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Yorodumi- PDB-7ptr: Structure of hexameric S-layer protein from Haloferax volcanii archaea -
+Open data
-Basic information
Entry | Database: PDB / ID: 7ptr | ||||||||||||
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Title | Structure of hexameric S-layer protein from Haloferax volcanii archaea | ||||||||||||
Components | Cell surface glycoprotein | ||||||||||||
Keywords | STRUCTURAL PROTEIN / S-layer csg | ||||||||||||
Function / homology | Function and homology information S-layer / cell wall organization / extracellular region / plasma membrane Similarity search - Function | ||||||||||||
Biological species | Haloferax volcanii DS2 (archaea) | ||||||||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.46 Å | ||||||||||||
Authors | von Kuegelgen, A. / Bharat, T.A.M. | ||||||||||||
Funding support | United Kingdom, 3items
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Citation | Journal: 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 |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | Molecule: MolmilJmol/JSmol |
-Downloads & links
-Download
PDBx/mmCIF format | 7ptr.cif.gz | 689 KB | Display | PDBx/mmCIF format |
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PDB format | pdb7ptr.ent.gz | 572.6 KB | Display | PDB format |
PDBx/mmJSON format | 7ptr.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 7ptr_validation.pdf.gz | 1.7 MB | Display | wwPDB validaton report |
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Full document | 7ptr_full_validation.pdf.gz | 1.8 MB | Display | |
Data in XML | 7ptr_validation.xml.gz | 114 KB | Display | |
Data in CIF | 7ptr_validation.cif.gz | 170 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/pt/7ptr ftp://data.pdbj.org/pub/pdb/validation_reports/pt/7ptr | HTTPS FTP |
-Related structure data
Related structure data | 13634MC 7ptpC 7pttC 7ptuC M: map data used to model this data C: citing same article (ref.) |
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Similar structure data |
-Links
-Assembly
Deposited unit |
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1 |
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-Components
#1: Protein | Mass: 81755.602 Da / Num. of mol.: 6 / Source method: isolated from a natural source / Source: (natural) Haloferax volcanii DS2 (archaea) / Plasmid details: Allers et al 2004 / References: UniProt: P25062 #2: Chemical | ChemComp-CA / #3: Sugar | ChemComp-BGC / Has ligand of interest | Y | Has protein modification | Y | |
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-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: 2D ARRAY / 3D reconstruction method: single particle reconstruction |
-Sample preparation
Component | Name: Structure of hexameric S-layer protein csg / Type: COMPLEX / Details: Structure of hexameric S-layer protein csg / Entity ID: #1 / Source: NATURAL | |||||||||||||||||||||||||
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Molecular weight | Experimental value: NO | |||||||||||||||||||||||||
Source (natural) | Organism: Haloferax volcanii DS2 (archaea) / Cellular location: Cell surface | |||||||||||||||||||||||||
Buffer solution | pH: 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. 15 mM Calcium chloride was added 15 minutes before vitrification. | |||||||||||||||||||||||||
Buffer component |
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Specimen | Conc.: 3.2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES Details: Purified csg protein mixed with 15 mM CaCl2 after 15 minutes incubation. | |||||||||||||||||||||||||
Specimen support | Details: 20 seconds, 15 mA / Grid material: COPPER/RHODIUM / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R2/2 | |||||||||||||||||||||||||
Vitrification | Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 283.15 K Details: Vitrobot options: Blot time 4.5 seconds, Blot force -10,1, Wait time 10 seconds, Drain time 0.5 seconds |
-Electron microscopy imaging
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
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Microscopy | Model: FEI TITAN KRIOS Details: EPU software with faster acquisition mode AFIS (Aberration Free Image Shift). |
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM |
Electron lens | Mode: BRIGHT FIELD / 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 holder | Cryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 70 K / Temperature (min): 70 K |
Image recording | Average exposure time: 3.4 sec. / Electron dose: 51.441 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 2 / Num. of real images: 18468 Details: Images were collected in two sessions movie-mode and subjected to 3.4 seconds of exposure where a total dose of 49 or 51.441 e-/A2 was applied, and 40 frames were recorded per movie. A total ...Details: Images were collected in two sessions movie-mode and subjected to 3.4 seconds of exposure where a total dose of 49 or 51.441 e-/A2 was applied, and 40 frames were recorded per movie. A total of 18468 movies were collected in two sessions with the same microscope and settings. |
EM imaging optics | Energyfilter name: GIF Quantum LS / Energyfilter slit width: 20 eV |
Image scans | Width: 5760 / Height: 4092 |
-Processing
Software | Name: PHENIX / Version: 1.19_4092: / Classification: refinement | ||||||||||||||||||||||||||||||||||||||||||||||||||
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EM software |
