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- EMDB-11591: Bacillus endospore appendages form a novel family of disulfide-li... -

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Basic information

Entry
Database: EMDB / ID: EMD-11591
TitleBacillus endospore appendages form a novel family of disulfide-linked pili
Map data
Sample
  • Organelle or cellular component: Ena1B
    • Protein or peptide: DUF3992 domain-containing protein
Function / homologyEndospore appendages core / Endospore appendages / DUF3992 domain-containing protein
Function and homology information
Biological speciesBacillus cereus (bacteria)
Methodhelical reconstruction / cryo EM / Resolution: 3.0 Å
AuthorsPradhan B / Liedtke J / Sleutel M / Lindback T / Llarena AK / Brynildsrud O / Aspholm M / Remaut H
Funding support Belgium, 1 items
OrganizationGrant numberCountry
Research Foundation - Flanders (FWO)G0G0818N Belgium
Citation
Journal: EMBO J / Year: 2021
Title: Endospore Appendages: a novel pilus superfamily from the endospores of pathogenic Bacilli.
Authors: Brajabandhu Pradhan / Janine Liedtke / Mike Sleutel / Toril Lindbäck / Ephrem Debebe Zegeye / Kristin O Sullivan / Ann-Katrin Llarena / Ola Brynildsrud / Marina Aspholm / Han Remaut /
Abstract: Bacillus cereus sensu lato is a group of Gram-positive endospore-forming bacteria with high ecological diversity. Their endospores are decorated with micrometer-long appendages of unknown identity ...Bacillus cereus sensu lato is a group of Gram-positive endospore-forming bacteria with high ecological diversity. Their endospores are decorated with micrometer-long appendages of unknown identity and function. Here, we isolate endospore appendages (Enas) from the food poisoning outbreak strain B. cereus NVH 0075-95 and find proteinaceous fibers of two main morphologies: S- and L-Ena. By using cryoEM and 3D helical reconstruction of S-Enas, we show these to represent a novel class of Gram-positive pili. S-Enas consist of single domain subunits with jellyroll topology that are laterally stacked by β-sheet augmentation. S-Enas are longitudinally stabilized by disulfide bonding through N-terminal connector peptides that bridge the helical turns. Together, this results in flexible pili that are highly resistant to heat, drought, and chemical damage. Phylogenomic analysis reveals a ubiquitous presence of the ena-gene cluster in the B. cereus group, which include species of clinical, environmental, and food importance. We propose Enas to represent a new class of pili specifically adapted to the harsh conditions encountered by bacterial spores.
#1: Journal: Biorxiv / Year: 2020
Title: Bacillus endospore appendages form a novel family of disulfide-linked pili
Authors: Pradhan B / Liedtke J / Sleutel M / Lindback T / Llarena AK / Brynildsrud O / Aspholm M / Remaut H
History
DepositionAug 6, 2020-
Header (metadata) releaseApr 28, 2021-
Map releaseApr 28, 2021-
UpdateSep 15, 2021-
Current statusSep 15, 2021Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 1.5
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 1.5
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7a02
  • Surface level: 1.5
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-7a02
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_11591.png

Downloads & links

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Map

FileDownload / File: emd_11591.map.gz / Format: CCP4 / Size: 14.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.784 Å
Density
Contour LevelBy AUTHOR: 0.65 / Movie #1: 1.5
Minimum - Maximum-5.84897 - 8.823653
Average (Standard dev.)9.952629e-12 (±1.0)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderZYX
Origin746674
Dimensions152167152
Spacing152152167
CellA: 119.168 Å / B: 119.168 Å / C: 130.928 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.7840.7840.784
M x/y/z152152167
origin x/y/z0.0000.0000.000
length x/y/z119.168119.168130.928
α/β/γ90.00090.00090.000
start NX/NY/NZ747466
NX/NY/NZ152152167
MAP C/R/S321
start NC/NR/NS667474
NC/NR/NS167152152
D min/max/mean-5.8498.8240.000

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Supplemental data

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Sample components

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Entire : Ena1B

EntireName: Ena1B
Components
  • Organelle or cellular component: Ena1B
    • Protein or peptide: DUF3992 domain-containing protein

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Supramolecule #1: Ena1B

SupramoleculeName: Ena1B / type: organelle_or_cellular_component / ID: 1 / Parent: 0 / Macromolecule list: all
Details: In vitro assembled Bacillus endospore appendage comprising the subunit Ena1B
Source (natural)Organism: Bacillus cereus (bacteria) / Strain: NVH 0075-95 / Organelle: endospore appendage
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant plasmid: pET28a

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Macromolecule #1: DUF3992 domain-containing protein

MacromoleculeName: DUF3992 domain-containing protein / type: protein_or_peptide / ID: 1 / Number of copies: 23 / Enantiomer: LEVO
Source (natural)Organism: Bacillus cereus (bacteria)
Molecular weightTheoretical: 12.162675 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MGNCSTNLSC CANGQKTIVQ DKVCIDWTAA ATAAIIYADN ISQDIYASGY LKVDTGTGPV TIVFYSGGVT GTAVETIVVA TGSSASFTV RRFDTVTILG TAAAETGEFC MTIRYTLS

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Experimental details

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Structure determination

Methodcryo EM
Processinghelical reconstruction
Aggregation statehelical array

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Sample preparation

BufferpH: 7 / Component - Formula: H2O
GridModel: Quantifoil R2/1 / Material: COPPER / Support film - Material: GRAPHENE OXIDE / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: OTHER
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 298.15 K / Instrument: GATAN CRYOPLUNGE 3

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Electron microscopy

MicroscopeJEOL CRYO ARM 300
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 1 / Number real images: 3000 / Average electron dose: 64.66 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated defocus max: 3.5 µm / Calibrated defocus min: 0.5 µm / Calibrated magnification: 60000 / Illumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELD
Sample stageSpecimen holder model: JEOL 3200FSC CRYOHOLDER

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Image processing

Final reconstructionApplied symmetry - Helical parameters - Δz: 32.3504 Å
Applied symmetry - Helical parameters - Δ&Phi: 3.43721 °
Applied symmetry - Helical parameters - Axial symmetry: C1 (asymmetric)
Resolution.type: BY AUTHOR / Resolution: 3.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0) / Number images used: 65466
CTF correctionSoftware - Name: CTFFIND (ver. 4.2)
Segment selectionNumber selected: 100495 / Software - Name: RELION (ver. 3.0)
Final angle assignmentType: NOT APPLICABLE
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: AB INITIO MODEL / Overall B value: 27.4
Output model

PDB-7a02:
Bacillus endospore appendages form a novel family of disulfide-linked pili

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