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- EMDB-11827: Nonameric cytoplasmic domain of FlhA from Vibrio parahaemolyticus -

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Basic information

Entry
Database: EMDB / ID: EMD-11827
TitleNonameric cytoplasmic domain of FlhA from Vibrio parahaemolyticus
Map datapostprocessed map
Sample
  • Complex: Nonamer of the FlhA cytoplasmic domain from Vibrio parahaemolyticus
    • Protein or peptide: Flagellar biosynthesis protein FlhA
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)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.75 Å
AuthorsKuhlen L / Johnson S / Lea S
Funding support United Kingdom, 5 items
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, 2020-
Header (metadata) releaseJun 2, 2021-
Map releaseJun 2, 2021-
UpdateJul 7, 2021-
Current statusJul 7, 2021Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.01
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.01
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  • Surface view with fitted model
  • Atomic models: PDB-7amy
  • Surface level: 0.01
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-7amy
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
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Supplemental images

emd_11827.png

Downloads & links

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Map

FileDownload / File: emd_11827.map.gz / Format: CCP4 / Size: 290.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationpostprocessed map
Voxel sizeX=Y=Z: 0.822 Å
Density
Contour LevelBy AUTHOR: 0.0146 / Movie #1: 0.01
Minimum - Maximum-0.022297744 - 0.047841974
Average (Standard dev.)9.652649e-06 (±0.0016556889)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions424424424
Spacing424424424
CellA=B=C: 348.528 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.8220.8220.822
M x/y/z424424424
origin x/y/z0.0000.0000.000
length x/y/z348.528348.528348.528
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ400400400
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS424424424
D min/max/mean-0.0220.0480.000

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Supplemental data

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Mask #1

Fileemd_11827_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half map 1

Fileemd_11827_half_map_1.map
Annotationhalf map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half map 2

Fileemd_11827_half_map_2.map
Annotationhalf map 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Nonamer of the FlhA cytoplasmic domain from Vibrio parahaemolyticus

EntireName: Nonamer of the FlhA cytoplasmic domain from Vibrio parahaemolyticus
Components
  • Complex: Nonamer of the FlhA cytoplasmic domain from Vibrio parahaemolyticus
    • Protein or peptide: Flagellar biosynthesis protein FlhA

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Supramolecule #1: Nonamer of the FlhA cytoplasmic domain from Vibrio parahaemolyticus

SupramoleculeName: Nonamer of the FlhA cytoplasmic domain from Vibrio parahaemolyticus
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Vibrio parahaemolyticus (bacteria)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
Molecular weightTheoretical: 700 KDa

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Macromolecule #1: Flagellar biosynthesis protein FlhA

MacromoleculeName: Flagellar biosynthesis protein FlhA / type: protein_or_peptide / ID: 1 / Number of copies: 9 / Enantiomer: LEVO
Source (natural)Organism: Vibrio parahaemolyticus (bacteria)
Molecular weightTheoretical: 76.55582 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MLTRLKQLQT STKGYIGIPI VLLMILAMVI LPLPPLLLDA LFTFNIVLAI LVLLVSTTAK RPLDFSVFPT ILLVATLLRL TLNVASTRI VLLEGHNGGD AAGKVIQAFG EVVIGGNYVV GMVVFIILMI INFVVITKGG ERISEVSARF TLDALPGKQM A IDADLNAG ...String:
MLTRLKQLQT STKGYIGIPI VLLMILAMVI LPLPPLLLDA LFTFNIVLAI LVLLVSTTAK RPLDFSVFPT ILLVATLLRL TLNVASTRI VLLEGHNGGD AAGKVIQAFG EVVIGGNYVV GMVVFIILMI INFVVITKGG ERISEVSARF TLDALPGKQM A IDADLNAG LIDQETARLR RKEVANEADF HGSMDGASKF VRGDAVAGLL ILFINIIGGI SIGVFEHGLP ASEAFKTYAL LT IGDGLVA QIPSLLLATA AAIIVTRIND SDNGMSETMQ KQLLATPATL FTVAGIMAVI GMVPGMPHLA FFAFAGALGF AGW RQSKKP VQDTQIEQVE ALSQAMQEED TPLTWDDIPH VHTLSLALGY RLVHLVNKDQ GAPLSQRIRG VRRNLSEQVG FLLP EVRIR DNLSLKPNQY TISLNGEVIE QGFIEPERLM AIAVGDTYGE IDGILGSDPA YQLPAVWIEH QDKAKALNMG YQVVD DGTV IATHISKIMK TNLAELFTHD DVEAMTQRLT QQAPKLAEAL AAALNPAQQL KVYRQLLLDQ VPLKDIRTIA NTMLES SEN TKDPILLAAD VRCALKRTLV NLIAGQKPEL NVYALSDELE QMLLTSLQQA QASGTVVLDS FPIEPNILGQ FQQNLPL IR QQLKQQGLPP ILLVMPQLRP LLARYARTFT QGLAVLSYNE IPENKQINVV GNLGENLYFQ

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 8
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Average electron dose: 48.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Initial angle assignmentType: NOT APPLICABLE
Final 3D classificationSoftware - Name: RELION
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionApplied symmetry - Point group: C9 (9 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 3.75 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Number images used: 9756
FSC plot (resolution estimation)

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