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- EMDB-4774: Heterodimeric ABC exporter TmrAB in inward-facing wide conformati... -

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Basic information

Entry
Database: EMDB / ID: EMD-4774
TitleHeterodimeric ABC exporter TmrAB in inward-facing wide conformation under turnover conditions
Map dataTmrAB in IF wide conformation under turnover conditions
Sample
  • Complex: TmrAB in inward-facing wide conformation
    • Complex: TmrAB
      • Protein or peptide: Multidrug resistance ABC transporter ATP-binding and permease protein
      • Protein or peptide: Multidrug resistance ABC transporter ATP-binding and permease protein
    • Complex: nanobody
      • Protein or peptide: Nanobody Nb9F10
  • Ligand: MAGNESIUM ION
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE
KeywordsATP-binding cassette transporter / membrane protein / heterodimer / exporter / TRANSPORT PROTEIN
Function / homology
Function and homology information


ABC-type transporter activity / ATP hydrolysis activity / ATP binding / membrane
Similarity search - Function
Type 1 protein exporter / ABC transporter transmembrane region / ABC transporter type 1, transmembrane domain / ABC transporter integral membrane type-1 fused domain profile. / ABC transporter type 1, transmembrane domain superfamily / 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. ...Type 1 protein exporter / ABC transporter transmembrane region / ABC transporter type 1, transmembrane domain / ABC transporter integral membrane type-1 fused domain profile. / ABC transporter type 1, transmembrane domain superfamily / 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
Multidrug resistance ABC transporter ATP-binding and permease protein / Multidrug resistance ABC transporter ATP-binding and permease protein
Similarity search - Component
Biological speciesThermus thermophilus (bacteria) / Vicugna pacos (alpaca)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.2 Å
AuthorsJanuliene D / Hofmann S / Medhdipour AR / Thomas C / Hummer G / Tampe R / Moeller A
Funding support Germany, 3 items
OrganizationGrant numberCountry
German Research FoundationMo2752/2 Germany
German Research FoundationSFB 807 Germany
German Research FoundationEXC 115 Germany
CitationJournal: Nature / Year: 2019
Title: Conformation space of a heterodimeric ABC exporter under turnover conditions.
Authors: Susanne Hofmann / Dovile Januliene / Ahmad R Mehdipour / Christoph Thomas / Erich Stefan / Stefan Brüchert / Benedikt T Kuhn / Eric R Geertsma / Gerhard Hummer / Robert Tampé / Arne Moeller /
Abstract: Cryo-electron microscopy (cryo-EM) has the capacity to capture molecular machines in action. ATP-binding cassette (ABC) exporters are highly dynamic membrane proteins that extrude a wide range of ...Cryo-electron microscopy (cryo-EM) has the capacity to capture molecular machines in action. ATP-binding cassette (ABC) exporters are highly dynamic membrane proteins that extrude a wide range of substances from the cytosol and thereby contribute to essential cellular processes, adaptive immunity and multidrug resistance. Despite their importance, the coupling of nucleotide binding, hydrolysis and release to the conformational dynamics of these proteins remains poorly resolved, especially for heterodimeric and/or asymmetric ABC exporters that are abundant in humans. Here we present eight high-resolution cryo-EM structures that delineate the full functional cycle of an asymmetric ABC exporter in a lipid environment. Cryo-EM analysis under active turnover conditions reveals distinct inward-facing (IF) conformations-one of them with a bound peptide substrate-and previously undescribed asymmetric post-hydrolysis states with dimerized nucleotide-binding domains and a closed extracellular gate. By decreasing the rate of ATP hydrolysis, we could capture an outward-facing (OF) open conformation-an otherwise transient state vulnerable to substrate re-entry. The ATP-bound pre-hydrolysis and vanadate-trapped states are conformationally equivalent; both comprise co-existing OF conformations with open and closed extracellular gates. By contrast, the post-hydrolysis states from the turnover experiment exhibit asymmetric ATP and ADP occlusion after phosphate release from the canonical site and display a progressive separation of the nucleotide-binding domains and unlocking of the intracellular gate. Our findings reveal that phosphate release, not ATP hydrolysis, triggers the return of the exporter to the IF conformation. By mapping the conformational landscape during active turnover, aided by mutational and chemical modulation of kinetic rates to trap the key intermediates, we resolved fundamental steps of the substrate translocation cycle of asymmetric ABC transporters.
History
Header (metadata) releaseNov 8, 2017-
DepositionApr 6, 2019-
Map releaseJul 31, 2019-
UpdateOct 23, 2024-
Current statusOct 23, 2024Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.5
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 0.5
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6rag
  • Surface level: 0.5
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_4774.png

