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- PDB-7qgq: Extended H/L (SLPH/SLPL) complex from C. difficile (CD630 strain)... -

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Basic information

Entry
Database: PDB / ID: 7qgq
TitleExtended H/L (SLPH/SLPL) complex from C. difficile (CD630 strain) fit into R20291 S-layer negative stain map
Components(Precursor of the S-layer proteins) x 2
KeywordsSTRUCTURAL PROTEIN / Bacterial surface / S-layer
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 / ell wall binding domain 2 (CWB2) / identical protein binding / Precursor of the S-layer proteins
Function and homology information
Biological speciesClostridioides difficile 630 (bacteria)
MethodELECTRON CRYSTALLOGRAPHY / electron crystallography / negative staining
AuthorsBanerji, O. / Wilson, J.S. / Bullough, P.A.
Funding support United Kingdom, 1items
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, 2021Deposition site: PDBE / Processing site: PDBE
Revision 1.0Apr 6, 2022Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
B: Precursor of the S-layer proteins
C: Precursor of the S-layer proteins
J: Precursor of the S-layer proteins
O: Precursor of the S-layer proteins
D: Precursor of the S-layer proteins
T: Precursor of the S-layer proteins
A: Precursor of the S-layer proteins
P: Precursor of the S-layer proteins
Q: Precursor of the S-layer proteins
E: Precursor of the S-layer proteins
K: Precursor of the S-layer proteins
L: Precursor of the S-layer proteins
U: Precursor of the S-layer proteins
V: Precursor of the S-layer proteins
F: Precursor of the S-layer proteins
R: Precursor of the S-layer proteins
S: Precursor of the S-layer proteins
G: Precursor of the S-layer proteins
M: Precursor of the S-layer proteins
N: Precursor of the S-layer proteins
W: Precursor of the S-layer proteins
H: Precursor of the S-layer proteins
I: Precursor of the S-layer proteins
X: Precursor of the S-layer proteins


Theoretical massNumber of molelcules
Total (without water)881,33824
Polymers881,33824
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)321.0, 261.0, 345.312
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number1
Space group name H-MP1

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Components

#1: Protein
Precursor of the S-layer proteins


Mass: 39495.035 Da / Num. of mol.: 12 / Source method: isolated from a natural source / Source: (natural) Clostridioides difficile 630 (bacteria) / Plasmid details: RT012, SLCT7 / Strain: 630 / References: UniProt: Q183M8
#2: Protein
Precursor of the S-layer proteins


Mass: 33949.785 Da / Num. of mol.: 12 / Source method: isolated from a natural source / Source: (natural) Clostridioides difficile 630 (bacteria) / Plasmid details: RT012, SLCT7 / Strain: 630 / References: UniProt: Q183M8

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Experimental details

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Experiment

ExperimentMethod: ELECTRON CRYSTALLOGRAPHY
EM experimentAggregation state: 2D ARRAY / 3D reconstruction method: electron crystallography

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Sample preparation

ComponentName: 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 / Entity ID: all / Source: NATURAL
Molecular weightExperimental value: NO
Source (natural)Organism: Clostridioides difficile R20291 (bacteria)
Buffer solutionpH: 7.4
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: YES / Vitrification applied: NO
EM stainingType: NEGATIVE
Details: Negatively stained EM samples were prepared by depositing sample on continuous carbon layer and staining with 2 % Uranyl Formate with blotting
Material: Uranyl Formate
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Homemade

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Data collection

MicroscopyModel: FEI/PHILIPS CM200FEG
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2200 nm / Nominal defocus min: 800 nm
Image recordingElectron dose: 0.1 e/Å2 / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Num. of real images: 36 / Details: Images binned by 2 before processing
Image scansSampling size: 15 µm / Width: 2048 / Height: 2048
EM diffractionCamera length: 0.1 mm
EM diffraction shellResolution: 18.25→78.79 Å / Fourier space coverage: 0.1 % / Multiplicity: 0.1 / Num. of structure factors: 710 / Phase residual: 21.9 °
EM diffraction statsFourier space coverage: 0.1 % / High resolution: 18.25 Å / Num. of intensities measured: 1085 / Num. of structure factors: 710 / Phase error: 22.3 ° / Phase residual: 21.9 °
Phase error rejection criteria: Structure factors with phase errors higher than 45 degrees were omitted from refinement
Rmerge: 0.193

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Processing

EM software
IDNameVersionCategoryDetails
3CTFFIND3CTF correction
6ISOLDE1.1.0model fittingIsolde was used with reduced map weighting, torsion and distance restraints
10MRC IMAGE PROCESSING PACKAGElattice distortion correction
11MRC IMAGE PROCESSING PACKAGEsymmetry determination
12MRC IMAGE PROCESSING PACKAGEcrystallography merging
EM 3D crystal entity∠α: 90 ° / ∠β: 90 ° / ∠γ: 100 ° / A: 80 Å / B: 80 Å / C: 160 Å / Space group name: P112 / Space group num: 13
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution method: DIFFRACTION PATTERN/LAYERLINES / Symmetry type: 3D CRYSTAL
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
RefinementCross valid method: THROUGHOUT
Displacement parametersBiso max: 104.01 Å2 / Biso mean: 57.857 Å2 / Biso min: 34.65 Å2

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