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- EMDB-16492: In situ structure of the Nitrosopumilus maritimus S-layer - Compo... -

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Basic information

Entry
Database: EMDB / ID: EMD-16492
TitleIn situ structure of the Nitrosopumilus maritimus S-layer - Composite map between C2 and C6
Map dataComposite map between C2 and C6
Sample
  • Organelle or cellular component: Nitrosopumilus maritimus S-layer
    • Protein or peptide: Cell surface protein
KeywordsNmar_1547 S-layer / STRUCTURAL PROTEIN
Function / homologymembrane / Uncharacterized protein
Function and homology information
Biological speciesNitrosopumilus maritimus SCM1 (archaea)
Methodsubtomogram averaging / cryo EM / Resolution: 3.5 Å
Authorsvon Kuegelgen A / Bharat T
Funding support United Kingdom, 2 items
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, 2023-
Header (metadata) releaseApr 17, 2024-
Map releaseApr 17, 2024-
UpdateNov 20, 2024-
Current statusNov 20, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_16492.png

Downloads & links

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Map

FileDownload / File: emd_16492.map.gz / Format: CCP4 / Size: 30.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationComposite map between C2 and C6
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.33 Å/pix.
x 200 pix.
= 265.4 Å		1.33 Å/pix.
x 200 pix.
= 265.4 Å		1.33 Å/pix.
x 200 pix.
= 265.4 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.327 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 1.45621
Minimum - Maximum-6.78924 - 12.055393
Average (Standard dev.)0.0009801809 (±0.58209246)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions200200200
Spacing200200200
CellA=B=C: 265.4 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : Nitrosopumilus maritimus S-layer

EntireName: Nitrosopumilus maritimus S-layer
Components
  • Organelle or cellular component: Nitrosopumilus maritimus S-layer
    • Protein or peptide: Cell surface protein

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Supramolecule #1: Nitrosopumilus maritimus S-layer

SupramoleculeName: Nitrosopumilus maritimus S-layer / type: organelle_or_cellular_component / ID: 1 / Parent: 0 / Macromolecule list: all / Details: Nitrosopumilus maritimus S-layer C2 symmetrised
Source (natural)Organism: Nitrosopumilus maritimus SCM1 (archaea) / Strain: SCM1 / Location in cell: extracellular

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Macromolecule #1: Cell surface protein

MacromoleculeName: Cell surface protein / type: protein_or_peptide / ID: 1 / Number of copies: 6 / Enantiomer: LEVO
Source (natural)Organism: Nitrosopumilus maritimus SCM1 (archaea) / Strain: SCM1
Molecular weightTheoretical: 183.156 KDa
SequenceString: 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

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Experimental details

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Structure determination

Methodcryo EM
Processingsubtomogram averaging
Aggregation statecell

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Sample preparation

BufferpH: 7.4
Component:
ConcentrationFormulaName
10.0 mMC8H18N2O4SHEPES
444.9 mMNaClNaCl
20.286 mMMgSO4MgSO4
24.59 mMMgCl2MgCl2
10.2 mMCaCl2CaCl2
1.0 mMNH4ClNH4Cl
14.696 microMKH2PO4KH2PO4
7.5 microMFeNaC10H12N2O8FeNaEDTA
0.5 microMH3BO3H3BO3
0.5 microMMnCl2MnCl2
0.8 microMCoCl2CoCl2
0.1 microMNiCl2NiCl2
0.01 microMCuCl2CuCl2
0.5 microMZnSO4ZnSO4
0.15 microMNa2MoO4Na2MoO4

Details: Modified Syntheic Crenarchaeota medium
GridModel: 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
VitrificationCryogen 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.
DetailsNitrosopumilus maritimus cells

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Electron microscopy

MicroscopeFEI TITAN KRIOS
TemperatureMin: 70.0 K / Max: 70.0 K
Specialist opticsSpherical aberration corrector: not used / Chromatic aberration corrector: not used / Energy filter - Name: GIF Quantum LS / Energy filter - Slit width: 20 eV
Image recordingFilm 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)
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 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 / Cs: 2.7 mm / Nominal defocus max: 5.0 µm / Nominal defocus min: 2.0 µm / Nominal magnification: 105000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

DetailsMovies 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.
Final reconstructionAlgorithm: 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
ExtractionNumber tomograms: 153 / Number images used: 138532 / Reference model: None / Software - Name: RELION (ver. 4.0.0)
Final 3D classificationSoftware - Name: RELION (ver. 4.0.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 4.0.0) / Details: RELION 4.0.0

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Atomic model buiding 1

RefinementSpace: RECIPROCAL / Protocol: AB INITIO MODEL / Target criteria: Best Fit
Output model

PDB-8c8r:
In situ structure of the Nitrosopumilus maritimus S-layer - Composite map between C2 and C6

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