Downloads & links

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Map

FileDownload / File: emd_4774.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationTmrAB in IF wide conformation under turnover conditions
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.08 Å/pix.
x 256 pix.
= 275.712 Å		1.08 Å/pix.
x 256 pix.
= 275.712 Å		1.08 Å/pix.
x 256 pix.
= 275.712 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.077 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.5 / Movie #1: 0.5
Minimum - Maximum-1.0850275 - 2.0163908
Average (Standard dev.)0.003428961 (±0.047778234)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 275.712 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0771.0771.077
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z275.712275.712275.712
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-1.0852.0160.003

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Supplemental data

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Mask #1

Fileemd_4774_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: None

Fileemd_4774_half_map_1.map
AnnotationNone
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: None

Fileemd_4774_half_map_2.map
AnnotationNone
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : TmrAB in inward-facing wide conformation

EntireName: TmrAB in inward-facing wide conformation
Components
  • Complex: TmrAB in inward-facing wide conformation
    • Complex: TmrAB
      • Protein or peptide: Multidrug resistance ABC transporter ATP-binding and permease protein
      • Protein or peptide: Multidrug resistance ABC transporter ATP-binding and permease protein
    • Complex: nanobody
      • Protein or peptide: Nanobody Nb9F10
  • Ligand: MAGNESIUM ION
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE

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Supramolecule #1: TmrAB in inward-facing wide conformation

SupramoleculeName: TmrAB in inward-facing wide conformation / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Molecular weightTheoretical: 150 KDa

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Supramolecule #2: TmrAB

SupramoleculeName: TmrAB / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1-#2
Source (natural)Organism: Thermus thermophilus (bacteria)

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Supramolecule #3: nanobody

SupramoleculeName: nanobody / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #3
Source (natural)Organism: Vicugna pacos (alpaca)

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Macromolecule #1: Multidrug resistance ABC transporter ATP-binding and permease protein

MacromoleculeName: Multidrug resistance ABC transporter ATP-binding and permease protein
type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Thermus thermophilus (bacteria)
Molecular weightTheoretical: 70.664797 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MTEDTYSKAF DRALFARILR YVWPYRLQVV LALLFLLVVT LAAAATPLFF KWAIDLALVP TEPRPLAERF HLLLWISLGF LAVRAVHFA ATYGETYLIQ WVGQRVLFDL RSDLFAKLMR LHPGFYDRNP VGRLMTRVTS DVDAINQFIT GGLVGVIADL F TLVGLLGF ...String:
MTEDTYSKAF DRALFARILR YVWPYRLQVV LALLFLLVVT LAAAATPLFF KWAIDLALVP TEPRPLAERF HLLLWISLGF LAVRAVHFA ATYGETYLIQ WVGQRVLFDL RSDLFAKLMR LHPGFYDRNP VGRLMTRVTS DVDAINQFIT GGLVGVIADL F TLVGLLGF MLFLSPKLTL VVLLVAPVLL AVTTWVRLGM RSAYREMRLR LARVNAALQE NLSGVETIQL FVKEREREEK FD RLNRDLF RAWVEIIRWF ALFFPVVGFL GDFAVASLVY YGGGEVVRGA VSLGLLVAFV DYTRQLFQPL QDLSDKFNLF QGA MASAER IFGVLDTEEE LKDPEDPTPI RGFRGEVEFR DVWLAYTPKG VEPTEKDWVL KGVSFRVRPG EKVALVGATG AGKT SVVSL IARFYDPQRG CVFLDGVDVR RYRQEELRRH VGIVLQEPFL FSGTVLDNLR LFDPSVPPER VEEVARFLGA HEFIL RLPK GYQTVLGERG AGLSTGEKQL LALVRALLAS PDILLILDEA TASVDSETEK RLQEALYKAM EGRTSLIIAH RLSTIR HVD RILVFRKGRL VEEGSHEELL AKGGYYAALY RLQFQEAKLG GGGENLYFQG HHHHHHHHHH

