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- EMDB-49393: In-situ cryo-EM structure of outer membrane cap (OMC) of the Dot/... -

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Basic information

Entry
Database: EMDB / ID: EMD-49393
TitleIn-situ cryo-EM structure of outer membrane cap (OMC) of the Dot/Icm machine
Map dataOverall map
Sample
  • Complex: outer membrane cap (OMC)
    • Protein or peptide: DotD
    • Protein or peptide: LphA (DotK)
    • Protein or peptide: Putative auto-transporter adhesin head GIN domain-containing protein
    • Protein or peptide: Neurogenic locus notch protein homolog
    • Protein or peptide: IcmG (DotF)
    • Protein or peptide: IcmK (DotH)
    • Protein or peptide: Outer membrane protein, OmpA family protein
    • Protein or peptide: DotC
    • Protein or peptide: Secreted protein
KeywordsType IVB Dot/Icm Secretion Machine / PROTEIN TRANSPORT
Function / homology
Function and homology information


Putative auto-transporter adhesin, head GIN domain / Putative auto-transporter adhesin, head GIN domain / Putative type IV secretory system protein / Type IV secretory system, conjugal DNA-protein transfer / DotD protein / DotD superfamily / DotD protein / Putative outer membrane core complex of type IVb secretion / Putative outer membrane core complex of type IVb secretion / : ...Putative auto-transporter adhesin, head GIN domain / Putative auto-transporter adhesin, head GIN domain / Putative type IV secretory system protein / Type IV secretory system, conjugal DNA-protein transfer / DotD protein / DotD superfamily / DotD protein / Putative outer membrane core complex of type IVb secretion / Putative outer membrane core complex of type IVb secretion / : / OmpA-like domain profile. / OmpA-like domain superfamily / OmpA family / OmpA-like domain / Prokaryotic membrane lipoprotein lipid attachment site profile.
Similarity search - Domain/homology
Secreted protein / Putative auto-transporter adhesin head GIN domain-containing protein / DotC / DotD / Neurogenic locus notch protein homolog / Outer membrane protein, OmpA family protein / IcmG (DotF) / IcmK (DotH) / LphA (DotK)
Similarity search - Component
Biological speciesLegionella pneumophila subsp. pneumophila (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.96 Å
AuthorsYue J / Jun L
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/Office of the DirectorR01AI152421 United States
CitationJournal: bioRxiv / Year: 2025
Title: structures of the Dot/Icm T4SS identify the DotA-IcmX complex as the gatekeeper for effector translocation.
Authors: Jian Yue / Samira Heydari / Donghyun Park / David Chetrit / Shoichi Tachiyama / Wangbiao Guo / Jack M Botting / Shenping Wu / Craig R Roy / Jun Liu
Abstract: The Dot/Icm machine in is one of the most versatile type IV secretion systems (T4SSs), with a remarkable capacity to translocate over 330 different effector proteins across the bacterial envelope ...The Dot/Icm machine in is one of the most versatile type IV secretion systems (T4SSs), with a remarkable capacity to translocate over 330 different effector proteins across the bacterial envelope into host cells. At least 27 Dot and Icm proteins are required for assembly and function of the system, yet the architecture and activation mechanism remain poorly understood at the molecular level. Here, we deploy cryo-electron microscopy to reveal structures of the Dot/Icm machine at near-atomic resolution. Importantly, two proteins essential for effector translocation, DotA and IcmX, form a pentameric protochannel at an inactive state. Upon activation, the DotA-IcmX protochannel undergoes extensive rearrangements to form an extended transenvelope passage capable of transporting effector proteins from the bacterial cytoplasm into host cells as revealed by cryo-electron tomography. Collectively, our findings suggest that the DotA-IcmX complex functions as the gatekeeper for effector translocation of the Dot/Icm T4SS.
History
DepositionFeb 22, 2025-
Header (metadata) releaseSep 17, 2025-
Map releaseSep 17, 2025-
UpdateSep 17, 2025-
Current statusSep 17, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_49393.png

