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- PDB-8b14: T5 Receptor Binding Protein pb5 in complex with its E. coli recep... -

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Basic information

Entry
Database: PDB / ID: 8b14
TitleT5 Receptor Binding Protein pb5 in complex with its E. coli receptor FhuA
Components
  • FhuA iron-ferrichrome transporter
  • pb5 bacteriophage T5 receptor binding protein
KeywordsVIRAL PROTEIN / bacteriophage / receptor / complex / RPB
Function / homology
Function and homology information


siderophore transmembrane transport / siderophore uptake transmembrane transporter activity / virus tail / virion binding / transmembrane transporter complex / toxic substance binding / cell outer membrane / signaling receptor activity / intracellular iron ion homeostasis / entry receptor-mediated virion attachment to host cell ...siderophore transmembrane transport / siderophore uptake transmembrane transporter activity / virus tail / virion binding / transmembrane transporter complex / toxic substance binding / cell outer membrane / signaling receptor activity / intracellular iron ion homeostasis / entry receptor-mediated virion attachment to host cell / receptor-mediated virion attachment to host cell / symbiont entry into host cell / iron ion binding / protein domain specific binding / membrane
Similarity search - Function
TonB-dependent receptor (TBDR) proteins signature 1. / TonB box, conserved site / TonB-dependent siderophore receptor / TonB-dependent receptor, conserved site / TonB-dependent receptor (TBDR) proteins signature 2. / TonB-dependent receptor (TBDR) proteins profile. / Vitamin B12 transporter BtuB-like / TonB-dependent receptor-like, beta-barrel / TonB dependent receptor-like, beta-barrel / TonB-dependent receptor, plug domain superfamily ...TonB-dependent receptor (TBDR) proteins signature 1. / TonB box, conserved site / TonB-dependent siderophore receptor / TonB-dependent receptor, conserved site / TonB-dependent receptor (TBDR) proteins signature 2. / TonB-dependent receptor (TBDR) proteins profile. / Vitamin B12 transporter BtuB-like / TonB-dependent receptor-like, beta-barrel / TonB dependent receptor-like, beta-barrel / TonB-dependent receptor, plug domain superfamily / TonB-dependent receptor, plug domain / TonB-dependent receptor-like, beta-barrel domain superfamily / TonB-dependent Receptor Plug Domain
Similarity search - Domain/homology
DECYLAMINE-N,N-DIMETHYL-N-OXIDE / Chem-LU9 / Ferrichrome outer membrane transporter/phage receptor / Receptor-binding protein pb5
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
Escherichia phage T5 (virus)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.6 Å
AuthorsDegroux, S. / Effantin, G. / Linares, R. / Schoehn, G. / Breyton, C.
Funding support France, 2items
OrganizationGrant numberCountry
Agence Nationale de la Recherche (ANR)ANR-16-CE11-0027 France
Agence Nationale de la Recherche (ANR)ANR-21-CE11-0023 France
CitationJournal: J Virol / Year: 2023
Title: Deciphering Bacteriophage T5 Host Recognition Mechanism and Infection Trigger.
Authors: Séraphine Degroux / Grégory Effantin / Romain Linares / Guy Schoehn / Cécile Breyton /
Abstract: Bacteriophages, viruses infecting bacteria, recognize their host with high specificity, binding to either saccharide motifs or proteins of the cell wall of their host. In the majority of ...Bacteriophages, viruses infecting bacteria, recognize their host with high specificity, binding to either saccharide motifs or proteins of the cell wall of their host. In the majority of bacteriophages, this host recognition is performed by receptor binding proteins (RBPs) located at the extremity of a tail. Interaction between the RBPs and the host is the trigger for bacteriophage infection, but the molecular details of the mechanisms are unknown for most bacteriophages. Here, we present the electron cryomicroscopy (cryo-EM) structure of bacteriophage T5 RBP in complex with its Escherichia coli receptor, the iron ferrichrome transporter FhuA. Monomeric RBP is located at the extremity of T5's long flexible tail, and its irreversible binding to FhuA commits T5 to infection. Analysis of the structure of RBP within the complex, comparison with its AlphaFold2-predicted structure, and its fit into a previously determined map of the T5 tail tip in complex with FhuA allow us to propose a mechanism of transmission of the RBP receptor binding to the straight fiber, initiating the cascade of events that commits T5 to DNA ejection. Tailed bacteriophages specifically recognize their bacterial host by interaction of their receptor binding protein(s) (RBPs) with saccharides and/or proteins located at the surface of their prey. This crucial interaction commits the virus to infection, but the molecular details of this mechanism are unknown for the majority of bacteriophages. We determined the structure of bacteriophage T5 RBP in complex with its E. coli receptor, FhuA, by cryo-EM. This first structure of an RBP bound to its protein receptor allowed us to propose a mechanism of transmission of host recognition to the rest of the phage, ultimately opening the capsid and perforating the cell wall and, thus, allowing safe channeling of the DNA into the host cytoplasm.
History
DepositionSep 9, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0Feb 8, 2023Provider: repository / Type: Initial release
Revision 1.1Feb 21, 2024Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / citation / citation_author / em_3d_fitting_list
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID / _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: FhuA iron-ferrichrome transporter
B: pb5 bacteriophage T5 receptor binding protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)150,8574
Polymers148,6602
Non-polymers2,1982
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area9510 Å2
ΔGint-35 kcal/mol
Surface area40810 Å2

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Components

#1: Protein FhuA iron-ferrichrome transporter


Mass: 79876.945 Da / Num. of mol.: 1
Mutation: Presence of an Histag after P405, added residues : SHHHHHHGS
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Production host: Escherichia coli (E. coli) / Strain (production host): AW740 / References: UniProt: P06971
#2: Protein pb5 bacteriophage T5 receptor binding protein


