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- PDB-9nh0: In situ cryo-EM structure of PR and DotA-IcmX of the Legionella D... -

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Basic information

Entry
Database: PDB / ID: 9nh0
TitleIn situ cryo-EM structure of PR and DotA-IcmX of the Legionella Dot/Icm T4SS machine at C1 symmetry
Components
  • (unknown peptide ...) x 4
  • DotA
  • IcmE (DotG)
  • IcmG (DotF)
  • IcmK (DotH)
  • IcmX (IcmY)
KeywordsPROTEIN TRANSPORT / Type IVB Dot/Icm Secretion Machine
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 / Putative outer membrane core complex of type IVb secretion / Putative outer membrane core complex of type IVb secretion / Type IV secretion system, VirB10/TrbI / Bacterial conjugation TrbI-like protein ...Phagosome trafficking protein DotA / Conjugal transfer/type IV secretion protein DotA/TraY / Phagosome trafficking protein DotA / : / : / Decapeptide repeat region / Putative outer membrane core complex of type IVb secretion / Putative outer membrane core complex of type IVb secretion / Type IV secretion system, VirB10/TrbI / Bacterial conjugation TrbI-like protein / Type IV secretion system, VirB10 / TraB / TrbI / Prokaryotic membrane lipoprotein lipid attachment site profile.
Similarity search - Domain/homology
IcmX (IcmY) / DotA / IcmG (DotF) / IcmE (DotG) / IcmK (DotH)
Similarity search - Component
Biological speciesLegionella pneumophila subsp. pneumophila (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.63 Å
AuthorsYue, J. / Liu, J.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/Office of the DirectorR01AI152421 United States
Citation
Journal: Proc Natl Acad Sci U S A / Year: 2025
Title: In situ 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 of is among the most versatile type IV secretion systems (T4SSs), capable of translocating more than 330 distinct effector proteins across the bacterial envelope into host cells. ...The Dot/Icm machine of is among the most versatile type IV secretion systems (T4SSs), capable of translocating more than 330 distinct effector proteins across the bacterial envelope into host cells. Assembly and function of the system require at least 27 Dot and Icm proteins, yet its architecture and activation mechanism remain poorly understood at the molecular level. Here, we deploy in situ single-particle cryoelectron microscopy to determine near-atomic structures of the Dot/Icm machine and its intimate association with three distinct outer membrane porins in intact bacteria. Notably, two essential yet enigmatic components, DotA and IcmX, form a pentameric protochannel in an inactive state at the central axis of the Dot/Icm machine. Upon Dot/Icm activation with host lysate, this protochannel undergoes extensive rearrangements to generate an extended transenvelope conduit, as visualized by cryoelectron tomography (cryo-ET) and subtomogram averaging. Furthermore, a combination of cryo-ET and cryo-FIB milling of macrophages infected with reveals tethering of the Dot/Icm machine to the host membrane, suggesting direct translocation of effector proteins from the bacterial cytoplasm into the host. Together, our studies identify the DotA-IcmX complex as a gatekeeper for effector translocation and provide a molecular framework for understanding the assembly and activation of the elaborate Dot/Icm T4SS.
#1: Journal: 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 23, 2025Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 17, 2025Provider: repository / Type: Initial release
Revision 1.1Nov 12, 2025Group: Data collection / Database references / Category: citation / citation_author / em_admin / Item: _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
Aa: unknown peptide E
Ab: unknown peptide F
Ac: unknown peptide G
Ad: unknown peptide H
Ae: IcmG (DotF)
Af: IcmK (DotH)
Ag: unknown peptide E
Ah: unknown peptide F
Ai: unknown peptide G
Aj: unknown peptide H
Ak: IcmG (DotF)
Al: IcmK (DotH)
Am: unknown peptide E
An: unknown peptide F
Ao: unknown peptide G
Ap: unknown peptide H
Aq: IcmG (DotF)
Ar: IcmK (DotH)
As: unknown peptide E
At: unknown peptide F
Au: unknown peptide G
Av: unknown peptide H
Aw: IcmG (DotF)
Ax: IcmK (DotH)
Ay: unknown peptide E
Az: unknown peptide F
Ba: unknown peptide G
Bb: unknown peptide H
Bc: IcmG (DotF)
Bd: IcmK (DotH)
Be: unknown peptide E
Bf: unknown peptide F
Bg: unknown peptide G
Bh: unknown peptide H
Bi: IcmG (DotF)
Bj: IcmK (DotH)
Bk: unknown peptide E
Bl: unknown peptide F
Bm: unknown peptide G
Bn: unknown peptide H
Bo: IcmG (DotF)
Bp: IcmK (DotH)
Bq: unknown peptide E
Br: unknown peptide F
Bs: unknown peptide G
Bt: unknown peptide H
Bu: IcmG (DotF)
Bv: IcmK (DotH)
Bw: unknown peptide E
Bx: unknown peptide F
By: unknown peptide G
Bz: unknown peptide H
Ca: IcmG (DotF)
Cb: IcmK (DotH)
Cc: unknown peptide E
Cd: unknown peptide F
Ce: unknown peptide G
Cf: unknown peptide H
Cg: IcmG (DotF)
Ch: IcmK (DotH)
Ci: unknown peptide E
Cj: unknown peptide F
Ck: unknown peptide G
Cl: unknown peptide H
Cm: IcmG (DotF)
Cn: IcmK (DotH)
Co: unknown peptide E
Cp: unknown peptide F
Cq: unknown peptide G
Cr: unknown peptide H
Cs: IcmG (DotF)
Ct: IcmK (DotH)
Cu: unknown peptide E
Cv: unknown peptide F
Cw: unknown peptide G
Cx: unknown peptide H
Cy: IcmG (DotF)
Cz: IcmK (DotH)
Da: unknown peptide E
Db: unknown peptide F
Dc: unknown peptide G
Dd: unknown peptide H
De: IcmG (DotF)
Df: IcmK (DotH)
Dg: unknown peptide E
Dh: unknown peptide F
Di: unknown peptide G
Dj: unknown peptide H
Dk: IcmG (DotF)
Dl: IcmK (DotH)
Dm: unknown peptide E
Dn: unknown peptide F
Do: unknown peptide G
Dp: unknown peptide H
Dq: IcmG (DotF)
Dr: IcmK (DotH)
Ds: unknown peptide E
Dt: unknown peptide F
Du: unknown peptide G
Dv: unknown peptide H
Dw: IcmG (DotF)
Dx: IcmK (DotH)
Dy: unknown peptide E
Dz: unknown peptide F
Ea: unknown peptide G
Eb: unknown peptide H
Ec: IcmG (DotF)
Ed: IcmK (DotH)
Ee: IcmE (DotG)
Ef: IcmE (DotG)
Eg: IcmE (DotG)
Eh: IcmE (DotG)
Ei: IcmE (DotG)
Ej: IcmE (DotG)
Ek: IcmE (DotG)
El: IcmE (DotG)
Em: IcmE (DotG)
En: IcmE (DotG)
Eo: IcmE (DotG)
Ep: IcmE (DotG)
Eq: IcmE (DotG)
Er: IcmE (DotG)
Es: IcmE (DotG)
Et: IcmE (DotG)
Eu: IcmE (DotG)
Ev: IcmE (DotG)
Ew: IcmX (IcmY)
Ex: IcmX (IcmY)
Ey: IcmX (IcmY)
Ez: IcmX (IcmY)
Fa: IcmX (IcmY)
Fb: DotA
Fc: DotA
Fd: DotA
Fe: DotA
Ff: DotA
Fg: IcmE (DotG)
Fh: IcmE (DotG)
Fi: IcmE (DotG)
Fj: IcmE (DotG)
Fk: IcmE (DotG)


