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- EMDB-10187: Structure of protomer 1 of the ESX-3 core complex -

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Basic information

Entry
Database: EMDB / ID: EMD-10187
TitleStructure of protomer 1 of the ESX-3 core complex
Map dataStructure of protomer 1 of the ESX-3 secretion system
Sample
  • Complex: Protomer1 of the ESX-3 core complex
    • Protein or peptide: ESX-3 secretion system EccB3
    • Protein or peptide: ESX-3 secretion system protein EccD3
    • Protein or peptide: ESX-3 secretion system protein EccC3
    • Protein or peptide: ESX-3 secretion system protein EccE3
Function / homology
Function and homology information


Hydrolases; Acting on acid anhydrides / hydrolase activity / ATP hydrolysis activity / DNA binding / ATP binding / plasma membrane
Similarity search - Function
Type VII secretion system protein EccE / Putative type VII ESX secretion system translocon, EccE / Type VII secretion system membrane protein EccD / YukD-like / WXG100 protein secretion system (Wss), protein YukD / EccCa-like, Actinobacteria / EccCb-like, Actinobacteria / Type VII secretion system EccB, repeat 1 domain / Type VII secretion system EccB / Type VII secretion system EccB, repeat 3 domain ...Type VII secretion system protein EccE / Putative type VII ESX secretion system translocon, EccE / Type VII secretion system membrane protein EccD / YukD-like / WXG100 protein secretion system (Wss), protein YukD / EccCa-like, Actinobacteria / EccCb-like, Actinobacteria / Type VII secretion system EccB, repeat 1 domain / Type VII secretion system EccB / Type VII secretion system EccB, repeat 3 domain / Type VII secretion system ESX-1, transport TM domain B / FtsK domain / FtsK/SpoIIIE family / FtsK domain profile. / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
ESX-3 secretion system ATPase EccB3 / ESX-3 secretion system protein EccC3 / ESX-3 secretion system protein EccD3 / ESX-3 secretion system protein EccE3
Similarity search - Component
Biological speciesMycolicibacterium smegmatis MC2 155 (bacteria) / Mycobacterium smegmatis (strain ATCC 700084 / mc(2)155) (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.7 Å
AuthorsRivera-Calzada A / Famelis N / Geibel S / Llorca O
Funding support Spain, Germany, 5 items
OrganizationGrant numberCountry
Spanish Ministry of Science, Innovation, and UniversitiesSAF2017-82632-P o-funded by the European Regional Development Fund Spain
European Regional Development FundP2018/NMT4443 co-funded by Autonomous Region of Madrid Spain
European Regional Development FundY2018/BIO4747 co-funded by the Autonomous Region of Madrid Spain
Bavarian State Ministry of Science and the Arts Funding OrganisationN-BM-2013-246 Germany
European UnionHorizon 2020, iNEXT (PID2907) , Grant number 653706 Spain
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.
History
DepositionAug 5, 2019-
Header (metadata) releaseAug 28, 2019-
Map releaseOct 9, 2019-
UpdateDec 25, 2019-
Current statusDec 25, 2019Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.03
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 0.03
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6sgx
  • Surface level: 0.03
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_10187.map.gz / Format: CCP4 / Size: 274.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationStructure of protomer 1 of the ESX-3 secretion system
Voxel sizeX=Y=Z: 1.0635 Å
Density
Contour LevelBy AUTHOR: 0.03 / Movie #1: 0.03
Minimum - Maximum-0.1438568 - 0.2192611
Average (Standard dev.)0.00000150013 (±0.003856069)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions416416416
Spacing416416416
CellA=B=C: 442.41602 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.06351.06351.0635
M x/y/z416416416
origin x/y/z0.0000.0000.000
length x/y/z442.416442.416442.416
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS416416416
D min/max/mean-0.1440.2190.000

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Supplemental data

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Sample components

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Entire : Protomer1 of the ESX-3 core complex

EntireName: Protomer1 of the ESX-3 core complex
Components
  • Complex: Protomer1 of the ESX-3 core complex
    • Protein or peptide: ESX-3 secretion system EccB3
    • Protein or peptide: ESX-3 secretion system protein EccD3
    • Protein or peptide: ESX-3 secretion system protein EccC3
    • Protein or peptide: ESX-3 secretion system protein EccE3

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Supramolecule #1: Protomer1 of the ESX-3 core complex

SupramoleculeName: Protomer1 of the ESX-3 core complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
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
Source (natural)Organism: Mycolicibacterium smegmatis MC2 155 (bacteria)
Recombinant expressionOrganism: Mycolicibacterium smegmatis MC2 155 (bacteria) / Recombinant plasmid: pMyNT
Molecular weightExperimental: 650 KDa

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Macromolecule #1: ESX-3 secretion system EccB3

MacromoleculeName: ESX-3 secretion system EccB3 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: Hydrolases; Acting on acid anhydrides
Source (natural)Organism: Mycobacterium smegmatis (strain ATCC 700084 / mc(2)155) (bacteria)
Molecular weightTheoretical: 8.875269 KDa
Recombinant expressionOrganism: Mycolicibacterium smegmatis MC2 155 (bacteria)
SequenceString:
RSFSSRTPVN ENPDGVQYRR GFVTRHQVSG WRFVMRRIAS GVALHDTRML VDPLRTQSRA VLTGALILVT GLVGCFIFS

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Macromolecule #2: ESX-3 secretion system protein EccD3

