus / cytosol / cytoplasm
Similarity search - Function
WD repeat-containing protein 41 / Guanine nucleotide exchange factor C9orf72 / C9orf72-like protein family / Tripartite DENN C9ORF72-type domain profile. / Folliculin/SMCR8, longin domain / Folliculin/SMCR8, tripartite DENN domain / Vesicle coat protein involved in Golgi to plasma membrane transport / Tripartite DENN FLCN/SMCR8-type domain profile. / G-protein beta WD-40 repeat / WD40 repeat, conserved site ...WD repeat-containing protein 41 / Guanine nucleotide exchange factor C9orf72 / C9orf72-like protein family / Tripartite DENN C9ORF72-type domain profile. / Folliculin/SMCR8, longin domain / Folliculin/SMCR8, tripartite DENN domain / Vesicle coat protein involved in Golgi to plasma membrane transport / Tripartite DENN FLCN/SMCR8-type domain profile. / G-protein beta WD-40 repeat / WD40 repeat, conserved site / Trp-Asp (WD) repeats signature. / Trp-Asp (WD) repeats profile. / Trp-Asp (WD) repeats circular profile. / WD domain, G-beta repeat / WD40 repeats / WD40 repeat / WD40-repeat-containing domain superfamily / WD40/YVTN repeat-like-containing domain superfamily
Similarity search - Domain/homology
Guanine nucleotide exchange protein SMCR8 / Guanine nucleotide exchange factor C9orf72 / WD repeat-containing protein 41
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsTang, D. / Sheng, J. / Xu, L. / Zhan, X. / Yan, C. / Qi, S.
Funding support China, 3items
OrganizationGrant numberCountry
Ministry of Science and Technology (MoST, China)2017YFA0506300 China
Ministry of Science and Technology (MoST, China)2018YFC1004601 China
National Science Foundation (NSF, China)81671388 China
CitationJournal: Proc Natl Acad Sci U S A / Year: 2020
Title: Cryo-EM structure of C9ORF72-SMCR8-WDR41 reveals the role as a GAP for Rab8a and Rab11a.
Authors: Dan Tang / Jingwen Sheng / Liangting Xu / Xiechao Zhan / Jiaming Liu / Hui Jiang / Xiaoling Shu / Xiaoyu Liu / Tizhong Zhang / Lan Jiang / Cuiyan Zhou / Wenqi Li / Wei Cheng / Zhonghan Li / ...Authors: Dan Tang / Jingwen Sheng / Liangting Xu / Xiechao Zhan / Jiaming Liu / Hui Jiang / Xiaoling Shu / Xiaoyu Liu / Tizhong Zhang / Lan Jiang / Cuiyan Zhou / Wenqi Li / Wei Cheng / Zhonghan Li / Kunjie Wang / Kefeng Lu / Chuangye Yan / Shiqian Qi /
Abstract: A massive intronic hexanucleotide repeat (GGGGCC) expansion in is a genetic origin of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Recently, C9ORF72, together with SMCR8 ...A massive intronic hexanucleotide repeat (GGGGCC) expansion in is a genetic origin of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Recently, C9ORF72, together with SMCR8 and WDR41, has been shown to regulate autophagy and function as Rab GEF. However, the precise function of C9ORF72 remains unclear. Here, we report the cryogenic electron microscopy (cryo-EM) structure of the human C9ORF72-SMCR8-WDR41 complex at a resolution of 3.2 Å. The structure reveals the dimeric assembly of a heterotrimer of C9ORF72-SMCR8-WDR41. Notably, the C-terminal tail of C9ORF72 and the DENN domain of SMCR8 play critical roles in the dimerization of the two protomers of the C9ORF72-SMCR8-WDR41 complex. In the protomer, C9ORF72 and WDR41 are joined by SMCR8 without direct interaction. WDR41 binds to the DENN domain of SMCR8 by the C-terminal helix. Interestingly, the prominent structural feature of C9ORF72-SMCR8 resembles that of the FLNC-FNIP2 complex, the GTPase activating protein (GAP) of RagC/D. Structural comparison and sequence alignment revealed that Arg147 of SMCR8 is conserved and corresponds to the arginine finger of FLCN, and biochemical analysis indicated that the Arg147 of SMCR8 is critical to the stimulatory effect of the C9ORF72-SMCR8 complex on Rab8a and Rab11a. Our study not only illustrates the basis of C9ORF72-SMCR8-WDR41 complex assembly but also reveals the GAP activity of the C9ORF72-SMCR8 complex.
History
DepositionJan 21, 2020Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Apr 15, 2020Provider: repository / Type: Initial release
Revision 1.1May 6, 2020Group: Database references / Category: citation / Item: _citation.pdbx_database_id_PubMed
Revision 1.2May 27, 2020Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.3Mar 27, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

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  • Deposited structure unit
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Assembly

Deposited unit
A: WD repeat-containing protein 41
B: Guanine nucleotide exchange protein SMCR8
C: Guanine nucleotide exchange C9orf72
D: WD repeat-containing protein 41
E: Guanine nucleotide exchange protein SMCR8
F: Guanine nucleotide exchange C9orf72


Theoretical massNumber of molelcules
Total (without water)422,6496
Polymers422,6496
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area18520 Å2
ΔGint-101 kcal/mol
Surface area102390 Å2

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Components

#1: Protein WD repeat-containing protein 41


Mass: 51783.805 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: WDR41, MSTP048 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q9HAD4
#2: Protein Guanine nucleotide exchange protein SMCR8 / Smith-Magenis syndrome chromosomal region candidate gene 8 protein


Mass: 105149.094 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: SMCR8 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q8TEV9
#3: Protein Guanine nucleotide exchange C9orf72


Mass: 54391.477 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: C9orf72 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q96LT7

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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: Dimer of C9-ORF72-SMCR8-WDR41 / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.4 MDa / Experimental value: YES
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm)
Buffer solutionpH: 8
Buffer component
IDConc.NameBuffer-ID
125 mMTirs1
2150 mMNaCl1
30.5 mMTCEP1
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: monodisperse
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationCryogen name: ETHANE

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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: DIFFRACTION
Specimen holderCryogen: HELIUM / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 0.25 sec. / Electron dose: 5.3 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 5332
Image scansMovie frames/image: 32

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Processing

SoftwareName: PHENIX / Version: 1.11.1_2575: / Classification: refinement
EM software
IDNameCategoryDetails
1Gautomatchparticle selection
2Auto3DEMimage acquisitionatuoEMation
4GctfCTF correction
7Cootmodel fitting
9PHENIXmodel refinement
10RELIONinitial Euler assignment
11RELIONfinal Euler assignment
12RELIONclassification
13RELION3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 347925 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Details: The atomic coordinates of the CSW complex was generated by combining homology modelling and de novo model building.
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0059068
ELECTRON MICROSCOPYf_angle_d1.06312259
ELECTRON MICROSCOPYf_dihedral_angle_d10.0675402
ELECTRON MICROSCOPYf_chiral_restr0.0581419
ELECTRON MICROSCOPYf_plane_restr0.0071529

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