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- EMDB-11784: OpuA inhibited inward facing -

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Basic information

Entry
Database: EMDB / ID: EMD-11784
TitleOpuA inhibited inward facing
Map data
Sample
  • Complex: OpuA inhibited inward facing
    • Protein or peptide: ABC transporter permease subunit
    • Protein or peptide: ABC-type proline/glycine betaine transport system ATPase component
  • Ligand: (2R,3R,3aS,5R,7aR,9R,10R,10aS,12R,14aR)-2,9-bis(6-amino-9H-purin-9-yl)octahydro-2H,7H-difuro[3,2-d:3',2'-j][1,3,7,9,2,8 ]tetraoxadiphosphacyclododecine-3,5,10,12-tetrol 5,12-dioxide
Keywordsosmoregulation / ABC-transporter / glycine betaine uptake system / MEMBRANE PROTEIN / cyclic-di-AMP
Function / homology
Function and homology information


ABC-type quaternary amine transporter / amine transmembrane transporter activity / ABC-type quaternary ammonium compound transporting activity / carnitine transmembrane transporter activity / glycine betaine transport / choline transport / peptide transport / amino acid transport / ATP-binding cassette (ABC) transporter complex / protein transport ...ABC-type quaternary amine transporter / amine transmembrane transporter activity / ABC-type quaternary ammonium compound transporting activity / carnitine transmembrane transporter activity / glycine betaine transport / choline transport / peptide transport / amino acid transport / ATP-binding cassette (ABC) transporter complex / protein transport / ATP hydrolysis activity / ATP binding / membrane / plasma 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 ...: / 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
Quaternary amine transport ATP-binding protein / ABC transporter permease/substrate binding protein / Glycine betaine transport ATP-binding protein OpuAA
Similarity search - Component
Biological speciesLactococcus lactis subsp. lactis (lactic acid bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.1 Å
AuthorsSikkema HR / Rheinberger J
Funding support Netherlands, 3 items
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, 2020-
Header (metadata) releaseNov 25, 2020-
Map releaseNov 25, 2020-
UpdateMay 1, 2024-
Current statusMay 1, 2024Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0355
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.0355
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7ahe
  • Surface level: 0.0355
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_11784.png

Downloads & links

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Map

FileDownload / File: emd_11784.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.01 Å/pix.
x 256 pix.
= 259.072 Å		1.01 Å/pix.
x 256 pix.
= 259.072 Å		1.01 Å/pix.
x 256 pix.
= 259.072 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.012 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0355 / Movie #1: 0.0355
Minimum - Maximum-0.050819404 - 0.09918038
Average (Standard dev.)0.0002836347 (±0.004397728)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 259.072 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0121.0121.012
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z259.072259.072259.072
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-0.0510.0990.000

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Supplemental data

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Mask #1

Fileemd_11784_msk_1.map
Projections & Slices
AxesZYX

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Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_11784_half_map_1.map
Projections & Slices
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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_11784_half_map_2.map
Projections & Slices
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Histogram (log scale)

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Sample components

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Entire : OpuA inhibited inward facing

EntireName: OpuA inhibited inward facing
Components
  • Complex: OpuA inhibited inward facing
    • Protein or peptide: ABC transporter permease subunit
    • Protein or peptide: ABC-type proline/glycine betaine transport system ATPase component
  • Ligand: (2R,3R,3aS,5R,7aR,9R,10R,10aS,12R,14aR)-2,9-bis(6-amino-9H-purin-9-yl)octahydro-2H,7H-difuro[3,2-d:3',2'-j][1,3,7,9,2,8 ]tetraoxadiphosphacyclododecine-3,5,10,12-tetrol 5,12-dioxide

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Supramolecule #1: OpuA inhibited inward facing

SupramoleculeName: OpuA inhibited inward facing / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Lactococcus lactis subsp. lactis (lactic acid bacteria)

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Macromolecule #1: ABC transporter permease subunit

