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- EMDB-10389: Structure of the RsaA N-terminal domain bound to LPS -

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Basic information

Entry
Database: EMDB / ID: EMD-10389
TitleStructure of the RsaA N-terminal domain bound to LPS
Map datacryo-EM map of the RsaA protein N-terminal domain
Sample
  • Complex: Structure of RsaA N-terminal domain bound to LPS
    • Protein or peptide: S-layer protein
  • Ligand: CALCIUM ION
KeywordsS-layer LPS RsaA / STRUCTURAL PROTEIN
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 vibrioides CB15 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.7 Å
Authorsvon Kuegelgen A / Bharat TAM
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) releaseNov 6, 2019-
Map releaseJan 15, 2020-
UpdateMay 22, 2024-
Current statusMay 22, 2024Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0195
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.0195
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6t72
  • Surface level: 0.0195
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-6t72
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_10389.png

Downloads & links

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Map

FileDownload / File: emd_10389.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationcryo-EM map of the RsaA protein N-terminal domain
Voxel sizeX=Y=Z: 1.08 Å
Density
Contour LevelBy AUTHOR: 0.0195 / Movie #1: 0.0195
Minimum - Maximum-0.06003098 - 0.107363954
Average (Standard dev.)0.000029459738 (±0.0039067115)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 324.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.081.081.08
M x/y/z300300300
origin x/y/z0.0000.0000.000
length x/y/z324.000324.000324.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS300300300
D min/max/mean-0.0600.1070.000

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Supplemental data

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Sample components

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Entire : Structure of RsaA N-terminal domain bound to LPS

EntireName: Structure of RsaA N-terminal domain bound to LPS
Components
  • Complex: Structure of RsaA N-terminal domain bound to LPS
    • Protein or peptide: S-layer protein
  • Ligand: CALCIUM ION

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Supramolecule #1: Structure of RsaA N-terminal domain bound to LPS

SupramoleculeName: Structure of RsaA N-terminal domain bound to LPS / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1 / Details: Structure of RsaA N-terminal domain bound to LPS
Source (natural)Organism: Caulobacter vibrioides CB15 (bacteria) / Strain: YB1001
Molecular weightTheoretical: 577 KDa

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Macromolecule #1: S-layer protein

MacromoleculeName: S-layer protein / type: protein_or_peptide / ID: 1 / Details: LPS O-antigen bound to protein / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Caulobacter vibrioides CB15 (bacteria)
Molecular weightTheoretical: 25.820354 KDa
Recombinant expressionOrganism: Caulobacter vibrioides CB15 (bacteria)
SequenceString: AYTTAQLVTA YTNANLGKAP DAATTLTLDA YATQTQTGGL SDAAALTNTL KLVNSTTAVA IQTYQFFTGV APSAAGLDFL VDSTTNTND LNDAYYSKFA QENRFINFSI NLATGAGAGA TAFAAAYTGV SYAQTVATAY DKIIGNAVAT AAGVDVAAAV A FLSRQANI ...String:
AYTTAQLVTA YTNANLGKAP DAATTLTLDA YATQTQTGGL SDAAALTNTL KLVNSTTAVA IQTYQFFTGV APSAAGLDFL VDSTTNTND LNDAYYSKFA QENRFINFSI NLATGAGAGA TAFAAAYTGV SYAQTVATAY DKIIGNAVAT AAGVDVAAAV A FLSRQANI DYLTAFVRAN TPFTAAADID LAVKAALIGT ILNAATVSGI GGYATATAAM INDLSDGALS TDNAAGVNLF TA YPSSGVS GSENLYFQ

UniProtKB: S-layer protein

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Macromolecule #3: CALCIUM ION

MacromoleculeName: CALCIUM ION / type: ligand / ID: 3 / Number of copies: 3 / Formula: CA
Molecular weightTheoretical: 40.078 Da

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration2.5 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
25.0 mMC8H18N2O4SHEPES
100.0 mMNaClsodium chloride
1.0 mMMgCl2magnesium chloride
1.0 mMCaCl2calcium chloride

