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- EMDB-13930: 3D reconstruction of the membrane domains of the sialic acid TRAP... -

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Basic information

Entry
Database: EMDB / ID: EMD-13930
Title3D reconstruction of the membrane domains of the sialic acid TRAP transporter HiSiaQM from Haemophilus influenzae in lipid nanodiscs bound to a high affinity megabody
Map data3D reconstruction of HiSiaQM with a megabody bound to the periplasmic side
Sample
  • Complex: HiSiaQM/Megabody complex
    • Protein or peptide: Sialic acid TRAP transporter permease protein SiaT
    • Protein or peptide: Megabody3
Keywordsmembrane transporter / TRAP / sialic acid / elevator / TRANSPORT PROTEIN
Function / homologyTRAP transporter large membrane protein DctM / 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 / plasma membrane / Sialic acid TRAP transporter permease protein SiaT
Function and homology information
Biological speciesHaemophilus influenzae (bacteria) / Vicugna pacos (alpaca)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.7 Å
AuthorsPeter MF / Hagelueken G
Funding support Germany, 2 items
OrganizationGrant numberCountry
German Research Foundation (DFG)HA 6805/5-1 Germany
German Research Foundation (DFG)HA 6805/4-1 Germany
CitationJournal: Nat Commun / Year: 2022
Title: Structural and mechanistic analysis of a tripartite ATP-independent periplasmic TRAP transporter.
Authors: Martin F Peter / Jan A Ruland / Peer Depping / Niels Schneberger / Emmanuele Severi / Jonas Moecking / Karl Gatterdam / Sarah Tindall / Alexandre Durand / Veronika Heinz / Jan Peter ...Authors: Martin F Peter / Jan A Ruland / Peer Depping / Niels Schneberger / Emmanuele Severi / Jonas Moecking / Karl Gatterdam / Sarah Tindall / Alexandre Durand / Veronika Heinz / Jan Peter Siebrasse / Paul-Albert Koenig / Matthias Geyer / Christine Ziegler / Ulrich Kubitscheck / Gavin H Thomas / Gregor Hagelueken /
Abstract: Tripartite ATP-independent periplasmic (TRAP) transporters are found widely in bacteria and archaea and consist of three structural domains, a soluble substrate-binding protein (P-domain), and two ...Tripartite ATP-independent periplasmic (TRAP) transporters are found widely in bacteria and archaea and consist of three structural domains, a soluble substrate-binding protein (P-domain), and two transmembrane domains (Q- and M-domains). HiSiaPQM and its homologs are TRAP transporters for sialic acid and are essential for host colonization by pathogenic bacteria. Here, we reconstitute HiSiaQM into lipid nanodiscs and use cryo-EM to reveal the structure of a TRAP transporter. It is composed of 16 transmembrane helices that are unexpectedly structurally related to multimeric elevator-type transporters. The idiosyncratic Q-domain of TRAP transporters enables the formation of a monomeric elevator architecture. A model of the tripartite PQM complex is experimentally validated and reveals the coupling of the substrate-binding protein to the transporter domains. We use single-molecule total internal reflection fluorescence (TIRF) microscopy in solid-supported lipid bilayers and surface plasmon resonance to study the formation of the tripartite complex and to investigate the impact of interface mutants. Furthermore, we characterize high-affinity single variable domains on heavy chain (VHH) antibodies that bind to the periplasmic side of HiSiaQM and inhibit sialic acid uptake, providing insight into how TRAP transporter function might be inhibited in vivo.
History
DepositionDec 1, 2021-
Header (metadata) releaseJul 27, 2022-
Map releaseJul 27, 2022-
UpdateNov 20, 2024-
Current statusNov 20, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_13930.png

Downloads & links

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Map

FileDownload / File: emd_13930.map.gz / Format: CCP4 / Size: 27 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotation3D reconstruction of HiSiaQM with a megabody bound to the periplasmic side
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.72 Å/pix.
x 192 pix.
= 330.24 Å		1.72 Å/pix.
x 192 pix.
= 330.24 Å		1.72 Å/pix.
x 192 pix.
= 330.24 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.72 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.2
Minimum - Maximum-0.70691705 - 1.3967427
Average (Standard dev.)0.0009237686 (±0.023707028)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions192192192
Spacing192192192
CellA=B=C: 330.24 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Half map B

Fileemd_13930_half_map_1.map
AnnotationHalf map B
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map B

Fileemd_13930_half_map_2.map
AnnotationHalf map B
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : HiSiaQM/Megabody complex

EntireName: HiSiaQM/Megabody complex
Components
  • Complex: HiSiaQM/Megabody complex
    • Protein or peptide: Sialic acid TRAP transporter permease protein SiaT
    • Protein or peptide: Megabody3

