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- EMDB-16483: In vitro structure of the Nitrosopumilus maritimus S-layer - Two-... -

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Basic information

Entry
Database: EMDB / ID: EMD-16483
TitleIn vitro structure of the Nitrosopumilus maritimus S-layer - Two-fold symmetry (C2)
Map dataPostProcessed map with B-factor sharpening
Sample
  • Organelle or cellular component: Nitrosopumilus maritimus S-layer
    • Protein or peptide: Cell surface proteinCell membrane
KeywordsNmar_1547 S-layer / STRUCTURAL PROTEIN
Function / homologymembrane / Uncharacterized protein
Function and homology information
Biological speciesNitrosopumilus maritimus SCM1 (archaea)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.71 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: To Be Published
Title: Membrane-less channels sieve cations in ammonia-oxidising marine archaea
Authors: von Kuegelgen A / Cassidy CK / van Dorst S / Pagani LL / Ford Z / Loewe J / Stansfeld PJ / Bharat TAM
History
DepositionJan 20, 2023-
Header (metadata) releaseApr 10, 2024-
Map releaseApr 10, 2024-
UpdateApr 10, 2024-
Current statusApr 10, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_16483.png

Downloads & links

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Map

FileDownload / File: emd_16483.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationPostProcessed map with B-factor sharpening
Voxel sizeX=Y=Z: 1.092 Å
Density
Contour LevelBy AUTHOR: 0.01471
Minimum - Maximum-0.059272207 - 0.108998545
Average (Standard dev.)0.000040543782 (±0.0058679176)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 349.44 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_16483_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Full map with out b-factor sharpening

Fileemd_16483_additional_1.map
AnnotationFull map with out b-factor sharpening
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map 2

Fileemd_16483_half_map_1.map
AnnotationHalf map 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map 1

Fileemd_16483_half_map_2.map
AnnotationHalf map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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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 proteinCell membrane

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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 / Details: In-vitro isolated S-layer / 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
Processingsingle particle reconstruction
Aggregation state2D array

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Sample preparation

BufferpH: 7.5
Component:
ConcentrationFormulaName
50.0 mMC8H18N2O4SHEPES
500.0 mMNaClSodium chlorideNaClSodium chloride
50.0 mMMgCl2MgCl2
10.0 mMCaCl2CaCl2

Details: 50 mM HEPES/NaOH pH=7.5, 500 mM NaCl, 50 mM MgCl2, 10 mM CaCl2
GridModel: Quantifoil R2/2 / Material: COPPER/RHODIUM / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY ARRAY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 20 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.
DetailsIn vitro isolate S-layer cell envelopes

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Electron microscopy

MicroscopeFEI TITAN KRIOS
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: 81000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 5.0 µm / Nominal defocus min: 2.0 µm / Nominal magnification: 81000
Specialist opticsSpherical aberration corrector: not used / Chromatic aberration corrector: not used / Energy filter - Name: GIF Quantum LS / Energy filter - Slit width: 20 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
TemperatureMin: 70.0 K / Max: 70.0 K
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Digitization - Dimensions - Width: 5760 pixel / Digitization - Dimensions - Height: 4092 pixel / Number grids imaged: 3 / Number real images: 12557 / Average exposure time: 4.2 sec. / Average electron dose: 48.5 e/Å2
Details: collected over three sessions with one session with the stage tilted by 33 degrees (alpha tilt)
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber 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.
Startup modelType of model: NONE
Details: All side views and a subset of top and bottom views were used for initial model generation in RELION-3.1. The scaled and lowpass filtered output was then used as a starting model for 3D auto ...Details: All side views and a subset of top and bottom views were used for initial model generation in RELION-3.1. The scaled and lowpass filtered output was then used as a starting model for 3D auto refinement in a 512x512 pixel box.
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1) / Details: Angle assignment was performed within RELION3.1
Final 3D classificationSoftware - Name: RELION (ver. 3.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1) / Details: Angle assignment was performed within RELION3.1
Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 2.71 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1)
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
Number images used: 354860
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.
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: RECIPROCAL / Protocol: AB INITIO MODEL / Overall B value: 41.4 / Target criteria: Best Fit
Output model

PDB-8c8l:
In vitro structure of the Nitrosopumilus maritimus S-layer - Two-fold symmetry (C2)

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