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- PDB-6m3x: Cryo-EM structure of sulfur oxygenase reductase from Sulfurisphae... -

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Basic information

Entry
Database: PDB / ID: 6m3x
TitleCryo-EM structure of sulfur oxygenase reductase from Sulfurisphaera tokodaii
ComponentsSulfur oxygenase/reductase
KeywordsOXIDOREDUCTASE / spherical homo 24-mer
Function / homologysulfur oxygenase/reductase / sulfur oxygenase/reductase activity / Sulphur oxygenase reductase / Sulphur oxygenase reductase / Dimeric alpha-beta barrel / : / Sulfur oxygenase/reductase
Function and homology information
Biological speciesSulfurisphaera tokodaii (archaea)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.24 Å
AuthorsSato, Y. / Adachi, N. / Moriya, T. / Arakawa, T. / Kawasaki, M. / Yamada, C. / Senda, T. / Fushinobu, S.
Funding support Japan, 2items
OrganizationGrant numberCountry
Japan Society for the Promotion of Science (JSPS)24580136 Japan
Japan Agency for Medical Research and Development (AMED)JP20am0101071 Japan
CitationJournal: J Struct Biol X / Year: 2020
Title: Crystallographic and cryogenic electron microscopic structures and enzymatic characterization of sulfur oxygenase reductase from .
Authors: Yuta Sato / Takashi Yabuki / Naruhiko Adachi / Toshio Moriya / Takatoshi Arakawa / Masato Kawasaki / Chihaya Yamada / Toshiya Senda / Shinya Fushinobu / Takayoshi Wakagi /
Abstract: Sulfur oxygenase reductases (SORs) are present in thermophilic and mesophilic archaea and bacteria, and catalyze oxygen-dependent oxygenation and disproportionation of elemental sulfur. SOR has a ...Sulfur oxygenase reductases (SORs) are present in thermophilic and mesophilic archaea and bacteria, and catalyze oxygen-dependent oxygenation and disproportionation of elemental sulfur. SOR has a hollow, spherical homo-24-mer structure and reactions take place at active sites inside the chamber. The crystal structures of SORs from species have been reported. However, the states of the active site components (mononuclear iron and cysteines) and the entry and exit paths of the substrate and products are still in dispute. Here, we report the biochemical and structural characterizations of SORs from the thermoacidophilic archaeon (StSOR) and present high-resolution structures determined by X-ray crystallography and cryogenic electron microscopy (cryo-EM). The crystal structure of StSOR was determined at 1.73 Å resolution. At the catalytic center, iron is ligated to His86, His90, Glu114, and two water molecules. Three conserved cysteines in the cavity are located 9.5-13 Å from the iron and were observed as free thiol forms. A mutational analysis indicated that the iron and one of the cysteines (Cys31) were essential for both activities. The cryo-EM structure was determined at 2.24 Å resolution using an instrument operating at 200 kV. The two structures determined by different methodologies showed similar main chain traces, but the maps exhibited different features at catalytically important components. A possible role of StSOR in the sulfur metabolism of (an obligate aerobe) is discussed based on this study. Given the high resolution achieved in this study, StSOR was shown to be a good benchmark sample for cryo-EM.
History
DepositionMar 4, 2020Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Jul 15, 2020Provider: repository / Type: Initial release
Revision 1.1Aug 26, 2020Group: Database references / Derived calculations / Category: citation / struct_conn
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _struct_conn.pdbx_dist_value / _struct_conn.ptnr1_auth_asym_id / _struct_conn.ptnr1_auth_comp_id / _struct_conn.ptnr1_auth_seq_id / _struct_conn.ptnr1_label_asym_id / _struct_conn.ptnr1_label_atom_id / _struct_conn.ptnr1_label_comp_id / _struct_conn.ptnr1_label_seq_id / _struct_conn.ptnr2_auth_asym_id / _struct_conn.ptnr2_auth_comp_id / _struct_conn.ptnr2_auth_seq_id / _struct_conn.ptnr2_label_asym_id / _struct_conn.ptnr2_label_atom_id / _struct_conn.ptnr2_label_comp_id
Revision 1.2Mar 27, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Assembly

