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- EMDB-49396: In-situ cryo-EM structure of protochannel of the Dot/Icm machine -

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Basic information

Entry
Database: EMDB / ID: EMD-49396
TitleIn-situ cryo-EM structure of protochannel of the Dot/Icm machine
Map datastructure of protochannel of the Dot/Icm machine
Sample
  • Complex: protochannel
    • Protein or peptide: IcmX (IcmY)
    • Protein or peptide: DotA
    • Protein or peptide: IcmE (DotG)
KeywordsType IVB Dot/Icm Secretion Machine / PROTEIN TRANSPORT
Function / homology
Function and homology information


Phagosome trafficking protein DotA / Conjugal transfer/type IV secretion protein DotA/TraY / Phagosome trafficking protein DotA / : / : / Decapeptide repeat region / Type IV secretion system, VirB10/TrbI / Bacterial conjugation TrbI-like protein / Type IV secretion system, VirB10 / TraB / TrbI
Similarity search - Domain/homology
IcmX (IcmY) / DotA / IcmE (DotG)
Similarity search - Component
Biological speciesLegionella pneumophila subsp. pneumophila (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.63 Å
AuthorsYue J / Liu J
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_49396.png

Downloads & links

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Map

FileDownload / File: emd_49396.map.gz / Format: CCP4 / Size: 512 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationstructure of protochannel of the Dot/Icm machine
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.25 Å/pix.
x 512 pix.
= 640.819 Å		1.25 Å/pix.
x 512 pix.
= 640.819 Å		1.25 Å/pix.
x 512 pix.
= 640.819 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.2516 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.2
Minimum - Maximum-0.52556473 - 0.8181758
Average (Standard dev.)0.0023984343 (±0.027851958)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions512512512
Spacing512512512
CellA=B=C: 640.8192 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Half Map A

Fileemd_49396_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_49396_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 : protochannel

EntireName: protochannel
Components
  • Complex: protochannel
    • Protein or peptide: IcmX (IcmY)
    • Protein or peptide: DotA
    • Protein or peptide: IcmE (DotG)

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Supramolecule #1: protochannel

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

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Macromolecule #1: IcmX (IcmY)

MacromoleculeName: IcmX (IcmY) / type: protein_or_peptide / ID: 1 / Number of copies: 5 / Enantiomer: LEVO
Source (natural)Organism: Legionella pneumophila subsp. pneumophila (bacteria)
Molecular weightTheoretical: 50.712422 KDa
SequenceString: MKVLPKLALA NLVCFTTLSV FAQPADPSGQ QTSTNTSNLV TYLTNLGKYL GYDITQSSKA PNPPYSQLFN SNVVQLVQNY AYNTFLGAI PVDAMSQSLM NFVTDKVQGN SLINNLANTT FKYQNFSAPS SGADGKITAN GLIDQSTASA QTPTTPGIFS A PQITTSGT ...String:
MKVLPKLALA NLVCFTTLSV FAQPADPSGQ QTSTNTSNLV TYLTNLGKYL GYDITQSSKA PNPPYSQLFN SNVVQLVQNY AYNTFLGAI PVDAMSQSLM NFVTDKVQGN SLINNLANTT FKYQNFSAPS SGADGKITAN GLIDQSTASA QTPTTPGIFS A PQITTSGT YLNDPVSQAV FNILGTPDYS FCMDNEQKNW LPNCNLMYQN LVMQNVIGTL PPAQTSGSTP AFYSYKYNQP LI SQLNSNS LIAPLLMDTS ASQSGQDSGL TAKSQAQQAL NFIRYASAQV TPPSLPKLSA YSELWNQATA KPNSAGYNEV QQK QAAATL SSYFNNLRVY AAQTSVGVSN LYYILSKRLP QNMSADQSNA NITSQALNEF NMATRRLFDP TASNTPGQPN QQWI KQIND ASPATVQKEI AILLAEINYQ MYLDRQIQER ILLTNSIMLL QNLKAAQPTA DFSSQGSPSE Q

UniProtKB: IcmX (IcmY)

