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- PDB-7amy: Nonameric cytoplasmic domain of FlhA from Vibrio parahaemolyticus -

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Basic information

Entry
Database: PDB / ID: 7amy
TitleNonameric cytoplasmic domain of FlhA from Vibrio parahaemolyticus
ComponentsFlagellar biosynthesis protein FlhA
KeywordsPROTEIN TRANSPORT / T3SS / export apparatus / secretion / protein export / motility
Function / homology
Function and homology information


bacterial-type flagellum assembly / protein secretion / membrane => GO:0016020 / plasma membrane
Similarity search - Function
Flagellar biosynthesis protein FlhA / FHIPEP, domain 1 / FHIPEP conserved site / Bacterial export FHIPEP family signature. / Type III secretion system FHIPEP / FHIPEP, domain 3 / FHIPEP, domain 4 / FHIPEP family
Similarity search - Domain/homology
Flagellar biosynthesis protein FlhA
Similarity search - Component
Biological speciesVibrio parahaemolyticus (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.75 Å
AuthorsKuhlen, L. / Johnson, S. / Lea, S.
Funding support United Kingdom, 5items
OrganizationGrant numberCountry
Wellcome Trust109136 United Kingdom
Wolfson FoundationWL160052 United Kingdom
Wellcome Trust100298 United Kingdom
Wellcome Trust219477 United Kingdom
Medical Research Council (MRC, United Kingdom)S021264 United Kingdom
CitationJournal: PLoS One / Year: 2021
Title: Nonameric structures of the cytoplasmic domain of FlhA and SctV in the context of the full-length protein.
Authors: Lucas Kuhlen / Steven Johnson / Jerry Cao / Justin C Deme / Susan M Lea /
Abstract: Type three secretion is the mechanism of protein secretion found in bacterial flagella and injectisomes. At its centre is the export apparatus (EA), a complex of five membrane proteins through which ...Type three secretion is the mechanism of protein secretion found in bacterial flagella and injectisomes. At its centre is the export apparatus (EA), a complex of five membrane proteins through which secretion substrates pass the inner membrane. While the complex formed by four of the EA proteins has been well characterised structurally, little is known about the structure of the membrane domain of the largest subunit, FlhA in flagella, SctV in injectisomes. Furthermore, the biologically relevant nonameric assembly of FlhA/SctV has been infrequently observed and differences in conformation of the cytoplasmic portion of FlhA/SctV between open and closed states have been suggested to reflect secretion system specific differences. FlhA has been shown to bind to chaperone-substrate complexes in an open state, but in previous assembled ring structures, SctV is in a closed state. Here, we identify FlhA and SctV homologues that can be recombinantly produced in the oligomeric state and study them using cryo-electron microscopy. The structures of the cytoplasmic domains from both FlhA and SctV are in the open state and we observe a conserved interaction between a short stretch of residues at the N-terminus of the cytoplasmic domain, known as FlhAL/SctVL, with a groove on the adjacent protomer's cytoplasmic domain, which stabilises the nonameric ring assembly.
History
DepositionOct 9, 2020Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jun 2, 2021Provider: repository / Type: Initial release
Revision 1.1Jul 7, 2021Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year

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Structure visualization

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Assembly

Deposited unit
A: Flagellar biosynthesis protein FlhA
B: Flagellar biosynthesis protein FlhA
C: Flagellar biosynthesis protein FlhA
D: Flagellar biosynthesis protein FlhA
E: Flagellar biosynthesis protein FlhA
F: Flagellar biosynthesis protein FlhA
G: Flagellar biosynthesis protein FlhA
H: Flagellar biosynthesis protein FlhA
I: Flagellar biosynthesis protein FlhA


Theoretical massNumber of molelcules
Total (without water)689,0029
Polymers689,0029
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area23770 Å2
ΔGint-76 kcal/mol
Surface area146240 Å2
MethodPISA

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Components

#1: Protein
Flagellar biosynthesis protein FlhA


Mass: 76555.820 Da / Num. of mol.: 9
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Vibrio parahaemolyticus (bacteria)
Gene: flhA, C1S91_05000, C9I78_24000, CGI34_13080, D5E78_16030, E4P16_05430, F0L99_07770, WR32_16185
Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: A0A0F5SXE4

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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: Nonamer of the FlhA cytoplasmic domain from Vibrio parahaemolyticus
Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.7 MDa / Experimental value: NO
Source (natural)Organism: Vibrio parahaemolyticus (bacteria)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 8
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 %

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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: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy
Image recordingElectron dose: 48 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.18rc1_3769: / Classification: refinement
EM software
IDNameCategory
12RELIONclassification
13RELION3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C9 (9 fold cyclic)
3D reconstructionResolution: 3.75 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 9756 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0125254
ELECTRON MICROSCOPYf_angle_d0.84934371
ELECTRON MICROSCOPYf_dihedral_angle_d23.4473402
ELECTRON MICROSCOPYf_chiral_restr0.0514086
ELECTRON MICROSCOPYf_plane_restr0.0054509

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