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- EMDB-13638: Structure of pentameric S-layer protein from Halofaerax volcanii -

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Basic information

Entry
Database: EMDB / ID: EMD-13638
TitleStructure of pentameric S-layer protein from Halofaerax volcanii
Map dataNon-postprocessed, non-sharpened full map.
Sample
  • Complex: Structure of pentameric S-layer protein csg
    • Protein or peptide: Cell surface glycoprotein
  • Ligand: beta-D-glucopyranoseGlucose
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)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.87 Å
Authorsvon Kuegelgen A / Bharat TAM
Funding support United Kingdom, 3 items
OrganizationGrant numberCountry
Wellcome Trust202231/Z/16/Z United Kingdom
Leverhulme TrustPhilip Leverhulme Prize United Kingdom
Other privateVallee Scholarship 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: 0.0114
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 0.0114
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7ptu
  • Surface level: 0.0114
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-7ptu
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_13638.png

Downloads & links

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Map

FileDownload / File: emd_13638.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationNon-postprocessed, non-sharpened full map.
Voxel sizeX=Y=Z: 1.1 Å
Density
Contour LevelBy AUTHOR: 0.0114 / Movie #1: 0.0114
Minimum - Maximum-0.010560917 - 0.039823484
Average (Standard dev.)7.4053205e-05 (±0.0014097756)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 352.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.11.11.1
M x/y/z320320320
origin x/y/z0.0000.0000.000
length x/y/z352.000352.000352.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS320320320
D min/max/mean-0.0110.0400.000

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Supplemental data

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Mask #1

Fileemd_13638_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Postprocessed, sharpened map with an applied B-factor of -170 A^2

Fileemd_13638_additional_1.map
AnnotationPostprocessed, sharpened map with an applied B-factor of -170 A^2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map 1, non-postprocessed, non sharpened.

Fileemd_13638_half_map_1.map
AnnotationHalf map 1, non-postprocessed, non sharpened.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map 2, non-postprocessed, non sharpened.

Fileemd_13638_half_map_2.map
AnnotationHalf map 2, non-postprocessed, non sharpened.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Structure of pentameric S-layer protein csg

EntireName: Structure of pentameric S-layer protein csg
Components
  • Complex: Structure of pentameric S-layer protein csg
    • Protein or peptide: Cell surface glycoprotein
  • Ligand: beta-D-glucopyranoseGlucose

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Supramolecule #1: Structure of pentameric S-layer protein csg

SupramoleculeName: Structure of pentameric S-layer protein csg / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1 / Details: Structure of pentameric S-layer protein csg
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: 5 / 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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Macromolecule #2: beta-D-glucopyranose

MacromoleculeName: beta-D-glucopyranose / type: ligand / ID: 2 / Number of copies: 5 / Formula: BGC
Molecular weightTheoretical: 180.156 Da
Chemical component information

ChemComp-BGC:
beta-D-glucopyranose / Glucose

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration2.5 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
20.0 mMC8H18N2O4SHEPES
150.0 mMMgCl2magnesium chloride
1.75 mMHoCl3holmium chloride
0.65 % (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. 1.75 mM holmium chloride was added 2 hours before vitrification.
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 5 seconds, Blot force -10,1, Wait time 10 seconds, Drain time 0.5 seconds.
DetailsPurified csg protein mixed with 1.75 mM HoCl3 after 2 hour incubation.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
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: 81000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 4.0 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 81000
Specialist opticsEnergy 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
DetailsEPU software with faster acquisition mode AFIS (Aberration Free Image Shift).
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Digitization - Dimensions - Width: 5760 pixel / Digitization - Dimensions - Height: 4092 pixel / Number grids imaged: 2 / Number real images: 11871 / Average exposure time: 3.6 sec. / Average electron dose: 53.9 e/Å2
Details: Images were collected in two sessions in movie-mode and subjected to 3.6 seconds of exposure where a total dose of 53.45 or 53.9 e-/A2 was applied, and 40 frames were recorded per movie. A ...Details: Images were collected in two sessions in movie-mode and subjected to 3.6 seconds of exposure where a total dose of 53.45 or 53.9 e-/A2 was applied, and 40 frames were recorded per movie. A total of 11871 movies were collected in two sessions with the same microscope and settings.
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 1773652
Details: Particles were initially picking using the Laplacian-of gaussian algorithm implemented in RELION3.0 (Zivanov et al., 2018). Particles were extracted in 8x down-sampled in 50x50 pixel boxes ...Details: Particles were initially picking using the Laplacian-of gaussian algorithm implemented in RELION3.0 (Zivanov et al., 2018). Particles were extracted in 8x down-sampled in 50x50 pixel boxes and classified using reference-free 2D classification inside RELION3.0.
CTF correctionSoftware - Name: CTFFIND (ver. 4.1.13)
Software - details: CTFFIND4 was used as implemented in RELION 3.1
Details: RELION refinement with in-built CTF correction. The function is similar to a Wiener filter, so amplitude correction included.
Startup modelType of model: NONE
Details: Initial 3D reference model was prepared inside RELION3.0 with the stochastic gradient descent (SGD) algorithm.
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0) / Details: Angle assignment was performed within RELION3.0
Final 3D classificationNumber classes: 4 / Avg.num./class: 95500 / Software - Name: RELION (ver. 3.1) / Details: 3D-Classification using RELION3.0
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1) / Details: Angle assignment was performed within RELION3.1
Final reconstructionNumber classes used: 3 / Applied symmetry - Point group: C5 (5 fold cyclic) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.87 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1)
Details: The final map (RELION3.1) was obtained from 382,105 particles and post-processed using a soft mask focused on the entire pentameric map yielding a global resolution of 3.87 angstrom with ...Details: The final map (RELION3.1) was obtained from 382,105 particles and post-processed using a soft mask focused on the entire pentameric map yielding a global resolution of 3.87 angstrom with resolution anisotropy from 3.49-8.11 angstrom from the central C5 axis near domains D1-D3 (well resolved) to the more flexible domains D4 (partially resolved) and D5-D6 (not resolved).
Number images used: 382105
DetailsImported movies were motion-corrected, dose weighted, and Fourier cropped (2x) with MotionCor2 (Zheng et al., 2017) implemented in RELION3.1 (Zivanov et al., 2018). Contrast transfer functions (CTFs) of the resulting motion-corrected micrographs were estimated using CTFFIND4 (Rohou and Grigorieff, 2015).

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Atomic model buiding 1

DetailsThe initial manual build of D1-D2 was performed independently using the csg pentameric cryo-EM map, which served as an additional validation of the manual building performed in the csg hexamer in the related deposition. The manual building exercise yielded a nearly identical result to the hexamer; thus, the final refined hexameric structures of D1-D2, along with D3-D4 were taken and fitted into the pentameric map (~3.87 angstrom resolution in D1-D3, lower in D4 which is partially resolved). Five copies of these D1-D4 were used for refinement and model building as for the hexamer, except D3 and D4 was restrained in position, due to steadily deteriorating resolution in this part of the map. D5-D6 were not resolved in the pentameric structure and were thus not included in the refinements. Model validation was performed in PHENIX and CCP-EM.
RefinementSpace: REAL / Protocol: AB INITIO MODEL / Overall B value: 179.68 / Target criteria: Best Fit
Output model

PDB-7ptu:
Structure of pentameric S-layer protein from Halofaerax volcanii

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