+Open data
-Basic information
Entry | Database: PDB / ID: 8rwy | ||||||
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Title | DtpB hexamer from Streptomyces lividans | ||||||
Components | Dyp-type peroxidase family | ||||||
Keywords | OXIDOREDUCTASE / Heme / iron / dye-type peroxidase / METAL BINDING PROTEIN | ||||||
Function / homology | Function and homology information | ||||||
Biological species | Streptomyces lividans (bacteria) | ||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.02 Å | ||||||
Authors | Worrall, J.A.R. / Chaplin, A.K. / Allport, T. | ||||||
Funding support | 1items
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Citation | Journal: Protein Sci / Year: 2024 Title: The oligomeric states of dye-decolorizing peroxidases from Streptomyces lividans and their implications for mechanism of substrate oxidation. Authors: Marina Lučić / Thomas Allport / Thomas A Clarke / Lewis J Williams / Michael T Wilson / Amanda K Chaplin / Jonathan A R Worrall / Abstract: A common evolutionary mechanism in biology to drive function is protein oligomerization. In prokaryotes, the symmetrical assembly of repeating protein units to form homomers is widespread, yet ...A common evolutionary mechanism in biology to drive function is protein oligomerization. In prokaryotes, the symmetrical assembly of repeating protein units to form homomers is widespread, yet consideration in vitro of whether such assemblies have functional or mechanistic consequences is often overlooked. Dye-decolorizing peroxidases (DyPs) are one such example, where their dimeric α + β barrel units can form various oligomeric states, but the oligomer influence, if any, on mechanism and function has received little attention. In this work, we have explored the oligomeric state of three DyPs found in Streptomyces lividans, each with very different mechanistic behaviors in their reactions with hydrogen peroxide and organic substrates. Using analytical ultracentrifugation, we reveal that except for one of the A-type DyPs where only a single sedimenting species is detected, oligomer states ranging from homodimers to dodecamers are prevalent in solution. Using cryo-EM on preparations of the B-type DyP, we determined a 3.02 Å resolution structure of a hexamer assembly that corresponds to the dominant oligomeric state in solution as determined by analytical ultracentrifugation. Furthermore, cryo-EM data detected sub-populations of higher-order oligomers, with one of these formed by an arrangement of two B-type DyP hexamers to give a dodecamer assembly. Our solution and structural insights of these oligomer states provide a new framework to consider previous mechanistic studies of these DyP members and are discussed in terms of long-range electron transfer for substrate oxidation and in the "storage" of oxidizable equivalents on the heme until a two-electron donor is available. | ||||||
History |
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-Structure visualization
Structure viewer | Molecule: MolmilJmol/JSmol |
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-Downloads & links
-Download
PDBx/mmCIF format | 8rwy.cif.gz | 308.4 KB | Display | PDBx/mmCIF format |
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PDB format | pdb8rwy.ent.gz | 254.5 KB | Display | PDB format |
PDBx/mmJSON format | 8rwy.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 8rwy_validation.pdf.gz | 2 MB | Display | wwPDB validaton report |
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Full document | 8rwy_full_validation.pdf.gz | 2.1 MB | Display | |
Data in XML | 8rwy_validation.xml.gz | 71.7 KB | Display | |
Data in CIF | 8rwy_validation.cif.gz | 102.8 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/rw/8rwy ftp://data.pdbj.org/pub/pdb/validation_reports/rw/8rwy | HTTPS FTP |
-Related structure data
Related structure data | 19568MC M: map data used to model this data C: citing same article (ref.) |
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Similar structure data | Similarity search - Function & homologyF&H Search |
-Links
-Assembly
Deposited unit |
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1 |
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-Components
#1: Protein | Mass: 34172.223 Da / Num. of mol.: 6 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Streptomyces lividans (bacteria) / Gene: SSPG_00656 / Production host: Escherichia coli (E. coli) / References: UniProt: A0A7U8UU09 #2: Chemical | ChemComp-HEM / Has ligand of interest | Y | |
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-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
-Sample preparation
Component | Name: Hexamer complex of Dye-type peroxidase B from Streptomyces Lividans Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT |
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Source (natural) | Organism: Streptomyces lividans (bacteria) |
Source (recombinant) | Organism: Escherichia coli (E. coli) |
Buffer solution | pH: 7 |
Specimen | Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES |
Specimen support | Grid material: COPPER / Grid mesh size: 300 divisions/in. |
Vitrification | Cryogen name: ETHANE |
-Electron microscopy imaging
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
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Microscopy | Model: FEI TITAN KRIOS |
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM |
Electron lens | Mode: BRIGHT FIELD / Nominal magnification: 130000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 700 nm |
Specimen holder | Cryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER |
Image recording | Electron dose: 47.19 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) |
-Processing
EM software |
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||
Symmetry | Point symmetry: D3 (2x3 fold dihedral) | ||||||||||||||||||||||||
3D reconstruction | Resolution: 3.02 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 24402 / Symmetry type: POINT | ||||||||||||||||||||||||
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