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- EMDB-20504: 10 Angstrom structure of the asymmetric flagellar filament purifi... -

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Entry
Database: EMDB / ID: EMD-20504
Title10 Angstrom structure of the asymmetric flagellar filament purified from Leptospira biflexa Patoc WT cells resolved via subtomogram averaging
Map dataMasked full EM map resulting from subtomogram averaging in emClarity of flagellar filaments purified from wildtype Leptospira biflexa serovar Patoc strain Patoc 1.
Sample
  • Organelle or cellular component: Flagellar filament purified from the periplasm of the Spirochete bacterium, Leptospira biflexa
    • Protein or peptide: Flagellin B1 (FlaB1)
    • Protein or peptide: Flagellar coiling protein A (FcpA)
    • Protein or peptide: Flagellar coiling protein B (FcpB)
Keywordsbacterial flagella / FcpA / FcpB / FlaA / FlaB / Leptospira / STRUCTURAL PROTEIN
Function / homology
Function and homology information


periplasmic flagellum / bacterial-type flagellum filament / bacterial-type flagellum-dependent cell motility / structural molecule activity
Similarity search - Function
Flagellin, C-terminal domain, subdomain 2 / Bacterial flagellin C-terminal helical region / Flagellin / Flagellin, N-terminal domain / Bacterial flagellin N-terminal helical region
Similarity search - Domain/homology
Uncharacterized protein / Flagellin / Uncharacterized protein
Similarity search - Component
Biological speciesLeptospira biflexa serovar Patoc strain 'Patoc 1 (Paris)' (bacteria) / Leptospira biflexa serovar Patoc (strain Patoc 1 / ATCC 23582 / Paris) (bacteria)
Methodsubtomogram averaging / cryo EM / Resolution: 9.83 Å
AuthorsGibson KH / Sindelar CV
Funding support United States, Uruguay, 10 items
OrganizationGrant numberCountry
National Institutes of Health/National Library of Medicine (NIH/NLM)R01 GM 110530 United States
National Institutes of Health/National Library of Medicine (NIH/NLM)U01 AI088752 United States
National Institutes of Health/National Library of Medicine (NIH/NLM)R01 TW009504 United States
National Institutes of Health/National Library of Medicine (NIH/NLM)R01 AI052473 United States
National Institutes of Health/National Library of Medicine (NIH/NLM)R01 298 AI121207 United States
Agencia Nacional de Investigacion e Innovacion (ANII)FCE_3_2016_1_126797Uruguay
Agencia Nacional de Investigacion e Innovacion (ANII)ALI_1_2014_1_4982Uruguay
Agencia Nacional de Investigacion e Innovacion (ANII)FCE_3_2016_1_126797Uruguay
French National Research AgencyANR-18-CE15-0027-1Uruguay
French National Research AgencyANR-08-300 MIE-018Uruguay
CitationJournal: Front Cell Infect Microbiol / Year: 2018
Title: FcpB Is a Surface Filament Protein of the Endoflagellum Required for the Motility of the Spirochete .
Authors: Elsio A Wunder / Leyla Slamti / David N Suwondo / Kimberley H Gibson / Zhiguo Shang / Charles V Sindelar / Felipe Trajtenberg / Alejandro Buschiazzo / Albert I Ko / Mathieu Picardeau /
Abstract: The spirochete endoflagellum is a unique motility apparatus among bacteria. Despite its critical importance for pathogenesis, the full composition of the flagellum remains to be determined. We have ...The spirochete endoflagellum is a unique motility apparatus among bacteria. Despite its critical importance for pathogenesis, the full composition of the flagellum remains to be determined. We have recently reported that FcpA is a novel flagellar protein and a major component of the sheath of the filament of the spirochete . By screening a library of random transposon mutants in the spirochete , we found a motility-deficient mutant harboring a disruption in a hypothetical gene of unknown function. Here, we show that this gene encodes a surface component of the endoflagellar filament and is required for typical hook- and spiral-shaped ends of the cell body, coiled structure of the endoflagella, and high velocity phenotype. We therefore named the gene for flagellar-coiling protein B. is conserved in all members of the genus, but not present in other organisms including other spirochetes. Complementation of the mutant restored the wild-type morphology and motility phenotypes. Immunoblotting with anti-FcpA and anti-FcpB antisera and cryo-electron microscopy of the filament indicated that FcpB assembled onto the surface of the sheath of the filament and mostly located on the outer (convex) side of the coiled filament. We provide evidence that FcpB, together with FcpA, are -specific novel components of the sheath of the filament, key determinants of the coiled and asymmetric structure of the endoflagella and are essential for high velocity. Defining the components of the endoflagella and their functions in these atypical bacteria should greatly enhance our understanding of the mechanisms by which these bacteria produce motility.
History
DepositionJul 22, 2019-
Header (metadata) releaseDec 25, 2019-
Map releaseMar 25, 2020-
UpdateMar 20, 2024-
Current statusMar 20, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 3.15
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 3.15
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6pwb
  • Surface level: 3.15
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_20504.png

