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- EMDB-16694: Deinococcus radidurans HPI S-layer -

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Basic information

Entry
Database: EMDB / ID: EMD-16694
TitleDeinococcus radidurans HPI S-layer
Map dataPostProcessed map with B-factor sharpening.
Sample
  • Organelle or cellular component: Structure of Hexagonally Packed Intermediate-layer (HPI) protein
    • Protein or peptide: Hexagonally packed intermediate-layer surface protein
Function / homologyS-layer / cell wall organization / Prokaryotic membrane lipoprotein lipid attachment site profile. / extracellular region / Hexagonally packed intermediate-layer surface protein
Function and homology information
Biological speciesDeinococcus radiodurans (radioresistant) / Deinococcus radiodurans (strain ATCC 13939 / DSM 20539 / JCM 16871 / LMG 4051 / NBRC 15346 / NCIMB 9279 / R1 / VKM B-1422) (radioresistant)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.52 Å
Authorsvon Kuegelgen A / Yamashita K / Murshudov G / 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: Proc Natl Acad Sci U S A / Year: 2023
Title: Interdigitated immunoglobulin arrays form the hyperstable surface layer of the extremophilic bacterium .
Authors: Andriko von Kügelgen / Sofie van Dorst / Keitaro Yamashita / Danielle L Sexton / Elitza I Tocheva / Garib Murshudov / Vikram Alva / Tanmay A M Bharat /
Abstract: is an atypical diderm bacterium with a remarkable ability to tolerate various environmental stresses, due in part to its complex cell envelope encapsulated within a hyperstable surface layer (S- ... is an atypical diderm bacterium with a remarkable ability to tolerate various environmental stresses, due in part to its complex cell envelope encapsulated within a hyperstable surface layer (S-layer). Despite decades of research on this cell envelope, atomic structural details of the S-layer have remained obscure. In this study, we report the electron cryomicroscopy structure of the S-layer, showing how it is formed by the Hexagonally Packed Intermediate-layer (HPI) protein arranged in a planar hexagonal lattice. The HPI protein forms an array of immunoglobulin-like folds within the S-layer, with each monomer extending into the adjacent hexamer, resulting in a highly interconnected, stable, sheet-like arrangement. Using electron cryotomography and subtomogram averaging from focused ion beam-milled cells, we have obtained a structure of the cellular S-layer, showing how this HPI S-layer coats native membranes on the surface of cells. Our S-layer structure from the diderm bacterium shows similarities to immunoglobulin-like domain-containing S-layers from monoderm bacteria and archaea, highlighting common features in cell surface organization across different domains of life, with connotations on the evolution of immunoglobulin-based molecular recognition systems in eukaryotes.
History
DepositionFeb 14, 2023-
Header (metadata) releaseApr 19, 2023-
Map releaseApr 19, 2023-
UpdateApr 26, 2023-
Current statusApr 26, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_16694.png

Downloads & links

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Map

FileDownload / File: emd_16694.map.gz / Format: CCP4 / Size: 244.1 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: 0.01187
Minimum - Maximum-0.02526621 - 0.065192774
Average (Standard dev.)-2.4295787e-05 (±0.002974129)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions400400400
Spacing400400400
CellA=B=C: 436.8 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_16694_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map 2

Fileemd_16694_half_map_1.map
AnnotationHalf map 2
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map 1

Fileemd_16694_half_map_2.map
AnnotationHalf map 1
Projections & Slices
AxesZYX

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Slices (1/2)
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Histogram

Histogram (log scale)

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Sample components

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Entire : Structure of Hexagonally Packed Intermediate-layer (HPI) protein

EntireName: Structure of Hexagonally Packed Intermediate-layer (HPI) protein
Components
  • Organelle or cellular component: Structure of Hexagonally Packed Intermediate-layer (HPI) protein
    • Protein or peptide: Hexagonally packed intermediate-layer surface protein

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Supramolecule #1: Structure of Hexagonally Packed Intermediate-layer (HPI) protein

SupramoleculeName: Structure of Hexagonally Packed Intermediate-layer (HPI) protein
type: organelle_or_cellular_component / ID: 1 / Parent: 0 / Macromolecule list: all
Details: Structure of Hexagonally Packed Intermediate-layer (HPI) protein
Source (natural)Organism: Deinococcus radiodurans (radioresistant) / Location in cell: extracellular

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Macromolecule #1: Hexagonally packed intermediate-layer surface protein

