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

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Basic information

Entry
Database: PDB / ID: 7ptu
TitleStructure of pentameric S-layer protein from Halofaerax volcanii
ComponentsCell surface glycoprotein
KeywordsSTRUCTURAL PROTEIN / S-layer csg
Function / homology
Function and homology information


S-layer / cell wall organization / extracellular region / plasma membrane
Similarity search - Function
Surface glycoprotein signal peptide / Major cell surface glycoprotein / PGF-CTERM archaeal protein-sorting signal / PGF-CTERM motif
Similarity search - Domain/homology
beta-D-glucopyranose / Cell surface glycoprotein
Similarity search - Component
Biological speciesHaloferax volcanii DS2 (archaea)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.87 Å
Authorsvon Kuegelgen, A. / Bharat, T.A.M.
Funding support United Kingdom, 3items
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, 2021Deposition site: PDBE / Processing site: PDBE
Revision 1.0Dec 15, 2021Provider: repository / Type: Initial release

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Structure visualization

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  • Deposited structure unit
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  • Simplified surface model + fitted atomic model
  • EMDB-13638
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  • Superimposition on EM map
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Structure viewerMolecule:
MolmilJmol/JSmol

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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
hetero molecules


Theoretical massNumber of molelcules
Total (without water)409,67910
Polymers408,7785
Non-polymers9015
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area9210 Å2
ΔGint-6 kcal/mol
Surface area102930 Å2

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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 DS2 (archaea) / Plasmid details: Allers et al 2004 / References: UniProt: P25062
#2: Sugar
ChemComp-BGC / beta-D-glucopyranose / beta-D-glucose / D-glucose / glucose / Glucose


Type: D-saccharide, beta linking / Mass: 180.156 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: C6H12O6 / Feature type: SUBJECT OF INVESTIGATION
IdentifierTypeProgram
DGlcpbCONDENSED IUPAC CARBOHYDRATE SYMBOLGMML 1.0
b-D-glucopyranoseCOMMON NAMEGMML 1.0
b-D-GlcpIUPAC CARBOHYDRATE SYMBOLPDB-CARE 1.0
GlcSNFG CARBOHYDRATE SYMBOLGMML 1.0
Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Structure of pentameric S-layer protein csg / Type: COMPLEX / Details: Structure of pentameric S-layer protein csg / Entity ID: #1 / Source: NATURAL
Molecular weightExperimental value: NO
Source (natural)Organism: Haloferax volcanii DS2 (archaea) / Cellular location: Cell surface
Buffer solutionpH: 7.5
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.
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMHEPESC8H18N2O4S1
2150 mMmagnesium chlorideMgCl21
31.75 mMholmium chlorideHoCl31
40.65 % (w/v)CHAPS detergentC32H58N2O7S1
SpecimenConc.: 2.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: Purified csg protein mixed with 1.75 mM HoCl3 after 2 hour incubation.
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 5 seconds, Blot force -10,1, Wait time 10 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: EPU software with faster acquisition mode AFIS (Aberration Free Image Shift).
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 81000 X / Calibrated magnification: 81000 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: 3.6 sec. / Electron dose: 53.9 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 2 / Num. of real images: 11871
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.
EM imaging opticsEnergyfilter name: GIF Quantum LS / Energyfilter slit width: 20 eV
Image scansWidth: 5760 / Height: 4092

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Processing

SoftwareName: PHENIX / Version: 1.19_4092: / Classification: refinement
EM software
IDNameVersionCategoryDetails
2RELION3particle selectionAutoPicking
3EPUimage acquisition
5CTFFIND4.1.13CTF correctionCTFFIND4 was used as implemented in RELION 3.1
8Coot0.9.2-premodel fitting
10RELION3initial Euler assignment
11RELION3.1final Euler assignment
12RELION3.1classification
13RELION3.13D reconstruction
14PHENIX1.19-4092model refinement
Image processingDetails: Imported 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 ...Details: Imported 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).
CTF correctionDetails: RELION refinement with in-built CTF correction. The function is similar to a Wiener filter, so amplitude correction included.
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: 3.87 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 382105 / Algorithm: FOURIER SPACE
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).
Num. of class averages: 3 / Symmetry type: POINT
Atomic model buildingB value: 179.68 / Protocol: AB INITIO MODEL / Space: REAL / Target criteria: Best Fit
Details: The 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 ...Details: The 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.

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