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Macromolecule #2: DotA

MacromoleculeName: DotA / type: protein_or_peptide / ID: 2 / Number of copies: 5 / Enantiomer: LEVO
Source (natural)Organism: Legionella pneumophila subsp. pneumophila (bacteria)
Molecular weightTheoretical: 113.23557 KDa
SequenceString: MNKLAITVLL CFFPALALAE NGALSFAPPA SDLSVVFLGN LFGVVDGVLH GTGSQIMGNM FGVFNSAVLA LGGIIIMYTL MVSTMNTAH EGQMLGQKWS SIWIPLRSTF GLALLIPKAS GYCMMQVFFM WVIVQGVGAA DKIWEAALSY LNRGGVIIQA Q ADPTKSLQ ...String:
MNKLAITVLL CFFPALALAE NGALSFAPPA SDLSVVFLGN LFGVVDGVLH GTGSQIMGNM FGVFNSAVLA LGGIIIMYTL MVSTMNTAH EGQMLGQKWS SIWIPLRSTF GLALLIPKAS GYCMMQVFFM WVIVQGVGAA DKIWEAALSY LNRGGVIIQA Q ADPTKSLQ AAGSSSSGVA KGALTILGGQ ICMLGLQKQL QAQRDLYLSQ SKSPPCGGNP TPEMNTFCRT AIPDFISTVN FV KKQNDDT PKDLTANQPA SFELDMPNFD KSSPFYFLNG ICGTVKWNNI SALNSTNQSD NKGLVTVGGA GSNSSMGANS LNI TSSQLQ TARLSRAIAI QQMYVTLSTV AQVMVNNDPA FSTTTSTGNS KNDFSAIAKQ QFGVPYKSSG EVCTEYQQVC QTWG SVPSS TGSTTGVLFN GTEFLGAIND YNGIMMPTLN LIRQATSKEF DKKSRDFIAE ANAKGWIMAG SYFFDLVKLN GSATE FADQ FDTGTGLDKS SFDPTQLTKP FGKTCQDPYS LLCTWFQNKS DKLIQIQSLI DGVPALGQDG VKQPDLSDNP QRQSVS GPL SSTVYGFVNN SMMVQLPGQP GIKPLTFANL INFKVDTSLY YMKHQDFDCG RVKILFFSFC LGRMMGDLFY NYVFRYV YN FFLAIFGEMI NSIVMAFLMI PLQGMKDIFI VGVQTLTQPG INPIVALANM GTMYINFSGT LWLTLLNMAV VSSLIPLF G IFIFALIMMA MPLLMAWIGT MVSIGFVTAY YIPVLPYMIF TFGSFAWLIA VIEAMVAAPI VALGVTHPEG NEAFGKGEF AIMILVNVFL RPSLMIIGYI AAIALSYVGV WILNAGFDHA ISYIQSDQGI ETWEKGTSSN DAKRFFQSAG NWTGAKMDTS QWDNFKDKL TGDYQSTALE GGYTGWAGVY AFFFSILIYT SMYLIIVQKA FTLIAHLPDK VLRWIGGSPE SFGQETMQWG E EAKGRVEK AGEATYGAEK AIGDKLGAKG QEMLGKLGPQ AKKISENAGD VSGKGKNSGG SQTGPSSKPD TGQQLGGSDS GT PTSTPPP E

UniProtKB: DotA

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Macromolecule #3: IcmE (DotG)

MacromoleculeName: IcmE (DotG) / type: protein_or_peptide / ID: 3 / Number of copies: 5 / Enantiomer: LEVO
Source (natural)Organism: Legionella pneumophila subsp. pneumophila (bacteria)
Molecular weightTheoretical: 107.911891 KDa
SequenceString: MASKKENLKS LFSNTRTRVI IIFTAALLII AVVIGFFKIR GATTGSIAAA EVSTVPGGIQ SIPGVLDPTA QYAKLQEEQN ITQAQVAEK TGGSAIPTII RTQALGEGVG VIGSQSGVGF AALAQEELGG PQRSLWIQEL QDGSCSKSVI TKVVNQGAQL T DLKAACSC ...String:
MASKKENLKS LFSNTRTRVI IIFTAALLII AVVIGFFKIR GATTGSIAAA EVSTVPGGIQ SIPGVLDPTA QYAKLQEEQN ITQAQVAEK TGGSAIPTII RTQALGEGVG VIGSQSGVGF AALAQEELGG PQRSLWIQEL QDGSCSKSVI TKVVNQGAQL T DLKAACSC VQLKDSGYGL QELEQVCECK ELKSAGYNAR QLKEAGYSAG RLRNCGFDAC ELRNAGFTAQ EMKDGGFSDG EL KGAGFSD AEIAKASGLP DGITADDVRK AGCGAAALAK LRQAGVSASA IRKISGCTAE QLKAAGYTAK ELKDAGFSAA DLR RAGFSA AELKDAGFTA RDLLNAGFTP ADLAKAGFSD AQIKAAQAEL PPGITPQDVK NAGCDVEALK KEREAGVSAA LIRQ YAGCS AQALKAAGFT DADLANAGFT PAQISAATPL SDAEIKAAGC DPDKLKKLFS AGVSAKRIKE LNGCSAEALK AAGYD AQSL LAAGFTPQEL LAAGFTPKQL EDAGLNPVSI IADGRVADCS VESLKKARAA GVSALTIKQT LGCSAAALKA AGYTAK ELK DAGFTAAELK AAGFSAKELK DAGFTAKELR DAGFSAQELK DVGFSAKDLK DAGFSAAELK AAGFTAAQLK AAGFSAK DL KDAGFSAAEL KAAGFSAKEL KDAGFSASDL KNAGFSAKEL KDAGFSASDL KSAGFSASEL KNAGYSADEL KKAGYTSA E LRNAGFSPQE SAVAGLQGPD LQQLDSSITG IPSIPGATPR PTTSDAASSA EQLQAILQKQ NEQLAEQKYQ QEIQQRTSD MLTAATQLVQ DWKQVETQVY TEGTEETKTS GGESAVPGTG TGTGSNNQPV DQGAVSAQNQ AIIKTGDIMF AVLDTSVNSD EPGPILATI VTGKLKGSKL IGSFNLPSNA DKMVITFNTM SIPGAEKTIS ISAYAIDPNT ARTALASRTN HHYLMRYGSL F ASSFLQGF GNAFQSANTT ITIGGTGGGN NITVANGVGR STLENAVIGL ATVGKAWSQQ AQQLFNTPTT VEVYSGTGLG IL FTQDVTT I

UniProtKB: IcmE (DotG)

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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: 3.63 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 37503
Initial angle assignmentType: NOT APPLICABLE
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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