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- EMDB-13957: Extended H/L (SLPH/SLPL) complex from C. difficile (CD630 strain)... -

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Basic information

Entry
Database: EMDB / ID: EMD-13957
TitleExtended H/L (SLPH/SLPL) complex from C. difficile (CD630 strain) fit into R20291 S-layer negative stain map
Map dataElectron crystallographic reconstruction of R20291 S-layer, extended to cover 12 molecules of SlpA for flexible fitting of X-ray structure to map.
Sample
  • Complex: Electron crystallographic reconstruction of R20291 S-layer, extended to cover 12 molecules of SlpA for flexible fitting of X-ray structure to map
    • Protein or peptide: Precursor of the S-layer proteins
    • Protein or peptide: Precursor of the S-layer proteins
Function / homologyLow molecular weight S layer protein, N-terminal / Low molecular weight S layer protein, N-terminal, subdomain / Low molecular weight S layer protein N terminal / Putative cell wall binding repeat 2 / Cell wall binding domain 2 (CWB2) / outer membrane-bounded periplasmic space / identical protein binding / Precursor of the S-layer proteins
Function and homology information
Biological speciesClostridioides difficile R20291 (bacteria) / Clostridioides difficile 630 (bacteria)
Methodelectron crystallography / negative staining
AuthorsBanerji O / Wilson JS / Bullough PA
Funding support United Kingdom, 1 items
OrganizationGrant numberCountry
Wellcome Trust204877/Z/16/Z United Kingdom
CitationJournal: Nat Commun / Year: 2022
Title: Structure and assembly of the S-layer in C. difficile.
Authors: Paola Lanzoni-Mangutchi / Oishik Banerji / Jason Wilson / Anna Barwinska-Sendra / Joseph A Kirk / Filipa Vaz / Shauna O'Beirne / Arnaud Baslé / Kamel El Omari / Armin Wagner / Neil F ...Authors: Paola Lanzoni-Mangutchi / Oishik Banerji / Jason Wilson / Anna Barwinska-Sendra / Joseph A Kirk / Filipa Vaz / Shauna O'Beirne / Arnaud Baslé / Kamel El Omari / Armin Wagner / Neil F Fairweather / Gillian R Douce / Per A Bullough / Robert P Fagan / Paula S Salgado /
Abstract: Many bacteria and archaea possess a two-dimensional protein array, or S-layer, that covers the cell surface and plays crucial roles in cell physiology. Here, we report the crystal structure of SlpA, ...Many bacteria and archaea possess a two-dimensional protein array, or S-layer, that covers the cell surface and plays crucial roles in cell physiology. Here, we report the crystal structure of SlpA, the main S-layer protein of the bacterial pathogen Clostridioides difficile, and use electron microscopy to study S-layer organisation and assembly. The SlpA crystal lattice mimics S-layer assembly in the cell, through tiling of triangular prisms above the cell wall, interlocked by distinct ridges facing the environment. Strikingly, the array is very compact, with pores of only ~10 Å in diameter, compared to other S-layers (30-100 Å). The surface-exposed flexible ridges are partially dispensable for overall structure and assembly, although a mutant lacking this region becomes susceptible to lysozyme, an important molecule in host defence. Thus, our work gives insights into S-layer organisation and provides a basis for development of C. difficile-specific therapeutics.
History
DepositionDec 9, 2021-
Header (metadata) releaseApr 6, 2022-
Map releaseApr 6, 2022-
UpdateApr 6, 2022-
Current statusApr 6, 2022Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_13957.png

Downloads & links

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Map

FileDownload / File: emd_13957.map.gz / Format: CCP4 / Size: 70.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationElectron crystallographic reconstruction of R20291 S-layer, extended to cover 12 molecules of SlpA for flexible fitting of X-ray structure to map.
Voxel sizeX: 1 Å / Y: 1 Å / Z: 1.5625 Å
Density
Contour LevelBy AUTHOR: 22.1
Minimum - Maximum-250.0 - 213.45927
Average (Standard dev.)-0.27080622 (±37.640175)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-89-94-110
Dimensions261321221
Spacing321261221
CellA: 321.0 Å / B: 261.0 Å / C: 345.3125 Å
α: 90.0 ° / β: 90.0 ° / γ: 100.0 °

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Supplemental data

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Sample components

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Entire : Electron crystallographic reconstruction of R20291 S-layer, exten...

EntireName: Electron crystallographic reconstruction of R20291 S-layer, extended to cover 12 molecules of SlpA for flexible fitting of X-ray structure to map
Components
  • Complex: Electron crystallographic reconstruction of R20291 S-layer, extended to cover 12 molecules of SlpA for flexible fitting of X-ray structure to map
    • Protein or peptide: Precursor of the S-layer proteins
    • Protein or peptide: Precursor of the S-layer proteins

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Supramolecule #1: Electron crystallographic reconstruction of R20291 S-layer, exten...

