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- PDB-6r69: Improved map of the FliPQR complex that forms the core of the Sal... -

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Basic information

Entry
Database: PDB / ID: 6r69
TitleImproved map of the FliPQR complex that forms the core of the Salmonella type III secretion system export apparatus.
Components
  • Flagellar biosynthetic protein FliP
  • Flagellar biosynthetic protein FliQ
  • Flagellar biosynthetic protein FliR
KeywordsPROTEIN TRANSPORT / T3SS / Flagella / Cryo-EM / Export / Membrane protein
Function / homology
Function and homology information


bacterial-type flagellum organization / bacterial-type flagellum basal body / bacterial-type flagellum assembly / protein secretion / protein targeting / membrane => GO:0016020 / plasma membrane
Similarity search - Function
Flagellar biosynthesis protein FliQ / Flagellar biosynthesis protein FliR / Flagellar transport protein FliP / Type III secretion system inner membrane R protein / Bacterial export protein family 3 / Bacterial export proteins, family 1 / Bacterial export proteins, family 3 / Flagella transport protein fliP family signature 1. / Type III secretion system inner membrane P protein / FliP family / Flagella transport protein fliP family signature 2.
Similarity search - Domain/homology
Flagellar biosynthetic protein FliQ / Flagellar biosynthetic protein FliQ / Flagellar biosynthetic protein FliP / Flagellar biosynthetic protein FliR
Similarity search - Component
Biological speciesSalmonella enterica subsp. enterica (bacteria)
Salmonella enterica subsp. enterica serovar Typhimurium (bacteria)
Salmonella enterica (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.65 Å
AuthorsJohnson, S. / Kuhlen, L. / Abrusci, P. / Lea, S.M.
Funding support United Kingdom, 5items
OrganizationGrant numberCountry
Medical Research Council (United Kingdom)M011984 United Kingdom
Wellcome Trust100298 United Kingdom
Wellcome Trust201536 United Kingdom
Wolfson FoundationWL160052 United Kingdom
Wellcome Trust109136 United Kingdom
Citation
Journal: mBio / Year: 2019
Title: The Structure of an Injectisome Export Gate Demonstrates Conservation of Architecture in the Core Export Gate between Flagellar and Virulence Type III Secretion Systems.
Authors: Steven Johnson / Lucas Kuhlen / Justin C Deme / Patrizia Abrusci / Susan M Lea /
Abstract: Export of proteins through type III secretion systems (T3SS) is critical for motility and virulence of many major bacterial pathogens. Proteins are exported through a genetically defined export gate ...Export of proteins through type III secretion systems (T3SS) is critical for motility and virulence of many major bacterial pathogens. Proteins are exported through a genetically defined export gate complex consisting of three proteins. We have recently shown at 4.2 Å that the flagellar complex of these three putative membrane proteins (FliPQR in flagellar systems, SctRST in virulence systems) assembles into an extramembrane helical assembly that likely seeds correct assembly of the rod. Here we present the structure of an equivalent complex from the virulence system at 3.5 Å by cryo-electron microscopy. This higher-resolution structure yields a more precise description of the structure and confirms the prediction of structural conservation in this core complex. Analysis of particle heterogeneity also suggests how the SctS/FliQ subunits sequentially assemble in the complex. Although predicted on the basis of sequence conservation, the work presented here formally demonstrates that all classes of type III secretion systems, flagellar or virulence, share the same architecture at the level of the core structures. This absolute conservation of the unusual extramembrane structure of the core export gate complex now allows work to move to focusing on both mechanistic studies of type III but also on fundamental studies of how such a complex is assembled.
#1: Journal: Nat Struct Mol Biol / Year: 2018
Title: Structure of the core of the type III secretion system export apparatus.
Authors: Lucas Kuhlen / Patrizia Abrusci / Steven Johnson / Joseph Gault / Justin Deme / Joseph Caesar / Tobias Dietsche / Mehari Tesfazgi Mebrhatu / Tariq Ganief / Boris Macek / Samuel Wagner / ...Authors: Lucas Kuhlen / Patrizia Abrusci / Steven Johnson / Joseph Gault / Justin Deme / Joseph Caesar / Tobias Dietsche / Mehari Tesfazgi Mebrhatu / Tariq Ganief / Boris Macek / Samuel Wagner / Carol V Robinson / Susan M Lea /
Abstract: Export of proteins through type III secretion systems is critical for motility and virulence of many major bacterial pathogens. Three putative integral membrane proteins (FliP, FliQ, FliR) are ...Export of proteins through type III secretion systems is critical for motility and virulence of many major bacterial pathogens. Three putative integral membrane proteins (FliP, FliQ, FliR) are suggested to form the core of an export gate in the inner membrane, but their structure, assembly and location within the final nanomachine remain unclear. Here, we present the cryoelectron microscopy structure of the Salmonella Typhimurium FliP-FliQ-FliR complex at 4.2 Å. None of the subunits adopt canonical integral membrane protein topologies, and common helix-turn-helix structural elements allow them to form a helical assembly with 5:4:1 stoichiometry. Fitting of the structure into reconstructions of intact secretion systems, combined with cross-linking, localize the export gate as a core component of the periplasmic portion of the machinery. This study thereby identifies the export gate as a key element of the secretion channel and implies that it primes the helical architecture of the components assembling downstream.
History
DepositionMar 26, 2019Deposition site: PDBE / Processing site: PDBE
Revision 1.0May 29, 2019Provider: repository / Type: Initial release
Revision 1.1Jul 10, 2019Group: Data collection / Database references
Category: citation / citation_author ...citation / citation_author / em_admin / pdbx_database_proc
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID / _citation_author.name / _em_admin.last_update
Revision 1.2Oct 30, 2019Group: Data collection / Database references / Category: em_image_scans / pdbx_database_related / Item: _pdbx_database_related.content_type
Revision 1.3Nov 6, 2019Group: Data collection / Refinement description / Category: software / Item: _software.name
Revision 1.4Dec 18, 2019Group: Other / Category: atom_sites
Item: _atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] / _atom_sites.fract_transf_matrix[3][3]

