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- EMDB-10388: In situ structure of the Caulobacter crescentus S-layer -

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Basic information

Entry
Database: EMDB / ID: EMD-10388
TitleIn situ structure of the Caulobacter crescentus S-layer
Map dataIn situ structure of the Caulobacter crescentus S-layer
Sample
  • Organelle or cellular component: Caulobacter crescentus S-layer
Function / homologyRsaA N-terminal domain / S-layer / RTX calcium-binding nonapeptide repeat / RTX calcium-binding nonapeptide repeat (4 copies) / Serralysin-like metalloprotease, C-terminal / calcium ion binding / extracellular region / S-layer protein
Function and homology information
Biological speciesCaulobacter crescentus NA1000 (bacteria)
Methodsubtomogram averaging / cryo EM / Resolution: 4.8 Å
AuthorsBharat T / von Kuegelgen A
Funding support United Kingdom, 1 items
OrganizationGrant numberCountry
Wellcome Trust202231/Z/16/Z United Kingdom
CitationJournal: Cell / Year: 2020
Title: In Situ Structure of an Intact Lipopolysaccharide-Bound Bacterial Surface Layer.
Authors: Andriko von Kügelgen / Haiping Tang / Gail G Hardy / Danguole Kureisaite-Ciziene / Yves V Brun / Phillip J Stansfeld / Carol V Robinson / Tanmay A M Bharat /
Abstract: Most bacterial and all archaeal cells are encapsulated by a paracrystalline, protective, and cell-shape-determining proteinaceous surface layer (S-layer). On Gram-negative bacteria, S-layers are ...Most bacterial and all archaeal cells are encapsulated by a paracrystalline, protective, and cell-shape-determining proteinaceous surface layer (S-layer). On Gram-negative bacteria, S-layers are anchored to cells via lipopolysaccharide. Here, we report an electron cryomicroscopy structure of the Caulobacter crescentus S-layer bound to the O-antigen of lipopolysaccharide. Using native mass spectrometry and molecular dynamics simulations, we deduce the length of the O-antigen on cells and show how lipopolysaccharide binding and S-layer assembly is regulated by calcium. Finally, we present a near-atomic resolution in situ structure of the complete S-layer using cellular electron cryotomography, showing S-layer arrangement at the tip of the O-antigen. A complete atomic structure of the S-layer shows the power of cellular tomography for in situ structural biology and sheds light on a very abundant class of self-assembling molecules with important roles in prokaryotic physiology with marked potential for synthetic biology and surface-display applications.
History
DepositionOct 21, 2019-
Header (metadata) releaseJan 15, 2020-
Map releaseJan 15, 2020-
UpdateJul 29, 2020-
Current statusJul 29, 2020Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.1
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 0.1
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6z7p
  • Surface level: 0.1
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-6z7p
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_10388.png

Downloads & links

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Map

FileDownload / File: emd_10388.map.gz / Format: CCP4 / Size: 30.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationIn situ structure 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: 0.1 / Movie #1: 0.1
Minimum - Maximum-0.15512758 - 0.3130494
Average (Standard dev.)0.00001737453 (±0.03268774)
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-0.1550.3130.000

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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

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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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Experimental details

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Structure determination

Methodcryo EM
Processingsubtomogram averaging
Aggregation statecell

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Sample preparation

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 - Atmosphere: AIR / Details: 15 mA
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 LS / Energy filter - Slit width: 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, JSB)
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 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 4.8 Å / Resolution method: FSC 0.143 CUT-OFF
Details: Tomogram generation in IMOD using the NovaCTF implementation of Turonova and Briggs. Sub-tomogram averaging performed using the AV3 package (Friedrich Foerster) applied to tubular specimens ...Details: Tomogram generation in IMOD using the NovaCTF implementation of Turonova and Briggs. Sub-tomogram averaging performed using the AV3 package (Friedrich Foerster) applied to tubular specimens (Bharat et al, PLoS Biology, 2011)
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
CTF correctionDetails: Following Turonova and Briggs NovaCTF
Final 3D classificationNumber classes: 1 / Avg.num./class: 1 / Details: No classification done
Final angle assignmentType: OTHER / Details: Using the AV3 package (Foerster et al, PNAS 2006)

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Atomic model buiding 1

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

PDB-6z7p:
Composite model of the Caulobacter crescentus S-layer bound to the O-antigen of lipopolysaccharide

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