Downloads & links

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Map

FileDownload / File: emd_20504.map.gz / Format: CCP4 / Size: 27 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationMasked full EM map resulting from subtomogram averaging in emClarity of flagellar filaments purified from wildtype Leptospira biflexa serovar Patoc strain Patoc 1.
Voxel sizeX=Y=Z: 2.604 Å
Density
Contour LevelBy AUTHOR: 3.15 / Movie #1: 3.15
Minimum - Maximum-3.8043377 - 10.010209
Average (Standard dev.)0.15763956 (±0.89477086)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-96-96-96
Dimensions192192192
Spacing192192192
CellA=B=C: 499.96802 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.6042.6042.604
M x/y/z192192192
origin x/y/z0.0000.0000.000
length x/y/z499.968499.968499.968
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS-96-96-96
NC/NR/NS192192192
D min/max/mean-3.80410.0100.158

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Supplemental data

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Additional map: Masked half map 1 resulting from subtomogram averaging...

Fileemd_20504_additional_1.map
AnnotationMasked half map 1 resulting from subtomogram averaging in emClarity of flagellar filaments purified from wildtype Leptospira biflexa serovar Patoc strain Patoc 1.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Unmasked full EM map resulting from subtomogram averaging...

Fileemd_20504_additional_2.map
AnnotationUnmasked full EM map resulting from subtomogram averaging in emClarity of flagellar filaments purified from wildtype Leptospira biflexa serovar Patoc strain Patoc 1.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Masked half map 2 resulting from subtomogram averaging...

Fileemd_20504_additional_3.map
AnnotationMasked half map 2 resulting from subtomogram averaging in emClarity of flagellar filaments purified from wildtype Leptospira biflexa serovar Patoc strain Patoc 1.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Symmetrised map generated by rotational symmetrization of the...

Fileemd_20504_additional_4.map
AnnotationSymmetrised map generated by rotational symmetrization of the L. biflexa Patoc WT filament core resulting from subtomogram averaging. Map used to calculate helical parameters for FlaB core model.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Unmasked-half map 1 resulting from subtomogram averaging in...

Fileemd_20504_half_map_1.map
AnnotationUnmasked-half map 1 resulting from subtomogram averaging in emClarity of flagellar filaments purified from wildtype Leptospira biflexa serovar Patoc strain Patoc 1.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Unmasked-half map 2 resulting from subtomogram averaging in...

Fileemd_20504_half_map_2.map
AnnotationUnmasked-half map 2 resulting from subtomogram averaging in emClarity of flagellar filaments purified from wildtype Leptospira biflexa serovar Patoc strain Patoc 1.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Flagellar filament purified from the periplasm of the Spirochete ...

EntireName: Flagellar filament purified from the periplasm of the Spirochete bacterium, Leptospira biflexa
Components
  • Organelle or cellular component: Flagellar filament purified from the periplasm of the Spirochete bacterium, Leptospira biflexa
    • Protein or peptide: Flagellin B1 (FlaB1)
    • Protein or peptide: Flagellar coiling protein A (FcpA)
    • Protein or peptide: Flagellar coiling protein B (FcpB)

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Supramolecule #1: Flagellar filament purified from the periplasm of the Spirochete ...

SupramoleculeName: Flagellar filament purified from the periplasm of the Spirochete bacterium, Leptospira biflexa
type: organelle_or_cellular_component / ID: 1 / Parent: 0 / Macromolecule list: all
Details: Filaments were purified from Leptospira biflexa wild type cells.
Source (natural)Organism: Leptospira biflexa serovar Patoc strain 'Patoc 1 (Paris)' (bacteria)
Organelle: flagellar filament / Location in cell: periplasm

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Macromolecule #1: Flagellin B1 (FlaB1)