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Structure visualization

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Structure viewerMolecule:
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Assembly

Deposited unit
A: Flagellar biosynthetic protein FliP
B: Flagellar biosynthetic protein FliP
C: Flagellar biosynthetic protein FliP
D: Flagellar biosynthetic protein FliP
E: Flagellar biosynthetic protein FliP
F: Flagellar biosynthetic protein FliR
G: Flagellar biosynthetic protein FliQ
H: Flagellar biosynthetic protein FliQ
I: Flagellar biosynthetic protein FliQ
J: Flagellar biosynthetic protein FliQ


Theoretical massNumber of molelcules
Total (without water)205,34110
Polymers205,34110
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by software
  • Evidence: light scattering
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area43020 Å2
ΔGint-542 kcal/mol
Surface area69010 Å2
MethodPISA

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Components

#1: Protein
Flagellar biosynthetic protein FliP


Mass: 26769.021 Da / Num. of mol.: 5
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Salmonella enterica subsp. enterica (bacteria)
Gene: fliP, LTSERUB_0568 / Production host: Escherichia coli (E. coli) / Strain (production host): MT56 / References: UniProt: G5QE81
#2: Protein Flagellar biosynthetic protein FliR


Mass: 33069.180 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Salmonella enterica subsp. enterica serovar Typhimurium (bacteria)
Strain: LT2 / SGSC1412 / ATCC 700720 / Gene: fliR, flaP, STM1981 / Production host: Escherichia coli (E. coli) / Strain (production host): MT56 / References: UniProt: P54702
#3: Protein
Flagellar biosynthetic protein FliQ


Mass: 9606.758 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Salmonella enterica (bacteria)
Gene: fliQ, fliQ_1, fliQ_2, A7D45_07890, A7S51_18675, AL463_17545, BH006_19970, BUJ17_0011480, BUJ19_005435, C4801_20645, C4860_05670, CBI64_04070, CHC34_11945, CHD23_08750, CHE19_09990, CIC26_24535, ...Gene: fliQ, fliQ_1, fliQ_2, A7D45_07890, A7S51_18675, AL463_17545, BH006_19970, BUJ17_0011480, BUJ19_005435, C4801_20645, C4860_05670, CBI64_04070, CHC34_11945, CHD23_08750, CHE19_09990, CIC26_24535, CRE05_10505, IN95_15645, NCTC10252_03215, NCTC10718_02756, NCTC6385_02264, NCTC9854_01819
Production host: Escherichia coli (E. coli) / Strain (production host): MT56 / References: UniProt: A0A0M0QTK6, UniProt: A0A0F7J7J8*PLUS

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Experimental details

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Experiment

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

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Sample preparation

ComponentName: Complex of the flagellar type III secretion system export apparatus components FliP, FliQ and FliRType three secretion system
Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 195 kDa/nm / Experimental value: YES
Source (natural)Organism: Salmonella enterica (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli) / Strain: Mt56
Buffer solutionpH: 8
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: SPOT SCAN
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 4000 nm / Nominal defocus min: 500 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recording
IDImaging-IDElectron dose (e/Å2)Detector modeFilm or detector modelNum. of real images
1147COUNTINGGATAN K2 SUMMIT (4k x 4k)401
2150COUNTINGFEI FALCON III (4k x 4k)1687

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Processing

Software
NameVersionClassificationNB
PHENIXdev_3406refinement
PHENIXdev_3406refinement
EM software
IDNameVersionCategory
1Gautomatchparticle selection
4CTFFIND4.1.8CTF correction
7PHENIXDEV_3406model fitting
9PHENIXDEV_3406model refinement
10RELION3initial Euler assignment
11RELION3final Euler assignment
13RELION33D reconstruction
Image processingDetails: Images from K2 were also added.
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.65 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 97719 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Atomic model buildingPDB-ID: 6F2D

6f2d

RefinementHighest resolution: 3.65 Å
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.005312820
ELECTRON MICROSCOPYf_angle_d0.777417476
ELECTRON MICROSCOPYf_chiral_restr0.04472189
ELECTRON MICROSCOPYf_plane_restr0.00712130
ELECTRON MICROSCOPYf_dihedral_angle_d3.49797731

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