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- PDB-8c8n: In situ structure of the Nitrosopumilus maritimus S-layer - Two-f... -

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Basic information

Entry
Database: PDB / ID: 8c8n
TitleIn situ structure of the Nitrosopumilus maritimus S-layer - Two-fold symmetry (C2)
ComponentsCell surface proteinCell membrane
KeywordsSTRUCTURAL PROTEIN / Nmar_1547 S-layer
Function / homologymembrane / Uncharacterized protein
Function and homology information
Biological speciesNitrosopumilus maritimus SCM1 (archaea)
MethodELECTRON MICROSCOPY / subtomogram averaging / cryo EM / Resolution: 3.4 Å
Authorsvon Kuegelgen, A. / Bharat, T.
Funding support United Kingdom, 2items
OrganizationGrant numberCountry
Wellcome Trust202231/Z/16/Z United Kingdom
Medical Research Council (MRC, United Kingdom)MC_UP_1201/31 United Kingdom
CitationJournal: Nature / Year: 2024
Title: Membraneless channels sieve cations in ammonia-oxidizing marine archaea.
Authors: Andriko von Kügelgen / C Keith Cassidy / Sofie van Dorst / Lennart L Pagani / Christopher Batters / Zephyr Ford / Jan Löwe / Vikram Alva / Phillip J Stansfeld / Tanmay A M Bharat /
Abstract: Nitrosopumilus maritimus is an ammonia-oxidizing archaeon that is crucial to the global nitrogen cycle. A critical step for nitrogen oxidation is the entrapment of ammonium ions from a dilute marine ...Nitrosopumilus maritimus is an ammonia-oxidizing archaeon that is crucial to the global nitrogen cycle. A critical step for nitrogen oxidation is the entrapment of ammonium ions from a dilute marine environment at the cell surface and their subsequent channelling to the cell membrane of N. maritimus. Here we elucidate the structure of the molecular machinery responsible for this process, comprising the surface layer (S-layer), using electron cryotomography and subtomogram averaging from cells. We supplemented our in situ structure of the ammonium-binding S-layer array with a single-particle electron cryomicroscopy structure, revealing detailed features of this immunoglobulin-rich and glycan-decorated S-layer. Biochemical analyses showed strong ammonium binding by the cell surface, which was lost after S-layer disassembly. Sensitive bioinformatic analyses identified similar S-layers in many ammonia-oxidizing archaea, with conserved sequence and structural characteristics. Moreover, molecular simulations and structure determination of ammonium-enriched specimens enabled us to examine the cation-binding properties of the S-layer, revealing how it concentrates ammonium ions on its cell-facing side, effectively acting as a multichannel sieve on the cell membrane. This in situ structural study illuminates the biogeochemically essential process of ammonium binding and channelling, common to many marine microorganisms that are fundamental to the nitrogen cycle.
History
DepositionJan 20, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Apr 10, 2024Provider: repository / Type: Initial release
Revision 1.1Jun 5, 2024Group: Database references / Category: citation
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.title / _citation.year
Revision 1.2Jun 12, 2024Group: Database references / Category: citation / citation_author / Item: _citation.pdbx_database_id_PubMed / _citation.title

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Cell surface protein
B: Cell surface protein
C: Cell surface protein
D: Cell surface protein
E: Cell surface protein
F: Cell surface protein


Theoretical massNumber of molelcules
Total (without water)1,098,9366
Polymers1,098,9366
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area11500 Å2
ΔGint-20 kcal/mol
Surface area84840 Å2

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Components

#1: Protein
Cell surface protein / Cell membrane


Mass: 183156.000 Da / Num. of mol.: 6 / Source method: isolated from a natural source / Source: (natural) Nitrosopumilus maritimus SCM1 (archaea) / Strain: SCM1 / References: UniProt: A9A4Y9

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: CELL / 3D reconstruction method: subtomogram averaging

