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- PDB-8y5i: Cryo-EM structure of E.coli spermidine transporter PotD-PotABC in... -

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Basic information

Entry
Database: PDB / ID: 8y5i
TitleCryo-EM structure of E.coli spermidine transporter PotD-PotABC in translocation intermidiate state
Components
  • (Spermidine/putrescine transport system permease protein ...) x 2
  • Putrescine-binding periplasmic protein
  • Spermidine/putrescine import ATP-binding protein PotA
KeywordsTRANSPORT PROTEIN / ABC transporter
Function / homology
Function and homology information


ABC-type polyamine transporter / ABC-type putrescine transporter activity / spermidine transmembrane transport / polyamine binding / polyamine transport / putrescine transport / ABC-type polyamine transporter activity / ATP-binding cassette (ABC) transporter complex / outer membrane-bounded periplasmic space / nucleotide binding ...ABC-type polyamine transporter / ABC-type putrescine transporter activity / spermidine transmembrane transport / polyamine binding / polyamine transport / putrescine transport / ABC-type polyamine transporter activity / ATP-binding cassette (ABC) transporter complex / outer membrane-bounded periplasmic space / nucleotide binding / ATP hydrolysis activity / ATP binding / membrane / plasma membrane
Similarity search - Function
Spermidine/putrescine import ATP-binding protein potA family profile. / Spermidine/putrescine ABC transporter, ATP-binding subunit / PotA, ATP-binding domain / : / : / Spermidine/putrescine-binding periplasmic protein / Transport-associated OB, type 2 / TOBE domain / Molybdate/tungstate binding, C-terminal / ABC transporter type 1, transmembrane domain MetI-like ...Spermidine/putrescine import ATP-binding protein potA family profile. / Spermidine/putrescine ABC transporter, ATP-binding subunit / PotA, ATP-binding domain / : / : / Spermidine/putrescine-binding periplasmic protein / Transport-associated OB, type 2 / TOBE domain / Molybdate/tungstate binding, C-terminal / ABC transporter type 1, transmembrane domain MetI-like / MetI-like superfamily / Binding-protein-dependent transport system inner membrane component / ABC transporter integral membrane type-1 domain profile. / Bacterial extracellular solute-binding protein / Bacterial extracellular solute-binding protein / ABC transporter-like, conserved site / ABC transporters family signature. / ABC transporter / ABC transporter-like, ATP-binding domain / ATP-binding cassette, ABC transporter-type domain profile. / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
ADENOSINE-5'-TRIPHOSPHATE / Putrescine-binding periplasmic protein / Spermidine/putrescine transport system permease protein PotC / Spermidine/putrescine import ATP-binding protein PotA
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3 Å
AuthorsQiao, Z. / Gao, Y.G.
Funding support Singapore, 1items
OrganizationGrant numberCountry
Ministry of Education (MoE, Singapore) Singapore
CitationJournal: Sci Adv / Year: 2024
Title: Structural insights into polyamine spermidine uptake by the ABC transporter PotD-PotABC.
Authors: Zhu Qiao / Phong Hoa Do / Joshua Yi Yeo / Rya Ero / Zhuowen Li / Liying Zhan / Sandip Basak / Yong-Gui Gao /
Abstract: Polyamines, characterized by their polycationic nature, are ubiquitously present in all organisms and play numerous cellular functions. Among polyamines, spermidine stands out as the predominant type ...Polyamines, characterized by their polycationic nature, are ubiquitously present in all organisms and play numerous cellular functions. Among polyamines, spermidine stands out as the predominant type in both prokaryotic and eukaryotic cells. The PotD-PotABC protein complex in , belonging to the adenosine triphosphate-binding cassette transporter family, is a spermidine-preferential uptake system. Here, we report structural details of the polyamine uptake system PotD-PotABC in various states. Our analyses reveal distinct "inward-facing" and "outward-facing" conformations of the PotD-PotABC transporter, as well as conformational changes in the "gating" residues (F222, Y223, D226, and K241 in PotB; Y219 and K223 in PotC) controlling spermidine uptake. Therefore, our structural analysis provides insights into how the PotD-PotABC importer recognizes the substrate-binding protein PotD and elucidates molecular insights into the spermidine uptake mechanism of bacteria.
History
DepositionJan 31, 2024Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Oct 9, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
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Assembly

Deposited unit
A: Spermidine/putrescine import ATP-binding protein PotA
B: Spermidine/putrescine transport system permease protein PotB
C: Spermidine/putrescine transport system permease protein PotC
D: Spermidine/putrescine import ATP-binding protein PotA
E: Putrescine-binding periplasmic protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)187,4679
Polymers186,4045
Non-polymers1,0634
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

