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- EMDB-41266: Cryo-EM structure of the Tripartite ATP-independent Periplasmic (... -

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Basic information

Entry
Database: EMDB / ID: EMD-41266
TitleCryo-EM structure of the Tripartite ATP-independent Periplasmic (TRAP) transporter SiaQM from Haemophilus influenzae (antiparallel dimer)
Map data
Sample
  • Complex: HiSiaQM transporter protein solubilised in amphipol A8-35
    • Protein or peptide: Sialic acid TRAP transporter permease protein SiaT
  • Ligand: SODIUM IONSodium
  • Ligand: PHOSPHATIDYLETHANOLAMINE
  • Ligand: (1S)-2-{[{[(2R)-2,3-DIHYDROXYPROPYL]OXY}(HYDROXY)PHOSPHORYL]OXY}-1-[(PALMITOYLOXY)METHYL]ETHYL STEARATE
KeywordsSugar transport / sialic acid / TRAP transporter / secondary active transport / ion transporter superfamily / TRANSPORT PROTEIN
Function / homologyTRAP 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 / transmembrane transporter activity / carbohydrate transport / plasma membrane / Sialic acid TRAP transporter permease protein SiaT
Function and homology information
Biological speciesHaemophilus influenzae Rd KW20 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.99 Å
AuthorsDavies JS / Currie MC / Dobson RCJ / North RA
Funding support New Zealand, 2 items
OrganizationGrant numberCountry
Marsden FundUOC1506 New Zealand
Ministry of Business, Innovation and Employment (New Zealand)UOCX1706 New Zealand
Citation
Journal: Elife / Year: 2024
Title: Structural and biophysical analysis of a tripartite ATP-independent periplasmic (TRAP) transporter.
Authors: Michael J Currie / James S Davies / Mariafrancesca Scalise / Ashutosh Gulati / Joshua D Wright / Michael C Newton-Vesty / Gayan S Abeysekera / Ramaswamy Subramanian / Weixiao Y Wahlgren / ...Authors: Michael J Currie / James S Davies / Mariafrancesca Scalise / Ashutosh Gulati / Joshua D Wright / Michael C Newton-Vesty / Gayan S Abeysekera / Ramaswamy Subramanian / Weixiao Y Wahlgren / Rosmarie Friemann / Jane R Allison / Peter D Mace / Michael D W Griffin / Borries Demeler / Soichi Wakatsuki / David Drew / Cesare Indiveri / Renwick C J Dobson / Rachel A North /
Abstract: Tripartite ATP-independent periplasmic (TRAP) transporters are secondary-active transporters that receive their substrates via a soluble-binding protein to move bioorganic acids across bacterial or ...Tripartite ATP-independent periplasmic (TRAP) transporters are secondary-active transporters that receive their substrates via a soluble-binding protein to move bioorganic acids across bacterial or archaeal cell membranes. Recent cryo-electron microscopy (cryo-EM) structures of TRAP transporters provide a broad framework to understand how they work, but the mechanistic details of transport are not yet defined. Here we report the cryo-EM structure of the -acetylneuraminate TRAP transporter (SiaQM) at 2.99 Å resolution (extending to 2.2 Å at the core), revealing new features. The improved resolution (the previous SiaQM structure is 4.7 Å resolution) permits accurate assignment of two Na sites and the architecture of the substrate-binding site, consistent with mutagenic and functional data. Moreover, rather than a monomer, the SiaQM structure is a homodimer. We observe lipids at the dimer interface, as well as a lipid trapped within the fusion that links the SiaQ and SiaM subunits. We show that the affinity () for the complex between the soluble SiaP protein and SiaQM is in the micromolar range and that a related SiaP can bind SiaQM. This work provides key data that enhances our understanding of the 'elevator-with-an-operator' mechanism of TRAP transporters.
#1: Journal: Elife / Year: 2023
Title: Structural and biophysical analysis of a Haemophilus influenzae tripartite ATP-independent periplasmic (TRAP) transporter
Authors: Currie MJ / Davies JS / Scalise M / Gulati A / Wright JD / Newton-Vesty MC / Abeysekera GS / Subramanian R / Wahlgren WY / Friemann R / Allison JR / Mace PD / Griffin MDW / Demeler B / ...Authors: Currie MJ / Davies JS / Scalise M / Gulati A / Wright JD / Newton-Vesty MC / Abeysekera GS / Subramanian R / Wahlgren WY / Friemann R / Allison JR / Mace PD / Griffin MDW / Demeler B / Wakatsuki S / Drew D / Indiveri C / Dobson RCJ / North RA
History
DepositionJul 16, 2023-
Header (metadata) releaseNov 22, 2023-
Map releaseNov 22, 2023-
UpdateFeb 28, 2024-
Current statusFeb 28, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_41266.png

