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- PDB-6sgx: Structure of protomer 1 of the ESX-3 core complex -

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Basic information

Entry
Database: PDB / ID: 6sgx
TitleStructure of protomer 1 of the ESX-3 core complex
Components
  • ESX-3 secretion system EccB3
  • ESX-3 secretion system protein EccC3
  • ESX-3 secretion system protein EccD3
  • ESX-3 secretion system protein EccE3
KeywordsMEMBRANE PROTEIN / Type VII Secretion System ESX-3 secretion system T7SS ESX-3 Mycobacterium smegmatis
Function / homology
Function and homology information


Hydrolases; Acting on acid anhydrides / hydrolase activity / DNA binding / integral component of membrane / ATP binding / plasma membrane
Putative type VII ESX secretion system translocon, EccE / Type VII secretion system EccB / WXG100 protein secretion system (Wss), protein YukD / Type VII secretion system EccB, repeat 3 domain / P-loop containing nucleoside triphosphate hydrolase / YukD-like / EccCb-like, Actinobacteria / Type VII secretion system protein EccE / EccCa-like, Actinobacteria / Type VII secretion system membrane protein EccD ...Putative type VII ESX secretion system translocon, EccE / Type VII secretion system EccB / WXG100 protein secretion system (Wss), protein YukD / Type VII secretion system EccB, repeat 3 domain / P-loop containing nucleoside triphosphate hydrolase / YukD-like / EccCb-like, Actinobacteria / Type VII secretion system protein EccE / EccCa-like, Actinobacteria / Type VII secretion system membrane protein EccD / AAA+ ATPase domain / FtsK domain
ESX-3 secretion system ATPase EccB3 / ESX-3 secretion system protein EccC3 / ESX-3 secretion system protein EccD3 / ESX-3 secretion system protein EccE3
Biological speciesMycobacterium smegmatis (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.7 Å
AuthorsFamelis, N. / Rivera-Calzada, A. / Llorca, O. / Geibel, S.
Funding support Spain, Germany, 5items
OrganizationGrant numberCountry
Spanish Ministry of Science, Innovation, and UniversitiesSAF2017-82632-P o-funded by the European Regional Development Fund Spain
European Regional Development FundY2018/BIO4747 co-funded by the Autonomous Region of Madrid Spain
European Regional Development FundP2018/NMT4443 co-funded by Autonomous Region of Madrid Spain
European UnionHorizon 2020, iNEXT (PID2907) , Grant number 653706 Spain
Bavarian State Ministry of Science and the Arts Funding OrganisationN-BM-2013-246 Germany
CitationJournal: Nature / Year: 2019
Title: Architecture of the mycobacterial type VII secretion system.
Authors: Nikolaos Famelis / Angel Rivera-Calzada / Gianluca Degliesposti / Maria Wingender / Nicole Mietrach / J Mark Skehel / Rafael Fernandez-Leiro / Bettina Böttcher / Andreas Schlosser / Oscar ...Authors: Nikolaos Famelis / Angel Rivera-Calzada / Gianluca Degliesposti / Maria Wingender / Nicole Mietrach / J Mark Skehel / Rafael Fernandez-Leiro / Bettina Böttcher / Andreas Schlosser / Oscar Llorca / Sebastian Geibel /
Abstract: Host infection by pathogenic mycobacteria, such as Mycobacterium tuberculosis, is facilitated by virulence factors that are secreted by type VII secretion systems. A molecular understanding of the ...Host infection by pathogenic mycobacteria, such as Mycobacterium tuberculosis, is facilitated by virulence factors that are secreted by type VII secretion systems. A molecular understanding of the type VII secretion mechanism has been hampered owing to a lack of three-dimensional structures of the fully assembled secretion apparatus. Here we report the cryo-electron microscopy structure of a membrane-embedded core complex of the ESX-3/type VII secretion system from Mycobacterium smegmatis. The core of the ESX-3 secretion machine consists of four protein components-EccB3, EccC3, EccD3 and EccE3, in a 1:1:2:1 stoichiometry-which form two identical protomers. The EccC3 coupling protein comprises a flexible array of four ATPase domains, which are linked to the membrane through a stalk domain. The domain of unknown function (DUF) adjacent to the stalk is identified as an ATPase domain that is essential for secretion. EccB3 is predominantly periplasmatic, but a small segment crosses the membrane and contacts the stalk domain. This suggests that conformational changes in the stalk domain-triggered by substrate binding at the distal end of EccC3 and subsequent ATP hydrolysis in the DUF-could be coupled to substrate secretion to the periplasm. Our results reveal that the architecture of type VII secretion systems differs markedly from that of other known secretion machines, and provide a structural understanding of these systems that will be useful for the design of antimicrobial strategies that target bacterial virulence.
Validation Report
SummaryFull reportAbout validation report
History
DepositionAug 5, 2019Deposition site: PDBE / Processing site: PDBE
Revision 1.0Oct 9, 2019Provider: repository / Type: Initial release
Revision 1.1Oct 23, 2019Group: Data collection / Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID
Revision 1.2Dec 25, 2019Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last