MacromoleculeName: Flagellin B1 (FlaB1) / type: protein_or_peptide / ID: 1 / Number of copies: 84 / Enantiomer: LEVO
Source (natural)Organism: Leptospira biflexa serovar Patoc (strain Patoc 1 / ATCC 23582 / Paris) (bacteria)
Strain: Patoc 1 / ATCC 23582 / Paris / Cell: bacteria
Molecular weightTheoretical: 29.467033 KDa
SequenceString: AAINSHRVLK FQNEEVSKNM EKLSSGMRIN RAGDDASGLA VSEKMRTQVN GLRQAERNTE DGMSLIQTTE GFLQESNDII QRIRTLAIQ SSNGIYTEED RQMIQVEVSQ LIDEVDRIAS QAEFNKMNLL QGDFARGSRA TSMWFHIGPN MHQRERVFIA T MTARSLNL ...String:
AAINSHRVLK FQNEEVSKNM EKLSSGMRIN RAGDDASGLA VSEKMRTQVN GLRQAERNTE DGMSLIQTTE GFLQESNDII QRIRTLAIQ SSNGIYTEED RQMIQVEVSQ LIDEVDRIAS QAEFNKMNLL QGDFARGSRA TSMWFHIGPN MHQRERVFIA T MTARSLNL KGQSGELLSL STADKSNDAI GTLDAALTRI SKQRANLGAY FNRLEHAAKG LMNAYENTQA SESRIRDADM AE ETVAFTK NQILVQSGTA MLAQANVR

UniProtKB: Flagellin

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Macromolecule #2: Flagellar coiling protein A (FcpA)

MacromoleculeName: Flagellar coiling protein A (FcpA) / type: protein_or_peptide / ID: 2 / Number of copies: 47 / Enantiomer: LEVO
Source (natural)Organism: Leptospira biflexa serovar Patoc (strain Patoc 1 / ATCC 23582 / Paris) (bacteria)
Strain: Patoc 1 / ATCC 23582 / Paris / Cell: bacteria
Molecular weightTheoretical: 28.931002 KDa
SequenceString: LTEDQKKKKK EIMEQESLWK NPDFKGYNKT FQELHQLSKT FANNQFRLAL SNYQSGVNTI MKNRDWVEQY RKEEAEKKRL DEKWYWQKV DRKAREERVV YREKMKAKQD ALNYFSKAIN HLDEIKNPDL RERPEFKRLL SDVYRSWIMA EYDLQNLPQT I PILELYIE ...String:
LTEDQKKKKK EIMEQESLWK NPDFKGYNKT FQELHQLSKT FANNQFRLAL SNYQSGVNTI MKNRDWVEQY RKEEAEKKRL DEKWYWQKV DRKAREERVV YREKMKAKQD ALNYFSKAIN HLDEIKNPDL RERPEFKRLL SDVYRSWIMA EYDLQNLPQT I PILELYIE IDDNEKEYPA HKYLASAYSF EENMIKKTKG PDDMLFKYRY KKNVHLLRAT ELKYGKDSPE YKHIVNVIN

UniProtKB: Uncharacterized protein

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Macromolecule #3: Flagellar coiling protein B (FcpB)

MacromoleculeName: Flagellar coiling protein B (FcpB) / type: protein_or_peptide / ID: 3 / Number of copies: 32 / Enantiomer: LEVO
Source (natural)Organism: Leptospira biflexa serovar Patoc (strain Patoc 1 / ATCC 23582 / Paris) (bacteria)
Strain: Patoc 1 / ATCC 23582 / Paris / Cell: bacteria
Molecular weightTheoretical: 25.539666 KDa
SequenceString: SGKSMADTEK ELDDNISEVN KRLRLHTVLF KMKVRTLPHK TVLYKGKPSA DGERCEAADK QEAQDNTCLH LEVFDFVGSE DGKSSKNLG AKFKKMELFF EGSNNADPDP RKEQPRNLTK IRTYIYQNNF LLEDKVISVI ADVAPNGEPA HNDKIELFYQ H DDYPVWGT ...String:
SGKSMADTEK ELDDNISEVN KRLRLHTVLF KMKVRTLPHK TVLYKGKPSA DGERCEAADK QEAQDNTCLH LEVFDFVGSE DGKSSKNLG AKFKKMELFF EGSNNADPDP RKEQPRNLTK IRTYIYQNNF LLEDKVISVI ADVAPNGEPA HNDKIELFYQ H DDYPVWGT PETPSEKGVG KYILSNVENT KSNPIRNNFK KQFYFKNLDY FDKLFTKIFD YND