Theoretical massNumber of molelcules
Total (without water)4,625,800141
Polymers4,625,800141
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

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Unknown peptide ... , 4 types, 72 molecules AaAgAmAsAyBeBkBqBwCcCiCoCuDaDgDmDsDyAbAhAnAtAzBfBlBrBxCdCjCp...

#1: Protein/peptide
unknown peptide E


Mass: 1679.849 Da / Num. of mol.: 18 / Source method: isolated from a natural source
Source: (natural) Legionella pneumophila subsp. pneumophila (bacteria)
#2: Protein/peptide
unknown peptide F


Mass: 910.095 Da / Num. of mol.: 18 / Source method: isolated from a natural source
Source: (natural) Legionella pneumophila subsp. pneumophila (bacteria)
#3: Protein/peptide
unknown peptide G


Mass: 1775.978 Da / Num. of mol.: 18 / Source method: isolated from a natural source
Source: (natural) Legionella pneumophila subsp. pneumophila (bacteria)
#4: Protein/peptide
unknown peptide H


Mass: 505.521 Da / Num. of mol.: 18 / Source method: isolated from a natural source
Source: (natural) Legionella pneumophila subsp. pneumophila (bacteria)

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Protein , 5 types, 69 molecules AeAkAqAwBcBiBoBuCaCgCmCsCyDeDkDqDwEcAfAlArAxBdBjBpBvCbChCnCt...

#5: Protein
IcmG (DotF)


Mass: 29729.969 Da / Num. of mol.: 18 / Source method: isolated from a natural source
Source: (natural) Legionella pneumophila subsp. pneumophila (bacteria)
References: UniProt: Q5ZYC0
#6: Protein
IcmK (DotH)


Mass: 38958.926 Da / Num. of mol.: 18 / Source method: isolated from a natural source
Source: (natural) Legionella pneumophila subsp. pneumophila (bacteria)
References: UniProt: Q5ZYC2
#7: Protein ...
IcmE (DotG)


Mass: 107911.891 Da / Num. of mol.: 23 / Source method: isolated from a natural source
Source: (natural) Legionella pneumophila subsp. pneumophila (bacteria)
References: UniProt: Q5ZYC1
#8: Protein
IcmX (IcmY)


Mass: 50712.422 Da / Num. of mol.: 5 / Source method: isolated from a natural source
Source: (natural) Legionella pneumophila subsp. pneumophila (bacteria)
References: UniProt: Q5ZS30
#9: Protein
DotA


Mass: 113235.570 Da / Num. of mol.: 5 / Source method: isolated from a natural source
Source: (natural) Legionella pneumophila subsp. pneumophila (bacteria)
References: UniProt: Q5ZS33

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Details

Has protein modificationY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: CELL / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: PR-protoChannel / Type: COMPLEX / Entity ID: all / Source: NATURAL
Molecular weightExperimental value: NO
Source (natural)Organism: Legionella pneumophila subsp. pneumophila (bacteria)
Buffer solutionpH: 6.7
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

MicroscopyModel: FEI TECNAI 12
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2000 nm / Nominal defocus min: 800 nm
Image recordingElectron dose: 73 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

EM softwareName: PHENIX / Version: 1.21.2_5419 / Category: model refinement
CTF correctionType: PHASE FLIPPING ONLY
3D reconstructionResolution: 4.63 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 37503 / Symmetry type: POINT
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 260.1 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.005295926
ELECTRON MICROSCOPYf_angle_d1.2066130297
ELECTRON MICROSCOPYf_chiral_restr0.060514884
ELECTRON MICROSCOPYf_plane_restr0.00716764
ELECTRON MICROSCOPYf_dihedral_angle_d6.606413099

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