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Yorodumi- EMDB-13634: Structure of hexameric S-layer protein from Haloferax volcanii archaea -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-13634 | ||||||||||||
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Title | Structure of hexameric S-layer protein from Haloferax volcanii archaea | ||||||||||||
Map data | Full map, non-sharpened, non post-processed | ||||||||||||
Sample |
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Keywords | S-layer csg / STRUCTURAL PROTEIN | ||||||||||||
Function / homology | Surface 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 species | Haloferax volcanii DS2 (archaea) | ||||||||||||
Method | single particle reconstruction / cryo EM / Resolution: 3.46 Å | ||||||||||||
Authors | von Kuegelgen A / Bharat TAM | ||||||||||||
Funding support | United Kingdom, 3 items
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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 | EM map: SurfViewMolmilJmol/JSmol |
Supplemental images |
-Downloads & links
-EMDB archive
Map data | emd_13634.map.gz | 188.8 MB | EMDB map data format | |
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Header (meta data) | emd-13634-v30.xml emd-13634.xml | 28.6 KB 28.6 KB | Display Display | EMDB header |
Images | emd_13634.png | 235.4 KB | ||
Masks | emd_13634_msk_1.map | 244.1 MB | Mask map | |
Filedesc metadata | emd-13634.cif.gz | 8.9 KB | ||
Others | emd_13634_additional_1.map.gz emd_13634_half_map_1.map.gz emd_13634_half_map_2.map.gz | 212 MB 191.3 MB 191.3 MB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-13634 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-13634 | HTTPS FTP |
-Validation report
Summary document | emd_13634_validation.pdf.gz | 785.3 KB | Display | EMDB validaton report |
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Full document | emd_13634_full_validation.pdf.gz | 784.9 KB | Display | |
Data in XML | emd_13634_validation.xml.gz | 15.3 KB | Display | |
Data in CIF | emd_13634_validation.cif.gz | 18.2 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-13634 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-13634 | HTTPS FTP |
-Related structure data
Related structure data | 7ptrMC 7ptpC 7pttC 7ptuC M: atomic model generated by this map C: citing same article (ref.) |
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Similar structure data |
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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-Map
File | Download / File: emd_13634.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | Full map, non-sharpened, non post-processed | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Projections & slices | Image control
Images are generated by Spider. | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 1.1 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Mask #1
File | emd_13634_msk_1.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Additional map: Density modified map using deepEMhancer (Sanchez-Garcia et al 2021)
File | emd_13634_additional_1.map | ||||||||||||
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Annotation | Density modified map using deepEMhancer (Sanchez-Garcia et al 2021) | ||||||||||||
Projections & Slices |
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Density Histograms |
-Half map: Half map1, non-sharpened, non post-processed
File | emd_13634_half_map_1.map | ||||||||||||
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Annotation | Half map1, non-sharpened, non post-processed | ||||||||||||
Projections & Slices |
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Density Histograms |
-Half map: Half map2, non-sharpened, non post-processed
File | emd_13634_half_map_2.map | ||||||||||||
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Annotation | Half map2, non-sharpened, non post-processed | ||||||||||||
Projections & Slices |
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Density Histograms |
-Sample components
-Entire : Structure of hexameric S-layer protein csg
Entire | Name: Structure of hexameric S-layer protein csg |
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Components |
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-Supramolecule #1: Structure of hexameric S-layer protein csg
Supramolecule | Name: Structure of hexameric S-layer protein csg / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1 / Details: Structure of hexameric S-layer protein csg |
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Source (natural) | Organism: Haloferax volcanii DS2 (archaea) / Location in cell: Cell surface |
-Macromolecule #1: Cell surface glycoprotein
Macromolecule | Name: Cell surface glycoprotein / type: protein_or_peptide / ID: 1 / Number of copies: 6 / Enantiomer: LEVO |
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Source (natural) | Organism: Haloferax volcanii DS2 (archaea) |
Molecular weight | Theoretical: 81.755602 KDa |
Sequence | String: 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 UniProtKB: Cell surface glycoprotein |
-Macromolecule #2: CALCIUM ION
Macromolecule | Name: CALCIUM ION / type: ligand / ID: 2 / Number of copies: 18 / Formula: CA |
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Molecular weight | Theoretical: 40.078 Da |
-Macromolecule #3: beta-D-glucopyranose
Macromolecule | Name: beta-D-glucopyranose / type: ligand / ID: 3 / Number of copies: 18 / Formula: BGC |
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Molecular weight | Theoretical: 180.156 Da |
Chemical component information | ChemComp-BGC: |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | 2D array |
-Sample preparation
Concentration | 3.2 mg/mL | |||||||||||||||
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Buffer | pH: 7.5 Component:
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. | |||||||||||||||
Grid | Model: Quantifoil R2/2 / Material: COPPER/RHODIUM / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 20 sec. / Pretreatment - Atmosphere: AIR / Details: 20 seconds, 15 mA | |||||||||||||||
Vitrification | Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 283.15 K / Instrument: FEI VITROBOT MARK IV Details: Vitrobot options: Blot time 4.5 seconds, Blot force -10,1, Wait time 10 seconds, Drain time 0.5 seconds. | |||||||||||||||
Details | Purified csg protein mixed with 15 mM CaCl2 after 15 minutes incubation. |
-Electron microscopy
Microscope | FEI TITAN KRIOS |
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Temperature | Min: 70.0 K / Max: 70.0 K |
Specialist optics | Energy filter - Name: GIF Quantum LS / Energy filter - Slit width: 20 eV |
Details | EPU software with faster acquisition mode AFIS (Aberration Free Image Shift). |
Image recording | Film 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: 18468 / Average exposure time: 3.4 sec. / Average electron dose: 51.441 e/Å2 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. |
Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | C2 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 FIELD / Cs: 2.7 mm / Nominal defocus max: 4.0 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 81000 |
Sample stage | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN |
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
+Image processing
-Atomic model buiding 1
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. |
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Refinement | Space: REAL / Protocol: AB INITIO MODEL / Overall B value: 143.26 / Target criteria: Best Fit |
Output model | PDB-7ptr: |