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- EMDB-39765: Homomeric 35mer of the Vibrio flagellar MS-ring protein FliF with... -

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Entry
Database: EMDB / ID: EMD-39765
TitleHomomeric 35mer of the Vibrio flagellar MS-ring protein FliF without symmetry imposition
Map data
Sample
  • Complex: Homomeric 35mer of FliFG fusion protein of Vibrio alginolyticus
    • Protein or peptide: FliFG fusion protein
KeywordsComplex / Rotor / MOTOR PROTEIN
Biological speciesVibrio alginolyticus (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.76 Å
AuthorsTakekawa N / Nishikino T / Kishikawa J / Hirose M / Kato T / Imada K / Homma M
Funding support Japan, 4 items
OrganizationGrant numberCountry
Japan Society for the Promotion of Science (JSPS)21H00393 Japan
Japan Society for the Promotion of Science (JSPS)21H01772 Japan
Japan Society for the Promotion of Science (JSPS)22K18943 Japan
Japan Society for the Promotion of Science (JSPS)20H03220 Japan
CitationJournal: mBio / Year: 2024
Title: Structural analysis of S-ring composed of FliFG fusion proteins in marine polar flagellar motor.
Authors: Norihiro Takekawa / Tatsuro Nishikino / Jun-Ichi Kishikawa / Mika Hirose / Miki Kinoshita / Seiji Kojima / Tohru Minamino / Takayuki Uchihashi / Takayuki Kato / Katsumi Imada / Michio Homma /
Abstract: The marine bacterium possesses a polar flagellum driven by a sodium ion flow. The main components of the flagellar motor are the stator and rotor. The C-ring and MS-ring, which are composed of FliG ...The marine bacterium possesses a polar flagellum driven by a sodium ion flow. The main components of the flagellar motor are the stator and rotor. The C-ring and MS-ring, which are composed of FliG and FliF, respectively, are parts of the rotor. Here, we purified an MS-ring composed of FliF-FliG fusion proteins and solved the near-atomic resolution structure of the S-ring-the upper part of the MS-ring-using cryo-electron microscopy. This is the first report of an S-ring structure from , whereas, previously, only those from have been reported. The S-ring structure reveals novel features compared with that of , such as tilt angle differences of the RBM3 domain and the β-collar region, which contribute to the vertical arrangement of the upper part of the β-collar region despite the diversity in the RBM3 domain angles. Additionally, there is a decrease of the inter-subunit interaction between RBM3 domains, which influences the efficiency of the MS-ring formation in different bacterial species. Furthermore, although the inner-surface electrostatic properties of and S-rings are altered, the residues potentially interacting with other flagellar components, such as FliE and FlgB, are well structurally conserved in the S-ring. These comparisons clarified the conserved and non-conserved structural features of the MS-ring across different species.IMPORTANCEUnderstanding the structure and function of the flagellar motor in bacterial species is essential for uncovering the mechanisms underlying bacterial motility and pathogenesis. Our study revealed the structure of the S-ring, a part of its polar flagellar motor, and highlighted its unique features compared with the well-studied S-ring. The observed differences in the inter-subunit interactions and in the tilt angles between the and S-rings highlighted the species-specific variations and the mechanism for the optimization of MS-ring formation in the flagellar assembly. By concentrating on the region where the S-ring and the rod proteins interact, we uncovered conserved residues essential for the interaction. Our research contributes to the advancement of bacterial flagellar biology.
History
DepositionApr 17, 2024-
Header (metadata) releaseSep 4, 2024-
Map releaseSep 4, 2024-
UpdateMar 19, 2025-
Current statusMar 19, 2025Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_39765.png

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Map

FileDownload / File: emd_39765.map.gz / Format: CCP4 / Size: 824 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

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AxesZ (Sec.)Y (Row.)X (Col.)
1.14 Å/pix.
x 600 pix.
= 684. Å		1.14 Å/pix.
x 600 pix.
= 684. Å		1.14 Å/pix.
x 600 pix.
= 684. Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.14 Å
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Histogram (log scale)
Contour LevelBy AUTHOR: 0.246
Minimum - Maximum-0.5758347 - 1.1125981
Average (Standard dev.)-0.0005394753 (±0.039683726)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions600600600
Spacing600600600
CellA=B=C: 684.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_39765_msk_1.map
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Half map: #1

