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- EMDB-13968: Cryo-EM structure of the Tripartite ATP-independent Periplasmic (... -

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Basic information

Entry
Database: EMDB / ID: EMD-13968
TitleCryo-EM structure of the Tripartite ATP-independent Periplasmic (TRAP) transporter SiaQM from Photobacterium profundum in amphipol
Map data
Sample
  • Complex: SiaQM with megabody
    • Complex: SiaQM
      • Protein or peptide: Putative TRAP-type C4-dicarboxylate transport system, small permease component
      • Protein or peptide: Putative TRAP-type C4-dicarboxylate transport system, large permease component
    • Complex: Megabody
      • Protein or peptide: Megabody c7HopQ
  • Ligand: SODIUM IONSodium
Function / homology
Function and homology information


TRAP transporter large membrane protein DctM / TRAP transporter, small membrane protein DctQ / TRAP C4-dicarboxylate transport system permease DctM subunit / Tripartite ATP-independent periplasmic transporters, DctQ component / Tripartite ATP-independent periplasmic transporter, DctM component / SabA, N-terminal extracellular adhesion domain / SabA N-terminal extracellular adhesion domain / Outer membrane protein, Helicobacter / Helicobacter outer membrane protein
Similarity search - Domain/homology
Outer membrane protein / TRAP transporter small permease protein / TRAP transporter large permease protein
Similarity search - Component
Biological speciesPhotobacterium profundum (bacteria) / Helicobacter pylori (bacteria) / Photobacterium profundum SS9 (bacteria) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.97 Å
AuthorsNorth RA / Davies JS / Morado D / Dobson RCJ
Funding support New Zealand, 2 items
OrganizationGrant numberCountry
Marsden FundUOC1506 New Zealand
Ministry of Business, Innovation and Employment (New Zealand)UOCX1706 New Zealand
CitationJournal: Nat Commun / Year: 2023
Title: Structure and mechanism of a tripartite ATP-independent periplasmic TRAP transporter.
Authors: James S Davies / Michael J Currie / Rachel A North / Mariafrancesca Scalise / Joshua D Wright / Jack M Copping / Daniela M Remus / Ashutosh Gulati / Dustin R Morado / Sam A Jamieson / ...Authors: James S Davies / Michael J Currie / Rachel A North / Mariafrancesca Scalise / Joshua D Wright / Jack M Copping / Daniela M Remus / Ashutosh Gulati / Dustin R Morado / Sam A Jamieson / Michael C Newton-Vesty / Gayan S Abeysekera / Subramanian Ramaswamy / Rosmarie Friemann / Soichi Wakatsuki / Jane R Allison / Cesare Indiveri / David Drew / Peter D Mace / Renwick C J Dobson /
Abstract: In bacteria and archaea, tripartite ATP-independent periplasmic (TRAP) transporters uptake essential nutrients. TRAP transporters receive their substrates via a secreted soluble substrate-binding ...In bacteria and archaea, tripartite ATP-independent periplasmic (TRAP) transporters uptake essential nutrients. TRAP transporters receive their substrates via a secreted soluble substrate-binding protein. How a sodium ion-driven secondary active transporter is strictly coupled to a substrate-binding protein is poorly understood. Here we report the cryo-EM structure of the sialic acid TRAP transporter SiaQM from Photobacterium profundum at 2.97 Å resolution. SiaM comprises a "transport" domain and a "scaffold" domain, with the transport domain consisting of helical hairpins as seen in the sodium ion-coupled elevator transporter VcINDY. The SiaQ protein forms intimate contacts with SiaM to extend the size of the scaffold domain, suggesting that TRAP transporters may operate as monomers, rather than the typically observed oligomers for elevator-type transporters. We identify the Na and sialic acid binding sites in SiaM and demonstrate a strict dependence on the substrate-binding protein SiaP for uptake. We report the SiaP crystal structure that, together with docking studies, suggest the molecular basis for how sialic acid is delivered to the SiaQM transporter complex. We thus propose a model for substrate transport by TRAP proteins, which we describe herein as an 'elevator-with-an-operator' mechanism.
History
DepositionDec 11, 2021-
Header (metadata) releaseDec 21, 2022-
Map releaseDec 21, 2022-
UpdateMar 22, 2023-
Current statusMar 22, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_13968.png

Downloads & links

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Map

FileDownload / File: emd_13968.map.gz / Format: CCP4 / Size: 52.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 240 pix.
= 199.35 Å		0.83 Å/pix.
x 240 pix.
= 199.35 Å		0.83 Å/pix.
x 240 pix.
= 199.35 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.83063 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.084
Minimum - Maximum-0.12569457 - 0.32063696
Average (Standard dev.)0.0009114622 (±0.0112039335)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions240240240
Spacing240240240
CellA=B=C: 199.35 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_13968_msk_1.map
Projections & Slices
AxesZYX

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Slices (1/2)
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Histogram

Histogram (log scale)

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Half map: #2

Fileemd_13968_half_map_1.map
Projections & Slices
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Histogram

Histogram (log scale)

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Half map: #1

Fileemd_13968_half_map_2.map
Projections & Slices
AxesZYX

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Histogram (log scale)

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Sample components

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Entire : SiaQM with megabody

EntireName: SiaQM with megabody
Components
  • Complex: SiaQM with megabody
    • Complex: SiaQM
      • Protein or peptide: Putative TRAP-type C4-dicarboxylate transport system, small permease component
      • Protein or peptide: Putative TRAP-type C4-dicarboxylate transport system, large permease component
    • Complex: Megabody
      • Protein or peptide: Megabody c7HopQ
  • Ligand: SODIUM IONSodium

