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- EMDB-13639: In-vitro structure of inverted S-layer tube -

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Basic information

Entry
Database: EMDB / ID: EMD-13639
TitleIn-vitro structure of inverted S-layer tube
Map dataPostprocessed map without B-factor sharpening
Sample
  • Complex: In-vitro structure of inverted S-layer tube
    • Protein or peptide: S-layer protein
Function / homologySurface glycoprotein signal peptide / Major cell surface glycoprotein / PGF-CTERM archaeal protein-sorting signal / PGF-CTERM motif / S-layer / cell wall organization / extracellular region / plasma membrane / Cell surface glycoprotein
Function and homology information
Biological speciesHaloferax volcanii DS2 (archaea)
Methodsubtomogram averaging / cryo EM / Resolution: 15.87 Å
Authorsvon Kuegelgen A / Bharat TAM
Funding support United Kingdom, 1 items
OrganizationGrant numberCountry
Wellcome Trust202231/Z/16/Z United Kingdom
CitationJournal: Cell Rep / Year: 2021
Title: Complete atomic structure of a native archaeal cell surface.
Authors: Andriko von Kügelgen / Vikram Alva / Tanmay A M Bharat /
Abstract: Many prokaryotic cells are covered by an ordered, proteinaceous, sheet-like structure called a surface layer (S-layer). S-layer proteins (SLPs) are usually the highest copy number macromolecules in ...Many prokaryotic cells are covered by an ordered, proteinaceous, sheet-like structure called a surface layer (S-layer). S-layer proteins (SLPs) are usually the highest copy number macromolecules in prokaryotes, playing critical roles in cellular physiology such as blocking predators, scaffolding membranes, and facilitating environmental interactions. Using electron cryomicroscopy of two-dimensional sheets, we report the atomic structure of the S-layer from the archaeal model organism Haloferax volcanii. This S-layer consists of a hexagonal array of tightly interacting immunoglobulin-like domains, which are also found in SLPs across several classes of archaea. Cellular tomography reveal that the S-layer is nearly continuous on the cell surface, completed by pentameric defects in the hexagonal lattice. We further report the atomic structure of the SLP pentamer, which shows markedly different relative arrangements of SLP domains needed to complete the S-layer. Our structural data provide a framework for understanding cell surfaces of archaea at the atomic level.
History
DepositionSep 27, 2021-
Header (metadata) releaseDec 15, 2021-
Map releaseDec 15, 2021-
UpdateDec 15, 2021-
Current statusDec 15, 2021Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.04262
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 0.04262
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_13639.png

Downloads & links

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Map

FileDownload / File: emd_13639.map.gz / Format: CCP4 / Size: 8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationPostprocessed map without B-factor sharpening
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.24 Å/pix.
x 128 pix.
= 286.464 Å		2.24 Å/pix.
x 128 pix.
= 286.464 Å		2.24 Å/pix.
x 128 pix.
= 286.464 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2.238 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.04262 / Movie #1: 0.04262
Minimum - Maximum-0.032700524 - 0.11928224
Average (Standard dev.)0.0026785338 (±0.015977861)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions128128128
Spacing128128128
CellA=B=C: 286.464 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.2382.2382.238
M x/y/z128128128
origin x/y/z0.0000.0000.000
length x/y/z286.464286.464286.464
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS128128128
D min/max/mean-0.0330.1190.003

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Supplemental data

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Mask #1

Fileemd_13639_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Full map without post-processing and B-factor sharpening

Fileemd_13639_additional_1.map
AnnotationFull map without post-processing and B-factor sharpening
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : In-vitro structure of inverted S-layer tube

EntireName: In-vitro structure of inverted S-layer tube
Components
  • Complex: In-vitro structure of inverted S-layer tube
    • Protein or peptide: S-layer protein

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Supramolecule #1: In-vitro structure of inverted S-layer tube

SupramoleculeName: In-vitro structure of inverted S-layer tube / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all / Details: In-vitro structure of inverted S-layer tube
Source (natural)Organism: Haloferax volcanii DS2 (archaea) / Location in cell: Cell surface

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Macromolecule #1: S-layer protein