Downloads & links

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Map

FileDownload / File: emd_49393.map.gz / Format: CCP4 / Size: 343 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationOverall map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.34 Å/pix.
x 448 pix.
= 598.08 Å		1.34 Å/pix.
x 448 pix.
= 598.08 Å		1.34 Å/pix.
x 448 pix.
= 598.08 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.335 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.2
Minimum - Maximum-0.9953161 - 1.6558901
Average (Standard dev.)0.0066420673 (±0.057351112)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions448448448
Spacing448448448
CellA=B=C: 598.08 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: half map A

Fileemd_49393_half_map_1.map
Annotationhalf map A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half map B

Fileemd_49393_half_map_2.map
Annotationhalf map B
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : outer membrane cap (OMC)

EntireName: outer membrane cap (OMC)
Components
  • Complex: outer membrane cap (OMC)
    • Protein or peptide: DotD
    • Protein or peptide: LphA (DotK)
    • Protein or peptide: Putative auto-transporter adhesin head GIN domain-containing protein
    • Protein or peptide: Neurogenic locus notch protein homolog
    • Protein or peptide: IcmG (DotF)
    • Protein or peptide: IcmK (DotH)
    • Protein or peptide: Outer membrane protein, OmpA family protein
    • Protein or peptide: DotC
    • Protein or peptide: Secreted protein

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Supramolecule #1: outer membrane cap (OMC)

SupramoleculeName: outer membrane cap (OMC) / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Legionella pneumophila subsp. pneumophila (bacteria)

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Macromolecule #1: DotD

MacromoleculeName: DotD / type: protein_or_peptide / ID: 1 / Number of copies: 26 / Enantiomer: LEVO
Source (natural)Organism: Legionella pneumophila subsp. pneumophila (bacteria)
Molecular weightTheoretical: 17.860678 KDa
SequenceString:
MNNNKIVIMF IFSALLAGCA GTMKFKKPPI NNPSDDATIK LAEAAVSVSD SMLEMAKVEK VITPPSKDNT LTIPNAYNLQ ARASVDWSG PIEELTARIA KAAHFRFRVL GKSPSVPVLI SISTKDESLA EILRDIDYQA GKKASIHVYP NSQVVELRYA K IYS

UniProtKB: DotD

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Macromolecule #2: LphA (DotK)

MacromoleculeName: LphA (DotK) / type: protein_or_peptide / ID: 2 / Number of copies: 13 / Enantiomer: LEVO
Source (natural)Organism: Legionella pneumophila subsp. pneumophila (bacteria)
Molecular weightTheoretical: 21.096492 KDa
SequenceString:
MRSLRTNYIY VLFKTTGLLF LLLLSACNRS GYIPENEVPK LPCRVDGACD ATIIKMMTDL NKKGIKVASV GQNYLISIPA SALFADQSP RLNWASYSLL NEIAAFLKQF RKIAITVTSY SSKYVSVKRE RALTLARSRV VSEYLWSQGV DSRIIFTQGL G SDKPITSY TLGGDRSPNA RVEITFRRAV A

UniProtKB: LphA (DotK)

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Macromolecule #3: Putative auto-transporter adhesin head GIN domain-containing protein

MacromoleculeName: Putative auto-transporter adhesin head GIN domain-containing protein
type: protein_or_peptide / ID: 3 / Number of copies: 13 / Enantiomer: LEVO
Source (natural)Organism: Legionella pneumophila subsp. pneumophila (bacteria)
Molecular weightTheoretical: 36.095367 KDa
SequenceString: MLKRCYLLIL LMFVLASCAH HKPQTPPAEV KKQGTSSTRQ FRQVSSFNQI VVQGRLNVNL HTGYNKPEVM LRGDPRDLVQ VRTIVKQNT LYVSLGQGYP DYGAVTVDIK TKFLNRFRYE GAGVVTGNNL RTSYLDLYLA NEGTTRLAGN IGLQKLEAVG N GVTQINGV ...String:
MLKRCYLLIL LMFVLASCAH HKPQTPPAEV KKQGTSSTRQ FRQVSSFNQI VVQGRLNVNL HTGYNKPEVM LRGDPRDLVQ VRTIVKQNT LYVSLGQGYP DYGAVTVDIK TKFLNRFRYE GAGVVTGNNL RTSYLDLYLA NEGTTRLAGN IGLQKLEAVG N GVTQINGV SSRNLQIVLK GDPKVLISGF VNLRQLDMYG KGTLSLYWIK SDTLTIRAKK AAKIQLAGIV NRLDVELWDF AQ FKGKYLR AQRSFVKTHD KSVAEISAVN HQSSLATDAS DIYYYNLSKT RADFMAFNGS VLDMREWGQS DLKDFDRYNK QFP

