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- PDB-7ahc: OpuA apo inward-facing -

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Basic information

Entry
Database: PDB / ID: 7ahc
TitleOpuA apo inward-facing
Components
  • ABC transporter permease subunit
  • ABC-type proline/glycine betaine transport system ATPase component
KeywordsMEMBRANE PROTEIN / osmoregulation / ABC-transporter / glycine betaine uptake system
Function / homology
Function and homology information


ABC-type quaternary amine transporter / ABC-type quaternary ammonium compound transporting activity / glycine betaine transport / peptide transport / amino acid transport / response to stimulus / transmembrane transporter activity / ATP-binding cassette (ABC) transporter complex / protein transport / ATP hydrolysis activity ...ABC-type quaternary amine transporter / ABC-type quaternary ammonium compound transporting activity / glycine betaine transport / peptide transport / amino acid transport / response to stimulus / transmembrane transporter activity / ATP-binding cassette (ABC) transporter complex / protein transport / ATP hydrolysis activity / ATP binding / membrane
Similarity search - Function
Glycine betaine transport ATP-binding subunit / ABC-type glycine betaine transport system, substrate-binding domain / Substrate binding domain of ABC-type glycine betaine transport system / ABC transporter type 1, transmembrane domain MetI-like / MetI-like superfamily / Binding-protein-dependent transport system inner membrane component / ABC transporter integral membrane type-1 domain profile. / CBS domain superfamily / CBS domain / CBS domain ...Glycine betaine transport ATP-binding subunit / ABC-type glycine betaine transport system, substrate-binding domain / Substrate binding domain of ABC-type glycine betaine transport system / ABC transporter type 1, transmembrane domain MetI-like / MetI-like superfamily / Binding-protein-dependent transport system inner membrane component / ABC transporter integral membrane type-1 domain profile. / CBS domain superfamily / CBS domain / CBS domain / CBS domain profile. / 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
ABC-type proline/glycine betaine transport system ATPase component / ABC-type proline/glycine betaine transport system permease component / Glycine betaine transport ATP-binding protein OpuAA
Similarity search - Component
Biological speciesLactococcus lactis subsp. lactis (lactic acid bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsSikkema, H.R. / Rheinberger, J. / Paulino, C. / Poolman, B.
Funding support Netherlands, 3items
OrganizationGrant numberCountry
European Research Council (ERC)670578 Netherlands
Netherlands Organisation for Scientific Research (NWO)722.017.001 Netherlands
Netherlands Organisation for Scientific Research (NWO)740.018.016 Netherlands
CitationJournal: Sci Adv / Year: 2020
Title: Gating by ionic strength and safety check by cyclic-di-AMP in the ABC transporter OpuA.
Authors: Hendrik R Sikkema / Marco van den Noort / Jan Rheinberger / Marijn de Boer / Sabrina T Krepel / Gea K Schuurman-Wolters / Cristina Paulino / Bert Poolman /
Abstract: (Micro)organisms are exposed to fluctuating environmental conditions, and adaptation to stress is essential for survival. Increased osmolality (hypertonicity) causes outflow of water and loss of ...(Micro)organisms are exposed to fluctuating environmental conditions, and adaptation to stress is essential for survival. Increased osmolality (hypertonicity) causes outflow of water and loss of turgor and is dangerous if the cell is not capable of rapidly restoring its volume. The osmoregulatory adenosine triphosphate-binding cassette transporter OpuA restores the cell volume by accumulating large amounts of compatible solute. OpuA is gated by ionic strength and inhibited by the second messenger cyclic-di-AMP, a molecule recently shown to affect many cellular processes. Despite the master regulatory role of cyclic-di-AMP, structural and functional insights into how the second messenger regulates (transport) proteins on the molecular level are lacking. Here, we present high-resolution cryo-electron microscopy structures of OpuA and in vitro activity assays that show how the osmoregulator OpuA is activated by high ionic strength and how cyclic-di-AMP acts as a backstop to prevent unbridled uptake of compatible solutes.
History
DepositionSep 24, 2020Deposition site: PDBE / Processing site: PDBE
Revision 1.0Nov 25, 2020Provider: repository / Type: Initial release
Revision 1.1Dec 2, 2020Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.pdbx_database_id_PubMed ..._citation.journal_volume / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _citation_author.name

