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- EMDB-13355: Structure of the Caulobacter crescentus S-layer protein RsaA N-te... -

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Basic information

Entry
Database: EMDB / ID: EMD-13355
TitleStructure of the Caulobacter crescentus S-layer protein RsaA N-terminal domain bound to LPS and soaked with Holmium
Map data
Sample
  • Complex: Structure of the Caulobacter crescentus S-layer protein RsaA N-terminal domain bound to LPS and soaked with Holmium
    • Protein or peptide: S-layer protein
  • Ligand: CALCIUM IONCalcium
  • Ligand: HOLMIUM ATOM
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 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.37 Å
Authorsvon Kugelgen A / Bharat TAM
Funding support United Kingdom, 2 items
OrganizationGrant numberCountry
Wellcome Trust202231/Z/16/Z United Kingdom
Leverhulme TrustPhilip Leverhulme Prize United Kingdom
CitationJournal: Structure / Year: 2022
Title: High-resolution mapping of metal ions reveals principles of surface layer assembly in Caulobacter crescentus cells.
Authors: Matthew Herdman / Andriko von Kügelgen / Danguole Kureisaite-Ciziene / Ramona Duman / Kamel El Omari / Elspeth F Garman / Andreas Kjaer / Dimitrios Kolokouris / Jan Löwe / Armin Wagner / ...Authors: Matthew Herdman / Andriko von Kügelgen / Danguole Kureisaite-Ciziene / Ramona Duman / Kamel El Omari / Elspeth F Garman / Andreas Kjaer / Dimitrios Kolokouris / Jan Löwe / Armin Wagner / Phillip J Stansfeld / Tanmay A M Bharat /
Abstract: Surface layers (S-layers) are proteinaceous crystalline coats that constitute the outermost component of most prokaryotic cell envelopes. In this study, we have investigated the role of metal ions in ...Surface layers (S-layers) are proteinaceous crystalline coats that constitute the outermost component of most prokaryotic cell envelopes. In this study, we have investigated the role of metal ions in the formation of the Caulobacter crescentus S-layer using high-resolution structural and cell biology techniques, as well as molecular simulations. Utilizing optical microscopy of fluorescently tagged S-layers, we show that calcium ions facilitate S-layer lattice formation and cell-surface binding. We report all-atom molecular dynamics simulations of the S-layer lattice, revealing the importance of bound metal ions. Finally, using electron cryomicroscopy and long-wavelength X-ray diffraction experiments, we mapped the positions of metal ions in the S-layer at near-atomic resolution, supporting our insights from the cellular and simulations data. Our findings contribute to the understanding of how C. crescentus cells form a regularly arranged S-layer on their surface, with implications on fundamental S-layer biology and the synthetic biology of self-assembling biomaterials.
History
DepositionAug 11, 2021-
Header (metadata) releaseDec 1, 2021-
Map releaseDec 1, 2021-
UpdateFeb 16, 2022-
Current statusFeb 16, 2022Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.00765
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.00765
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7peo
  • Surface level: 0.00765
  • Imaged by UCSF Chimera
  • Download
  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-7peo
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_13355.png

Downloads & links

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Map

FileDownload / File: emd_13355.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.08 Å
Density
Contour LevelBy AUTHOR: 0.00765 / Movie #1: 0.00765
Minimum - Maximum-0.0063713784 - 0.030576872
Average (Standard dev.)2.5826846e-05 (±0.0015256229)
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
start NX/NY/NZ000
NX/NY/NZ300300300
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS300300300
D min/max/mean-0.0060.0310.000

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Supplemental data

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Sample components

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Entire : Structure of the Caulobacter crescentus S-layer protein RsaA N-te...

EntireName: Structure of the Caulobacter crescentus S-layer protein RsaA N-terminal domain bound to LPS and soaked with Holmium
Components
  • Complex: Structure of the Caulobacter crescentus S-layer protein RsaA N-terminal domain bound to LPS and soaked with Holmium
    • Protein or peptide: S-layer protein
  • Ligand: CALCIUM IONCalcium
  • Ligand: HOLMIUM ATOM

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Supramolecule #1: Structure of the Caulobacter crescentus S-layer protein RsaA N-te...