MacromoleculeName: S-layer protein / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Haloferax volcanii DS2 (archaea) / Strain: H26
SequenceString: ERGNLDADSE SFNKTIQSGD RVFLGE EIS TDAGLGASNP LLTGTAGNSE GVSLDLSSPI PQTTENQPLG TYDVDGSGSA TTPNVTL LA PRITDSEILT SSGGDVTGSA ISSSDAGNLY VNADYNYESA EKVEVTVEDP SGTDITNE V LSGTDTFVDD GSIGSTSSTG ...String:
ERGNLDADSE SFNKTIQSGD RVFLGE EIS TDAGLGASNP LLTGTAGNSE GVSLDLSSPI PQTTENQPLG TYDVDGSGSA TTPNVTL LA PRITDSEILT SSGGDVTGSA ISSSDAGNLY VNADYNYESA EKVEVTVEDP SGTDITNE V LSGTDTFVDD GSIGSTSSTG GGVGIDMSDQ DAGEYTIILE GAEDLDFGDA TETMTLTIS SQDEIGIELD SESVTQGTDV QYTVTNGIDG NEHVVAMDLS DLQNDATTEQ AKEVFRNIGD TSEVGIANS SATNTSGSST GPTVETADIA YAVVEIDGAS AVGGIETQYL DDSEVDLEVY D AGVSATAA VGQDATNDIT LTIEEGGTTL SSPTGQYVVG SEVDINGTAT SSDSVAIYVR DD GDWQLLE IGGDNEISVD SDDTFEEEDI ALSGLSGDGS SILSLTGTYR IGVIDASDAD VGG DGSVDD SLTTSEFTSG VSSSNSIRVT DQALTGQFTT INGQVAPVET GTVDINGTAS GANS VLVIF VDERGNVNYQ EVSVDSDGTY DEDDITVGLT QGRVTAHILS VGRDSAIGDG SLPSG PSNG ATLNDLTGYL DTLDQNNNNG EQINELIASE TVDETASDDL IVTETFRLAE SSTSID SIY PDAAEAAGIN PVATGETMVI AGSTNLKPDD NTISIEVTNE DGTSVALEDT DEWNNDG QW MVEIDTTDFE TGTFTVEADD GDNTDTVNVE VVSEREDTTT SSDNATDTTT TTDGPTET T TTAEPTETTE EPTEETTTSS NTPGFGIAVA LVALVGAALL ALRREN

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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: 7.5
Component:
ConcentrationFormulaName
25.0 mMC8H18N2O4SHEPES
150.0 mMMgCl2magnesium chloride
15.0 mMCaCl2calcium chloride
56.3 % (w/v)C32H58N2O7SCHAPS detergent

Details: Buffer solutions were prepared fresh from sterile filtered concentrated stocksolutions. Solutions were filtered through a 0.22 um filter to avoid microbial contamination and degassed using a ...Details: Buffer solutions were prepared fresh from sterile filtered concentrated stocksolutions. Solutions were filtered through a 0.22 um filter to avoid microbial contamination and degassed using a vacuum fold pump. The pH of the HEPES stock solution was adjusted with sodium hydroxide at 4 deg C. 15 mM Calcium chloride was added 15 minutes before vitrification.
GridModel: Quantifoil R2/2 / Material: COPPER/RHODIUM / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY ARRAY / Pretreatment - Atmosphere: AIR / Details: 20 seconds, 15 mA
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 283.15 K / Instrument: FEI VITROBOT MARK IV
Details: Vitrobot options: Blot time 2.0 seconds, Blot force -15,1, Wait time 0 seconds, Drain time 0.5 seconds.
DetailsHaloferax volcanii vesicles

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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
DetailsSerialEM Dose symmetric scheme
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 3838 pixel / Digitization - Dimensions - Height: 3710 pixel / Number grids imaged: 2 / Average exposure time: 2.4 sec. / Average electron dose: 3.0 e/Å2
Details: Dose symmetric tilt scheme (Hagen et al, JSB); 2.4 seconds of exposure with 6 saved frames.
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Calibrated defocus max: 0.4 µm / Calibrated defocus min: 0.1 µm / Calibrated magnification: 64000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 4.0 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 64000
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

Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C2 (2 fold cyclic) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 15.87 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number subtomograms used: 25843
ExtractionNumber tomograms: 24 / Number images used: 25843 / Reference model: Ab initio / Method: RELION / Software - Name: RELION (ver. 3.1) / Details: RELION subtomogram averaging
CTF correctionSoftware - Name: CTFFIND (ver. 4.1.13)
Software - details: CTFFIND4 was used as implemented in RELION 3.1
Details: RELION subtomogram averaging (Bharat & Scheres 2016)
Final 3D classificationSoftware - Name: RELION (ver. 3.1) / Details: 3D-Classification using RELION3.1
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1) / Details: Angle assignment was performed within RELION3.1

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Atomic model buiding 1

DetailsRigid body fit inside coot of D1-D6 domains and real space refinement with restraints of the original model obtained by single particle analysis in PHENIX.
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Target criteria: Best Fit

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