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- EMDB-10189: ESX-3 core complex centred at the cytoplasmic region, conformation 1. -

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Basic information

Entry
Database: EMDB / ID: EMD-10189
TitleESX-3 core complex centred at the cytoplasmic region, conformation 1.
Map dataESX-3 core dimer centred at the cytoplasmic region, conformation 1.
Sample
  • Complex: ESX-3 core complex, conformation 1
    • Protein or peptide: EccB3
    • Protein or peptide: EccD3
    • Protein or peptide: EccC3
    • Protein or peptide: EccE3
Biological speciesMycolicibacterium smegmatis MC2 155 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 5.4 Å
AuthorsRivera-Calzada A / Famelis N / Geibel S / Llorca O
Funding support Germany, Spain, 6 items
OrganizationGrant numberCountry
German Research FoundationElite Network of Bavaria (N-BM-2013-246), Bavarian State Ministry of Science and the Arts Germany
European Regional Development FundY2018/BIO4747 co-funded by the Autonomous Region of Madrid Spain
Spanish Ministry of Science, Innovation, and UniversitiesSAF2017-82632-P o-funded by the European Regional Development Fund Spain
European UnionHorizon 2020, iNEXT (PID2907) , Grant number 653706 Spain
European Regional Development FundP2018/NMT4443 co-funded by Autonomous Region of Madrid 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.
History
DepositionAug 5, 2019-
Header (metadata) releaseOct 9, 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: 0.0085
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 0.0085
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_10189.png

Downloads & links

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Map

FileDownload / File: emd_10189.map.gz / Format: CCP4 / Size: 274.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationESX-3 core dimer centred at the cytoplasmic region, conformation 1.
Voxel sizeX=Y=Z: 1.0635 Å
Density
Contour LevelBy AUTHOR: 0.0085 / Movie #1: 0.0085
Minimum - Maximum-0.037227653 - 0.08501123
Average (Standard dev.)-0.00000052725 (±0.0022724236)
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
start NX/NY/NZ-200-200-200
NX/NY/NZ401401401
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS416416416
D min/max/mean-0.0370.085-0.000

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Supplemental data

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Sample components

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Entire : ESX-3 core complex, conformation 1

EntireName: ESX-3 core complex, conformation 1
Components
  • Complex: ESX-3 core complex, conformation 1
    • Protein or peptide: EccB3
    • Protein or peptide: EccD3
    • Protein or peptide: EccC3
    • Protein or peptide: EccE3

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Supramolecule #1: ESX-3 core complex, conformation 1

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

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Macromolecule #1: EccB3

MacromoleculeName: EccB3 / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Mycolicibacterium smegmatis MC2 155 (bacteria)
Recombinant expressionOrganism: Mycolicibacterium smegmatis MC2 155 (bacteria)
SequenceString: MTGPVNPDDR RSFSSRTPVN ENPDGVQYRR GFVTRHQVSG WRFVMRRIAS GVALHDTRML VDPLRTQSR AVLTGALILV TGLVGCFIFS LFRPGGVPGN NAILADRSTS ALYVRVGEQL H PVLNLTSA RLISGSPDNP TMVKTSEIDK FPRGNLLGIP GAPERMVQNA ...String:
MTGPVNPDDR RSFSSRTPVN ENPDGVQYRR GFVTRHQVSG WRFVMRRIAS GVALHDTRML VDPLRTQSR AVLTGALILV TGLVGCFIFS LFRPGGVPGN NAILADRSTS ALYVRVGEQL H PVLNLTSA RLISGSPDNP TMVKTSEIDK FPRGNLLGIP GAPERMVQNA ATDAEWTVCD AV GGANPGV TVIAGPLGAD GERAAPLPPD HAVLVHSDAE PNPGDWLLWD GKRSPIDLAD RAV TDALGL GGQALAPRPI AAGLFNAVPA APALTAPVIP DAGAAPQFEL SLPVPVGAVV VAYD ADNTA RYYAVLSDGL QPISPVLAAI LRNTDSHGFA QPPRLGPDEV ARTPMSRGLD TSAYP DNPV TLVEASAHPV TCAHWTKPSD AAESSLSVLS GAVLPLAEGL HTVDLVGAGA GGAANR VAL TPGTGYFVQT VGAEPGSPTA GSMFWVSDTG VRYGIDTAED DKVVAALGLS TSPLPVP WS VLSQFAAGPA LSRGDALVAH DAVSTNPNSA RMEASR

