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- PDB-6raj: Heterodimeric ABC exporter TmrAB in vanadate trapped outward-faci... -
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Open data
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Basic information
Entry | Database: PDB / ID: 6raj | ||||||||||||
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Title | Heterodimeric ABC exporter TmrAB in vanadate trapped outward-facing open conformation | ||||||||||||
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![]() | TRANSPORT PROTEIN / ATP-binding cassette transporter / membrane protein / heterodimer / exporter | ||||||||||||
Function / homology | ![]() ATPase-coupled lipid transmembrane transporter activity / ABC-type transporter activity / ATP hydrolysis activity / ATP binding / membrane Similarity search - Function | ||||||||||||
Biological species | ![]() ![]() ![]() ![]() ![]() ![]() | ||||||||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.5 Å | ||||||||||||
![]() | Thomas, C. / Januliene, D. / Mehdipour, A.R. / Hofmann, S. / Hummer, G. / Moeller, A. / Tampe, R. | ||||||||||||
Funding support | ![]()
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![]() | ![]() 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 |
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Structure visualization
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Structure viewer | Molecule: ![]() ![]() |
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Downloads & links
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PDBx/mmCIF format | ![]() | 230.3 KB | Display | ![]() |
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PDB format | ![]() | 189.3 KB | Display | ![]() |
PDBx/mmJSON format | ![]() | Tree view | ![]() | |
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-Validation report
Arichive directory | ![]() ![]() | HTTPS FTP |
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-Related structure data
Related structure data | ![]() 4777MC ![]() 4773C ![]() 4774C ![]() 4775C ![]() 4776C ![]() 4778C ![]() 4779C ![]() 4780C ![]() 4781C ![]() 6rafC ![]() 6ragC ![]() 6rahC ![]() 6raiC ![]() 6rakC ![]() 6ralC ![]() 6ramC ![]() 6ranC M: map data used to model this data C: citing same article ( |
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Similar structure data |
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Links
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Assembly
Deposited unit | ![]()
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Components
-Multidrug resistance ABC transporter ATP-binding and permease ... , 2 types, 2 molecules AB
#1: Protein | Mass: 70664.797 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() ![]() ![]() |
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#2: Protein | Mass: 64634.457 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() ![]() ![]() |
-Antibody , 1 types, 1 molecules C
#3: Antibody | Mass: 14594.405 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() ![]() ![]() |
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-Non-polymers , 3 types, 4 molecules ![](data/chem/img/AOV.gif)
![](data/chem/img/ATP.gif)
![](data/chem/img/MG.gif)
![](data/chem/img/ATP.gif)
![](data/chem/img/MG.gif)
#4: Chemical | ChemComp-AOV / |
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#5: Chemical | ChemComp-ATP / |
#6: Chemical |
-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
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Sample preparation
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Molecular weight | Value: 0.15 MDa | ||||||||||||||||||||||||||||||
Source (natural) |
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Source (recombinant) |
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Buffer solution | pH: 7.5 | ||||||||||||||||||||||||||||||
Specimen | Conc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES | ||||||||||||||||||||||||||||||
Specimen support | Grid material: GOLD / Grid type: Quantifoil, UltrAuFoil | ||||||||||||||||||||||||||||||
Vitrification | Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K |
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Electron microscopy imaging
Experimental equipment | ![]() Model: Titan Krios / Image courtesy: FEI Company |
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Microscopy | Model: FEI TITAN KRIOS |
Electron gun | Electron source: ![]() |
Electron lens | Mode: BRIGHT FIELD / Cs: 2.7 mm |
Image recording | Average exposure time: 8 sec. / Electron dose: 64 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 QUANTUM (4k x 4k) |
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Processing
Software | Name: PHENIX / Version: 1.14_3260: / Classification: refinement | ||||||||||||||||||||||||||||
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EM software |
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CTF correction | Details: CTF correction was performed within 3D reconstruction in RELION-3 Type: NONE | ||||||||||||||||||||||||||||
Particle selection | Num. of particles selected: 1300000 Details: The particles were generously picked, using RELION-3 template picker | ||||||||||||||||||||||||||||
Symmetry | Point symmetry: C1 (asymmetric) | ||||||||||||||||||||||||||||
3D reconstruction | Resolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 44000 / Symmetry type: POINT |