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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
SampleStructure of the Caulobacter crescentus S-layer protein RsaA N-terminal domain bound to LPS and soaked with Holmium:
S-layer protein / (ligand) x 2
Function / homologyS-layer / RTX calcium-binding nonapeptide repeat / RTX calcium-binding nonapeptide repeat (4 copies) / Serralysin-like metalloprotease, C-terminal / cell wall / 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: 2021
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-
UpdateDec 1, 2021-
Current statusDec 1, 2021Processing 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

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)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.08 Å/pix.
x 300 pix.
= 324. Å
1.08 Å/pix.
x 300 pix.
= 324. Å
1.08 Å/pix.
x 300 pix.
= 324. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

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
Details: Structure of the Caulobacter crescentus S-layer protein RsaA N-terminal domain bound to LPS and soaked with Holmium
Number of Components: 4

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Component #1: protein, Structure of the Caulobacter crescentus S-layer protein ...

ProteinName: Structure of the Caulobacter crescentus S-layer protein RsaA N-terminal domain bound to LPS and soaked with Holmium
Details: Structure of the Caulobacter crescentus S-layer protein RsaA N-terminal domain bound to LPS and soaked with Holmium
Recombinant expression: No
SourceSpecies: Caulobacter vibrioides (bacteria) / Strain: YB1001

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Component #2: protein, S-layer protein

ProteinName: S-layer protein / Details: LPS O-antigen bound to the protein / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 25.820354 kDa
SourceSpecies: Caulobacter vibrioides (bacteria) / Strain: YB1001
Source (engineered)Expression System: Caulobacter vibrioides CB15 (bacteria)

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Component #3: ligand, CALCIUM ION

LigandName: CALCIUM IONCalcium / Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 4.007805 MDa

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Component #4: ligand, HOLMIUM ATOM

LigandName: HOLMIUM ATOM / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 0.16493 kDa

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Experimental details

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Sample preparation

SpecimenSpecimen State: Particle / Method: cryo EM
Sample solutionSpecimen conc.: 2.25 mg/mL
Buffer solution: 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 ...Buffer solution: 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.
pH: 7.5
Support film20 seconds, 15 mA
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen Name: ETHANE / Temperature: 283.15 K / Humidity: 100 %
Details: Vitrobot options: Blot time 4 seconds, Blot force -13,1, Wait time 10 seconds, Drain time 0.5 seconds,.

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
ImagingMicroscope: FEI TITAN KRIOS / Details: EPU software
Electron gunElectron Source: FIELD EMISSION GUN / Accelerating Voltage: 300 kV / Electron Dose: 44.8 e/Å2 / Illumination Mode: FLOOD BEAM
LensMagnification: 130000.0 X (nominal), 130000.0 X (calibrated)
Cs: 2.7 mm / Imaging Mode: BRIGHT FIELD / Defocus: -1000.0 - -4000.0 nm / Energy Filter: GIF Quantum LS
Specimen HolderModel: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature: (70.0 - 70.0 K)
CameraDetector: GATAN K2 SUMMIT (4k x 4k)

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Image acquisition

Image acquisitionNumber of Digital Images: 2038
Details: Two data collections: First: 0 degree stage tilt with 903 collected movies. Second: 30 degree stage tilt with 1135 collected movies

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Image processing

ProcessingMethod: single particle reconstruction / Applied Symmetry: C1 (asymmetric) / Number of Projections: 158430
Details: Movies 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 ...Details: Movies 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).
3D reconstructionAlgorithm: FOURIER SPACE / Software: RELION
CTF correction: RELION refinement with in-built CTF correction. The function is similar to a Wiener filter, so amplitude correction included.
Resolution: 4.37 Å / Resolution Method: FSC 0.143 CUT-OFF
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)
Euler angles: Angle assignment was performed within RELION 3.0.

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Atomic model buiding

Modeling #1Refinement protocol: rigid body / Refinement space: REAL
Details: The 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 ...Details: The 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).
Input PDB model: 6T72
Chain ID: A
Output model

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