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- EMDB-13637: In-situ structure of hexameric S-layer protein -

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Basic information

Entry
Database: EMDB / ID: EMD-13637
TitleIn-situ structure of hexameric S-layer protein
Map dataPostprocessed map without B-factor sharpening.
Sample
  • Organelle or cellular component: In-situ structure of hexameric S-layer of Haloferax volcanii
    • Protein or peptide: Cell surface glycoprotein
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: 7.968 Å
Authorsvon Kuegelgen A / Bharat TAM
Funding support United Kingdom, 3 items
OrganizationGrant numberCountry
Wellcome Trust202231/Z/16/Z United Kingdom
Other privateVallee Scholarship United Kingdom
Leverhulme TrustPhilip Leverhulme Prize 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: 0.0242
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 0.0242
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7ptt
  • Surface level: 0.0242
  • Imaged by UCSF Chimera
  • Download
  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-7ptt
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_13637.png

Downloads & links

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Map

FileDownload / File: emd_13637.map.gz / Format: CCP4 / Size: 27 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.)
1.33 Å/pix.
x 192 pix.
= 254.976 Å		1.33 Å/pix.
x 192 pix.
= 254.976 Å		1.33 Å/pix.
x 192 pix.
= 254.976 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.328 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0242 / Movie #1: 0.0242
Minimum - Maximum-0.047065843 - 0.08771319
Average (Standard dev.)0.0021711814 (±0.008803365)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions192192192
Spacing192192192
CellA=B=C: 254.97598 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.3281.3281.328
M x/y/z192192192
origin x/y/z0.0000.0000.000
length x/y/z254.976254.976254.976
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS192192192
D min/max/mean-0.0470.0880.002

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Supplemental data

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Mask #1

Fileemd_13637_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 postprocessing or B-factor sharpening

Fileemd_13637_additional_1.map
AnnotationFull map without postprocessing or 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-situ structure of hexameric S-layer of Haloferax volcanii

EntireName: In-situ structure of hexameric S-layer of Haloferax volcanii
Components
  • Organelle or cellular component: In-situ structure of hexameric S-layer of Haloferax volcanii
    • Protein or peptide: Cell surface glycoprotein

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Supramolecule #1: In-situ structure of hexameric S-layer of Haloferax volcanii

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

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Macromolecule #1: Cell surface glycoprotein

MacromoleculeName: Cell surface glycoprotein / type: protein_or_peptide / ID: 1 / Number of copies: 6 / Enantiomer: LEVO
Source (natural)Organism: Haloferax volcanii DS2 (archaea)
Molecular weightTheoretical: 81.755602 KDa
SequenceString: ERGNLDADSE SFNKTIQSGD RVFLGEEIST DAGLGASNPL LTGTAGNSEG VSLDLSSPIP QTTENQPLGT YDVDGSGSAT TPNVTLLAP RITDSEILTS SGGDVTGSAI SSSDAGNLYV NADYNYESAE KVEVTVEDPS GTDITNEVLS GTDTFVDDGS I GSTSSTGG ...String:
ERGNLDADSE SFNKTIQSGD RVFLGEEIST DAGLGASNPL LTGTAGNSEG VSLDLSSPIP QTTENQPLGT YDVDGSGSAT TPNVTLLAP RITDSEILTS SGGDVTGSAI SSSDAGNLYV NADYNYESAE KVEVTVEDPS GTDITNEVLS GTDTFVDDGS I GSTSSTGG GVGIDMSDQD AGEYTIILEG AEDLDFGDAT ETMTLTISSQ DEIGIELDSE SVTQGTDVQY TVTNGIDGNE HV VAMDLSD LQNDATTEQA KEVFRNIGDT SEVGIANSSA TNTSGSSTGP TVETADIAYA VVEIDGASAV GGIETQYLDD SEV DLEVYD AGVSATAAVG QDATNDITLT IEEGGTTLSS PTGQYVVGSE VDINGTATSS DSVAIYVRDD GDWQLLEIGG DNEI SVDSD DTFEEEDIAL SGLSGDGSSI LSLTGTYRIG VIDASDADVG GDGSVDDSLT TSEFTSGVSS SNSIRVTDQA LTGQF TTIN GQVAPVETGT VDINGTASGA NSVLVIFVDE RGNVNYQEVS VDSDGTYDED DITVGLTQGR VTAHILSVGR DSAIGD GSL PSGPSNGATL NDLTGYLDTL DQNNNNGEQI NELIASETVD ETASDDLIVT ETFRLAESST SIDSIYPDAA EAAGINP VA TGETMVIAGS TNLKPDDNTI SIEVTNEDGT SVALEDTDEW NNDGQWMVEI DTTDFETGTF TVEADDGDNT DTVNVEVV S EREDTTTSSD NATDTTTTTD GPTETTTTAE PTETTEEPTE ETTTSSNTPG FGIAVALVAL VGAALLALRR EN

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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
12.0 mMC4H11NO3Tris
2.4641 MNaClsodium chloride
88.5 mMMgCl2magnesium chloride
85.2 mMMgSO4magnesium sulfate
56.3 mMKClpotassium chloride
3.0 mMCaCl2calcium chloride

Details: 18 % (w/v) artificial sea water
GridModel: Quantifoil R2/2 / Material: COPPER/RHODIUM / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY ARRAY / Pretreatment - Type: GLOW DISCHARGE / 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 Hagen 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: 1 / Average exposure time: 1.0 sec. / Average electron dose: 2.9 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: 50.0 µm / Calibrated defocus max: 4.0 µm / Calibrated defocus min: 1.0 µm / Calibrated magnification: 105000 / 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: 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

Final reconstructionNumber classes used: 5 / Applied symmetry - Point group: C6 (6 fold cyclic) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 7.968 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number subtomograms used: 53063
ExtractionNumber tomograms: 127 / Number images used: 83713 / 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 classificationNumber classes: 6 / Software - 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
Output model

PDB-7ptt:
In-situ structure of hexameric S-layer protein

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