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Structure visualization

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  • Deposited structure unit
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Assembly

Deposited unit
A: ABC transporter permease subunit
B: ABC transporter permease subunit
D: ABC-type proline/glycine betaine transport system ATPase component
C: ABC-type proline/glycine betaine transport system ATPase component


Theoretical massNumber of molelcules
Total (without water)218,4434
Polymers218,4434
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area7480 Å2
ΔGint-63 kcal/mol
Surface area47570 Å2
MethodPISA

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Components

#1: Protein ABC transporter permease subunit / ABC-type proline/glycine betaine transport system permease component / Glycine betaine ABC ...ABC-type proline/glycine betaine transport system permease component / Glycine betaine ABC transport system permease and betaine-binding protein


Mass: 63437.812 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Lactococcus lactis subsp. lactis (lactic acid bacteria)
Gene: FEZ45_03205, FIB48_03540, GJI88_00475, GJI90_00975, LL275_1486, N42_0725
Production host: Lactococcus lactis subsp. lactis (lactic acid bacteria)
References: UniProt: A0A0V8ETW8
#2: Protein ABC-type proline/glycine betaine transport system ATPase component / Betaine/proline/choline family ABC transporter ATP-binding protein / Glycine betaine ABC transport ...Betaine/proline/choline family ABC transporter ATP-binding protein / Glycine betaine ABC transport system ATP-binding protein OpuAA / Glycine betaine/L-proline ABC transporter ATP-binding protein


Mass: 45783.684 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Lactococcus lactis subsp. lactis (lactic acid bacteria)
Gene: FEZ45_03200, GJI88_00480, GJI90_00980, KF282_2364, LL275_1487, M20_1239, N42_0726
Production host: Lactococcus lactis subsp. lactis (lactic acid bacteria)
References: UniProt: A0A0R2NIU5, UniProt: Q9KIF7*PLUS

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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

ComponentName: OpuA apo inward facing / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Lactococcus lactis subsp. lactis (lactic acid bacteria)
Source (recombinant)Organism: Lactococcus lactis subsp. lactis (lactic acid bacteria)
Buffer solutionpH: 7
Buffer component
IDConc.NameFormulaBuffer-ID
150 mMpotassium phosphateKPi1
2200 mMpotassium chlorideKCl1
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: at 5 mA / Grid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE-PROPANE / Humidity: 100 % / Chamber temperature: 295 K

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Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: FEI TALOS ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 130000 X / Calibrated magnification: 49407 X / Nominal defocus max: 2000 nm / Nominal defocus min: 500 nm / Calibrated defocus min: 500 nm / Calibrated defocus max: 2000 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 105 K / Temperature (min): 90 K
Image recordingAverage exposure time: 9 sec. / Electron dose: 53 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 6 / Num. of real images: 2861
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV
Image scansWidth: 3838 / Height: 3710 / Movie frames/image: 60 / Used frames/image: 1-60

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Processing

SoftwareName: PHENIX / Version: 1.17.1_3660: / Classification: refinement
EM software
IDNameCategory
2EPUimage acquisition
4CTFFINDCTF correction
7Cootmodel fitting
9PHENIXmodel refinement
10RELIONinitial Euler assignment
11RELIONfinal Euler assignment
13RELION3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1383502
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 78021 / Algorithm: BACK PROJECTION / Symmetry type: POINT
Atomic model buildingSpace: REAL
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0088313
ELECTRON MICROSCOPYf_angle_d0.5711243
ELECTRON MICROSCOPYf_dihedral_angle_d19.331114
ELECTRON MICROSCOPYf_chiral_restr0.0451361
ELECTRON MICROSCOPYf_plane_restr0.0041410

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