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Yorodumi- PDB-8the: Cryo-EM structure of Pseudomonas aeruginosa TonB-dependent transp... -
+Open data
-Basic information
Entry | Database: PDB / ID: 8the | ||||||
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Title | Cryo-EM structure of Pseudomonas aeruginosa TonB-dependent transporter PhuR in complex with synthetic antibody and heme | ||||||
Components |
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Keywords | MEMBRANE PROTEIN / beta barrel / outer membrane protein / heme binding protein / heme transport | ||||||
Function / homology | Function and homology information heme transport / siderophore transmembrane transport / heme transmembrane transporter activity / siderophore uptake transmembrane transporter activity / outer membrane / cellular response to iron ion / cell outer membrane Similarity search - Function | ||||||
Biological species | Pseudomonas aeruginosa (bacteria) Homo sapiens (human) | ||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.5 Å | ||||||
Authors | Knejski, P. / Erramilli, S.K. / Kossiakoff, A.A. | ||||||
Funding support | United States, 1items
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Citation | Journal: Structure / Year: 2024 Title: Chaperone-assisted cryo-EM structure of P. aeruginosa PhuR reveals molecular basis for heme binding. Authors: Paweł P Knejski / Satchal K Erramilli / Anthony A Kossiakoff / Abstract: Pathogenic bacteria, such as Pseudomonas aeruginosa, depend on scavenging heme for the acquisition of iron, an essential nutrient. The TonB-dependent transporter (TBDT) PhuR is the major heme uptake ...Pathogenic bacteria, such as Pseudomonas aeruginosa, depend on scavenging heme for the acquisition of iron, an essential nutrient. The TonB-dependent transporter (TBDT) PhuR is the major heme uptake protein in P. aeruginosa clinical isolates. However, a comprehensive understanding of heme recognition and TBDT transport mechanisms, especially PhuR, remains limited. In this study, we employed single-particle cryogenic electron microscopy (cryo-EM) and a phage display-generated synthetic antibody (sAB) as a fiducial marker to enable the determination of a high-resolution (2.5 Å) structure of PhuR with a bound heme. Notably, the structure reveals iron coordination by Y529 on a conserved extracellular loop, shedding light on the role of tyrosine in heme binding. Biochemical assays and negative-stain EM demonstrated that the sAB specifically targets the heme-bound state of PhuR. These findings provide insights into PhuR's heme binding and offer a template for developing conformation-specific sABs against outer membrane proteins (OMPs) for structure-function investigations. | ||||||
History |
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-Structure visualization
Structure viewer | Molecule: MolmilJmol/JSmol |
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-Downloads & links
-Download
PDBx/mmCIF format | 8the.cif.gz | 212.1 KB | Display | PDBx/mmCIF format |
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PDB format | pdb8the.ent.gz | Display | PDB format | |
PDBx/mmJSON format | 8the.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 8the_validation.pdf.gz | 1.4 MB | Display | wwPDB validaton report |
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Full document | 8the_full_validation.pdf.gz | 1.4 MB | Display | |
Data in XML | 8the_validation.xml.gz | 41.8 KB | Display | |
Data in CIF | 8the_validation.cif.gz | 59.7 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/th/8the ftp://data.pdbj.org/pub/pdb/validation_reports/th/8the | HTTPS FTP |
-Related structure data
Related structure data | 41255MC 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 |
Experimental dataset #1 | Data reference: 10.6019/EMPIAR-11627 / Data set type: EMPIAR |
-Links
-Assembly
Deposited unit |
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1 |
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-Components
-Protein , 1 types, 1 molecules A
#1: Protein | Mass: 86370.320 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Pseudomonas aeruginosa (bacteria) / Gene: phuR, PA4710 / Production host: Escherichia coli (E. coli) / References: UniProt: Q9HV88 |
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-Antibody , 2 types, 2 molecules HL
#2: Antibody | Mass: 25525.445 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Homo sapiens (human) / Production host: Escherichia coli (E. coli) |
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#3: Antibody | Mass: 23212.715 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Homo sapiens (human) / Production host: Escherichia coli (E. coli) |
-Non-polymers , 3 types, 83 molecules
#4: Chemical | ChemComp-HEM / |
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#5: Chemical | ChemComp-CTQ / ( |
#6: Water | ChemComp-HOH / |
-Details
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: Heme bound P. aeruginosa PhuR with bound synthetic antibody Type: COMPLEX / Entity ID: #1-#3 / Source: RECOMBINANT |
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Molecular weight | Experimental value: NO |
Source (natural) | Organism: Pseudomonas aeruginosa (bacteria) |
Source (recombinant) | Organism: Escherichia coli (E. coli) |
Buffer solution | pH: 7.5 |
Specimen | Conc.: 5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES |
Specimen support | Grid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3 |
Vitrification | Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 281 K |
-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 defocus max: 2500 nm / Nominal defocus min: 900 nm |
Image recording | Electron dose: 60 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of real images: 5031 |
-Processing
EM software |
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||||||||||
Symmetry | Point symmetry: C1 (asymmetric) | ||||||||||||||||||||||||||||||||
3D reconstruction | Resolution: 2.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 292512 / Symmetry type: POINT | ||||||||||||||||||||||||||||||||
Atomic model building | Source name: AlphaFold / Type: in silico model |