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- EMDB-41255: Cryo-EM structure of Pseudomonas aeruginosa TonB-dependent transp... -

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Entry
Database: EMDB / ID: EMD-41255
TitleCryo-EM structure of Pseudomonas aeruginosa TonB-dependent transporter PhuR in complex with synthetic antibody and heme
Map data
Sample
  • Complex: Heme bound P. aeruginosa PhuR with bound synthetic antibody
    • Protein or peptide: Heme/hemoglobin uptake outer membrane receptor PhuR
    • Protein or peptide: Synthetic Antibody Heavy Chain
    • Protein or peptide: Synthetic Antibody Light Chain
  • Ligand: PROTOPORPHYRIN IX CONTAINING FE
  • Ligand: (2~{R},4~{R},5~{S},6~{S})-6-[(1~{R})-1,2-bis(oxidanyl)ethyl]-2-[[(2~{R},3~{S},4~{S},5~{R},6~{S})-5-[[(3~{R})-3-dodecanoyloxytetradecanoyl]amino]-6-[[(2~{R},3~{R},4~{R},5~{S},6~{S})-3-oxidanyl-5-[[(3~{R})-3-oxidanyltetradecanoyl]amino]-4-[(3~{R})-3-oxidanyltetradecanoyl]oxy-6-phosphonooxy-oxan-2-yl]methoxy]-3-phosphonooxy-4-[(3~{S})-3-tetradecanoyloxytetradecanoyl]oxy-oxan-2-yl]methoxy]-4,5-bis(oxidanyl)oxane-2-carboxylic acid
  • Ligand: water
Keywordsbeta barrel / outer membrane protein / heme binding protein / heme transport / MEMBRANE PROTEIN
Function / homology
Function and homology information


heme transport / siderophore transmembrane transport / heme transmembrane transporter activity / siderophore uptake transmembrane transporter activity / outer membrane / cell outer membrane / cellular response to iron ion
Similarity search - Function
TonB-dependent haem/haemoglobin receptor / TonB-dependent haemoglobin/transferrin/lactoferrin receptor / Vitamin B12 transporter BtuB-like / TonB-dependent receptor-like, beta-barrel / TonB dependent receptor / 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
Heme/hemoglobin uptake outer membrane receptor PhuR
Similarity search - Component
Biological speciesPseudomonas aeruginosa (bacteria) / Homo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.5 Å
AuthorsKnejski P / Erramilli SK / Kossiakoff AA
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM117372 United States
CitationJournal: 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
DepositionJul 15, 2023-
Header (metadata) releaseJan 31, 2024-
Map releaseJan 31, 2024-
UpdateApr 17, 2024-
Current statusApr 17, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_41255.png

Downloads & links

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Map

FileDownload / File: emd_41255.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.068 Å
Density
Contour LevelBy AUTHOR: 0.35
Minimum - Maximum-2.1059093 - 3.2095459
Average (Standard dev.)0.0005146022 (±0.06505283)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions360360360
Spacing360360360
CellA=B=C: 384.47998 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_41255_msk_1.map
Projections & Slices
AxesZYX

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Slices (1/2)
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Half map: #2

Fileemd_41255_half_map_1.map
Projections & Slices
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Histogram

Histogram (log scale)

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Half map: #1

Fileemd_41255_half_map_2.map
Projections & Slices
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Sample components

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Entire : Heme bound P. aeruginosa PhuR with bound synthetic antibody

EntireName: Heme bound P. aeruginosa PhuR with bound synthetic antibody
Components
  • Complex: Heme bound P. aeruginosa PhuR with bound synthetic antibody
    • Protein or peptide: Heme/hemoglobin uptake outer membrane receptor PhuR
    • Protein or peptide: Synthetic Antibody Heavy Chain
    • Protein or peptide: Synthetic Antibody Light Chain
  • Ligand: PROTOPORPHYRIN IX CONTAINING FE
  • Ligand: (2~{R},4~{R},5~{S},6~{S})-6-[(1~{R})-1,2-bis(oxidanyl)ethyl]-2-[[(2~{R},3~{S},4~{S},5~{R},6~{S})-5-[[(3~{R})-3-dodecanoyloxytetradecanoyl]amino]-6-[[(2~{R},3~{R},4~{R},5~{S},6~{S})-3-oxidanyl-5-[[(3~{R})-3-oxidanyltetradecanoyl]amino]-4-[(3~{R})-3-oxidanyltetradecanoyl]oxy-6-phosphonooxy-oxan-2-yl]methoxy]-3-phosphonooxy-4-[(3~{S})-3-tetradecanoyloxytetradecanoyl]oxy-oxan-2-yl]methoxy]-4,5-bis(oxidanyl)oxane-2-carboxylic acid
  • Ligand: water