Deposited unit
A: Sulfur oxygenase/reductase
B: Sulfur oxygenase/reductase
C: Sulfur oxygenase/reductase
D: Sulfur oxygenase/reductase
E: Sulfur oxygenase/reductase
F: Sulfur oxygenase/reductase
G: Sulfur oxygenase/reductase
H: Sulfur oxygenase/reductase
I: Sulfur oxygenase/reductase
J: Sulfur oxygenase/reductase
K: Sulfur oxygenase/reductase
L: Sulfur oxygenase/reductase
M: Sulfur oxygenase/reductase
N: Sulfur oxygenase/reductase
O: Sulfur oxygenase/reductase
P: Sulfur oxygenase/reductase
Q: Sulfur oxygenase/reductase
R: Sulfur oxygenase/reductase
S: Sulfur oxygenase/reductase
T: Sulfur oxygenase/reductase
U: Sulfur oxygenase/reductase
V: Sulfur oxygenase/reductase
W: Sulfur oxygenase/reductase
X: Sulfur oxygenase/reductase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)859,66148
Polymers858,32024
Non-polymers1,34024
Water40,4082243
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration, > 16-mer
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein ...
Sulfur oxygenase/reductase /


Mass: 35763.344 Da / Num. of mol.: 24
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Sulfurisphaera tokodaii (strain DSM 16993 / JCM 10545 / NBRC 100140 / 7) (archaea)
Strain: DSM 16993 / JCM 10545 / NBRC 100140 / 7 / Gene: sor, ST1127, STK_11270 / Plasmid: pET-17b / Production host: Escherichia coli BL21 (bacteria) / Strain (production host): BL21 / Variant (production host): AI / References: UniProt: Q972K4, sulfur oxygenase/reductase
#2: Chemical...
ChemComp-FE / FE (III) ION / Iron


Mass: 55.845 Da / Num. of mol.: 24 / Source method: obtained synthetically / Formula: Fe / Feature type: SUBJECT OF INVESTIGATION
#3: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 2243 / Source method: isolated from a natural source / Formula: H2O
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: sulfur oxygenase reductase from Sulfurisphaera tokodaii
Type: ORGANELLE OR CELLULAR COMPONENT / Details: spherical homo 24-mer / Entity ID: #1 / Source: RECOMBINANT
Molecular weight
IDEntity assembly-IDValue (°)Experimental value
110.857 MDaNO
210.857 MDaNO
Source (natural)Organism: Sulfurisphaera tokodaii (archaea) / Strain: strain 7
Source (recombinant)Organism: Escherichia coli (E. coli) / Strain: BL21-AI
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMTris-HClTrisTris-HClTris1
2150 mMsodium chlorideNaClSodium chloride1
SpecimenConc.: 10 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: This sample was mono-disperse.
Specimen supportDetails: The grid was washed by acetone prior to use. / Grid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 291 K / Details: Blotting time was 5 seconds (blot force 20)

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Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: FEI TALOS ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 150000 X / Nominal defocus max: 1500 nm / Nominal defocus min: 300 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm
Specimen holderCryogen: NITROGEN
Image recordingAverage exposure time: 50.89 sec. / Electron dose: 50 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON III (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 2558

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Processing

EM software
IDNameVersionCategory
1RELION3particle selection
2EPUimage acquisition
4Gctf1.06CTF correction
7PHENIX1.14model fitting
9Coot0.8.9model refinement
10PHENIX1.14model refinement
11RELION3initial Euler assignment
12RELION3final Euler assignment
13RELION3classification
14RELION33D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 305182
SymmetryPoint symmetry: O (octahedral)
3D reconstructionResolution: 2.24 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 85621 / Symmetry type: POINT
Atomic model buildingB value: 20.65 / Protocol: AB INITIO MODEL / Space: REAL / Target criteria: CCmask

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