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- EMDB-45459: Structure of in vitro assembled B. anthracis S-layer protein Sap -

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Basic information

Entry
Database: EMDB / ID: EMD-45459
TitleStructure of in vitro assembled B. anthracis S-layer protein Sap
Map dataSharpened map from RELION postprocessing lowpass filtered to the final resolution (7.2 angstroms)
Sample
  • Complex: Multimeric array of B. anthracis S-layer protein Sap
    • Protein or peptide: S-layer protein Sap
KeywordsS-layer / surface protein / Bacillus anthracis / STRUCTURAL PROTEIN
Function / homology
Function and homology information


S-layer / extracellular region
Similarity search - Function
SbsA, Ig-like domain / Bacterial Ig-like domain / S-layer homology domain / S-layer homology domain / S-layer homology (SLH) domain profile. / Copper resistance protein CopC/internalin, immunoglobulin-like / Bacterial Ig-like domain (group 2) / Invasin/intimin cell-adhesion fragments / Bacterial Ig-like domain 2 / Bacterial Ig-like domain, group 2
Similarity search - Domain/homology
Biological speciesBacillus anthracis (anthrax bacterium)
Methodsubtomogram averaging / cryo EM / Resolution: 7.2 Å
AuthorsLeigh KE / Van der Verren SE / Remaut H / Kudryashev M
Funding support Germany, Belgium, 3 items
OrganizationGrant numberCountry
Alexander von Humboldt Foundation Germany
Vrije Universiteit Brussel Belgium
Research Foundation - Flanders (FWO) Belgium
CitationJournal: Proc Natl Acad Sci U S A / Year: 2024
Title: Architecture of the Sap S-layer of revealed by integrative structural biology.
Authors: Adrià Sogues / Kendra Leigh / Ethan V Halingstad / Sander E Van der Verren / Adam J Cecil / Antonella Fioravanti / Alexander J Pak / Misha Kudryashev / Han Remaut /
Abstract: is a spore-forming gram-positive bacterium responsible for anthrax, an infectious disease with a high mortality rate and a target of concern due to bioterrorism and long-term site contamination. The ... is a spore-forming gram-positive bacterium responsible for anthrax, an infectious disease with a high mortality rate and a target of concern due to bioterrorism and long-term site contamination. The entire surface of vegetative cells in exponential or stationary growth phase is covered in proteinaceous arrays called S-layers, composed of Sap or EA1 protein, respectively. The Sap S-layer represents an important virulence factor and cell envelope support structure whose paracrystalline nature is essential for its function. However, the spatial organization of Sap in its lattice state remains elusive. Here, we employed cryoelectron tomography and subtomogram averaging to obtain a map of the Sap S-layer from tubular polymers that revealed a conformational switch between the postassembly protomers and the previously available X-ray structure of the condensed monomers. To build and validate an atomic model of the lattice within this map, we used a combination of molecular dynamics simulations, X-ray crystallography, cross-linking mass spectrometry, and biophysics in an integrative structural biology approach. The Sap lattice model produced recapitulates a close-to-physiological arrangement, reveals high-resolution details of lattice contacts, and sheds light on the mechanisms underlying the stability of the Sap layer.
History
DepositionJun 23, 2024-
Header (metadata) releaseDec 18, 2024-
Map releaseDec 18, 2024-
UpdateDec 18, 2024-
Current statusDec 18, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_45459.png

Downloads & links

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Map

FileDownload / File: emd_45459.map.gz / Format: CCP4 / Size: 27 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSharpened map from RELION postprocessing lowpass filtered to the final resolution (7.2 angstroms)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.8 Å/pix.
x 192 pix.
= 345.6 Å		1.8 Å/pix.
x 192 pix.
= 345.6 Å		1.8 Å/pix.
x 192 pix.
= 345.6 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.8 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 2.56
Minimum - Maximum-5.63574 - 7.084151
Average (Standard dev.)-0.05793471 (±0.8336815)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions192192192
Spacing192192192
CellA=B=C: 345.59998 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_45459_msk_1.map
Projections & Slices
AxesZYX

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Slices (1/2)
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Histogram

Histogram (log scale)

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Additional map: Unfiltered unmasked map - average of the two half maps