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Protein , 2 types, 3 molecules ADE

#1: Protein Spermidine/putrescine import ATP-binding protein PotA


Mass: 43079.137 Da / Num. of mol.: 2 / Mutation: E173Q
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: potA, b1126, JW1112 / Production host: Escherichia coli (E. coli) / References: UniProt: P69874, ABC-type polyamine transporter
#4: Protein Putrescine-binding periplasmic protein


Mass: 38906.938 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli)
Gene: potD, potD_1, A9X72_15260, AC065_02235, ACN81_08960, ACU57_04395, AM464_23650, BF481_002097, BGM66_002097, BJI68_20810, BJJ90_15605, BMT50_26180, BMT91_12660, BvCmsSIP010_04197, C0P57_004320, ...Gene: potD, potD_1, A9X72_15260, AC065_02235, ACN81_08960, ACU57_04395, AM464_23650, BF481_002097, BGM66_002097, BJI68_20810, BJJ90_15605, BMT50_26180, BMT91_12660, BvCmsSIP010_04197, C0P57_004320, C1Q91_003201, C5N07_18640, CA593_22865, CCS08_25895, CF22_003053, CG692_05805, CG831_001508, CQ986_001050, CR538_15460, CR539_10310, CTR35_002408, CV83915_00461, D9H94_04145, DIV22_29210, DN627_23510, DS732_10680, DTL43_16095, E0I52_23195, E4K51_18510, E5H86_12360, E6D34_10765, EAN77_25155, ECs1499, EIZ93_12980, ERS139208_03585, F3P10_09120, FFF58_15800, FHD44_11245, FJQ40_01110, FN993_001405, FOI11_007330, FOI11_14300, FPS11_22525, FV293_24150, G3V95_08220, G4A38_16220, G4A47_15580, GIB53_10955, GJ11_06750, GKF89_00455, GNW61_10810, GOP14_002871, GOP25_17235, GP965_23750, GQM17_13980, GQM21_19780, GQN34_10415, GRW56_11575, GRW56_16830, H0P11_10030, HHH44_002984, HLQ92_15565, HMV95_16200, HV209_29265, HVY77_15605, HX136_15480, I6H00_08380, I6H02_24690, J0541_003203, J4S20_003291, J5U05_002927, JFD_02102, JNP96_10880, NCTC10090_00712, NCTC10865_03647, NCTC11127_00497, NCTC11181_00132, NCTC11341_04942, NCTC4450_04159, NCTC8960_00797, NCTC8985_01697, NCTC9071_02742, NCTC9706_00378, NEP60_22980, RG28_17065, SAMEA3472044_03468, SAMEA3752557_01589, SAMEA3753106_00760
Production host: Escherichia coli (E. coli) / References: UniProt: C3TDJ2

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Spermidine/putrescine transport system permease protein ... , 2 types, 2 molecules BC

#2: Protein Spermidine/putrescine transport system permease protein PotB


Mass: 32206.090 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: potB, b1125, JW1111 / Production host: Escherichia coli (E. coli)
#3: Protein Spermidine/putrescine transport system permease protein PotC


Mass: 29132.850 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: potC, b1124, JW1110 / Production host: Escherichia coli (E. coli) / References: UniProt: P0AFK6

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Non-polymers , 2 types, 4 molecules

#5: Chemical ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE


Mass: 507.181 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C10H16N5O13P3 / Feature type: SUBJECT OF INVESTIGATION / Comment: ATP, energy-carrying molecule*YM
#6: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION

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Details

Has ligand of interestY
Has protein modificationN

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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: ABC transporter / Type: COMPLEX / Entity ID: #1-#4 / Source: RECOMBINANT
Molecular weightValue: 186.1 kDa/nm / Experimental value: YES
Source (natural)Organism: Escherichia coli (E. coli)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
SpecimenConc.: 10 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: OTHER
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 1000 nm / Nominal defocus min: 500 nm
Image recordingElectron dose: 50 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k)

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Processing

EM softwareName: PHENIX / Version: 1.18.2_3874: / Category: model refinement
CTF correctionType: PHASE FLIPPING ONLY
3D reconstructionResolution: 3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 107909 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00612634
ELECTRON MICROSCOPYf_angle_d0.72417157
ELECTRON MICROSCOPYf_dihedral_angle_d6.1951680
ELECTRON MICROSCOPYf_chiral_restr0.0491974
ELECTRON MICROSCOPYf_plane_restr0.0072169

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