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- PDB-3j9o: CryoEM structure of a type VI secretion system -

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Basic information

Entry
Database: PDB / ID: 3j9o
TitleCryoEM structure of a type VI secretion system
Components
  • Intracellular growth locus protein A
  • Intracellular growth locus protein B
KeywordsSTRUCTURAL PROTEIN / T6SS
Function / homology
Function and homology information


host cell cytoplasm
Similarity search - Function
Type VI secretion system TssC-like / TssC1, N-terminal / TssC1, C-terminal / EvpB/VC_A0108, tail sheath N-terminal domain / EvpB/VC_A0108, tail sheath gpW/gp25-like domain / Type VI secretion system sheath protein TssB1 / Type VI secretion system, VipA, VC_A0107 or Hcp2
Similarity search - Domain/homology
Intracellular growth locus protein B / Intracellular growth locus protein A
Similarity search - Component
Biological speciesFrancisella tularensis subsp. novicida U112 (bacteria)
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 3.7 Å
AuthorsClemens, D.L. / Ge, P. / Lee, B.-Y. / Horwitz, M.A. / Zhou, Z.H.
CitationJournal: Cell / Year: 2015
Title: Atomic structure of T6SS reveals interlaced array essential to function.
Authors: Daniel L Clemens / Peng Ge / Bai-Yu Lee / Marcus A Horwitz / Z Hong Zhou /
Abstract: Type VI secretion systems (T6SSs) are newly identified contractile nanomachines that translocate effector proteins across bacterial membranes. The Francisella pathogenicity island, required for ...Type VI secretion systems (T6SSs) are newly identified contractile nanomachines that translocate effector proteins across bacterial membranes. The Francisella pathogenicity island, required for bacterial phagosome escape, intracellular replication, and virulence, was presumed to encode a T6SS-like apparatus. Here, we experimentally confirm the identity of this T6SS and, by cryo electron microscopy (cryoEM), show the structure of its post-contraction sheath at 3.7 Å resolution. We demonstrate the assembly of this T6SS by IglA/IglB and secretion of its putative effector proteins in response to environmental stimuli. The sheath has a quaternary structure with handedness opposite that of contracted sheath of T4 phage tail and is organized in an interlaced two-dimensional array by means of β sheet augmentation. By structure-based mutagenesis, we show that this interlacing is essential to secretion, phagosomal escape, and intracellular replication. Our atomic model of the T6SS will facilitate design of drugs targeting this highly prevalent secretion apparatus.
History
DepositionFeb 11, 2015Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 18, 2015Provider: repository / Type: Initial release
Revision 1.1Jul 18, 2018Group: Author supporting evidence / Data collection / Category: em_single_particle_entity / em_software / Item: _em_software.image_processing_id / _em_software.name
Revision 1.2Feb 21, 2024Group: Data collection / Database references / Derived calculations
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_struct_oper_list
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _pdbx_struct_oper_list.name / _pdbx_struct_oper_list.symmetry_operation / _pdbx_struct_oper_list.type

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Structure visualization

Movie
  • Biological unit as representative helical assembly
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  • Deposited structure unit
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  • Simplified surface model + fitted atomic model
  • EMDB-6266
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  • Superimposition on EM map
  • EMDB-6266
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  • EMDB-6266
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Structure viewerMolecule:
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Assembly

Deposited unit
A: Intracellular growth locus protein A
B: Intracellular growth locus protein B
C: Intracellular growth locus protein A
D: Intracellular growth locus protein B
E: Intracellular growth locus protein A
F: Intracellular growth locus protein B
G: Intracellular growth locus protein A
H: Intracellular growth locus protein B
I: Intracellular growth locus protein A
J: Intracellular growth locus protein B
K: Intracellular growth locus protein A
L: Intracellular growth locus protein B


Theoretical massNumber of molelcules
Total (without water)473,68312
Polymers473,68312
Non-polymers00
Water00
1
A: Intracellular growth locus protein A
B: Intracellular growth locus protein B
C: Intracellular growth locus protein A
D: Intracellular growth locus protein B
E: Intracellular growth locus protein A
F: Intracellular growth locus protein B
G: Intracellular growth locus protein A
H: Intracellular growth locus protein B
I: Intracellular growth locus protein A
J: Intracellular growth locus protein B
K: Intracellular growth locus protein A
L: Intracellular growth locus protein B
x 10


Theoretical massNumber of molelcules
Total (without water)4,736,831120
Polymers4,736,831120
Non-polymers00
Water0
TypeNameSymmetry operationNumber
helical symmetry operation9
identity operation1_555x,y,z1
2


  • Idetical with deposited unit
  • helical asymmetric unit
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
3


  • Idetical with deposited unit
  • helical asymmetric unit, std helical frame
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
SymmetryHelical symmetry: (Circular symmetry: 1 / Dyad axis: no / N subunits divisor: 1 / Num. of operations: 10 / Rise per n subunits: 20.8 Å / Rotation per n subunits: -33.4 °)

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Components

#1: Protein
Intracellular growth locus protein A / IglA


Mass: 21004.932 Da / Num. of mol.: 6 / Source method: isolated from a natural source
Source: (natural) Francisella tularensis subsp. novicida U112 (bacteria)
References: UniProt: A0Q7I5
#2: Protein
Intracellular growth locus protein B / IglB


Mass: 57942.254 Da / Num. of mol.: 6 / Source method: isolated from a natural source
Source: (natural) Francisella tularensis subsp. novicida U112 (bacteria)
References: UniProt: A0Q7I4

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: FILAMENT / 3D reconstruction method: helical reconstruction

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Sample preparation

Component
IDNameTypeDetailsParent-ID
1Contracted T6SS sheathCOMPLEXHelix by IglA/IglB dimers0
2IglA1
3IglB1
Buffer solutionName: TBS / pH: 7.5 / Details: 20 mM Tris, 0.9% NaCl
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: 200 mesh Quantifoil 1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Temp: 90 K / Humidity: 100 % / Details: Plunged into liquid ethane (FEI VITROBOT MARK IV)

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS / Date: Mar 1, 2014 / Details: K2 Summit in Counting mode
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 29000 X / Nominal defocus max: 3500 nm / Nominal defocus min: 1500 nm / Cs: 2.7 mm / Astigmatism: Software / Camera length: 0 mm
Specimen holderSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature: 80 K
Image recordingElectron dose: 25 e/Å2 / Film or detector model: GATAN K2 (4k x 4k)
Image scansNum. digital images: 1644
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthRelative weight: 1

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Processing

EM software
IDNameCategory
1IHRSR3D reconstruction
2RELION3D reconstruction
CTF correctionDetails: each particle
Helical symmertyAngular rotation/subunit: 33.4 ° / Axial rise/subunit: 20.8 Å / Axial symmetry: C6
Details: One asymmetric unit contains a heterodimer of IglA/IglB
3D reconstructionMethod: Relion / Resolution: 3.7 Å / Resolution method: FSC 0.143 CUT-OFF / Nominal pixel size: 1 Å / Actual pixel size: 1 Å / Details: (Helical Details: Relion-based IHRSR) / Symmetry type: HELICAL
Refinement stepCycle: LAST
ProteinNucleic acidLigandSolventTotal
Num. atoms27264 0 0 0 27264

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