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Macromolecule #2: EccD3

MacromoleculeName: EccD3 / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO
Source (natural)Organism: Mycolicibacterium smegmatis MC2 155 (bacteria)
Recombinant expressionOrganism: Mycolicibacterium smegmatis MC2 155 (bacteria)
SequenceString: MASWSHPQFE KGSMSENTVM PIVRVAVLAA GDDGGRLTEM ALPSELPLRE ILPAVQRIVQ PARENDGAAD PAAAPNPVRL SLAPIGGAPF SLDATLDTVG VVDGDLLALQ AVPSGPPAPR IVEDIADAAV IFSEARRRQW GPTHIARGAA LALIGLILVG TGLSVAHRVI ...String:
MASWSHPQFE KGSMSENTVM PIVRVAVLAA GDDGGRLTEM ALPSELPLRE ILPAVQRIVQ PARENDGAAD PAAAPNPVRL SLAPIGGAPF SLDATLDTVG VVDGDLLALQ AVPSGPPAPR IVEDIADAAV IFSEARRRQW GPTHIARGAA LALIGLILVG TGLSVAHRVI TGDLLGQFIV SGIALATVIA ALAVRNRSAV LATSLAVTAL VPVAAAFALG VPGDFGAPNV LLAAAGVAAW SLISMAGSPD DRGIAVFTAT AVTGVGVLLV AGAASLWVIS SDVIGCALVL LGLIVTVQAA QLSAMWARFP LPVIPAPGDP TPAARPLSVL ADLPRRVRVS QAHQTGVIAA GVLLGVAGSV ALVSSANASP WAWYIVVAAA AGAALRARVW DSAACKAWLL GHSYLLAVAL LVAFVIGDRY QAALWALAAL AVLVLVWIVA ALNPKIASPD TYSLPMRRMV GFLATGLDAS LIPVMALLVG LFSLVLDR