MacromoleculeName: Hexagonally packed intermediate-layer surface protein / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Deinococcus radiodurans (strain ATCC 13939 / DSM 20539 / JCM 16871 / LMG 4051 / NBRC 15346 / NCIMB 9279 / R1 / VKM B-1422) (radioresistant)
Molecular weightTheoretical: 99.424 KDa
SequenceString: MKKNIALMAL TGILTLASCG QNGTGTTPTA DACATANTCS VTVNISGVSS ADFDVTMDGK TTSMTLSNGQ KLPVAKTGTV TLTPKAKDG YTTPAAQSTT ISSTNLTPSV NFAYTTVPST GNGNGNGGTT PTQPFTLNIT SPTNGAAATT GTPIRVVFTS S VALSSATC ...String:
MKKNIALMAL TGILTLASCG QNGTGTTPTA DACATANTCS VTVNISGVSS ADFDVTMDGK TTSMTLSNGQ KLPVAKTGTV TLTPKAKDG YTTPAAQSTT ISSTNLTPSV NFAYTTVPST GNGNGNGGTT PTQPFTLNIT SPTNGAAATT GTPIRVVFTS S VALSSATC KIGNSAAVNA QVSSTGGYCD VTPTTAGGGL ITVTGTANGQ TVSSTVTVDV KAPVVDNRYG TVTPAGDQEL TL TNEGIVK DADNGWRRLG QGVSTPSDPN GNVDIYVKGT VNFSVNAAAG SKVEVFLART TGSDVPTNDD VQAGDVLRSV AST SGTETF SLDSRRLAEF DGVRKWIVVR INGTQVTYQP VIADNKGPQQ PDPELNGVQN AYSNILNNYN NSGLTYVRGD VNVF TGNPS LQDREFGQAP LGSSFVQRRP SGFESIRYYL VPETAFGNKA LQESDEMLRA KAIKSVATVV SAPVLEPGTV KATSF SRVI GSGATSTVTP KAQDNVTYRV YAISRDQLGN ETASATYELV RFDNVGPTIT GSVIRDTSDL PFASQEPERC LSDIAT ITL GGITDNAGGV GLNPGQGLTF TLGGRQIQAG QFDTNQLADG EYTIGFNSLT DALGNPVVSA PTNAKVYIDN TDPTVNF NR AVMQGTFASG ERVSVESDAS DGGCGVYETR LFWDTDNGVV DDATTTPAIG HPVQFARQRV TDGAKADSLN AGWNALQL P NGAGAVYLRA LVVDRAGNAT ISTTPIVVNA KITNQARPLL GGFDAFKRNA SAQFMSNSNA ISGVNGTAVT PNTTANSAL DNILSLDSVG TLTTNAYLPR GATETAITEK IRNVGAYGRF DATQWNRIRD YQLNTDPTLR SAYVNAGNLA NQRGNNWRIR TPWVELGSS DTANTQQKFD FNSDLLNDFY FGRTFGNNDN VNLFSYDQFN GIVSGTAGAY SFYGETVQK

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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
150.0 mMNaClNaCl
5.0 mMMgCl2MgCl2
1.0 mMCaCl2CaCl2

Details: 50 mM HEPES/NaOH pH=7.5, 150 mM NaCl, 5 mM MgCl2, 1 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 Hexagonally Packed Intermediate-layer (HPI) layer

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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 K3 BIOQUANTUM (6k x 4k) / Digitization - Dimensions - Width: 5760 pixel / Digitization - Dimensions - Height: 4092 pixel / Number grids imaged: 1 / Number real images: 1002 / Average exposure time: 3.43 sec. / Average electron dose: 53.245 e/Å2
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 FIELD / Cs: 2.7 mm / Nominal defocus max: 5.0 µm / Nominal defocus min: 2.0 µm / Nominal magnification: 81000
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.
Particle selectionNumber selected: 882866
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 40 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 40 Angstrom. Top and tilted views were manually picked at the central hexameric axis. Manually picked particles were extracted in 4x downsampled 100 x 100 boxes and classified using reference-free 2D classification inside RELION3.1. Class averages centered at a hexameric axis were used to automatically pick particles inside RELION3.1. Automatically picked particles were extracted in 4x downsampled 100x100 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 RELION3.1, which TOPAZ did not readily identify. Particles were extracted in 4x downsampled 100x100 pixel2 boxes and classified using reference-free 2D classification inside RELION3.1. 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 100x100 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 pixel2 box.
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C6 (6 fold cyclic) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 2.52 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 another round of focused 3D auto refinement and Bayesian particle ...Details: Per-particle defocus, anisotropy magnification, and higher-order aberrations were refined inside RELION3.1, followed by another round of focused 3D auto refinement and Bayesian particle polishing. The final map was obtained from 55,345 particles and post-processed using a soft mask focused on the central hexamer, including the dimeric bridge, yielding a global resolution of 2.52 Angstrom according to the gold standard Fourier shell correlation criterion of 0.143.
Number images used: 55345
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1) / Details: Angle assignment was performed within RELION3.1
Final 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)
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: RECIPROCAL / Protocol: AB INITIO MODEL / Overall B value: 19.48 / Target criteria: Best Fit
Output model

PDB-8cka:
Deinococcus radidurans HPI S-layer

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