Mass: 68782.562 Da / Num. of mol.: 1 / Mutation: Presence of an Histag in C-ter position
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia phage T5 (virus) / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P23207
#3: Chemical ChemComp-DDQ / DECYLAMINE-N,N-DIMETHYL-N-OXIDE


Mass: 201.349 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Formula: C12H27NO / Feature type: SUBJECT OF INVESTIGATION
#4: Chemical ChemComp-LU9 / [(2R,3S,4R,5R,6R)-2-[[(2R,4R,5R,6R)-6-[(1R)-1,2-bis(oxidanyl)ethyl]-4-[(2R,4R,5R,6R)-6-[(1R)-1,2-bis(oxidanyl)ethyl]-2-carboxy-4,5-bis(oxidanyl)oxan-2-yl]oxy-2-carboxy-5-oxidanyl-oxan-2-yl]oxymethyl]-5-[[(3R)-3-dodecanoyloxytetradecanoyl]amino]-4-(3-nonanoyloxypropanoyloxy)-6-[[(2R,3S,4R,5R,6R)-3-oxidanyl-4-[(3S)-3-oxidanyltetradecanoyl]oxy-5-[[(3R)-3-oxidanyltridecanoyl]amino]-6-phosphonatooxy-oxan-2-yl]methoxy]oxan-3-yl] phosphate / LIPOPOLYSACCHARIDE / Lipopolysaccharide


Mass: 1996.235 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C93H164N2O39P2 / Comment: toxin*YM
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

Component
IDNameTypeDetailsEntity IDParent-IDSource
1Complex between T5 Receptor Binding Protein pb5 and its E. coli receptor FhuA, stabilized with detergentCOMPLEXThe FhuA-pb5 complex was formed by adding equimolar amounts of the two proteins, which results in 100% complex formation. FhuA-RBPpb5 complex is stabilized with 1.6% C10DAO at a protein concentration of 4.3 mg/mL#1-#20RECOMBINANT
2E. coli receptor FhuACOMPLEX#11RECOMBINANT
3T5 Receptor Binding Protein pb5COMPLEX#21RECOMBINANT
Molecular weightValue: 0.15 MDa / Experimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
21Escherichia coli (E. coli)562
32Escherichia virus T52695836
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
21Escherichia coli (E. coli)562
32Escherichia coli BL21(DE3) (bacteria)469008
Buffer solutionpH: 8.5
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMTrisC4H11NO31
21.6 %C10DAO1
SpecimenConc.: 1.1111111111111 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: The FhuA-pb5 complex was formed by adding equimolar amounts of the two proteins, which results in 100% complex formation. FhuA-RBPpb5 complex is stabilized with 1.6% C10DAO at a protein ...Details: The FhuA-pb5 complex was formed by adding equimolar amounts of the two proteins, which results in 100% complex formation. FhuA-RBPpb5 complex is stabilized with 1.6% C10DAO at a protein concentration of 4.3 mg/ml
Specimen supportDetails: 25 mA / Grid material: COPPER/RHODIUM / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil R2/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 293.15 K
Details: 3.5 microliters of the FhuA-pb5 complex were deposited on a freshly glow discharged (25 mA, 30 sec) Cu/Rh 400 mesh Quantifoil R 2/1 EM grids and flash-frozen in nitrogen-cooled liquid ethane ...Details: 3.5 microliters of the FhuA-pb5 complex were deposited on a freshly glow discharged (25 mA, 30 sec) Cu/Rh 400 mesh Quantifoil R 2/1 EM grids and flash-frozen in nitrogen-cooled liquid ethane using a ThermoFisher Mark IV Vitrobot device (100% humidity, 293.15K, 2s blotting time, blot force 1).

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS / Details: calibrated pixel size = 1.052
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 130000 X / Nominal defocus max: 2600 nm / Nominal defocus min: 1200 nm / Cs: 2.7 mm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recording

Imaging-ID: 1 / Electron dose: 60 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1

IDNum. of real imagesDetails
18752
2777Unlike the first dataset, this one was acquired with a phase plate, close to focus (between -0.5 and -1.0 micrometer).
EM imaging opticsEnergyfilter name: GIF Quantum LS / Energyfilter slit width: 20 eV
Image scans
Movie frames/imageIDImage recording-IDEntry-ID
60228B14
118B14

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Processing

SoftwareName: PHENIX / Version: 1.20.1_4487: / Classification: refinement
EM software
IDNameVersionCategory
2EPUimage acquisition
4GctfCTF correction
7UCSF Chimeramodel fitting
9Coot0.9.2model refinement
10PHENIX1.18.2-3874model refinement
13RELION3classification
14RELION33D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 2191586
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 109350 / Algorithm: BACK PROJECTION / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL / Space: REAL
Details: The pb5 protein model was built de novo in the cryo-electron microscopy map. FhuA was adapted from the FhuA structure solved by X-ray crystallography (PDB 2GRX). The two structures were ...Details: The pb5 protein model was built de novo in the cryo-electron microscopy map. FhuA was adapted from the FhuA structure solved by X-ray crystallography (PDB 2GRX). The two structures were first refined separately using Coot (version 0.9.2) and Phenix (version 1.18.2-3874) softwares, then together. Structure validation was done using MolProbity.
Atomic model buildingPDB-ID: 2GRX

2grx


Accession code: 2GRX / Source name: PDB / Type: experimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0059777
ELECTRON MICROSCOPYf_angle_d0.51613278
ELECTRON MICROSCOPYf_dihedral_angle_d4.5921359
ELECTRON MICROSCOPYf_chiral_restr0.0451415
ELECTRON MICROSCOPYf_plane_restr0.0031753

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