Fileemd_45459_additional_1.map
AnnotationUnfiltered unmasked map - average of the two half maps
Projections & Slices
AxesZYX

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Slices (1/2)
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Histogram

Histogram (log scale)

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Half map: Unfiltered unmasked half map 1

Fileemd_45459_half_map_1.map
AnnotationUnfiltered unmasked half map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Unfiltered unmasked half map 2

Fileemd_45459_half_map_2.map
AnnotationUnfiltered unmasked half map 2
Projections & Slices
AxesZYX

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Histogram

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Sample components

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Entire : Multimeric array of B. anthracis S-layer protein Sap

EntireName: Multimeric array of B. anthracis S-layer protein Sap
Components
  • Complex: Multimeric array of B. anthracis S-layer protein Sap
    • Protein or peptide: S-layer protein Sap

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Supramolecule #1: Multimeric array of B. anthracis S-layer protein Sap

SupramoleculeName: Multimeric array of B. anthracis S-layer protein Sap / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Bacillus anthracis (anthrax bacterium)

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Macromolecule #1: S-layer protein Sap

MacromoleculeName: S-layer protein Sap / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Bacillus anthracis (anthrax bacterium)
Recombinant expressionOrganism: Escherichia coli BL21 (bacteria)
SequenceString: MSAKAVTTQK VEVKFSKAVE KLTKEDIKVT NKANNDKVLV KEVTLSEDKK SATVELYSNL AAKQTYTVDV NKVGKTEVAV GSLEAKTIEM ADQTVVADEP TALQFTVKDE NGTEVVSPEG IEFVTPAAEK INAKGEITLA KGTSTTVKAV YKKDGKVVAE SKEVKVSAEG ...String:
MSAKAVTTQK VEVKFSKAVE KLTKEDIKVT NKANNDKVLV KEVTLSEDKK SATVELYSNL AAKQTYTVDV NKVGKTEVAV GSLEAKTIEM ADQTVVADEP TALQFTVKDE NGTEVVSPEG IEFVTPAAEK INAKGEITLA KGTSTTVKAV YKKDGKVVAE SKEVKVSAEG AAVASISNWT VAEQNKADFT SKDFKQNNKV YEGDNAYVQV ELKDQFNAVT TGKVEYESLN TEVAVVDKAT GKVTVLSAGK APVKVTVKDS KGKELVSKTV EIEAFAQKAM KEIKLEKTNV ALSTKDVTDL KVKAPVLDQY GKEFTAPVTV KVLDKDGKEL KEQKLEAKYV NKELVLNAAG QEAGNYTVVL TAKSGEKEAK ATLALELKAP GAFSKFEVRG LEKELDKYVT EENQKNAMTV SVLPVDANGL VLKGAEAAEL KVTTTNKEGK EVDATDAQVT VQNNSVITVG QGAKAGETYK VTVVLDGKLI TTHSFKVVDT APTAKGLAVE FTSTSLKEVA PNADLKAALL NILSVDGVPA TTAKATVSNV EFVSADTNVV AENGTVGAKG ATSIYVKNLT VVKDGKEQKV EFDKAVQVAV SIKEAKPATK HHHHHH

UniProtKB: S-layer protein sap

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Experimental details

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Structure determination

Methodcryo EM
Processingsubtomogram averaging
Aggregation state2D array

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Sample preparation

BufferpH: 7.4 / Details: Phosphate buffered saline
GridModel: UltrAuFoil R2/2 / Material: GOLD / Support film - Material: GOLD / Support film - topology: HOLEY
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 295.15 K / Instrument: GATAN CRYOPLUNGE 3

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF Quantum SE / Energy filter - Slit width: 20 eV
Details: 70 micron objective aperture used in addition to the energy filter
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Number real images: 1 / Average electron dose: 3.8 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 3.5 µm / Nominal defocus min: 2.0 µm / Nominal magnification: 81000
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

Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 7.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software: (Name: Dynamo (ver. 1.1.478), RELION (ver. 3.1))
Details: Final half map reconstruction was done in Dynamo and resolution estimated using RELION 3.1 postprocessing.
Number subtomograms used: 10126
ExtractionNumber tomograms: 12 / Number images used: 117502 / Software - Name: Dynamo (ver. 1.1.401)
Final angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: Dynamo (ver. 1.1.478)
FSC plot (resolution estimation)

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