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- PDB-6rai: Heterodimeric ABC exporter TmrAB in ATP-bound outward-facing occl... -

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Basic information

Entry
Database: PDB / ID: 6rai
TitleHeterodimeric ABC exporter TmrAB in ATP-bound outward-facing occluded conformation
Components
  • (Multidrug resistance ABC transporter ATP-binding and permease ...) x 2
  • Nanobody Nb9F10
KeywordsTRANSPORT PROTEIN / ATP-binding cassette transporter / membrane protein / heterodimer / exporter
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. / P-loop containing nucleotide triphosphate hydrolases / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase / Rossmann fold / 3-Layer(aba) Sandwich / Alpha Beta
Similarity search - Domain/homology
ADENOSINE-5'-TRIPHOSPHATE / 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)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.9 Å
AuthorsThomas, C. / Januliene, D. / Mehdipour, A.R. / Hofmann, S. / Hummer, G. / Moeller, A. / Tampe, R.
Funding support Germany, 3items
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
DepositionApr 6, 2019Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jul 31, 2019Provider: repository / Type: Initial release
Revision 1.1Aug 7, 2019Group: Data collection / Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.2Aug 21, 2019Group: Data collection / Experimental preparation / Category: em_sample_support / Item: _em_sample_support.grid_type
Revision 1.3Dec 18, 2019Group: Other / Category: atom_sites
Item: _atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] / _atom_sites.fract_transf_matrix[3][3]

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Structure visualization

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Assembly

Deposited unit
A: Multidrug resistance ABC transporter ATP-binding and permease protein
B: Multidrug resistance ABC transporter ATP-binding and permease protein
C: Nanobody Nb9F10
hetero molecules


Theoretical massNumber of molelcules
Total (without water)150,9567
Polymers149,8933
Non-polymers1,0634
Water1086
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: microscopy, gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area19510 Å2
ΔGint-125 kcal/mol
Surface area50010 Å2
MethodPISA

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Components

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Multidrug resistance ABC transporter ATP-binding and permease ... , 2 types, 2 molecules AB

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


Mass: 70663.805 Da / Num. of mol.: 1 / Mutation: E523Q
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Thermus thermophilus (bacteria) / Gene: TT_C0976 / Production host: Escherichia coli (E. coli) / References: UniProt: Q72J05
#2: Protein Multidrug resistance ABC transporter ATP-binding and permease protein


Mass: 64634.457 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Thermus thermophilus (bacteria) / Gene: TT_C0977 / Production host: Escherichia coli (E. coli) / References: UniProt: Q72J04

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Antibody , 1 types, 1 molecules C

#3: Antibody Nanobody Nb9F10


Mass: 14594.405 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Vicugna pacos (alpaca) / Production host: Escherichia coli (E. coli)

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Non-polymers , 3 types, 10 molecules

#4: Chemical ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE / Adenosine triphosphate


Mass: 507.181 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C10H16N5O13P3 / Comment: ATP, energy-carrying molecule*YM
#5: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg
#6: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 6 / Source method: isolated from a natural source / Formula: H2O

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

Component
IDNameTypeEntity IDParent-IDSource
1TmrAB EQ mutant in ATP bound outward-occluded conformationCOMPLEX#1-#30MULTIPLE SOURCES
2TmrABCOMPLEX#1-#21RECOMBINANT
3nanobodySingle-domain antibodyCOMPLEX#31RECOMBINANT
Molecular weightValue: 0.15 MDa
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Thermus thermophilus (bacteria)274
33Vicugna pacos (alpaca)30538
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
22Escherichia coli (E. coli)562
33Escherichia coli (E. coli)562
Buffer solutionpH: 7.5
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid type: Quantifoil, UltrAuFoil
VitrificationInstrument: 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
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm
Image recordingAverage 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

SoftwareName: PHENIX / Version: 1.14_3260: / Classification: refinement
EM software
IDNameVersionCategory
2EPUimage acquisition
4GctfCTF correction
10RELION3initial Euler assignment
11RELION3final Euler assignment
12RELION3classification
13RELION33D reconstruction
CTF correctionDetails: CTF correction was performed within 3D reconstruction in RELION-3
Type: NONE
Particle selectionNum. of particles selected: 2200000
Details: The particles were generously picked, using RELION-3 template picker
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 94621 / Symmetry type: POINT

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