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Supramolecule #1: Heme bound P. aeruginosa PhuR with bound synthetic antibody

SupramoleculeName: Heme bound P. aeruginosa PhuR with bound synthetic antibody
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Source (natural)Organism: Pseudomonas aeruginosa (bacteria)

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Macromolecule #1: Heme/hemoglobin uptake outer membrane receptor PhuR

MacromoleculeName: Heme/hemoglobin uptake outer membrane receptor PhuR / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Pseudomonas aeruginosa (bacteria)
Molecular weightTheoretical: 86.37032 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MKYLLPTAAA GLLLLAAQPA MAMGHHHHHH GENLYFQGGN AVPLTPTTIT ATRTEQAVDS VPSTVSVQTR EQLDRQNVNN IKELVRYEP GVSVGGAGQR AGITGYNIRG IDGNRILTQI DGVELPNDFF SGPYAQTHRN YVDPDIVKRV EILRGPASAL Y GSNAIGGA ...String:
MKYLLPTAAA GLLLLAAQPA MAMGHHHHHH GENLYFQGGN AVPLTPTTIT ATRTEQAVDS VPSTVSVQTR EQLDRQNVNN IKELVRYEP GVSVGGAGQR AGITGYNIRG IDGNRILTQI DGVELPNDFF SGPYAQTHRN YVDPDIVKRV EILRGPASAL Y GSNAIGGA VSYFTLDPSD IIKDGKDVGA RLKAGYESAS HSWLTSATVA GRADDFDGLL HYGYRQGHET ESNGGHGGTG LS RSEANPE DADSYSLLGK LGWNYAEGSR FGLVFEKYKS DVDTDQKSAY GGPYDKGKPA IPPSMLPGGM YQWRKGNDTL TRE RYGLEH HFLLDSQVAD RIQWSLNYQL AKTDQATREF YYPITRKVLR TRDTTYKERL WVFDSQLDKS FAIGETEHLL SYGI NLKHQ KVTGMRSGTG TNLDTGADSP RDALERSSDF PDPTVKTYAL FAQDSISWND WTFTPGLRYD YTRMEPHITD EFLRT MKQS QNTAVDESDK KWHRVSPKFG VTYDFAQHYT WYGQYAQGFR TPTAKALYGR FENLQAGYHI EPNPNLKPEK SQSFET GLR GKFDEGSFGV AVFYNKYRDF IDEDALNTDS TGGNGQTFQS NNIERAVIKG VELKGRLELG AFGAPQGLYT QGSVAYA YG RNKDNGEPIN SVNPLTGVFG LGYDEADGNY GGLLSWTLVK RKDRVDDSTF HTPDGTASQF KTPGFGVLDL SAYYRLSK D LTLNAGLYNL TDKKYWLWDD VRGYDSVGEA SALAPANIDR LSQPGRNFAV NLVWDI

UniProtKB: Heme/hemoglobin uptake outer membrane receptor PhuR

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Macromolecule #2: Synthetic Antibody Heavy Chain

MacromoleculeName: Synthetic Antibody Heavy Chain / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 25.525445 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: EISEVQLVES GGGLVQPGGS LRLSCAASGF NFYYYSIHWV RQAPGKGLEW VASISPYYGS TYYADSVKGR FTISADTSKN TAYLQMNSL RAEDTAVYYC ARSMNWYYSS PYGMSQGMDY WGQGTLVTVS SASTKGPSVF PLAPSSKSTS GGTAALGCLV K DYFPEPVT ...String:
EISEVQLVES GGGLVQPGGS LRLSCAASGF NFYYYSIHWV RQAPGKGLEW VASISPYYGS TYYADSVKGR FTISADTSKN TAYLQMNSL RAEDTAVYYC ARSMNWYYSS PYGMSQGMDY WGQGTLVTVS SASTKGPSVF PLAPSSKSTS GGTAALGCLV K DYFPEPVT VSWNSGALTS GVHTFPAVLQ SSGLYSLSSV VTVPSSSLGT QTYICNVNHK PSNTKVDKKV EPKSCDKTHT

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Macromolecule #3: Synthetic Antibody Light Chain