UniProtKB: Uncharacterized protein

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Experimental details

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Structure determination

Methodcryo EM
Processingsubtomogram averaging
Aggregation statefilament

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Sample preparation

Concentration0.5 mg/mL
BufferpH: 6.8 / Details: Tris-HCl or ddH20, pH6.8 with <1% sodium azide as a preservative. FIDUCIAL MARKERS: Prior to vitrification, 1 uL of 6x concentrated Gold Tracer beads (10 nm colloidal gold) were mixed with 2 uL of purified flagella. To each grid, 3 uL of this mixture were applied.
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Support film - Film thickness: 600 / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Time: 30 sec. / Pretreatment - Atmosphere: OTHER
Details: Model 950 Solarus Advanced Plasma System manufactured by GATAN.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 291 K / Instrument: FEI VITROBOT MARK III
Details: 2 minute incubation time; blot time of 6-7.5 seconds; and blot offset of -2 mm.
DetailsLeptospira biflexa serovar Patoc strain Patoc I wild type, fcpA, and fcpB mutant cells cultured in Ellinghausen-McCullough-Johnson-Harris liquid medium until they reached logarithmic phase at 30C. The cells were pelleted and the periplasmic flagellar filaments were purified as described in Wunder et al., 2016; Wunder et al., 2018.

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Electron microscopy

MicroscopeFEI POLARA 300
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 19230 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.0 mm / Nominal defocus max: 5.0 µm / Nominal defocus min: 3.0 µm / Nominal magnification: 15000
Sample stageSpecimen holder model: SIDE ENTRY, EUCENTRIC / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 3710 pixel / Digitization - Dimensions - Height: 3838 pixel / Digitization - Frames/image: 1-12 / Number grids imaged: 1 / Average exposure time: 1.2 sec. / Average electron dose: 1.7 e/Å2
Details: Tilt series acquisition. A total of ~35 tilt angles per tilt stack were acquired. The total dose was ~ 60 e/Angstrom2.
Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company

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Image processing

ExtractionNumber tomograms: 62 / Number images used: 15000 / Reference model: de novo / Method: Manual selection
Software:
Namedetails
PEET (ver. 1.11)addModPts
RELION (ver. 2.1)preprocessing.py MODIFIED

Details: Using IMOD 3dmod, filament trajectories were traced by selecting particle points along a single filament. Each continuous filament was a single contour (3dmod). Using the addModPts program ...Details: Using IMOD 3dmod, filament trajectories were traced by selecting particle points along a single filament. Each continuous filament was a single contour (3dmod). Using the addModPts program in PEET, particle gaps along each filament trajectory contour were filled in with additional points according to a repeat spacing of 52 angstroms. The x,y,z coordinates were then imported into RELION using a modified version of the RELION preprocessing.py python script described in Bharat et. al. (2015). The script was modified to ensure that particles in one filament would be sorted into the same ODD or EVEN grouping to prevent over-estimation of FSC due to inclusion of the 2 or more overlapping particles in a filament in both half-datasets.
Final angle assignmentType: MAXIMUM LIKELIHOOD
Software:
Namedetails
RELION (ver. 2.1.b1-gcccuda-2016.10-cc37)3d-autorefinement
emClarity (ver. 1.0)averaging/alignment

Details: RELION 3d-autorefinment was used to refine angles. In-House particle (x, y, z, and euler angle) coordinate smoothing scripts. emClarity averaging and alignment
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 9.83 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: emClarity (ver. 1.1)
Details: emClarity: The two half-dataset volumes were combined with a B-170 factor of 0, and the Gold standard FSC at 0.143 was calculated to be 9.83 A with anisotropic resolutions ranging from 8.89-16.17 Angstrom.
Number subtomograms used: 10581
DetailsMotion correction was performed on movie stacks acquired at each angle in each tilt series using IMOD alignframes. Tilt series were aligned in IMOD eTomo usinf 10 nm fiducial gold markers. CTF estimation with phase flipping was carried out, along with gold bead subtraction in IMOD eTomo. Tilt series were reconstructed using Weighted Back Projection in Tomo3D.
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model
PDB IDChain

5wjt

source_name: PDB, initial_model_type: experimental model

6nqw

source_name: PDB, initial_model_type: experimental model

6nqz

source_name: PDB, initial_model_type: experimental model
DetailsEliminate minor clashes between FlaB1, FcpA, and FcpB
RefinementSpace: RECIPROCAL / Protocol: RIGID BODY FIT / Target criteria: Correlation coefficient
Output model

PDB-6pwb:
Rigid body fitting of flagellin FlaB, and flagellar coiling proteins, FcpA and FcpB, into a 10 Angstrom structure of the asymmetric flagellar filament purified from Leptospira biflexa Patoc WT cells resolved via subtomogram averaging

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