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Macromolecule #3: EccC3

MacromoleculeName: EccC3 / type: protein_or_peptide / ID: 3 / Enantiomer: LEVO
Source (natural)Organism: Mycolicibacterium smegmatis MC2 155 (bacteria)
Recombinant expressionOrganism: Mycolicibacterium smegmatis MC2 155 (bacteria)
SequenceString: MSRLIFEHQR RLTPPTTRKG TITIEPPPQL PRVVPPSLLR RVLPFLIVIL IVGMIVALFA TGMRLISPT MLFFPFVLLL AATALYRGGD NKMRTEEVDA ERADYLRYLS VVRDNVRAHA A EQRAALEW SHPEPEVLAT IPGTRRQWER DPRDRDFLVL RAGRHDVPLD ...String:
MSRLIFEHQR RLTPPTTRKG TITIEPPPQL PRVVPPSLLR RVLPFLIVIL IVGMIVALFA TGMRLISPT MLFFPFVLLL AATALYRGGD NKMRTEEVDA ERADYLRYLS VVRDNVRAHA A EQRAALEW SHPEPEVLAT IPGTRRQWER DPRDRDFLVL RAGRHDVPLD AALKVKDTAD EI DLEPVAH SALRGLLDVQ RTVRDAPTGL DVAKLARITV IGEADEARAA IRAWIAQAVT WHD PTMLGV ALAAPDLESG DWSWLKWLPH VDVPNEADGV GPARYLTTST AELRERLAPA LADR PLFPA ESGAALKHLL VVLDDPDADP DDIARKPGLT GVTVIHRTTE LPNREQYPDP ERPIL RVAD GRIERWQVGG WQPCVDVADA MSAAEAAHIA RRLSRWDSNP GYIRSTSTGS ATFTTL LGI PDASALDVAS LWAPRPRDEE LRVPIGVTST GEPLYFDLKD EAEGGMGPHG LMIGMTG SG KSQTLMSILL SLLTTHPADR LIVIYADFKG EAGADIFRHF PQVVAVISNM AEKRSLAD R FADTLRGEVA RREQILKEAG RRVQGSAFNS VAEYESAIAA GHDLPPMPTL FVVADEFTL MLAEHPEYAD LFDYVARKGR SFRIHLLFAS QTLDVGRIKD IDKNTSYRIG LKVASPSISR QIIGVEDAY HIESGREHKG EGFLVPAPGA VPIKFRSTYV DGIYDPPRAE KSIVVHALPQ P QVFTAGRV EPEPDTVIAT GDVEVHTAPP RKLIATIGDQ LAAYGPKAPQ LWLPPLDEPI AL ADVLAGA DVEPGQLRWP LGEIDKPFEM RRDVLVYDAH TAAANVLIHG GPRSGKSTAL QAF VLSAAA LHSPRAITFY CLDYGGGKLA DLADLAHVGS VATPLEPERI RRTFGELEQL LRAR QRQGA VNRTGSYTDG YGEVFLVIDN LYAFSRDNTD TFNTRNPLLA KVTELANSGL AYGIH VVIT TPNWLEVPLA MRDGLGLRLE LKLHDSHDSI VRVAGALRRP ADSVPADQPG RGLTMA AEH FLFAEPALSD IAVINARYPG VSAPPVRLLP TDLSPDALAP LYPAPETVVI GQREEDL AP VAVDFANHPL LMVFGDSKSG KTTLLRHIIR TVRENSTPDQ VAFTVIDRRL HLVDEPLF P DNEYTANIDR VLPAMLGLSA LIEKRRPPAG LSAQELSRWT YTGHTHYLIV DDVDQIPDT PAVSGPFVGQ RPWTNIVGLL AEAADLGLRV IVTARATGSA HAVMTAPLLR RLNDLQATTL MLSGNPTDS GKIRGHRFAR FPAGRGLLLT DTDTPDHIQL VNPLGDAALS GNIGNNGNHN R GGEYRGSM GGSHHHHHH

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Macromolecule #4: EccE3

MacromoleculeName: EccE3 / type: protein_or_peptide / ID: 4 / Enantiomer: LEVO
Source (natural)Organism: Mycolicibacterium smegmatis MC2 155 (bacteria)
Recombinant expressionOrganism: Mycolicibacterium smegmatis MC2 155 (bacteria)
SequenceString: MTARIALASL FVVAAVLAQP WQTTTQRWVL GVSIAAVIVL LAWWKGMFLT TRIGRALAMV RRNRAEDTV ETDAHRATVV LRVDPAAPAQ LPVVVGYLDR YGITCDKVRI THRDAGGTRR S WISLTVDA VDNLAALQAR SARIPLQDTT EVVGRRLADH LREQGWTVTV ...String:
MTARIALASL FVVAAVLAQP WQTTTQRWVL GVSIAAVIVL LAWWKGMFLT TRIGRALAMV RRNRAEDTV ETDAHRATVV LRVDPAAPAQ LPVVVGYLDR YGITCDKVRI THRDAGGTRR S WISLTVDA VDNLAALQAR SARIPLQDTT EVVGRRLADH LREQGWTVTV VEGVDTPLPV SG KETWRGV ADDAGVVAAY RVKVDDRLDE VLAEIGHLPA EETWTALEFT GSPAEPLLTV CAA VRTSDR PAAKAPLAGL TPARGRHRPA LAALNPLSTE RLDGTAVPLP AVVRTSVKGS VEHE AAQEA GHPA

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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
Details: Particles were extracted centred at the distal cytoplasmic tip instead than in the centre of mass in order to improve the resolution in that distal region.
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: 5.4 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Number images used: 80173
DetailsImages were collected in counting mode
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

4nh0


Chain - Chain ID: B / Chain - Residue range: 401-772
DetailsA homology model based on pdb 4NH0 was created using Phyre2 and fitted into the density using Chimera.
RefinementProtocol: RIGID BODY FIT / Target criteria: Map correlation coefficient

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