MacromoleculeName: Synthetic Antibody Light Chain / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 23.212715 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: SDIQMTQSPS SLSASVGDRV TITCRASQSV SSAVAWYQQK PGKAPKLLIY SASSLYSGVP SRFSGSRSGT DFTLTISSLQ PEDFATYYC QQGSSSPLTF GQGTKVEIKR TVAAPSVFIF PPSDSQLKSG TASVVCLLNN FYPREAKVQW KVDNALQSGN S QESVTEQD ...String:
SDIQMTQSPS SLSASVGDRV TITCRASQSV SSAVAWYQQK PGKAPKLLIY SASSLYSGVP SRFSGSRSGT DFTLTISSLQ PEDFATYYC QQGSSSPLTF GQGTKVEIKR TVAAPSVFIF PPSDSQLKSG TASVVCLLNN FYPREAKVQW KVDNALQSGN S QESVTEQD SKDSTYSLSS TLTLSKADYE KHKVYACEVT HQGLSSPVTK SFNRGEC

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Macromolecule #4: PROTOPORPHYRIN IX CONTAINING FE

MacromoleculeName: PROTOPORPHYRIN IX CONTAINING FE / type: ligand / ID: 4 / Number of copies: 1 / Formula: HEM
Molecular weightTheoretical: 616.487 Da
Chemical component information

ChemComp-HEM:
PROTOPORPHYRIN IX CONTAINING FE / Heme B

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Macromolecule #5: (2~{R},4~{R},5~{S},6~{S})-6-[(1~{R})-1,2-bis(oxidanyl)ethyl]-2-[[...

MacromoleculeName: (2~{R},4~{R},5~{S},6~{S})-6-[(1~{R})-1,2-bis(oxidanyl)ethyl]-2-[[(2~{R},3~{S},4~{S},5~{R},6~{S})-5-[[(3~{R})-3-dodecanoyloxytetradecanoyl]amino]-6-[[(2~{R},3~{R},4~{R},5~{S},6~{S})-3-oxidanyl-5- ...Name: (2~{R},4~{R},5~{S},6~{S})-6-[(1~{R})-1,2-bis(oxidanyl)ethyl]-2-[[(2~{R},3~{S},4~{S},5~{R},6~{S})-5-[[(3~{R})-3-dodecanoyloxytetradecanoyl]amino]-6-[[(2~{R},3~{R},4~{R},5~{S},6~{S})-3-oxidanyl-5-[[(3~{R})-3-oxidanyltetradecanoyl]amino]-4-[(3~{R})-3-oxidanyltetradecanoyl]oxy-6-phosphonooxy-oxan-2-yl]methoxy]-3-phosphonooxy-4-[(3~{S})-3-tetradecanoyloxytetradecanoyl]oxy-oxan-2-yl]methoxy]-4,5-bis(oxidanyl)oxane-2-carboxylic acid
type: ligand / ID: 5 / Number of copies: 1 / Formula: CTQ
Molecular weightTheoretical: 2.018542 KDa
Chemical component information

ChemComp-CTQ:
(2~{R},4~{R},5~{S},6~{S})-6-[(1~{R})-1,2-bis(oxidanyl)ethyl]-2-[[(2~{R},3~{S},4~{S},5~{R},6~{S})-5-[[(3~{R})-3-dodecanoyloxytetradecanoyl]amino]-6-[[(2~{R},3~{R},4~{R},5~{S},6~{S})-3-oxidanyl-5-[[(3~{R})-3-oxidanyltetradecanoyl]amino]-4-[(3~{R})-3-oxidanyltetradecanoyl]oxy-6-phosphonooxy-oxan-2-yl]methoxy]-3-phosphonooxy-4-[(3~{S})-3-tetradecanoyloxytetradecanoyl]oxy-oxan-2-yl]methoxy]-4,5-bis(oxidanyl)oxane-2-carboxylic acid

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Macromolecule #6: water

MacromoleculeName: water / type: ligand / ID: 6 / Number of copies: 81 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER / Water

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration5 mg/mL
BufferpH: 7.5
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 300
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 281 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2.5 µm / Nominal defocus min: 0.9 µm
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number real images: 5031 / Average electron dose: 60.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.3)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.3)
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 2.5 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.3) / Number images used: 292512

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Atomic model buiding 1

Initial modelChain - Source name: AlphaFold / Chain - Initial model type: in silico model
Output model

PDB-8the:
Cryo-EM structure of Pseudomonas aeruginosa TonB-dependent transporter PhuR in complex with synthetic antibody and heme

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