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Structure visualization

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Assembly

Deposited unit
A: ESX-3 secretion system EccB3
B: ESX-3 secretion system protein EccD3
C: ESX-3 secretion system protein EccD3
F: ESX-3 secretion system protein EccC3
G: ESX-3 secretion system protein EccE3


Theoretical massNumber of molelcules
Total (without water)178,5705
Polymers178,5705
Non-polymers00
Water0
1


TypeNameSymmetry operationNumber
identity operation1_5551
Buried area18810 Å2
ΔGint-131 kcal/mol
Surface area72810 Å2
MethodPISA

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Components

#1: Protein ESX-3 secretion system EccB3 / ESX conserved component B3 / Type VII secretion system protein EccB3 / T7SS protein EccB3


Mass: 8875.269 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycobacterium smegmatis (strain ATCC 700084 / mc(2)155) (bacteria)
Gene: eccB3, MSMEG_0616, MSMEI_0600
Production host: Mycolicibacterium smegmatis MC2 155 (bacteria)
References: UniProt: A0QQ39, Hydrolases; Acting on acid anhydrides
#2: Protein ESX-3 secretion system protein EccD3 / ESX conserved component D3 / Type VII secretion system protein EccD3 / T7SS protein EccD3


Mass: 47114.125 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycobacterium smegmatis (strain ATCC 700084 / mc(2)155) (bacteria)
Gene: eccD3, snm, MSMEG_0623, MSMEI_0607
Production host: Mycolicibacterium smegmatis MC2 155 (bacteria)
References: UniProt: A0QQ46
#3: Protein ESX-3 secretion system protein EccC3 / ESX conserved component C3 / Type VII secretion system protein EccC3 / T7SS protein EccC3


Mass: 44653.078 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycobacterium smegmatis (strain ATCC 700084 / mc(2)155) (bacteria)
Gene: eccC3, MSMEG_0617, MSMEI_0601
Production host: Mycolicibacterium smegmatis MC2 155 (bacteria)
References: UniProt: A0QQ40
#4: Protein ESX-3 secretion system protein EccE3 / ESX conserved component E3 / Type VII secretion system protein EccE3 / T7SS protein EccE3


Mass: 30813.355 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycobacterium smegmatis (strain ATCC 700084 / mc(2)155) (bacteria)
Gene: eccE3, MSMEG_0626, MSMEI_0609
Production host: Mycolicibacterium smegmatis MC2 155 (bacteria)
References: UniProt: A0QQ48

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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: Protomer1 of the ESX-3 core complex / Type: COMPLEX
Details: ESX-3 core complex The sample consists of four protein components, EccB3:EccC3:EccD3:EccE3 in a 1:1:2:1 stoichiometry Molecular weight of the complex without the amphipol micelle: 0.65 MDa
Entity ID: #1-#4 / Source: RECOMBINANT
Molecular weightValue: 0.65 MDa / Experimental value: YES
Source (natural)Organism: Mycolicibacterium smegmatis MC2 155 (bacteria)
Source (recombinant)Organism: Mycolicibacterium smegmatis MC2 155 (bacteria) / Plasmid: pMyNT
Buffer solutionpH: 8 / Details: 30 mM Hepes pH 8.0, 150 mM NaCl
SpecimenConc.: 0.3 mg/ml / Details: bound to Amphipol A8-35 / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 278 K / Details: Blotting time: 3s Blotting force: -10

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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 / Nominal magnification: 75000 X / Nominal defocus max: 2600 nm / Nominal defocus min: 1600 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN / Model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 50 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON III (4k x 4k) / Num. of real images: 11903
Details: Images acquired as 55 frames movies at a calibrated magnification of 1.0635 Angs/px

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Processing

SoftwareName: PHENIX / Version: 1.15.2_3472: / Classification: refinement
EM software
IDNameVersionCategoryDetails
1RELIONparticle selection
2EPUimage acquisition
4GctfCTF correction
9RELIONinitial Euler assignment
10RELIONfinal Euler assignment
11RELIONclassification
12RELION3D reconstruction
19PHENIX1.15.2-3472model refinementReal-space refinement
Image processingDetails: Images were collected in counting mode
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 2066007
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 126308
Details: In order to get the best possible resolution for protomer 1, the extra density not corresponding to this protomer was subtracted and masked out during the refinement.
Symmetry type: POINT
Atomic model buildingB value: 29.4 / Protocol: AB INITIO MODEL / Space: REAL / Target criteria: Map correlation coefficient
Refine LS restraints
Refinement-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00611436
ELECTRON MICROSCOPYf_angle_d1.00415655
ELECTRON MICROSCOPYf_dihedral_angle_d12.3386850
ELECTRON MICROSCOPYf_chiral_restr0.0591951
ELECTRON MICROSCOPYf_plane_restr0.0081974

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