Fileemd_39765_half_map_1.map
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Half map: #2

Fileemd_39765_half_map_2.map
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Sample components

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Entire : Homomeric 35mer of FliFG fusion protein of Vibrio alginolyticus

EntireName: Homomeric 35mer of FliFG fusion protein of Vibrio alginolyticus
Components
  • Complex: Homomeric 35mer of FliFG fusion protein of Vibrio alginolyticus
    • Protein or peptide: FliFG fusion protein

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Supramolecule #1: Homomeric 35mer of FliFG fusion protein of Vibrio alginolyticus

SupramoleculeName: Homomeric 35mer of FliFG fusion protein of Vibrio alginolyticus
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Vibrio alginolyticus (bacteria) / Strain: 138-2
Molecular weightTheoretical: 104 kDa/nm

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Macromolecule #1: FliFG fusion protein

MacromoleculeName: FliFG fusion protein / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
SequenceString: MNHKVHHHHH HIEGRHMADK STDLTVTEGG SDGALVASSD VDVESQNPDL EERSASKFDM AVGDLDLLRQ VVLVLSISIC VALIVMLFFW VKEPEMRPLG AYETEELIPV LDYLDQQKIN YKLDGNTISV ESSEYNSIKL GMVRSGVNQA TEAGDDILLQ DMGFGVSQRL ...String:
MNHKVHHHHH HIEGRHMADK STDLTVTEGG SDGALVASSD VDVESQNPDL EERSASKFDM AVGDLDLLRQ VVLVLSISIC VALIVMLFFW VKEPEMRPLG AYETEELIPV LDYLDQQKIN YKLDGNTISV ESSEYNSIKL GMVRSGVNQA TEAGDDILLQ DMGFGVSQRL EQERLKLSRE RQLAQAIEEM KQVRKARVLL ALPKHSVFVR HNQEASASVF LTLSTGTNLK QQEVDSIVDM VASAVPGMKT SRITVTDQHG RLLSSGSQDP ASAARRKEQE LERSQEQALR EKIDSVLLPI LGYGNYTAQV DIQMDFSAVE QTRKRFDPNT PATRSEYALE DYNNGNMVAG IPGALSNQPP ADASIPQDVA QMKDGSVMGQ GSVRKESTRN FELDTTISHE RKQTGTVARQ TVSVAIKDRR QVNPDTGEVT YTPMSESEIN AIRQVLIGTV GFDQGRGDLL NVLSVKFAEP EAEQLEEPPI WEHPNFSDWV RWFASALVII VVVLVLVRPA MKKLLNPTSD DEDEMYGPDG LPIGADGETS LIGSDIESSE LFEFGSSIDL PNLHKDEDVL KAVRALVANE PELAAQVVKN WMNDMANDIV PQDDNAAGMP VELDVSTITG EEKAAILLLS LNEQDAAGII RHLEPKQVQR VGSAMAKAKD LSQDKVSAVH RAFLEDIQKY TNIGMGSEDF MRNALVAALG EDKANNLVDQ ILLGTGSKGL DSLKWMDPRQ VASIIVNEHP QIQTIVLSYL EADQSAEILS QFPERVRLDL MMRIANLEEV QPSALAELNE IMEKQFAGQA GAQAAKISGL KAAAEIMNYL DNNVEGLLME QIRDQDEDMA TQIQDLMFVF ENLVEVDDQG IQKLLRDVPQ DVLQKALKGA DDSLREKVFK NMSKRAAEMM RDDIEAMPPV RVADVEAAQK EILAIARRMA DAGELMLSGG ADEFL

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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
Component:
ConcentrationFormulaName
20.0 mMC4H11NO3Tris-HCl
100.0 mMKClpotassium chloride
0.0025 %(w/v)C47H88O22LMNG
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K3 BIOCONTINUUM (6k x 4k) / Number real images: 6372 / Average exposure time: 7.3 sec. / Average electron dose: 49.7 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 1.7 µm / Nominal defocus min: 0.7000000000000001 µm / Nominal magnification: 64000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 2059366
Startup modelType of model: NONE
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.76 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 184915
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
FSC plot (resolution estimation)

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