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- EMDB-16492: In situ structure of the Nitrosopumilus maritimus S-layer - Compo... -
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Basic information
Entry | ![]() | |||||||||
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Title | In situ structure of the Nitrosopumilus maritimus S-layer - Composite map between C2 and C6 | |||||||||
![]() | Composite map between C2 and C6 | |||||||||
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![]() | Nmar_1547 S-layer / ![]() | |||||||||
Function / homology | ![]() ![]() | |||||||||
Biological species | ![]() ![]() | |||||||||
Method | subtomogram averaging / ![]() | |||||||||
![]() | 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
Supplemental images |
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Downloads & links
-EMDB archive
Map data | ![]() | 9.9 MB | ![]() | |
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Header (meta data) | ![]() ![]() | 20.5 KB 20.5 KB | Display Display | ![]() |
Images | ![]() | 241.7 KB | ||
Filedesc metadata | ![]() | 8.3 KB | ||
Archive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Related structure data | ![]() 8c8rMC ![]() 8c8kC ![]() 8c8lC ![]() 8c8mC ![]() 8c8nC ![]() 8c8oC C: citing same article ( M: atomic model generated by this map |
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Similar structure data | Similarity search - Function & homology ![]() |
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Links
EMDB pages | ![]() ![]() |
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Map
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Annotation | Composite map between C2 and C6 | ||||||||||||||||||||
Voxel size | X=Y=Z: 1.327 Å | ||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||
Details | EMDB XML:
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-Supplemental data
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Sample components
-Entire : Nitrosopumilus maritimus S-layer
Entire | Name: Nitrosopumilus maritimus S-layer |
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Components |
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-Supramolecule #1: Nitrosopumilus maritimus S-layer
Supramolecule | Name: Nitrosopumilus maritimus S-layer / type: organelle_or_cellular_component / ID: 1 / Parent: 0 / Macromolecule list: all / Details: Nitrosopumilus maritimus S-layer C2 symmetrised |
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Source (natural) | Organism: ![]() ![]() |
-Macromolecule #1: Cell surface protein
Macromolecule | Name: Cell surface protein / type: protein_or_peptide / ID: 1 / Number of copies: 6 / Enantiomer: LEVO |
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Source (natural) | Organism: ![]() ![]() |
Molecular weight | Theoretical: 183.156 KDa |
Sequence | String: MNNEIGRKIT SLTLMTIMVA GGLTFAIPGV MPEAMAANAN LFVSAENSQF DNYMSGPQVI EVVVIDSDIN DTDEAKGEPD VTVNGKVLR MVQAVDGNWY GYFADRDQAQ IADSTATTAD SGLDFGVFCA SSSGTAALGF STTETDGIAI PITIANATAT G NGTQTGSS ...String: MNNEIGRKIT SLTLMTIMVA GGLTFAIPGV MPEAMAANAN LFVSAENSQF DNYMSGPQVI EVVVIDSDIN DTDEAKGEPD VTVNGKVLR MVQAVDGNWY GYFADRDQAQ IADSTATTAD SGLDFGVFCA SSSGTAALGF STTETDGIAI PITIANATAT G NGTQTGSS SGGAITTTCA ANTLDASTAN GTINVVREAK DPVAASGSVS VGQIGLKNGT ANSGPNWPFI QLYELNPTGN VV VQYNKGG GVQSTTLTFD TVDQFAELSL DRTVFPRVSQ VHATITDLWL NIDPTDEDSW TFATNTKNTT SSFNVDTFYQ VFD ENGASG GSALTLRTTL SSLMCEDNCV LTLDVDAQSS GTPVVTIQDN GDSILTQLNA SSNTNANNAS AFGISTETAK LGTG SIPVT ITEQGPNSGV FGTYDESDKS VLKITDNAKR GTSASLDYNE TPQTILVGFS FASIDIQPVT DEWTSGQEIP VVIVD ADQN KNSRADEDLD LNNPDVTLIP ALRTGDPFTI DEGGTPSLIF TNGTNGDDSI FDTGAINNTS AGQVGNFTLN INVTRF SSA TNITSTESID TFSKRLISAQ TANSSANFDV DFAIIDLGSA TLETLKETVV DEDNTAVGFN FFNYDVRSLG ADTVSIA LL NTTGNILPWV NNDTRNVDKN NAILLVSNST NSQAYVDLTN AVSDAVYGST NTDSNVNIGF AMYFTGVGDL AAKEVIVM D FFSFGFTDDG VQSSERFANQ IIRIEAEETG DNTSTFEGSL EYVMVNQINI QDAGTFSGIT PIADDPSFIV IEDLTDEDA PRVNYNDLGA DGVTTPVSDQ EEAPSHSGVV SLNADSYKIA DTVVITVEDL