Downloads & links

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Map

FileDownload / File: emd_41266.map.gz / Format: CCP4 / Size: 421.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.6645 Å
Density
Contour LevelBy AUTHOR: 0.05
Minimum - Maximum-0.15204048 - 0.3413738
Average (Standard dev.)-0.000082717896 (±0.006268022)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions480480480
Spacing480480480
CellA=B=C: 318.96 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_41266_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_41266_half_map_2.map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : HiSiaQM transporter protein solubilised in amphipol A8-35

EntireName: HiSiaQM transporter protein solubilised in amphipol A8-35
Components
  • Complex: HiSiaQM transporter protein solubilised in amphipol A8-35
    • Protein or peptide: Sialic acid TRAP transporter permease protein SiaT
  • Ligand: SODIUM IONSodium
  • Ligand: PHOSPHATIDYLETHANOLAMINE
  • Ligand: (1S)-2-{[{[(2R)-2,3-DIHYDROXYPROPYL]OXY}(HYDROXY)PHOSPHORYL]OXY}-1-[(PALMITOYLOXY)METHYL]ETHYL STEARATE

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Supramolecule #1: HiSiaQM transporter protein solubilised in amphipol A8-35

SupramoleculeName: HiSiaQM transporter protein solubilised in amphipol A8-35
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Haemophilus influenzae Rd KW20 (bacteria)

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Macromolecule #1: Sialic acid TRAP transporter permease protein SiaT

MacromoleculeName: Sialic acid TRAP transporter permease protein SiaT / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Haemophilus influenzae Rd KW20 (bacteria)
Molecular weightTheoretical: 72.046812 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MGGSHHHHHH GMASMTGGQQ MGRDLYDDDD KDRWGSELEM KYINKLEEWL GGALFIAIFG ILIAQILSRQ VFHSPLIWSE ELAKLLFVY VGMLGISVAV RKQEHVFIDF LTNLMPEKIR KFTNTFVQLL VFICIFLFIH FGIRTFNGAS FPIDALGGIS E KWIFAALP ...String:
MGGSHHHHHH GMASMTGGQQ MGRDLYDDDD KDRWGSELEM KYINKLEEWL GGALFIAIFG ILIAQILSRQ VFHSPLIWSE ELAKLLFVY VGMLGISVAV RKQEHVFIDF LTNLMPEKIR KFTNTFVQLL VFICIFLFIH FGIRTFNGAS FPIDALGGIS E KWIFAALP VVAILMMFRF IQAQTLNFKT GKSYLPATFF IISAVILFAI LFFAPDWFKV LRISNYIKLG SSSVYVALLV WL IIMFIGV PVGWSLFIAT LLYFSMTRWN VVNAATEKLV YSLDSFPLLA VPFYILTGIL MNTGGITERI FNFAKALLGH YTG GMGHVN IGASLLFSGM SGSALADAGG LGQLEIKAMR DAGYDDDICG GITAASCIIG PLVPPSIAMI IYGVIANESI AKLF IAGFI PGVLITLALM AMNYRIAKKR GYPRTPKATR EQLCSSFKQS FWAILTPLLI IGGIFSGLFS PTESAIVAAA YSVII GKFV YKELTLKSLF NSCIEAMAIT GVVALMIMTV TFFGDMIARE QVAMRVADVF VAVADSPLTV LIMINALLLF LGMFID ALA LQFLVLPMLI PIAMQFNIDL IFFGVMTTLN MMVGILTPPM GMALFVVARV GNMSVSTVTK GVLPFLIPVF VTLVLIT IF PQIITFVPNL LIP

UniProtKB: Sialic acid TRAP transporter permease protein SiaT

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Macromolecule #2: SODIUM ION

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

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Macromolecule #3: PHOSPHATIDYLETHANOLAMINE

MacromoleculeName: PHOSPHATIDYLETHANOLAMINE / type: ligand / ID: 3 / Number of copies: 2 / Formula: PTY
Molecular weightTheoretical: 734.039 Da
Chemical component information

ChemComp-PTY:
PHOSPHATIDYLETHANOLAMINE / phospholipid*YM / Phosphatidylethanolamine

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Macromolecule #4: (1S)-2-{[{[(2R)-2,3-DIHYDROXYPROPYL]OXY}(HYDROXY)PHOSPHORYL]OXY}-...

MacromoleculeName: (1S)-2-{[{[(2R)-2,3-DIHYDROXYPROPYL]OXY}(HYDROXY)PHOSPHORYL]OXY}-1-[(PALMITOYLOXY)METHYL]ETHYL STEARATE
type: ligand / ID: 4 / Number of copies: 2 / Formula: PGT
Molecular weightTheoretical: 751.023 Da
Chemical component information

ChemComp-PGT:
(1S)-2-{[{[(2R)-2,3-DIHYDROXYPROPYL]OXY}(HYDROXY)PHOSPHORYL]OXY}-1-[(PALMITOYLOXY)METHYL]ETHYL STEARATE / phospholipid*YM / Phosphatidylglycerol

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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

Concentration4.1 mg/mL
BufferpH: 8
VitrificationCryogen name: ETHANE / 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) / Average electron dose: 73.1 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
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 2.99 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 225044
FSC plot (resolution estimation)

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