UniProtKB: Multidrug resistance ABC transporter ATP-binding and permease protein

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Macromolecule #2: Multidrug resistance ABC transporter ATP-binding and permease protein

MacromoleculeName: Multidrug resistance ABC transporter ATP-binding and permease protein
type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Thermus thermophilus (bacteria)
Molecular weightTheoretical: 64.634457 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MTGRSAAPLL RRLWPYVGRY RWRYLWAVLA GLVSIFFFVL TPYFLRLAVD AVQAGRGFGV YALAIVASAA LSGLLSYAMR RLAVVASRQ VEYDLRRDLL HHLLTLDRDF YHKHRVGDLM NRLNTDLSAV REMVGPGILM GSRLSFLVLL AFLSMYAVNA R LAFYLTLI ...String:
MTGRSAAPLL RRLWPYVGRY RWRYLWAVLA GLVSIFFFVL TPYFLRLAVD AVQAGRGFGV YALAIVASAA LSGLLSYAMR RLAVVASRQ VEYDLRRDLL HHLLTLDRDF YHKHRVGDLM NRLNTDLSAV REMVGPGILM GSRLSFLVLL AFLSMYAVNA R LAFYLTLI LPGIFLAMRF LLRLIDRRYR EAQEVFDRIS TLAQEAFSGI RVVKGYALER RMVAWFQDLN RLYVEKSLAL AR VEGPLHA LLGFLMGFAF LTVLWAGGAM VVRGELSVGE LVQFNAYLAQ LTWPILGLGW VMALYQRGLT SLRRLFELLD EKP AIRDED PLPLALEDLS GEVRFEGVGL KRDGRWLLRG LTLTIPEGMT LGITGRTGSG KSLLAALVPR LLDPSEGRVY VGGH EARRI PLAVLRKAVG VAPQEPFLFS ETILENIAFG LDEVDRERVE WAARLAGIHE EILAFPKGYE TVLGERGITL SGGQR QRVA LARALAKRPK ILILDDALSA VDAETEARIL QGLKTVLGKQ TTLLISHRTA ALRHADWIIV LDGGRIVEEG THESLL QAG GLYAEMDRLQ KEVEA

UniProtKB: Multidrug resistance ABC transporter ATP-binding and permease protein

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Macromolecule #3: Nanobody Nb9F10

MacromoleculeName: Nanobody Nb9F10 / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Vicugna pacos (alpaca)
Molecular weightTheoretical: 14.594405 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MAQLQLVESG GGLVQPGDSL RLSCAVSGSA LDYNAIGWFR QAPGKEREGV ACISKITGNT AYADSVKGRF TISRDNAKNT VHLQMNSLK PEDTAVYYCA TVTAVLLPGR CVPGKYWGQG TPVTVSSHHH HHHEPEA

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Macromolecule #4: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 4 / Number of copies: 2 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #5: ADENOSINE-5'-DIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-DIPHOSPHATE / type: ligand / ID: 5 / Number of copies: 1 / Formula: ADP
Molecular weightTheoretical: 427.201 Da
Chemical component information

ChemComp-ADP:
ADENOSINE-5'-DIPHOSPHATE / ADP, energy-carrying molecule*YM

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Macromolecule #6: ADENOSINE-5'-TRIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 6 / Number of copies: 1 / Formula: ATP
Molecular weightTheoretical: 507.181 Da
Chemical component information

ChemComp-ATP:
ADENOSINE-5'-TRIPHOSPHATE / ATP, energy-carrying molecule*YM

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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

Concentration1 mg/mL
BufferpH: 7.5
GridModel: Quantifoil, UltrAuFoil / Material: GOLD / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 QUANTUM (4k x 4k) / Detector mode: COUNTING / Average exposure time: 8.0 sec. / Average electron dose: 62.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 4000000
Details: The particles were generously picked, using RELION-3 template picker
Startup modelType of model: EMDB MAP
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 4.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 208674
Initial angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION (ver. 3)
Final angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: cryoSPARC
FSC plot (resolution estimation)

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