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- PDB-9g93: CryoET structure of the in vitro grown Bacillus anthracis Sap S-layer -

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Basic information

Entry
Database: PDB / ID: 9g93
TitleCryoET structure of the in vitro grown Bacillus anthracis Sap S-layer
ComponentsS-layer protein sap
KeywordsSTRUCTURAL PROTEIN / S-layer / exoskeleton / surface array
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 str. '34F2 '
MethodELECTRON MICROSCOPY / subtomogram averaging / cryo EM / Resolution: 7.2 Å
AuthorsSogues, A. / Leigh, K. / Van der Verren, S. / Kudryashev, M. / Pak, A. / Halingstad, E.V. / Cecil, A.J. / Fioravanti, A. / Remaut, H.
Funding support Belgium, European Union, Germany, United States, 6items
OrganizationGrant numberCountry
Research Foundation - Flanders (FWO)G065220N Belgium
European Molecular Biology Organization (EMBO)ALTF-709-2021European Union
H2020 Marie Curie Actions of the European CommissionEuropean Union
Alexander von Humboldt Foundation Germany
German Research Foundation (DFG) Germany
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)R21AI168838 United States
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
DepositionJul 24, 2024Deposition site: PDBE / Processing site: PDBE
Revision 1.0Dec 18, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: S-layer protein sap
B: S-layer protein sap
C: S-layer protein sap
D: S-layer protein sap
E: S-layer protein sap
F: S-layer protein sap
G: S-layer protein sap
I: S-layer protein sap
J: S-layer protein sap
L: S-layer protein sap
K: S-layer protein sap


Theoretical massNumber of molelcules
Total (without water)954,08111
Polymers954,08111
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
d_1ens_1chain "A"
d_2ens_1chain "B"
d_3ens_1chain "C"
d_4ens_1chain "D"
d_5ens_1chain "E"
d_6ens_1chain "F"
d_1ens_2chain "I"
d_2ens_2chain "K"

NCS domain segments:

Component-ID: 1 / Beg auth comp-ID: LYS / Beg label comp-ID: LYS / End auth comp-ID: GLU / End label comp-ID: GLU / Auth seq-ID: 214 - 808 / Label seq-ID: 213 - 808

Dom-IDEns-IDAuth asym-IDLabel asym-ID
d_1ens_1AA
d_2ens_1BB
d_3ens_1CC
d_4ens_1DD
d_5ens_1EE
d_6ens_1FF
d_1ens_2IH
d_2ens_2KK

NCS ensembles :
ID
ens_1
ens_2

NCS oper:
IDCodeMatrixVector
1given(-0.999811114072, 0.00349949816808, -0.0191177846691), (-0.0013509197222, -0.993790078759, -0.111262996441), (-0.0193884293843, -0.111216153834, 0.993607093338)339.116273074, 323.437870543, 21.8256266045
2given(0.999777459454, -0.0152069831604, 0.0146211911407), (0.010545103424, 0.960535177583, 0.2779585822), (-0.0182710799076, -0.277742543172, 0.960481778771)-27.6523115373, 70.4225190227, 43.0140992281
3given(-0.99972249711, -0.023428769534, 0.00245388071331), (0.0234731009929, -0.981965707902, 0.187596274042), (-0.00198552315821, 0.187601815723, 0.982243155453)312.991466318, 382.105886076, -35.5991677662
4given(0.999774417498, -0.0210316507328, 0.00296374494357), (0.0210548846388, 0.96303490929, -0.268552518741), (0.0027939129347, 0.2685543393, 0.963260484445)32.1728300775, -65.4428347802, -56.4560632884
5given(-0.999457100306, 0.0145845663362, -0.0295431053229), (-0.00265605908595, -0.929434067879, -0.368978669867), (-0.0328397624454, -0.368699883227, 0.928968215878)367.192369469, 246.583192684, 53.286627324
6given(-0.999892242554, -0.00468376737941, -0.0139127856039), (0.00599645975558, -0.995376615865, -0.0958615410845), (-0.0133994682926, -0.0959346387486, 0.995297442646)340.45452784, 320.991265875, 16.7180504112

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Components

#1: Protein
S-layer protein sap / Surface array protein / Surface layer protein


Mass: 86734.656 Da / Num. of mol.: 11
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Bacillus anthracis str. '34F2 (NMRC)' (bacteria)
Strain: 34F2 / Gene: sap, BA_0885, GBAA_0885, BAS0841 / Production host: Escherichia coli BL21 (bacteria) / References: UniProt: P49051
Has protein modificationN

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: 2D ARRAY / 3D reconstruction method: subtomogram averaging

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Sample preparation

ComponentName: in vitro S-layer of the Sap assembly domain / Type: COMPLEX
Details: 2D S-layer array of a fragment of the Sap protein lacking the SLH cell wall attachment domain, and corresponding to the S-layer assembly domain
Entity ID: all / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Bacillus anthracis str. '34F2 (NMRC)' (bacteria) / Strain: 34F2 / Organelle: S-layer
Source (recombinant)Organism: Escherichia coli (E. coli) / Strain: BL21
Buffer solutionpH: 8 / Details: 10 mM HEPES, pH 8 and 100 mM NaCl
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 81000 X / Nominal defocus max: 3500 nm / Nominal defocus min: 2000 nm
Image recordingElectron dose: 3.8 e/Å2 / Avg electron dose per subtomogram: 156 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of real images: 1

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Processing

EM software
IDNameVersionCategory
1Dynamo1.1.401volume selection
2SerialEMimage acquisition
4Gctf1.06CTF correction
5NOVACTFCTF correction
12Dynamo1.1.478final Euler assignment
14Dynamo1.1.4783D reconstruction
15RELION3.13D reconstruction
CTF correctionType: PHASE FLIPPING ONLY
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 7.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 10126
Details: Final half map reconstruction was done in Dynamo and resolution was estimated using RELION 3.1 postprocessing
Symmetry type: POINT
EM volume selectionNum. of tomograms: 12 / Num. of volumes extracted: 117502
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 50 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.008737499
ELECTRON MICROSCOPYf_angle_d1.019650710
ELECTRON MICROSCOPYf_chiral_restr0.06336358
ELECTRON MICROSCOPYf_plane_restr0.00546413
ELECTRON MICROSCOPYf_dihedral_angle_d17.62213900
Refine LS restraints NCS
Ens-IDDom-IDAsym-IDAuth asym-IDRefine-IDTypeRms dev position (Å)
ens_1d_2AAELECTRON MICROSCOPYNCS constraints0.0184177461378
ens_1d_3AAELECTRON MICROSCOPYNCS constraints0.00978801393067
ens_1d_4AAELECTRON MICROSCOPYNCS constraints0.709814636951
ens_1d_5AAELECTRON MICROSCOPYNCS constraints0.0147667351003
ens_1d_6AAELECTRON MICROSCOPYNCS constraints0.016403399054
ens_2d_2HIELECTRON MICROSCOPYNCS constraints0.0866346816967

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