SupramoleculeName: Electron crystallographic reconstruction of R20291 S-layer, extended to cover 12 molecules of SlpA for flexible fitting of X-ray structure to map
type: complex / Chimera: Yes / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Clostridioides difficile R20291 (bacteria)

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Macromolecule #1: Precursor of the S-layer proteins

MacromoleculeName: Precursor of the S-layer proteins / type: protein_or_peptide / ID: 1 / Number of copies: 12 / Enantiomer: LEVO
Source (natural)Organism: Clostridioides difficile 630 (bacteria) / Strain: 630
Molecular weightTheoretical: 39.495035 KDa
SequenceString: NDTIASQDTP AKVVIKANKL KDLKDYVDDL KTYNNTYSNV VTVAGEDRIE TAIELSSKYY NSDDKNAITD KAVNDIVLVG STSIVDGLV ASPLASEKTA PLLLTSKDKL DSSVKSEIKR VMNLKSDTGI NTSKKVYLAG GVNSISKDVE NELKNMGLKV T RLSGEDRY ...String:
NDTIASQDTP AKVVIKANKL KDLKDYVDDL KTYNNTYSNV VTVAGEDRIE TAIELSSKYY NSDDKNAITD KAVNDIVLVG STSIVDGLV ASPLASEKTA PLLLTSKDKL DSSVKSEIKR VMNLKSDTGI NTSKKVYLAG GVNSISKDVE NELKNMGLKV T RLSGEDRY ETSLAIADEI GLDNDKAFVV GGTGLADAMS IAPVASQLKD GDATPIVVVD GKAKEISDDA KSFLGTSDVD II GGKNSVS KEIEESIDSA TGKTPDRISG DDRQATNAEV LKEDDYFTDG EVVNYFVAKD GSTKEDQLVD ALAAAPIAGR FKE SPAPII LATDTLSSDQ NVAVSKAVPK DGGTNLVQVG KGIASSVINK MKDLLDM

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Macromolecule #2: Precursor of the S-layer proteins

MacromoleculeName: Precursor of the S-layer proteins / type: protein_or_peptide / ID: 2 / Number of copies: 12 / Enantiomer: LEVO
Source (natural)Organism: Clostridioides difficile 630 (bacteria) / Strain: 630
Molecular weightTheoretical: 33.949785 KDa
SequenceString: ATTGTQGYTV VKNDWKKAVK QLQDGLKDNS IGKITVSFND GVVGEVAPKS ANKKADRDAA AEKLYNLVNT QLDKLGDGDY VDFSVDYNL ENKIITNQAD AEAIVTKLNS LNEKTLIDIA TKDTFGMVSK TQDSEGKNVA ATKALKVKDV ATFGLKSGGS E DTGYVVEM ...String:
ATTGTQGYTV VKNDWKKAVK QLQDGLKDNS IGKITVSFND GVVGEVAPKS ANKKADRDAA AEKLYNLVNT QLDKLGDGDY VDFSVDYNL ENKIITNQAD AEAIVTKLNS LNEKTLIDIA TKDTFGMVSK TQDSEGKNVA ATKALKVKDV ATFGLKSGGS E DTGYVVEM KAGAVEDKYG KVGDSTAGIA INLPSTGLEY AGKGTTIDFN KTLKVDVTGG STPSAVAVSG FVTKDDTDLA KS GTINVRV INAKEESIDI DASSYTSAEN LAKRYVFDPD EISEAYKAIV ALQNDGIESN LVQLVNGKYQ VIFYPEGKRL E

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Experimental details

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Structure determination

Methodnegative staining
Processingelectron crystallography
Aggregation state2D array

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Sample preparation

BufferpH: 7.4
StainingType: NEGATIVE / Material: Uranyl Formate
Details: Negatively stained EM samples were prepared by depositing sample on continuous carbon layer and staining with 2 % Uranyl Formate with blotting
GridModel: Homemade / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR

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Electron microscopy

MicroscopeFEI/PHILIPS CM200FEG
Image recordingFilm or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Digitization - Dimensions - Width: 2048 pixel / Digitization - Dimensions - Height: 2048 pixel / Digitization - Sampling interval: 15.0 µm / Number real images: 36 / Average electron dose: 0.1 e/Å2 / Details: Images binned by 2 before processing
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.2 µm / Nominal defocus min: 0.8 µm / Camera length: 0.1 mm

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Image processing

Final reconstructionResolution method: DIFFRACTION PATTERN/LAYERLINES
Crystal parametersUnit cell - A: 80 Å / Unit cell - B: 80 Å / Unit cell - C: 160 Å / Unit cell - γ: 100 ° / Unit cell - α: 90 ° / Unit cell - β: 90 ° / Space group: P 1 1 2
CTF correctionSoftware - Name: CTFFIND (ver. 3)
Lattice distortion correction software listSoftware - Name: MRC IMAGE PROCESSING PACKAGE
Symmetry determination software listSoftware - Name: MRC IMAGE PROCESSING PACKAGE
Merging software listSoftware - Name: MRC IMAGE PROCESSING PACKAGE
Crystallography statisticsNumber intensities measured: 1085 / Number structure factors: 710 / Fourier space coverage: 0.1 / R merge: 0.193 / Overall phase error: 22.3 / Overall phase residual: 21.9
Phase error rejection criteria: Structure factors with phase errors higher than 45 degrees were omitted from refinement
High resolution: 18.25 Å / Shell - Shell ID: 1 / Shell - High resolution: 18.25 Å / Shell - Low resolution: 78.79 Å / Shell - Number structure factors: 710 / Shell - Phase residual: 21.9 / Shell - Fourier space coverage: 0.1 / Shell - Multiplicity: 0.1

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-7qgq:
Extended H/L (SLPH/SLPL) complex from C. difficile (CD630 strain) fit into R20291 S-layer negative stain map

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