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- PDB-5n97: Structure of the C. crescentus S-layer -

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Basic information

Entry
Database: PDB / ID: 5n97
TitleStructure of the C. crescentus S-layer
ComponentsS-layer protein rsaA
KeywordsSTRUCTURAL PROTEIN / S-layer / sub-tomogram averaging / bacteria / cell surface / caulobacter
Function / homologyRsaA N-terminal domain / RTX calcium-binding nonapeptide repeat / RTX calcium-binding nonapeptide repeat (4 copies) / Serralysin-like metalloprotease, C-terminal / calcium ion binding / S-layer protein rsaA
Function and homology information
Biological speciesCaulobacter crescentus NA1000 (bacteria)
MethodELECTRON MICROSCOPY / subtomogram averaging / cryo EM / Resolution: 7.4 Å
AuthorsBharat, T.A. / Hagen, W.J. / Briggs, J.A. / Lowe, J.
Funding support United Kingdom, Germany, 3items
OrganizationGrant numberCountry
Wellcome Trust095514/Z/11/Z United Kingdom
Medical Research Council (United Kingdom)U105184326 United Kingdom
European Molecular Biology OrganizationaALTF 778-2015 Germany
CitationJournal: Nat Microbiol / Year: 2017
Title: Structure of the hexagonal surface layer on Caulobacter crescentus cells.
Authors: Tanmay A M Bharat / Danguole Kureisaite-Ciziene / Gail G Hardy / Ellen W Yu / Jessica M Devant / Wim J H Hagen / Yves V Brun / John A G Briggs / Jan Löwe /
Abstract: Many prokaryotic cells are encapsulated by a surface layer (S-layer) consisting of repeating units of S-layer proteins. S-layer proteins are a diverse class of molecules found in Gram-positive and ...Many prokaryotic cells are encapsulated by a surface layer (S-layer) consisting of repeating units of S-layer proteins. S-layer proteins are a diverse class of molecules found in Gram-positive and Gram-negative bacteria and most archaea. S-layers protect cells from the outside, provide mechanical stability and also play roles in pathogenicity. In situ structural information about this highly abundant class of proteins is scarce, so atomic details of how S-layers are arranged on the surface of cells have remained elusive. Here, using purified Caulobacter crescentus' sole S-layer protein RsaA, we obtained a 2.7 Å X-ray structure that shows the hexameric S-layer lattice. We also solved a 7.4 Å structure of the S-layer through electron cryotomography and sub-tomogram averaging of cell stalks. The X-ray structure was docked unambiguously into the electron cryotomography map, resulting in a pseudo-atomic-level description of the in vivo S-layer, which agrees completely with the atomic X-ray lattice model. The cellular S-layer atomic structure shows that the S-layer is porous, with a largest gap dimension of 27 Å, and is stabilized by multiple Ca ions bound near the interfaces. This study spans different spatial scales from atoms to cells by combining X-ray crystallography with electron cryotomography and sub-nanometre-resolution sub-tomogram averaging.
History
DepositionFeb 24, 2017Deposition site: PDBE / Processing site: PDBE
Revision 1.0Apr 19, 2017Provider: repository / Type: Initial release
Revision 1.1Aug 2, 2017Group: Data collection / Derived calculations / Refinement description
Category: em_3d_fitting / em_image_scans ...em_3d_fitting / em_image_scans / em_imaging_optics / pdbx_struct_conn_angle
Item: _em_3d_fitting.target_criteria / _em_imaging_optics.energyfilter_name
Revision 1.2Oct 24, 2018Group: Data collection / Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year

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Structure visualization

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Assembly

Deposited unit
A: S-layer protein rsaA
B: S-layer protein rsaA
C: S-layer protein rsaA
D: S-layer protein rsaA
E: S-layer protein rsaA
F: S-layer protein rsaA
hetero molecules


Theoretical massNumber of molelcules
Total (without water)443,525120
Polymers438,9566
Non-polymers4,569114
Water0
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area9420 Å2
ΔGint-712 kcal/mol
Surface area169690 Å2
MethodPISA

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Components

#1: Protein
S-layer protein rsaA /


Mass: 73159.305 Da / Num. of mol.: 6 / Fragment: UNP residues 249-1026 / Source method: isolated from a natural source / Source: (natural) Caulobacter crescentus NA1000 (bacteria) / Variant: YB2811 / References: UniProt: A0A0H3C8J1
#2: Chemical...
ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 114 / Source method: obtained synthetically / Formula: Ca

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: subtomogram averaging
Crystal symmetry∠γ: 1 ° / C sampling length: 10 Å / A: 220 Å / B: 220 Å / C: 220 Å / Space group name H-M: P121

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Sample preparation

ComponentName: Caulobacter crescentus S-layer / Type: ORGANELLE OR CELLULAR COMPONENT / Details: Caulobacter crescentus S-layer / Entity ID: #1 / Source: NATURAL
Molecular weightExperimental value: NO
Source (natural)Organism: Caulobacter crescentus NA1000 (bacteria) / Strain: YB2811 / Cellular location: extra-cellular
Buffer solutionpH: 7 / Details: PYE medium
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: Caulobacter crescentus stalk
Specimen supportGrid material: COPPER/RHODIUM / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R2/2
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 283.15 K / Details: 1.5 s blot

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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 FIELDBright-field microscopy / Nominal magnification: 105000 X / Calibrated magnification: 105000 X / Nominal defocus max: 5000 nm / Nominal defocus min: 2000 nm / Calibrated defocus min: 2000 nm / Calibrated defocus max: 5000 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 70 K / Temperature (min): 70 K
Image recordingAverage exposure time: 1 sec. / Electron dose: 3.4 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Details: Dose symmetric tilt scheme (Hagen et al)
EM imaging opticsEnergyfilter name: GIF Quantum / Energyfilter upper: 20 eV / Energyfilter lower: 0 eV

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Processing

EM software
IDNameVersionCategory
1RELION1.4volume selection
2SerialEMimage acquisition
7UCSF Chimeramodel fitting
Crystal symmetry∠γ: 1 ° / C sampling length: 10 Å / A: 220 Å / B: 220 Å / C: 220 Å / Space group name H-M: P121
CTF correctionDetails: Following Schur et al, 2016 / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 7.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 51866 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: 2D CRYSTAL
EM volume selectionMethod: RELIONList of Walmart brands / Details: RELION subtomogram averaging / Num. of tomograms: 110 / Num. of volumes extracted: 51866 / Reference model: Ab initio
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL / Target criteria: Cross-correlation coefficient

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