UniProtKB: Putative auto-transporter adhesin head GIN domain-containing protein

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Macromolecule #4: Neurogenic locus notch protein homolog

MacromoleculeName: Neurogenic locus notch protein homolog / type: protein_or_peptide / ID: 4 / Number of copies: 13 / Enantiomer: LEVO
Source (natural)Organism: Legionella pneumophila subsp. pneumophila (bacteria)
Molecular weightTheoretical: 13.598854 KDa
SequenceString:
MLFLKIKTNQ RTTMNILKPK AFLLASVFVL SISPAFAADG CCSKMGGINY CDSSAGRLVC NNGFYSTCYC TRHAVMDLQF LMGCCLWHG GVYPQLNSSG LVVCNDGYVS EECSLQKPVE QISVY

UniProtKB: Neurogenic locus notch protein homolog

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Macromolecule #5: IcmG (DotF)

MacromoleculeName: IcmG (DotF) / type: protein_or_peptide / ID: 5 / Number of copies: 13 / Enantiomer: LEVO
Source (natural)Organism: Legionella pneumophila subsp. pneumophila (bacteria)
Molecular weightTheoretical: 29.729969 KDa
SequenceString: MMAEHDQNND EYKFAELDSY DMDQAGESDL DSEASYQSGK EGLTKKKDIK RNALIAIGAV VFIMVMYKII GWMFFSDKSS QVTSKPAIP PVTQVATPQP VQTIPTTTPI QQVQPTTIIE DDPDLKKKVS AIEMTQQSLR SEVNALSEQI NAVNNNIKNL N AQIVNLNQ ...String:
MMAEHDQNND EYKFAELDSY DMDQAGESDL DSEASYQSGK EGLTKKKDIK RNALIAIGAV VFIMVMYKII GWMFFSDKSS QVTSKPAIP PVTQVATPQP VQTIPTTTPI QQVQPTTIIE DDPDLKKKVS AIEMTQQSLR SEVNALSEQI NAVNNNIKNL N AQIVNLNQ IIGNMSNQIA RQSEVINVLM ARTTPKKVVK VSRPIVQARI IYYIQAVIPG RAWLIGSNGS TLTVREGSKI PG YGMVKLI DSLQGRILTS SGQVIKFSQE DS

UniProtKB: IcmG (DotF)

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Macromolecule #6: IcmK (DotH)

MacromoleculeName: IcmK (DotH) / type: protein_or_peptide / ID: 6 / Number of copies: 13 / Enantiomer: LEVO
Source (natural)Organism: Legionella pneumophila subsp. pneumophila (bacteria)
Molecular weightTheoretical: 38.958926 KDa
SequenceString: MMKKYDQLCK YCLVIGLTFS MSCSIYAADQ SDDAQQALQQ LRMLQQKLSQ NPSPDAQSGA GDGGDNAASD STQQPNQSGQ ANAPAANQT ATAGGDGQII SQDDAEVIDK KAFKDMTRNL YPLNPEQVVK LKQIYETSEY AKAATPGTPP KPTATSQFVN L SPGSTPPV ...String:
MMKKYDQLCK YCLVIGLTFS MSCSIYAADQ SDDAQQALQQ LRMLQQKLSQ NPSPDAQSGA GDGGDNAASD STQQPNQSGQ ANAPAANQT ATAGGDGQII SQDDAEVIDK KAFKDMTRNL YPLNPEQVVK LKQIYETSEY AKAATPGTPP KPTATSQFVN L SPGSTPPV IRLSQGFVSS LVFLDSTGAP WPIAAYDLGD PSSFNIQWDK TSNTLMIQAT KLYNYGNLAV RLRGLNTPVM LT LIPGQKA VDYRVDLRVQ GYGPNAKSMP TEEGIPPSAN DLLLHVLEGV PPPGSRRLVV SGGDARAWLS NEKMYVRTNL TIL SPGWLA SMTSADGTHA YEMQKSPVLL VSWHGKVMQL KVEGL