SupramoleculeName: Structure of the Caulobacter crescentus S-layer protein RsaA N-terminal domain bound to LPS and soaked with Holmium
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Details: Structure of the Caulobacter crescentus S-layer protein RsaA N-terminal domain bound to LPS and soaked with Holmium
Source (natural)Organism: Caulobacter vibrioides (bacteria) / Strain: YB1001

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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 the protein / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Caulobacter vibrioides (bacteria) / Strain: YB1001
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

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Macromolecule #3: CALCIUM ION

MacromoleculeName: CALCIUM ION / type: ligand / ID: 3 / Number of copies: 2 / Formula: CA
Molecular weightTheoretical: 40.078 Da

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Macromolecule #4: HOLMIUM ATOM

MacromoleculeName: HOLMIUM ATOM / type: ligand / ID: 4 / Number of copies: 1 / Formula: HO
Molecular weightTheoretical: 164.93 Da
Chemical component information

ChemComp-HO:
HOLMIUM ATOM

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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

Concentration2.25 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
25.0 mMC8H18N2O4SHEPES
100.0 mMNaClSodium chloridesodium chloride
1.0 mMMgCl2magnesium chloride
1.0 mMCaCl2calcium chloride
5.0 mMHoCl3holmium 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. 5 mM HoCl3 was added to the specimen 1.5 hours before vitrification.
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: 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 4 seconds, Blot force -13,1, Wait time 10 seconds, Drain time 0.5 seconds,.
DetailsRsaA N-terminal domain with LPS soaked with 5 mM HoCl3 for 1.5 h on ice before vitrification

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Electron microscopy

MicroscopeFEI TITAN KRIOS
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 FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: -4.0 µm / Nominal defocus min: -1.0 µm / Nominal magnification: 130000
Specialist opticsEnergy filter - Name: GIF Quantum LS / Energy filter - Slit width: 20 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
TemperatureMin: 70.0 K / Max: 70.0 K
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: 2 / Number real images: 2038 / Average exposure time: 8.0 sec. / Average electron dose: 44.8 e/Å2
Details: Two data collections: First: 0 degree stage tilt with 903 collected movies. Second: 30 degree stage tilt with 1135 collected movies
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 545533
Details: Initial Particles were extracted in a 2x down-sampled 150 pixel x 150 pixel box and classified using reference-free 2D-classification inside RELION 3.0.
CTF correctionSoftware - Name: CTFFIND (ver. 4.1.13)
Software - details: CTFFIND was used as implemented in Relion 3.0
Details: RELION refinement with in-built CTF correction. The function is similar to a Wiener filter, so amplitude correction included.
Startup modelType of model: EMDB MAP
EMDB ID:

10389


Details: 30 A lowpass filtered reference map of EMD-10389 was used as starting model.
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0) / Details: Angle assignment was performed within RELION 3.0.
Final 3D classificationNumber classes: 2 / Software - Name: RELION (ver. 3.0)
Details: Particles from classes showing high-resolution features from both datasets were merged, re-extracted in a 300 pixel x 300 pixel box and were subjected to 3D classification using a 30 A ...Details: Particles from classes showing high-resolution features from both datasets were merged, re-extracted in a 300 pixel x 300 pixel box and were subjected to 3D classification using a 30 A lowpass filtered reference map of EMD-10389 (von Kuegelgen et al., 2020).
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0) / Details: Angle assignment was performed within RELION 3.0.
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 4.37 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0)
Details: The final map was obtained from 158,430 particles and post-processed using a soft mask focused on the inner fourteen subunits yielding a resolution of 4.37 A according to the gold standard ...Details: The final map was obtained from 158,430 particles and post-processed using a soft mask focused on the inner fourteen subunits yielding a resolution of 4.37 A according to the gold standard Fourier shell correlation criterion of 0.143 (Scheres, 2012) with some anisotropy in Z as judged by directional FSCs (Tan et al., 2017)
Number images used: 158430
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).

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Atomic model buiding 1

Initial modelPDB ID:

6t72


Chain - Chain ID: A
DetailsThe atomic coordinates (PDB ID 6T72) of our previous cryo-EM structure (von Kugelgen et al., 2020) of the RsaANTD oligomer bound to the O-antigen of lipopolysaccharide (LPS) were rigid body fitted into the final post-processed map from Relion 3.0 (Zivanov et al., 2018) using UCSF Chimera (Pettersen et al., 2004). The resulting fitted model was subjected to real-space refinement using Refmac5 (Murshudov et al., 2011) inside the CCP-EM suite (Burnely et al., 2017), as described previously (von Kugelgen et al., 2020), using reference restraints of the initial structure (PDB ID 6T72) generated with PROSMART (Nicholls et al. 2012).
RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-7peo:
Structure of the Caulobacter crescentus S-layer protein RsaA N-terminal domain bound to LPS and soaked with Holmium

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