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- PDB-6sgy: Structure of EccB3 dimer from the ESX-3 core complex -

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Basic information

Entry
Database: PDB / ID: 6sgy
TitleStructure of EccB3 dimer from the ESX-3 core complex
ComponentsESX-3 secretion system protein EccB3
KeywordsMEMBRANE PROTEIN / Type VII Secretion System ESX-3 secretion system T7SS ESX-3 Mycobacterium smegmatis
Function / homologyType VII secretion system EccB, repeat 3 domain / Type VII secretion system EccB / Type VII secretion system ESX-1, transport TM domain B / Hydrolases, Acting on acid anhydrides / hydrolase activity / integral component of membrane / ATP binding / plasma membrane / ESX-3 secretion system ATPase EccB3
Function and homology information
Biological speciesMycobacterium smegmatis (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.6 Å
AuthorsFamelis, N. / Rivera-Calzada, A. / Llorca, O. / Geibel, S.
Funding support Germany, Spain, 6items
OrganizationGrant numberCountry
German Research FoundationElite Network of Bavaria (N-BM-2013-246), Bavarian State Ministry of Science and the Arts Germany
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 ESX-3/Type VII secretion system
Authors: Famelis, N. / Rivera-Calzada, A. / Degliesposti, G. / Wingender, M. / Mietrach, N. / Skehel, J.M. / Fernandez-Leiro, R. / Bottcher, B. / Schlosser, A. / Llorca, O. / Geibel, S.
Validation Report
SummaryFull reportAbout validation report
History
DepositionAug 5, 2019Deposition site: PDBE / Processing site: PDBE
Revision 1.0Oct 9, 2019Provider: repository / Type: Initial release

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Structure visualization

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Assembly

Deposited unit
A: ESX-3 secretion system protein EccB3
B: ESX-3 secretion system protein EccB3


Theoretical massNumber of molelcules
Total (without water)85,4942
Polymers85,4942
Non-polymers00
Water0
1


TypeNameSymmetry operationNumber
identity operation1_5551
Buried area2460 Å2
ΔGint-11 kcal/mol
Surface area38900 Å2
MethodPISA

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Components

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


Mass: 42746.754 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: eccB3, MSMEG_0616, MSMEI_0600
Production host: Mycolicibacterium smegmatis MC2 155 (bacteria)
References: UniProt: A0QQ39

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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: EccB3 dimeric structure from ESX-3/Type VII secretion system
Type: COMPLEX
Details: 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 / Source: RECOMBINANT
Molecular weightValue: 0.083 MDa / Experimental value: NO
Source (natural)Organism: Mycolicibacterium smegmatis MC2 155 (bacteria)
Source (recombinant)Organism: Mycolicibacterium smegmatis MC2 155 (bacteria)
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

EM software
IDNameVersionCategoryDetails
1RELIONparticle selection
2EPUimage acquisition
4GctfCTF correction
7PHENIX1.14model fittingReal-Space Refinement
9RELIONinitial Euler assignment
10RELIONfinal Euler assignment
11RELIONclassification
12RELION3D reconstruction
13PHENIX1.14model refinementMorphing
14UCSF Chimeramodel refinement
Image processingDetails: Images were collected in counting mode
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 2066007
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 4.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 55342
Details: Particles were extracted centred at extracellular density instead than in the centre of mass in order to improve the resolution in that distal region. Extra density not corresponding to the EccB3 dimer was subtracted
Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL / Space: REAL / Target criteria: Map correlation coefficient
Details: Two copies of the periplasmic domain of a EccB3 homology model (based on pdb 3X3M) were fitted into the density and then morphing was performed using PHENIX Real-Space Refinement.
Atomic model buildingPDB-ID: 3X3M
Pdb chain-ID: A / Pdb chain residue range: 100-518

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