MacromoleculeName: ESX-3 secretion system protein EccD3 / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Mycobacterium smegmatis (strain ATCC 700084 / mc(2)155) (bacteria)
Molecular weightTheoretical: 47.114125 KDa
Recombinant expressionOrganism: Mycolicibacterium smegmatis MC2 155 (bacteria)
SequenceString: PIVRVAVLAA GDDGGRLTEM ALPSELPLRE ILPAVQRIVQ PARENDGAAD PAAAPNPVRL SLAPIGGAPF SLDATLDTVG VVDGDLLAL QAVPSGPPAP RIVEDIADAA VIFSEARRRQ WGPTHIARGA ALALIGLILV GTGLSVAHRV ITGDLLGQFI V SGIALATV ...String:
PIVRVAVLAA GDDGGRLTEM ALPSELPLRE ILPAVQRIVQ PARENDGAAD PAAAPNPVRL SLAPIGGAPF SLDATLDTVG VVDGDLLAL QAVPSGPPAP RIVEDIADAA VIFSEARRRQ WGPTHIARGA ALALIGLILV GTGLSVAHRV ITGDLLGQFI V SGIALATV IAALAVRNRS AVLATSLAVT ALVPVAAAFA LGVPGDFGAP NVLLAAAGVA AWSLISMAGS PDDRGIAVFT AT AVTGVGV LLVAGAASLW VISSDVIGCA LVLLGLIVTV QAAQLSAMWA RFPLPVIPAP GDPTPAARPL SVLADLPRRV RVS QAHQTG VIAAGVLLGV AGSVALVSSA NASPWAWYIV VAAAAGAALR ARVWDSAACK AWLLGHSYLL AVALLVAFVI GDRY QAALW ALAALAVLVL VWIVAALNPK IASPDTYSLP MRRMVGFLAT GLDASLIPVM ALLVGLFSLV

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Macromolecule #3: ESX-3 secretion system protein EccC3

MacromoleculeName: ESX-3 secretion system protein EccC3 / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Mycobacterium smegmatis (strain ATCC 700084 / mc(2)155) (bacteria)
Molecular weightTheoretical: 44.653078 KDa
Recombinant expressionOrganism: Mycolicibacterium smegmatis MC2 155 (bacteria)
SequenceString: SRLIFEHQRR LTPPTTRKGT ITIEPPPQLP RVVPPSLLRR VLPFLIVILI VGMIVALFAT GMRLISPTML FFPFVLLLAA TALYRGGDN KMRTEEVDAE RADYLRYLSV VRDNVRAHAA EQRAALEWSH PEPEVLATIP GTRRQWERDP RDRDFLVLRA G RHDVPLDA ...String:
SRLIFEHQRR LTPPTTRKGT ITIEPPPQLP RVVPPSLLRR VLPFLIVILI VGMIVALFAT GMRLISPTML FFPFVLLLAA TALYRGGDN KMRTEEVDAE RADYLRYLSV VRDNVRAHAA EQRAALEWSH PEPEVLATIP GTRRQWERDP RDRDFLVLRA G RHDVPLDA ALKVKDTADE IDLEPVAHSA LRGLLDVQRT VRDAPTGLDV AKLARITVIG EADEARAAIR AWIAQAVTWH DP TMLGVAL AAPDLESGDW SWLKWLPHVD VPNEADGVGP ARYLTTSTAE LRERLAPALA DRPLFPAESG AALKHLLVVL DDP DADPDD IARKPGLTGV TVIHRTTELP NREQYPDPER PILRVADGRI ERWQVGGWQP CVDVADAMSA AEAAHIARRL SRWD S

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Macromolecule #4: ESX-3 secretion system protein EccE3

MacromoleculeName: ESX-3 secretion system protein EccE3 / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Mycobacterium smegmatis (strain ATCC 700084 / mc(2)155) (bacteria)
Molecular weightTheoretical: 30.813355 KDa
Recombinant expressionOrganism: Mycolicibacterium smegmatis MC2 155 (bacteria)
SequenceString: MTARIALASL FVVAAVLAQP WQTTTQRWVL GVSIAAVIVL LAWWKGMFLT TRIGRALAMV RRNRAEDTVE TDAHRATVVL RVDPAAPAQ LPVVVGYLDR YGITCDKVRI THRDAGGTRR SWISLTVDAV DNLAALQARS ARIPLQDTTE VVGRRLADHL R EQGWTVTV ...String:
MTARIALASL FVVAAVLAQP WQTTTQRWVL GVSIAAVIVL LAWWKGMFLT TRIGRALAMV RRNRAEDTVE TDAHRATVVL RVDPAAPAQ LPVVVGYLDR YGITCDKVRI THRDAGGTRR SWISLTVDAV DNLAALQARS ARIPLQDTTE VVGRRLADHL R EQGWTVTV VEGVDTPLPV SGKETWRGVA DDAGVVAAYR VKVDDRLDEV LAEIGHLPAE ETWTALEFTG SPAEPLLTVC AA VRTSDRP AAKAPLAGLT PARGRHRPAL AALNPLSTER LDGTAVPL

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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

Concentration0.3 mg/mL
BufferpH: 8 / Details: 30 mM Hepes pH 8.0, 150 mM NaCl
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 278 K / Instrument: FEI VITROBOT MARK IV / Details: Blotting time: 3s Blotting force: -10.
Detailsbound to Amphipol A8-35

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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: 2.7 mm / Nominal defocus max: 2.6 µm / Nominal defocus min: 1.6 µm / Nominal magnification: 75000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: COUNTING / Number real images: 11903 / Average electron dose: 50.0 e/Å2
Details: Images acquired as 55 frames movies at a calibrated magnification of 1.0635 Angs/px
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 2066007
CTF correctionSoftware - Name: Gctf
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.7 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION
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.
Number images used: 126308
DetailsImages were collected in counting mode
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: AB INITIO MODEL / Overall B value: 29.4 / Target criteria: Map correlation coefficient
Output model

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

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