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- PDB-7ptp: In-situ structure of pentameric S-layer protein -

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Basic information

Entry
Database: PDB / ID: 7ptp
TitleIn-situ structure of pentameric S-layer protein
ComponentsCell surface glycoprotein
KeywordsSTRUCTURAL PROTEIN / S-layer csg
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 (archaea)
MethodELECTRON MICROSCOPY / subtomogram averaging / cryo EM / Resolution: 11.58 Å
Authorsvon Kuegelgen, A. / Bharat, T.A.M.
Funding support United Kingdom, 1items
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, 2021Deposition site: PDBE / Processing site: PDBE
Revision 1.0Dec 15, 2021Provider: repository / Type: Initial release
Revision 1.1Jul 17, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond / em_admin / Item: _em_admin.last_update

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Structure visualization

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Assembly

Deposited unit
A: Cell surface glycoprotein
B: Cell surface glycoprotein
C: Cell surface glycoprotein
D: Cell surface glycoprotein
E: Cell surface glycoprotein


Theoretical massNumber of molelcules
Total (without water)408,7785
Polymers408,7785
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
Cell surface glycoprotein / S-layer glycoprotein


Mass: 81755.602 Da / Num. of mol.: 5 / Source method: isolated from a natural source
Source: (natural) Haloferax volcanii (strain ATCC 29605 / DSM 3757 / JCM 8879 / NBRC 14742 / NCIMB 2012 / VKM B-1768 / DS2) (archaea)
Plasmid details: Allers et al 2004
Strain: ATCC 29605 / DSM 3757 / JCM 8879 / NBRC 14742 / NCIMB 2012 / VKM B-1768 / DS2
References: UniProt: P25062

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: CELL / 3D reconstruction method: subtomogram averaging

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Sample preparation

ComponentName: In-situ structure of pentameric S-layer of Haloferax volcanii
Type: ORGANELLE OR CELLULAR COMPONENT
Details: In-situ structure of pentameric S-layer of Haloferax volcanii
Entity ID: all / Source: NATURAL
Molecular weightExperimental value: NO
Source (natural)Organism: Haloferax volcanii DS2 (archaea) / Cellular location: Cell surface
Buffer solutionpH: 7.5 / Details: 18 % (w/v) artificial sea water
Buffer component
IDConc.NameFormulaBuffer-ID
112 mMTrisC4H11NO31
22.4641 Msodium chlorideNaCl1
388.5 mMmagnesium chlorideMgCl21
485.2 mMmagnesium sulfateMgSO41
556.3 mMpotassium chlorideKCl1
63 mMcalcium chlorideCaCl21
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: Haloferax volcanii vesicles
Specimen supportDetails: 20 seconds, 15 mA / Grid material: COPPER/RHODIUM / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R2/2
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 283.15 K
Details: Vitrobot options: Blot time 2.0 seconds, Blot force -15,1, Wait time 0 seconds, Drain time 0.5 seconds

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS / Details: SerialEM Hagen Scheme
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 105000 X / Calibrated magnification: 105000 X / Nominal defocus max: 4000 nm / Nominal defocus min: 1000 nm / Calibrated defocus min: 1000 nm / Calibrated defocus max: 4000 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: ZEMLIN TABLEAU
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 70 K / Temperature (min): 70 K
Image recordingAverage exposure time: 1 sec. / Electron dose: 2.9 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Details: Dose symmetric tilt scheme (Hagen et al, JSB)
EM imaging opticsEnergyfilter name: GIF Quantum LS / Chromatic aberration corrector: not used / Energyfilter slit width: 20 eV / Spherical aberration corrector: not used
Image scansWidth: 3838 / Height: 3710

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Processing

SoftwareName: PHENIX / Version: 1.19_4092: / Classification: refinement
EM software
IDNameVersionCategoryDetails
1RELION3.1volume selection
2SerialEMimage acquisition
4CTFFIND4.1.13CTF correctionCTFFIND4 was used as implemented in RELION 3.1
7Coot0.9.2-premodel fitting
9RELION3initial Euler assignment
10RELION3.1final Euler assignment
11RELION3.1classification
12RELION3.13D reconstruction
13PHENIX1.19-4092model refinement
CTF correctionDetails: RELION subtomogram averaging (Bharat & Scheres 2016)
Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles 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.
SymmetryPoint symmetry: C5 (5 fold cyclic)
3D reconstructionResolution: 11.58 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 1640 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT
EM volume selectionMethod: RELION / Details: RELION subtomogram averaging / Num. of tomograms: 127 / Num. of volumes extracted: 3495 / Reference model: Ab initio
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL / Target criteria: Best Fit
Details: Rigid 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.
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00326490
ELECTRON MICROSCOPYf_angle_d0.60836225
ELECTRON MICROSCOPYf_dihedral_angle_d13.7259225
ELECTRON MICROSCOPYf_chiral_restr0.0464425
ELECTRON MICROSCOPYf_plane_restr0.0034950

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