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Supramolecule #1: HiSiaQM/Megabody complex

SupramoleculeName: HiSiaQM/Megabody complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Haemophilus influenzae (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: 1 / Enantiomer: LEVO
Source (natural)Organism: Haemophilus influenzae (bacteria) / Strain: ATCC 51907 / DSM 11121 / KW20 / Rd
Molecular weightTheoretical: 67.63607 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MKYINKLEEW LGGALFIAIF GILIAQILSR QVFHSPLIWS EELAKLLFVY VGMLGISVAV RKQEHVFIDF LTNLMPEKIR KFTNTFVQL LVFICIFLFI HFGIRTFNGA SFPIDALGGI SEKWIFAALP VVAILMMFRF IQAQTLNFKT GKSYLPATFF I ISAVILFA ...String:
MKYINKLEEW LGGALFIAIF GILIAQILSR QVFHSPLIWS EELAKLLFVY VGMLGISVAV RKQEHVFIDF LTNLMPEKIR KFTNTFVQL LVFICIFLFI HFGIRTFNGA SFPIDALGGI SEKWIFAALP VVAILMMFRF IQAQTLNFKT GKSYLPATFF I ISAVILFA ILFFAPDWFK VLRISNYIKL GSSSVYVALL VWLIIMFIGV PVGWSLFIAT LLYFSMTRWN VVNAATEKLV YS LDSFPLL AVPFYILTGI LMNTGGITER IFNFAKALLG HYTGGMGHVN IGASLLFSGM SGSALADAGG LGQLEIKAMR DAG YDDDIC GGITAASCII GPLVPPSIAM IIYGVIANES IAKLFIAGFI PGVLITLALM AMNYRIAKKR GYPRTPKATR EQLC SSFKQ SFWAILTPLL IIGGIFSGLF SPTESAIVAA AYSVIIGKFV YKELTLKSLF NSCIEAMAIT GVVALMIMTV TFFGD MIAR EQVAMRVADV FVAVADSPLT VLIMINALLL FLGMFIDALA LQFLVLPMLI PIAMQFNIDL IFFGVMTTLN MMVGIL TPP MGMALFVVAR VGNMSVSTVT KGVLPFLIPV FVTLVLITIF PQIITFVPNL LIP

UniProtKB: Sialic acid TRAP transporter permease protein SiaT

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Macromolecule #2: Megabody3

MacromoleculeName: Megabody3 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Vicugna pacos (alpaca)
Molecular weightTheoretical: 61.534348 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MQILFQGDSH NEIPIAYGSR WIVITRGPAG HGQVQLVESG GGLVQTKTTT SVIDTTNDAQ NLLTQAQTIV NTLKDYCPIL IAKSSSSNG GTNNANTPSW QTAGGGKNSC ATFGAEFSAA SDMINNAQKI VQETQQLSAN QPKNITQPHN LNLNSPSSLT A LAQKMLKN ...String:
MQILFQGDSH NEIPIAYGSR WIVITRGPAG HGQVQLVESG GGLVQTKTTT SVIDTTNDAQ NLLTQAQTIV NTLKDYCPIL IAKSSSSNG GTNNANTPSW QTAGGGKNSC ATFGAEFSAA SDMINNAQKI VQETQQLSAN QPKNITQPHN LNLNSPSSLT A LAQKMLKN AQSQAEILKL ANQVESDFNK LSSGHLKDYI GKCDASAISS ANMTMQNQKN NWGNGCAGVE ETQSLLKTSA AD FNNQTPQ INQAQNLANT LIQELGNNPF RASGGGSGGG GSGKLSDTYE QLSRLLTNDN GTNSKTSAQA INQAVNNLNE RAK TLAGGT TNSPAYQATL LALRSVLGLW NSMGYAVICG GYTKSPGENN QKDFHYTDEN GNGTTINCGG STNSNGTHSY NGTN TLKAD KNVSLSIEQY EKIHEAYQIL SKALKQAGLA PLNSKGEKLE AHVTTSYGSL RLSCTASRVT LDYHDIGWFR QAPGK EREG VSYISSSGGS TNYADSVKGR FTISRDNAKN TVYLQMNSLK PEDTAVYYCA RSSAYGSSWL NPSRYDYWGQ GTQVTV SSG GLPETGGHHH HHH

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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

BufferpH: 7.5
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 50.213 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.5 µm / Nominal defocus min: 0.8 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: INSILICO MODEL
Final reconstructionResolution.type: BY AUTHOR / Resolution: 4.7 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 215956
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.3.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.3.1)
FSC plot (resolution estimation)

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