Details: Buffer solutions were prepared fresh from sterile filtered concentrated stocksolutions. Solutions were filtered through a 0.22 um filter to avoid microbial contamination and degassed using a ...Details: Buffer solutions were prepared fresh from sterile filtered concentrated stocksolutions. Solutions were filtered through a 0.22 um filter to avoid microbial contamination and degassed using a vacuum fold pump. The pH of the HEPES stock solution was adjusted with sodium hydroxide at 4 deg C.
GridModel: Quantifoil R2/2 / Material: COPPER/RHODIUM / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY ARRAY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 20 sec. / Pretreatment - Atmosphere: AIR / Details: 20 seconds, 15 mA
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 283.15 K / Instrument: FEI VITROBOT MARK IV
Details: Vitrobot options: Blot time 3 seconds, Blot force -13,1, Wait time 10 seconds, Drain time 0.5 seconds,.
DetailsRsaA N-terminal domain with LPS

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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
DetailsEPU software
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 3838 pixel / Digitization - Dimensions - Height: 3710 pixel / Digitization - Frames/image: 1-20 / Number grids imaged: 1 / Number real images: 2422 / Average exposure time: 8.0 sec. / Average electron dose: 43.0 e/Å2
Details: Images were collected in movie-mode and subjected to 8 seconds of exposure where a total dose of 43 e-/A2 was applied, and 20 frames were recorded per movie.
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Calibrated defocus max: -4.0 µm / Calibrated defocus min: -1.0 µm / Calibrated magnification: 130000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: -4.0 µm / Nominal defocus min: -1.0 µm / Nominal magnification: 130000
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

DetailsMovies were motion corrected and dose weighted with MotionCor2 (Zheng et al., 2017) implemented in Relion 3.0 (Zivanov et al., 2018). Contrast transfer functions (CTFs) of the resulting motion corrected micrographs were estimated using CTFFIND4 (Rohou and Grigorieff, 2015).
Particle selectionNumber selected: 129633
Details: Particles were automatically picked from the motion and CTF corrected micrographs using the AutoPick function in Relion 3.0 (Zivanov et al., 2018). As particle reference a 3 dimensional ...Details: Particles were automatically picked from the motion and CTF corrected micrographs using the AutoPick function in Relion 3.0 (Zivanov et al., 2018). As particle reference a 3 dimensional reconstruction from an earlier dataset with different pixelsize was used which was reconstructed using an unbiased subtomogram average structure of the same sample.
Startup modelType of model: OTHER
Details: Initial model generation from cryo-ET data was performed using the Relion sub-tomogram averaging pipeline (Bharat et al., 2015; Bharat and Scheres, 2016). An unambiguous 3D reference was ...Details: Initial model generation from cryo-ET data was performed using the Relion sub-tomogram averaging pipeline (Bharat et al., 2015; Bharat and Scheres, 2016). An unambiguous 3D reference was generated and used in the single-particle EM pipeline.
Final reconstructionNumber classes used: 2 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.7 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0)
Details: Particles from two main 3D classes containing 21 or 20 RsaA subunits were combined for a focused 3D auto refinement on the central 14 subunits using the output from the 3D classification as ...Details: Particles from two main 3D classes containing 21 or 20 RsaA subunits were combined for a focused 3D auto refinement on the central 14 subunits using the output from the 3D classification as a starting model. The final map was obtained from 115,776 particles and post-processed using a soft mask focused on the inner fourteen subunits.
Number images used: 115776
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0) / Details: Angle assignment was performed within Relion 3.0.
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0) / Details: Angle assignment was performed within Relion 3.0.
Final 3D classificationNumber classes: 2 / Avg.num./class: 115776 / Software - Name: RELION (ver. 3.0)
Details: Particles from two main 3D classes containing 21 or 20 RsaA subunits were combined for a focused 3D auto refinement on the central 14 subunits using the output from the 3D classification as ...Details: Particles from two main 3D classes containing 21 or 20 RsaA subunits were combined for a focused 3D auto refinement on the central 14 subunits using the output from the 3D classification as a starting model. The final map was obtained from 115,776 particles and post-processed using a soft mask focused on the inner fourteen subunits.

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Atomic model buiding 1

DetailsThe carbon backbone of the RsaA protein was manually traced through a single subunit of the cryo-EM density using Coot (Emsley et al., 2010). Initially, side chains were assigned in regions with density corresponding to characteristic aromatic residues allowing us to deduce the register of the amino acid sequence in the map. Side chains for residues 2-243 of RsaA were thus assigned unambiguously and the structure was refined and manually rebuilt using Refmac5 (Murshudov et al., 2011) inside the CCP-EM (Burnley et al., 2017) software suite and Coot.
RefinementSpace: RECIPROCAL / Protocol: BACKBONE TRACE / Overall B value: 85.819 / Target criteria: Best fit
Output model

PDB-6t72:
Structure of the RsaA N-terminal domain bound to LPS

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