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- PDB-8c8k: In vitro structure of the Nitrosopumilus maritimus S-layer - Six-... -
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Open data
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Basic information
Entry | Database: PDB / ID: 8c8k | |||||||||
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Title | In vitro structure of the Nitrosopumilus maritimus S-layer - Six-fold symmetry (C6) | |||||||||
![]() | Cell surface protein | |||||||||
![]() | STRUCTURAL PROTEIN / Nmar_1547 S-layer | |||||||||
Function / homology | membrane / Uncharacterized protein![]() | |||||||||
Biological species | ![]() | |||||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.87 Å | |||||||||
![]() | von Kuegelgen, A. / Bharat, T. | |||||||||
Funding support | ![]()
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![]() | ![]() 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 |
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Structure visualization
Structure viewer | Molecule: ![]() ![]() |
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Downloads & links
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Download
PDBx/mmCIF format | ![]() | 440.8 KB | Display | ![]() |
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PDB format | ![]() | 267.6 KB | Display | ![]() |
PDBx/mmJSON format | ![]() | Tree view | ![]() | |
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-Validation report
Arichive directory | ![]() ![]() | HTTPS FTP |
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-Related structure data
Related structure data | ![]() 16482MC ![]() 8c8lC ![]() 8c8mC ![]() 8c8nC ![]() 8c8oC ![]() 8c8rC M: map data used to model this data C: citing same article ( |
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Similar structure data | Similarity search - Function & homology ![]() |
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Links
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Assembly
Deposited unit | ![]()
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Noncrystallographic symmetry (NCS) | NCS domain:
NCS domain segments:
NCS ensembles :
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Components
#1: Protein | Mass: 183156.000 Da / Num. of mol.: 6 / Source method: isolated from a natural source / Details: In-vitro isolated S-layer / Source: (natural) ![]() |
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-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: 2D ARRAY / 3D reconstruction method: single particle reconstruction |
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Sample preparation
Component | Name: Nitrosopumilus maritimus S-layer / Type: ORGANELLE OR CELLULAR COMPONENT / Details: Nitrosopumilus maritimus S-layer C6 symmetrised / Entity ID: all / Source: NATURAL | |||||||||||||||||||||||||
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Molecular weight | Experimental value: NO | |||||||||||||||||||||||||
Source (natural) | Organism: ![]() | |||||||||||||||||||||||||
Buffer solution | pH: 7.5 Details: 50 mM HEPES/NaOH pH=7.5, 500 mM NaCl, 50 mM MgCl2, 10 mM CaCl2 | |||||||||||||||||||||||||
Buffer component |
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Specimen | Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: In vitro isolate S-layer cell envelopes | |||||||||||||||||||||||||
Specimen support | Details: 15 mA / Grid material: COPPER/RHODIUM / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R2/2 | |||||||||||||||||||||||||
Vitrification | Instrument: 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 |
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Microscopy | Model: FEI TITAN KRIOS |
Electron gun | Electron source: ![]() |
Electron lens | Mode: BRIGHT FIELD / Nominal magnification: 81000 X / Calibrated magnification: 81000 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 holder | Cryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 70 K / Temperature (min): 70 K |
Image recording | Average exposure time: 4.2 sec. / Electron dose: 48.5 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 3 / Num. of real images: 12557 Details: collected over three sessions, two with no stage tilt and one session with stage tilted by 33 degrees (alpha tilt) |
EM imaging optics | Energyfilter name: GIF Quantum LS / Chromatic aberration corrector: not used / Energyfilter slit width: 20 eV / Spherical aberration corrector: not used |
Image scans | Width: 5760 / Height: 4092 |
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Processing
Software | Name: REFMAC / Version: 5.8.0267 / Classification: refinement | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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EM software |
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Image processing | Details: Movies collected at the scope were clustered into optics groups based on the XML meta-data of the data-collection software EPU (Thermo Fisher Scientific) using a k-means algorithm ...Details: Movies collected at the scope were clustered into optics groups based on the XML meta-data of the data-collection software EPU (Thermo Fisher Scientific) using a k-means algorithm implemented in EPU_group_AFIS (https://github.com/DustinMorado/EPU_group_AFIS). Imported movies were motion-corrected, dose-weighted, and Fourier cropped (2x) with MotionCor2 implemented in RELION-3.1. Contrast transfer functions (CTFs) of the resulting motion-corrected micrographs were estimated using CTFFIND4. | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
CTF correction | Details: RELION refinement with in-built CTF correction. The function is similar to a Wiener filter, so amplitude correction included. Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Particle selection | Num. 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. | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Symmetry | Point symmetry: C6 (6 fold cyclic) | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
3D reconstruction | Resolution: 2.87 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 354860 / Algorithm: FOURIER SPACE Details: Per-particle defocus, anisotropy magnification, and higher-order aberrations were refined inside RELION3.1, followed by three rounds of focused 3D auto refinement. Bayesian particle ...Details: Per-particle defocus, anisotropy magnification, and higher-order aberrations were refined inside RELION3.1, followed by three rounds of focused 3D auto refinement. Bayesian particle polishing was performed subsequently in a 640x640 pixel2 box followed by auto-refinement and symmetry relaxation. The final map was obtained from 354,860 particles and post-processed using a soft mask focused on the central hexamer, yielding a global resolution of 2.7 angstrom according to the Fourier shell correlation criterion between two independently refined half-maps at a threshold value at 0.143, and local resolution up to 2.5 angstrom Symmetry type: POINT | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Atomic model building | B value: 59.2 / Protocol: AB INITIO MODEL / Space: RECIPROCAL / Target criteria: Best Fit | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Refinement | Resolution: 2.87→2.87 Å / Cor.coef. Fo:Fc: 0.909 / SU B: 12.215 / SU ML: 0.232 / ESU R: 0.32 Stereochemistry target values: MAXIMUM LIKELIHOOD WITH PHASES Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
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Solvent computation | Solvent model: PARAMETERS FOR MASK CACLULATION | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Displacement parameters | Biso mean: 76.059 Å2
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Refinement step | Cycle: 1 / Total: 72246 | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Refine LS restraints |
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