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- EMDB-3604: Structure of the C. crescentus S-layer -

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Basic information

Entry
Database: EMDB / ID: EMD-3604
TitleStructure of the C. crescentus S-layer
Map dataSub-tomogram averaging of the Caulobacter crescentus S-layer
Sample
  • Organelle or cellular component: Caulobacter crescentus S-layer
    • Protein or peptide: S-layer protein rsaA
  • Ligand: CALCIUM ION
KeywordsS-layer / sub-tomogram averaging / bacteria / cell surface / caulobacter / Structural protein
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)
Methodsubtomogram averaging / cryo EM / Resolution: 7.4 Å
AuthorsBharat TA / Hagen WJ
Funding support United Kingdom, Germany, 3 items
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, 2017-
Header (metadata) releaseMar 29, 2017-
Map releaseApr 19, 2017-
UpdateMay 15, 2024-
Current statusMay 15, 2024Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.11
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 0.11
  • Imaged by UCSF Chimera
  • Download
  • Surface view with fitted model
  • Atomic models: PDB-5n97
  • Surface level: 0.11
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_3604.png

Downloads & links

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Map

FileDownload / File: emd_3604.map.gz / Format: CCP4 / Size: 30.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSub-tomogram averaging of the Caulobacter crescentus S-layer
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.35 Å/pix.
x 200 pix.
= 270. Å		1.35 Å/pix.
x 200 pix.
= 270. Å		1.35 Å/pix.
x 200 pix.
= 270. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.35 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.11 / Movie #1: 0.11
Minimum - Maximum-0.25166476 - 0.5049828
Average (Standard dev.)-0.009878825 (±0.0565414)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions200200200
Spacing200200200
CellA=B=C: 270.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.351.351.35
M x/y/z200200200
origin x/y/z0.0000.0000.000
length x/y/z270.000270.000270.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS200200200
D min/max/mean-0.2520.505-0.010

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Supplemental data

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Sample components

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Entire : Caulobacter crescentus S-layer

EntireName: Caulobacter crescentus S-layer
Components
  • Organelle or cellular component: Caulobacter crescentus S-layer
    • Protein or peptide: S-layer protein rsaA
  • Ligand: CALCIUM ION

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Supramolecule #1: Caulobacter crescentus S-layer

SupramoleculeName: Caulobacter crescentus S-layer / type: organelle_or_cellular_component / ID: 1 / Parent: 0 / Macromolecule list: #1 / Details: Caulobacter crescentus S-layer
Source (natural)Organism: Caulobacter crescentus NA1000 (bacteria) / Strain: YB2811 / Location in cell: extra-cellular

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Macromolecule #1: S-layer protein rsaA

MacromoleculeName: S-layer protein rsaA / type: protein_or_peptide / ID: 1 / Number of copies: 6 / Enantiomer: LEVO
Source (natural)Organism: Caulobacter crescentus NA1000 (bacteria)
Molecular weightTheoretical: 73.159305 KDa
SequenceString: GSTLSLTTGT DTLTGTANND TFVAGEVAGA ATLTVGDTLS GGAGTDVLNW VQAAAVTALP TGVTISGIET MNVTSGAAIT LNTSSGVTG LTALNTNTSG AAQTVTAGAG QNLTATTAAQ AANNVAVDGG ANVTVASTGV TSGTTTVGAN SAASGTVSVS V ANSSTTTT ...String:
GSTLSLTTGT DTLTGTANND TFVAGEVAGA ATLTVGDTLS GGAGTDVLNW VQAAAVTALP TGVTISGIET MNVTSGAAIT LNTSSGVTG LTALNTNTSG AAQTVTAGAG QNLTATTAAQ AANNVAVDGG ANVTVASTGV TSGTTTVGAN SAASGTVSVS V ANSSTTTT GAIAVTGGTA VTVAQTAGNA VNTTLTQADV TVTGNSSTTA VTVTQTAAAT AGATVAGRVN GAVTITDSAA AS ATTAGKI ATVTLGSFGA ATIDSSALTT VNLSGTGTSL GIGRGALTAT PTANTLTLNV NGLTTTGAIT DSEAAADDGF TTI NIAGST ASSTIASLVA ADATTLNISG DARVTITSHT AAALTGITVT NSVGATLGAE LATGLVFTGG AGADSILLGA TTKA IVMGA GDDTVTVSSA TLGAGGSVNG GDGTDVLVAN VNGSSFSADP AFGGFETLRV AGAAAQGSHN ANGFTALQLG ATAGA TTFT NVAVNVGLTV LAAPTGTTTV TLANATGTSD VFNLTLSSSA ALAAGTVALA GVETVNIAAT DTNTTAHVDT LTLQAT SAK SIVVTGNAGL NLTNTGNTAV TSFDASAVTG TGSAVTFVSA NTTVGEVVTI RGGAGADSLT GSATANDTII GGAGADT LV YTGGTDTFTG GTGADIFDIN AIGTSTAFVT ITDAAVGDKL DLVGISTNGA IADGAFGAAV TLGAAATLAQ YLDAAAAG D GSGTSVAKWF QFGGDTYVVV DSSAGATFVS GADAVIKLTG LVTLTTSAFA TEVLTLA

UniProtKB: S-layer protein rsaA

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Macromolecule #2: CALCIUM ION

MacromoleculeName: CALCIUM ION / type: ligand / ID: 2 / Number of copies: 114 / Formula: CA
Molecular weightTheoretical: 40.078 Da

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Experimental details

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Structure determination

Methodcryo EM
Processingsubtomogram averaging
Aggregation stateparticle

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Sample preparation

Concentration1 mg/mL
BufferpH: 7 / Details: PYE medium
GridModel: Quantifoil R2/2 / Material: COPPER/RHODIUM / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY ARRAY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec. / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 283.15 K / Instrument: FEI VITROBOT MARK IV / Details: 1.5 s blot.
DetailsCaulobacter crescentus stalk

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Electron microscopy

MicroscopeFEI TITAN KRIOS
TemperatureMin: 70.0 K / Max: 70.0 K
Specialist opticsEnergy filter - Name: GIF Quantum / Energy filter - Lower energy threshold: 0 eV / Energy filter - Upper energy threshold: 20 eV
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Number grids imaged: 1 / Average exposure time: 1.0 sec. / Average electron dose: 3.4 e/Å2 / Details: Dose symmetric tilt scheme (Hagen et al)
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Calibrated defocus max: 5.0 µm / Calibrated defocus min: 2.0 µm / Calibrated magnification: 105000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 5.0 µm / Nominal defocus min: 2.0 µm / Nominal magnification: 105000
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 reconstructionNumber classes used: 1 / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 7.4 Å / Resolution method: FSC 0.143 CUT-OFF / Number subtomograms used: 51866
ExtractionNumber tomograms: 110 / Number images used: 51866 / Reference model: Ab initio / Method: RELION / Software - Name: RELION (ver. 1.4) / Details: RELION subtomogram averaging
Final angle assignmentType: OTHER / Details: Following Schur et al, 2016
Crystal parametersUnit cell - A: 220 Å / Unit cell - B: 220 Å / Unit cell - C: 220 Å / Unit cell - C sampling length: 10 Å / Unit cell - γ: 1 ° / Plane group: P 1 21

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: RIGID BODY FIT / Target criteria: Cross-correlation coefficient
Output model

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

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