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Supramolecule #1: SiaQM with megabody

SupramoleculeName: SiaQM with megabody / type: complex / ID: 1 / Chimera: Yes / Parent: 0 / Macromolecule list: #1-#3

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Supramolecule #2: SiaQM

SupramoleculeName: SiaQM / type: complex / ID: 2 / Chimera: Yes / Parent: 1 / Macromolecule list: #1-#2
Source (natural)Organism: Photobacterium profundum (bacteria)

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Supramolecule #3: Megabody

SupramoleculeName: Megabody / type: complex / ID: 3 / Chimera: Yes / Parent: 1 / Macromolecule list: #3
Source (natural)Organism: Helicobacter pylori (bacteria)

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Macromolecule #1: Putative TRAP-type C4-dicarboxylate transport system, small perme...

MacromoleculeName: Putative TRAP-type C4-dicarboxylate transport system, small permease component
type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Photobacterium profundum SS9 (bacteria) / Strain: SS9
Molecular weightTheoretical: 19.748367 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MFRKIIDNIE EIITVPLMIA LLCILTWQIS SRWLFDSPSL WSEELARVLF LHMAIIGGAI AIKKDDHVKI TFFSDKLPRN FRYSLLFAL ELLVLITIVA MIYYGYAHVQ RTAFFELITL GISSSWMTYA LPVGGCFMLV RQCQKLYFVL IDWRINENKN T SHLTACDI NE

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Macromolecule #2: Putative TRAP-type C4-dicarboxylate transport system, large perme...

MacromoleculeName: Putative TRAP-type C4-dicarboxylate transport system, large permease component
type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Photobacterium profundum SS9 (bacteria) / Strain: SS9
Molecular weightTheoretical: 45.573605 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MFTSIVGWLG LLFAGMPVGF SLIFVGLAFL VLTESTGINF AAQQMIGGLD NFTLLAVPFF VLTGHLMNSA GITERIFNFA KAMVGHITG SLGHVNILAS LLFSGMSGSA LADAGGLGQL EIKSMRDAKY DDDFAGGLTA ASCIIGPLVP PSIPLVIYGV V SNTSIGAL ...String:
MFTSIVGWLG LLFAGMPVGF SLIFVGLAFL VLTESTGINF AAQQMIGGLD NFTLLAVPFF VLTGHLMNSA GITERIFNFA KAMVGHITG SLGHVNILAS LLFSGMSGSA LADAGGLGQL EIKSMRDAKY DDDFAGGLTA ASCIIGPLVP PSIPLVIYGV V SNTSIGAL FLAGAIPGLL CCIALCIMTY FIAKKRGYMT LPRASRKERL IAFRDAFLSL LTPFIIIGGI FSGKFTPTEA AI ISSLYAL FLGTVVYKSL TMDKFIKLVQ ETVTTTSVVA LMVMGVTVFG WIVAREQLPQ QLAELFLSIS DNPLILLLLI NLL LLFLGT FIESLALLLL LVPFLVPVAT SVGIDPVHFG VMAILNLMIG ILTPPMGMAL YVVSKVGNIP FHVLTRGVLP LLVP LFIVL GLIIVFPQIT LFLPQLVLGY GL

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Macromolecule #3: Megabody c7HopQ

MacromoleculeName: Megabody c7HopQ / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 54.867098 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: QVQLQESGGG LVQTKTTTSV IDTTNDAQNL LTQAQTIVNT LKDYCPILIA KSSSSNGGTN NANTPSWQTA GGGKNSCATF GAEFSAASD MINNAQKIVQ ETQQLSANQP KNITQPHNLN LNSPSSLTAL AQKMLKNAQS QAEILKLANQ VESDFNKLSS G HLKDYIGK ...String:
QVQLQESGGG LVQTKTTTSV IDTTNDAQNL LTQAQTIVNT LKDYCPILIA KSSSSNGGTN NANTPSWQTA GGGKNSCATF GAEFSAASD MINNAQKIVQ ETQQLSANQP KNITQPHNLN LNSPSSLTAL AQKMLKNAQS QAEILKLANQ VESDFNKLSS G HLKDYIGK CDASAISSAN MTMQNQKNNW GNGCAGVEET QSLLKTSAAD FNNQTPQINQ AQNLANTLIQ ELGNNDTYEQ LS RLLTNDN GTNSKTSAQA INQAVNNLNE RAKTLAGGTT NSPAYQATLL ALRSVLGLWN SMGYAVICGG YTKSPGENNQ KDF HYTDEN GNGTTINCGG STNSNGTHSY NGTNTLKADK NVSLSIEQYE KIHEAYQILS KALKQAGLAP LNSKGEKLEA HVTT SKYAG GSLRLSCAAS GNIFDRGYMG WYRQAPGKER ELVAGISYGG STYYADSVKG RFTISRDNAK NTVYLQMNSL KPEDT AVYY CAAYPLYDDP YYYWGQGTQV TVSSLE

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Macromolecule #4: SODIUM ION

MacromoleculeName: SODIUM ION / type: ligand / ID: 4 / Number of copies: 2
Molecular weightTheoretical: 22.99 Da

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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

Concentration2.9 mg/mL
BufferpH: 8
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 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: 2.0 µm / Nominal defocus min: 0.4 µm
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number grids imaged: 1 / Number real images: 10960 / Average exposure time: 1.9 sec. / Average electron dose: 71.3 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.97 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 624033
FSC plot (resolution estimation)

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