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Image processing | Details: Movies were clustered into optics groups based on the XML meta-data of the data-collection software EPU (ThermoFisher) using a k-means algorithm implemented in EPU_group_AFIS (https://github. ...Details: Movies were clustered into optics groups based on the XML meta-data of the data-collection software EPU (ThermoFisher) using a k-means algorithm implemented in EPU_group_AFIS (https://github.com/DustinMorado/EPU_group_AFIS). 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 correction | Details: 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 selection | Num. of particles selected: 10558369 Details: Top and tilted views were manually picked at the central hexameric axis. Manually picked particles were extracted in 4x downsampled 100 x 100 boxes and classified using reference-free 2D ...Details: Top and tilted views were manually picked at the central hexameric axis. Manually picked particles were extracted in 4x downsampled 100 x 100 boxes and classified using reference-free 2D classification inside RELION3.1 (Zivanov et al., 2020). Class averages centered at a hexameric axis were used to automatically pick particles inside RELION3.1. Automatically picked particles were extracted in 4x downsampled 100x100 pixel boxes and classified using reference-free 2D classification. Particle coordinates belonging to class averages centered at the hexameric axis were used to train TOPAZ (Bepler et al., 2019) in 5x downsampled micrographs with the neural network architecture ResNet8. For the final reconstruction, particles were picked using TOPAZ and the previously trained neural network above. Additionally, top and bottom views were picked using the reference-based autopicker inside RELION3.1, which were not readily identified by TOPAZ. Particles were extracted in 4x downsampled 100 x 100 boxes and classified using reference-free 2D classification inside RELION3.1. Particles belonging to class averages centered at the hexameric axis were combined, and particles within 100 angstrom were removed to prevent duplication after alignment. | ||||||||||||||||||||||||||||||||||||||||||||||||||
Symmetry | Point symmetry: C6 (6 fold cyclic) | ||||||||||||||||||||||||||||||||||||||||||||||||||
3D reconstruction | Resolution: 3.46 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 1087798 / Algorithm: FOURIER SPACE Details: Particles from classes with the same curvature were combined, re-extracted in 400 x 400 boxes and subjected to a focused 3D auto refinement on the central 6 subunits using the scaled and ...Details: Particles from classes with the same curvature were combined, re-extracted in 400 x 400 boxes and subjected to a focused 3D auto refinement on the central 6 subunits using the scaled and lowpass filtered output from the 3D classification as a starting model. Per-particle defocus, anisotropy magnification and higher-order aberrations were refined inside RELION3.1, followed by another round of focused 3D auto refinement and Bayesian particle polishing (Zivanov et al., 2020). Num. of class averages: 1 / Symmetry type: POINT | ||||||||||||||||||||||||||||||||||||||||||||||||||
Atomic model building | B value: 143.26 / Protocol: AB INITIO MODEL / Space: REAL / Target criteria: Best Fit Details: The boundaries of the six Ig-like domains, D1-D6, were predicted using HHpred (Steinegger et al., 2019) in default settings within the MPI Bioinformatics Toolkit (Zimmermann et al., 2018). ...Details: The boundaries of the six Ig-like domains, D1-D6, were predicted using HHpred (Steinegger et al., 2019) in default settings within the MPI Bioinformatics Toolkit (Zimmermann et al., 2018). Subsequently, structural models for these domains were built using the Robetta structure prediction server, employing the deep learning-based modelling method TrRosetta (Yang et al., 2020). The obtained structural models of domains D3-D6 resulted in an overall fit into the hexameric cryo-EM map of csg from the reconstituted sheets. D1-D2 deviated significantly from any obtained homology models, and for those domains, the carbon backbone of the csg protein was manually traced through a single subunit of the hexameric cryo-EM density using Coot (Emsley and Cowtan, 2004). Due to the edge effect of the box used in the refinement of the 3.5 angstrom map, parts of D6 displayed edge artefacts. These artefacts were removed using single-particle cryo-EM refinement in a larger box, which led to an overall slightly lower resolution (3.8 angstrom) but allowed fitting of the D6 homology model unambiguously. Following initial manual building (for D1-D2) or fitting in of structural models (for D3-D6), side chains were assigned in regions with density corresponding to characteristic aromatic residues allowing us to deduce the register of the amino acid sequence in the map. Another important check of the model building was the position of known glycan positions, which were readily assigned based on large unexplained densities on characteristic asparagine residues. The atomic model was then placed into the hexameric map in six copies and subjected to several rounds of refinement using refmac5 (Murshudov et al., 2011) inside the CCP-EM software suite (Burnley et al., 2017) and PHENIX (Liebschner et al., 2019), followed by manually rebuilding in Coot (Emsley and Cowtan, 2004). Model validation was performed in PHENIX and CCP-EM. |