UniProtKB: IcmK (DotH)

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Macromolecule #7: Outer membrane protein, OmpA family protein

MacromoleculeName: Outer membrane protein, OmpA family protein / type: protein_or_peptide / ID: 7 / Number of copies: 13 / Enantiomer: LEVO
Source (natural)Organism: Legionella pneumophila subsp. pneumophila (bacteria)
Molecular weightTheoretical: 27.582047 KDa
SequenceString: MRNLMRCLIM IKSLIKGVDM SRKLAKTRIL GYGLMICFLA GCFHPPYNNF QPDRRAVKRV GVDTGIGAVA GAIASGTASG TLIGAAAGG TVGLVASIYR DSKRKIIRDL QKQDIQYVEY GDTRTLIIPT DKYFMFSSPR LNEICYPGLN NVIRLLNFYP Q STIYVAGF ...String:
MRNLMRCLIM IKSLIKGVDM SRKLAKTRIL GYGLMICFLA GCFHPPYNNF QPDRRAVKRV GVDTGIGAVA GAIASGTASG TLIGAAAGG TVGLVASIYR DSKRKIIRDL QKQDIQYVEY GDTRTLIIPT DKYFMFSSPR LNEICYPGLN NVIRLLNFYP Q STIYVAGF TDNVGSRSHK RKLSQAQAET MMTFLWANGI AAKRLKAEGY GDKNAISDNA IIHGSAQNRR IEIQWFTSPA QP PQPQMAY VK

UniProtKB: Outer membrane protein, OmpA family protein

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Macromolecule #8: DotC

MacromoleculeName: DotC / type: protein_or_peptide / ID: 8 / Number of copies: 13 / Enantiomer: LEVO
Source (natural)Organism: Legionella pneumophila subsp. pneumophila (bacteria)
Molecular weightTheoretical: 34.162441 KDa
SequenceString: MRKFILSLSI LLSALLVACS SRNHYGDTGS LAGLQAMADS KYTRAQKKQK MGKIREMALK ETALSVGAQA GLAWRAKIID EQLNKQARN LDAIYDFNSL VLEHNILPPV LLEGRNTLNL ADAQSIRISD RTYKVAKQAH FITTPPTWRQ YLWMDYVKPE A PNVTLLPK ...String:
MRKFILSLSI LLSALLVACS SRNHYGDTGS LAGLQAMADS KYTRAQKKQK MGKIREMALK ETALSVGAQA GLAWRAKIID EQLNKQARN LDAIYDFNSL VLEHNILPPV LLEGRNTLNL ADAQSIRISD RTYKVAKQAH FITTPPTWRQ YLWMDYVKPE A PNVTLLPK TKAEKEIWCI YTERGWKNGI DQANTILEEN IARIKEDFGG MILYRKLLAM NMVSPPYVSH TDLGVTGDGS EI HIDDRVL RITALPELNV NSAEWRAAVA KDENALERFK NMEKLANQAK IVITNKSWQP IIAPVS

UniProtKB: DotC

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Macromolecule #9: Secreted protein

MacromoleculeName: Secreted protein / type: protein_or_peptide / ID: 9 / Number of copies: 13 / Enantiomer: LEVO
Source (natural)Organism: Legionella pneumophila subsp. pneumophila (bacteria)
Molecular weightTheoretical: 10.348009 KDa
SequenceString:
MKNKLAVSIL LFLLPTAAAM ANCDLTKFRW DCDLPVQVKP SPASSLLVYC GSSYGYITKQ QYDILARYQR ASVNMVLDIN GEYIDSPCI GAER

UniProtKB: Secreted protein

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation statecell

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Sample preparation

BufferpH: 6.7
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TECNAI 12
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 73.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.8 µm

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Image processing

CTF correctionType: PHASE FLIPPING ONLY
Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.96 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 76409
Initial angle assignmentType: NOT APPLICABLE
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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