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Sample preparation

ComponentName: Nitrosopumilus maritimus S-layer / Type: ORGANELLE OR CELLULAR COMPONENT / Details: Nitrosopumilus maritimus S-layer C2 symmetrised / Entity ID: all / Source: NATURAL
Molecular weightExperimental value: NO
Source (natural)Organism: Nitrosopumilus maritimus SCM1 (archaea) / Strain: SCM1 / Cellular location: extracellular
Buffer solutionpH: 7.4 / Details: Modified Syntheic Crenarchaeota medium
Buffer component
IDConc.NameFormulaBuffer-ID
110 mMHEPESC8H18N2O4S1
2444.9 mMNaClSodium chlorideNaClSodium chloride1
320.286 mMMgSO4MgSO41
424.59 mMMgCl2MgCl21
510.2 mMCaCl2CaCl21
61 mMNH4ClNH4Cl1
714.696 microMKH2PO4KH2PO41
87.5 microMFeNaEDTAFeNaC10H12N2O81
90.5 microMH3BO3H3BO31
100.5 microMMnCl2MnCl21
110.8 microMCoCl2CoCl21
120.1 microMNiCl2NiCl21
130.01 microMCuCl2CuCl21
140.5 microMZnSO4ZnSO41
150.15 microMNa2MoO4Na2MoO41
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: Nitrosopumilus maritimus cells
Specimen supportDetails: 15 mA / Grid material: COPPER/RHODIUM / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R2/2
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: NITROGEN / Humidity: 100 % / Chamber temperature: 283.15 K
Details: absorption for 60 sec and blotted for 5 sec with blot force -10

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 105000 X / Calibrated magnification: 105000 X / Nominal defocus max: 5000 nm / Nominal defocus min: 2000 nm / Calibrated defocus min: 2000 nm / Calibrated defocus max: 5000 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 70 K / Temperature (min): 70 K
Image recordingAverage exposure time: 0.9 sec. / Electron dose: 2.96 e/Å2 / Avg electron dose per subtomogram: 121 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of real images: 1 / Details: Dose-symmetric tilt scheme (Hagen et al)
EM imaging opticsEnergyfilter name: GIF Quantum LS / Chromatic aberration corrector: not used / Energyfilter slit width: 20 eV / Spherical aberration corrector: not used
Image scansMovie frames/image: 10 / Used frames/image: 1-10

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Processing

SoftwareName: PHENIX / Version: 1.19_4092: / Classification: refinement
EM software
IDNameVersionCategoryDetails
1RELION4.0.0volume selection
2SerialEMimage acquisition
4CTFFIND4.1.13CTF correctionCTF estimation
5RELION4.0.0CTF correctionCTF correction
8Coot0.9.2-premodel fitting
10PHENIX1.19-4092model refinement
11REFMACmodel refinement
12RELION3.1initial Euler assignment
13RELION4.0.0final Euler assignment
14RELION4.0.0classification
15RELION4.0.03D reconstruction
CTF correctionDetails: PseudoSubtomograms as described in Zivanov 2022 (https://elifesciences.org/articles/83724)
Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1971908
Details: Initially, side views of S-layer sheets were first manually picked along the edge of the lattice using the helical picking tab in RELION while setting the helical rise to 60 angstrom. Top ...Details: Initially, side views of S-layer sheets were first manually picked along the edge of the lattice using the helical picking tab in RELION while setting the helical rise to 60 angstrom. Top and tilted views were manually picked at the central hexameric axis. Manually picked particles were extracted in 4x downsampled 128x128 pixel2 boxes and classified using reference-free 2D classification inside RELION-3.1. Class averages centered at a hexameric axis were used to automatically pick particles inside RELION-3.1. Automatically picked particles were extracted in 4x downsampled 128x128 pixel2 boxes and classified using reference-free 2D classification. Particle coordinates belonging to class averages centered at the hexameric axis were used to train TOPAZ in 5x downsampled micrographs with the neural network architecture conv127. For the final reconstruction, particles were picked using TOPAZ and the previously trained neural network above. Additionally, top, bottom, and side views were picked using the reference-based autopicker inside RELION-3.1, which TOPAZ did not readily identify. Particles were extracted in 4x downsampled 128x128 pixel2 boxes and classified using reference-free 2D classification inside RELION-3.1. Particles belonging to class averages centered at the hexameric axis were combined, and particles within 30 angstrom were removed to prevent duplication after alignment. All resulting particles were then re-extracted in 4x downsampled 128x128 pixel2 boxes.
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 108621 / Algorithm: FOURIER SPACE / Symmetry type: POINT
EM volume selectionNum. of tomograms: 153 / Num. of volumes extracted: 138532 / Reference model: None
Atomic model buildingB value: 66.19 / Protocol: AB INITIO MODEL / Space: RECIPROCAL / Target criteria: Best Fit
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00218618
ELECTRON MICROSCOPYf_angle_d0.45825470
ELECTRON MICROSCOPYf_dihedral_angle_d4.2632592
ELECTRON MICROSCOPYf_chiral_restr0.0433084
ELECTRON MICROSCOPYf_plane_restr0.0033396

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