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- EMDB-16486: In vitro Nitrosopumilus maritimus S-layer with NH4Cl -

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Basic information

Entry
Database: EMDB / ID: EMD-16486
TitleIn vitro Nitrosopumilus maritimus S-layer with NH4Cl
Map dataPostProcessed map with B-factor sharpening
Sample
  • Organelle or cellular component: Nitrosopumilus maritimus S-layer
    • Protein or peptide: In vitro Nitrosopumilus maritimus S-layer with increased NH4Cl - C2 symmetrised
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: 3.05 Å
Authorsvon Kuegelgen A / van Dorst S / Bharat TAM
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_16486.png

Downloads & links

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Map

FileDownload / File: emd_16486.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: 0.0087
Minimum - Maximum-0.034883097 - 0.051341545
Average (Standard dev.)0.000017866323 (±0.003471657)
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_16486_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
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Histogram

Histogram (log scale)

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Additional map: Refine3D main map without B-factor sharpening

Fileemd_16486_additional_1.map
AnnotationRefine3D main map without B-factor sharpening
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map 1

Fileemd_16486_half_map_1.map
AnnotationHalf map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map 2

Fileemd_16486_half_map_2.map
AnnotationHalf map 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
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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: In vitro Nitrosopumilus maritimus S-layer with increased NH4Cl - C2 symmetrised

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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: In vitro Nitrosopumilus maritimus S-layer with increased NH4Cl - ...

MacromoleculeName: In vitro Nitrosopumilus maritimus S-layer with increased NH4Cl - C2 symmetrised
type: protein_or_peptide / ID: 1 / Details: NH4Cl (ammonium chloride) / Enantiomer: LEVO
Source (natural)Organism: Nitrosopumilus maritimus SCM1 (archaea) / Strain: SCM1
SequenceString: MNNEIGRKIT SLTLMTIMVA GGLTFAIPGV MPEAMAANAN LFVSAENSQF DNYMSGPQVI EVVVIDSDIN DTDEAKGEPD VTVNGKVLRM VQAVDGNWYG YFADRDQAQI ADSTATTADS GLDFGVFCAS SSGTAALGFS TTETDGIAIP ITIANATATG NGTQTGSSSG ...String:
MNNEIGRKIT SLTLMTIMVA GGLTFAIPGV MPEAMAANAN LFVSAENSQF DNYMSGPQVI EVVVIDSDIN DTDEAKGEPD VTVNGKVLRM VQAVDGNWYG YFADRDQAQI ADSTATTADS GLDFGVFCAS SSGTAALGFS TTETDGIAIP ITIANATATG NGTQTGSSSG GAITTTCAAN TLDASTANGT INVVREAKDP VAASGSVSVG QIGLKNGTAN SGPNWPFIQL YELNPTGNVV VQYNKGGGVQ STTLTFDTVD QFAELSLDRT VFPRVSQVHA TITDLWLNID PTDEDSWTFA TNTKNTTSSF NVDTFYQVFD ENGASGGSAL TLRTTLSSLM CEDNCVLTLD VDAQSSGTPV VTIQDNGDSI LTQLNASSNT NANNASAFGI STETAKLGTG SIPVTITEQG PNSGVFGTYD ESDKSVLKIT DNAKRGTSAS LDYNETPQTI LVGFSFASID IQPVTDEWTS GQEIPVVIVD ADQNKNSRAD EDLDLNNPDV TLIPALRTGD PFTIDEGGTP SLIFTNGTNG DDSIFDTGAI NNTSAGQVGN FTLNINVTRF SSATNITSTE SIDTFSKRLI SAQTANSSAN FDVDFAIIDL GSATLETLKE TVVDEDNTAV GFNFFNYDVR SLGADTVSIA LLNTTGNILP WVNNDTRNVD KNNAILLVSN STNSQAYVDL TNAVSDAVYG STNTDSNVNI GFAMYFTGVG DLAAKEVIVM DFFSFGFTDD GVQSSERFAN QIIRIEAEET GDNTSTFEGS LEYVMVNQIN IQDAGTFSGI TPIADDPSFI VIEDLTDEDA PRVNYNDLGA DGVTTPVSDQ EEAPSHSGVV SLNADSYKIA DTVVITVEDL DLNVDSDLID IFTVVSDNSK ATDDAVGSAT TQSLSFGELG RLLDVTFDDV IWSTPDGANN TATGNDSDTC STELSNAGIT DTGLGATGFT LVETGAATGV FVGDFQIPSF WCRVSDTTTT PYTYAGDEET TTGLDIEVNY VDFRDASGEI VEVGDSAGVR ANTGSVSLDR TVYPVPFGTI ADSSKAANAA PNGRSVFPIH ATGITSTIDS TEELPTGDLT IHVRINDPDF DENPAGEDAM DQDNALKISV IRGSDSVVLG YAGASERTGK IDVGGNNGTI SNIRSFGEMD EIAPDAGIFE LDVNIKFTDG PASAQCNSHD TLYTALDGTT GKADTNRFDD GAASGQEYCI LQGDILQVEY TDPADASGDA NTVTDSATFD LRNGVLQSDK SVYIIGSDMI LTLIEPDFDL DNDSAETYDL DLIEWDSDAA TTTMGNKGVT GAAAAFDPEP TDFRETGDST GIFQIVIEIP ESLSNDKLER GEEIILEYTD WGPSGSDYVG DEDEDVNLTI YTSNFGATVE LDQKVYSWTD KVYITIVAPD HNFDSDLVDE IGETDSDPIK VSTRGFDLDN YKLVETGTDT GIFTGEVILT GFTAHDADGD GNTGDATGTT SGSGPTDGLL ATDDDDGLTV SFEFSEDETI VGSALIRWNI GEVQWLEASY PASGTGVVRV IDPDMNLDPE AVDNFEVDVW SDSDAGGIDL TVTETNEATG IFEGTVFFTT LDESSGHRLR VSEGDTVTAE YEDNTLPDPY TTADELDITA TSLIGTVVPP LERAPAANLR TVDAFGNSLD SVSVDQQVQI SADLANGQDR EQSFAYLVQI QDANGVTVSL AWITGSLSSG QSFSPALSWI PTEAGTYTAT AFVWESVDNP TALSPPVSTT VNVS

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
2.5 mMNH4ClNH4Cl

Details: 50 mM HEPES/NaOH pH=7.5, 500 mM NaCl, 50 mM MgCl2, 10 mM CaCl2, 2.5 mM NHCl4
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: 9435 / Average exposure time: 4.2 sec. / Average electron dose: 49.863 e/Å2
Details: collected over two sessions, one session with the stage tilted by 30 deg
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 2100081
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: 3.05 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 217668
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: 52.5 / Target criteria: Best Fit

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