DLNVDSDLID IFTVVSDNSK ATDDAVGSAT TQSLSFGEL GRLLDVTFDD VIWSTPDGAN NTATGNDSDT CSTELSNAGI TDTGLGATGF TLVETGAATG VFVGDFQIPS F WCRVSDTT TTPYTYAGDE ETTTGLDIEV NYVDFRDASG EIVEVGDSAG VRANTGSVSL DRTVYPVPFG TIADSSKAAN AA PNGRSVF PIHATGITST IDSTEELPTG DLTIHVRIND PDFDENPAGE DAMDQDNALK ISVIRGSDSV VLGYAGASER TGK IDVGGN NGTISNIRSF GEMDEIAPDA GIFELDVNIK FTDGPASAQC NSHDTLYTAL DGTTGKADTN RFDDGAASGQ EYCI LQGDI LQVEYTDPAD ASGDANTVTD SATFDLRNGV LQSDKSVYII GSDMILTLIE PDFDLDNDSA ETYDLDLIEW DSDAA TTTM GNKGVTGAAA AFDPEPTDFR ETGDSTGIFQ IVIEIPESLS NDKLERGEEI ILEYTDWGPS GSDYVGDEDE DVNLTI YTS NFGATVELDQ KVYSWTDKVY ITIVAPDHNF DSDLVDEIGE TDSDPIKVST RGFDLDNYKL VETGTDTGIF TGEVILT GF TAHDADGDGN TGDATGTTSG SGPTDGLLAT DDDDGLTVSF EFSEDETIVG SALIRWNIGE VQWLEASYPA SGTGVVRV I DPDMNLDPEA VDNFEVDVWS DSDAGGIDLT VTETNEATGI FEGTVFFTTL DESSGHRLRV SEGDTVTAEY EDNTLPDPY TTADELDITA TSLIGTVVPP LERAPAANLR TVDAFGNSLD SVSVDQQVQI SADLANGQDR EQSFAYLVQI QDANGVTVSL AWITGSLSS GQSFSPALSW IPTEAGTYTA TAFVWESVDN PTALSPPVST TVNVS UniProtKB: Uncharacterized protein |
-Experimental details
-Structure determination
Method | ![]() |
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![]() | subtomogram averaging |
Aggregation state | cell |
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Sample preparation
Buffer | pH: 7.4 Component:
Details: Modified Syntheic Crenarchaeota medium | ||||||||||||||||||||||||||||||||||||||||||||||||
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Grid | Model: Quantifoil R2/2 / Material: COPPER/RHODIUM / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY ARRAY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 15 sec. / Pretreatment - Atmosphere: AIR / Details: 15 mA | ||||||||||||||||||||||||||||||||||||||||||||||||
Vitrification | Cryogen name: NITROGEN / Chamber humidity: 100 % / Chamber temperature: 283.15 K / Instrument: FEI VITROBOT MARK IV Details: absorption for 60 sec and blotted for 5 sec with blot force -10. | ||||||||||||||||||||||||||||||||||||||||||||||||
Details | Nitrosopumilus maritimus cells |
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Electron microscopy
Microscope | FEI TITAN KRIOS |
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Electron beam | Acceleration voltage: 300 kV / Electron source: ![]() |
Electron optics | C2 aperture diameter: 70.0 µm / Calibrated defocus max: 5.0 µm / Calibrated defocus min: 2.0 µm / Calibrated magnification: 105000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD![]() |
Specialist optics | Spherical aberration corrector: not used / Chromatic aberration corrector: not used / Energy filter - Name: GIF Quantum LS / Energy filter - Slit width: 20 eV |
Sample stage | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN |
Temperature | Min: 70.0 K / Max: 70.0 K |
Image recording | Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Frames/image: 1-10 / Number real images: 1 / Average exposure time: 0.9 sec. / Average electron dose: 2.96 e/Å2 / Details: Dose-symmetric tilt scheme (Hagen et al, JSB) |
Experimental equipment | ![]() Model: Titan Krios / Image courtesy: FEI Company |
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Image processing
Extraction | Number tomograms: 153 / Number images used: 138532 / Reference model: None / Software - Name: RELION (ver. 4.0.0) |
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Final 3D classification | Software - Name: RELION (ver. 4.0.0) |
Final angle assignment | Type: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 4.0.0) / Details: RELION 4.0.0 |
Final reconstruction | Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.5 Å / Resolution method: OTHER / Software - Name: RELION (ver. 4.0.0) / Details: Composite map between C2 and C6 / Number subtomograms used: 108621 |
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. |
-Atomic model buiding 1
Refinement | Space: RECIPROCAL / Protocol: AB INITIO MODEL / Target criteria: Best Fit |
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Output model | ![]() PDB-8c8r: |