MacromoleculeName: ABC transporter permease subunit / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Lactococcus lactis subsp. lactis (lactic acid bacteria)
Molecular weightTheoretical: 63.437812 KDa
Recombinant expressionOrganism: Lactococcus lactis subsp. lactis (lactic acid bacteria)
SequenceString: MIDLAIGQVP IANWVSSATD WITSTFSSGF DVIQKSGTVL MNGITGALTA VPFWLMIAVV TILAILVSGK KIAFPLFTFI GLSLIANQG LWSDLMSTIT LVLLSSLLSI IIGVPLGIWM AKSDLVAKIV QPILDFMQTM PGFVYLIPAV AFFGIGVVPG V FASVIFAL ...String:
MIDLAIGQVP IANWVSSATD WITSTFSSGF DVIQKSGTVL MNGITGALTA VPFWLMIAVV TILAILVSGK KIAFPLFTFI GLSLIANQG LWSDLMSTIT LVLLSSLLSI IIGVPLGIWM AKSDLVAKIV QPILDFMQTM PGFVYLIPAV AFFGIGVVPG V FASVIFAL PPTVRMTNLG IRQVSTELVE AADSFGSTAR QKLFKLEFPL AKGTIMAGVN QTIMLALSMV VIASMIGAPG LG RGVLAAV QSADIGKGFV SGISLVILAI IIDRFTQKLN VSPLEKQGNP TVKKWKRGIA LVSLLALIIG AFSGMSFGKT ASD KKVDLV YMNWDSEVAS INVLTQAMKE HGFDVKTTAL DNAVAWQTVA NGQADGMVSA WLPNTHKTQW QKYGKSVDLL GPNL KGAKV GFVVPSYMNV NSIEDLTNQA NKTITGIEPG AGVMAASEKT LNSYDNLKDW KLVPSSSGAM TVALGEAIKQ HKDIV ITGW SPHWMFNKYD LKYLADPKGT MGTSENINTI VRKGLKKENP EAYKVLDKFN WTTKDMEAVM LDIQNGKTPE EAAKNW IKD HQKEVDKWFK GSIEGRHHHH HH

UniProtKB: ABC transporter permease/substrate binding protein

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Macromolecule #2: ABC-type proline/glycine betaine transport system ATPase component

MacromoleculeName: ABC-type proline/glycine betaine transport system ATPase component
type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Lactococcus lactis subsp. lactis (lactic acid bacteria)
Molecular weightTheoretical: 45.783684 KDa
Recombinant expressionOrganism: Lactococcus lactis subsp. lactis (lactic acid bacteria)
SequenceString: MAVKIKIEHL TKIFGKRIKT ALTMVEKGEP KNEILKKTGA TVGVYDTNFE INEGEIFVIM GLSGSGKSTL LRLLNRLIEP TSGKIFIDN QDVATLNKED LLQVRRKTMS MVFQNFGLFP HRTILENTEY GLEVQNVPKE ERRKRAEKAL DNANLLDFKD Q YPKQLSGG ...String:
MAVKIKIEHL TKIFGKRIKT ALTMVEKGEP KNEILKKTGA TVGVYDTNFE INEGEIFVIM GLSGSGKSTL LRLLNRLIEP TSGKIFIDN QDVATLNKED LLQVRRKTMS MVFQNFGLFP HRTILENTEY GLEVQNVPKE ERRKRAEKAL DNANLLDFKD Q YPKQLSGG MQQRVGLARA LANDPEILLM DEAFSALDPL IRREMQDELL ELQAKFQKTI IFVSHDLNEA LRIGDRIAIM KD GKIMQIG TGEEILTNPA NDYVKTFVED VDRAKVITAE NIMIPALTTN IDVDGPSVAL KKMKTEEVSS LMAVDKKRQF RGV VTSEQA IAARKNNQPL KDVMTTDVGT VSKEMLVRDI LPIIYDAPTP LAVVDDNGFL KGVLIRGSVL EALADIPDED EVEE IEKEE ENK

UniProtKB: Quaternary amine transport ATP-binding protein

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Macromolecule #3: (2R,3R,3aS,5R,7aR,9R,10R,10aS,12R,14aR)-2,9-bis(6-amino-9H-purin-...

MacromoleculeName: (2R,3R,3aS,5R,7aR,9R,10R,10aS,12R,14aR)-2,9-bis(6-amino-9H-purin-9-yl)octahydro-2H,7H-difuro[3,2-d:3',2'-j][1,3,7,9,2,8 ]tetraoxadiphosphacyclododecine-3,5,10,12-tetrol 5,12-dioxide
type: ligand / ID: 3 / Number of copies: 1 / Formula: 2BA
Molecular weightTheoretical: 658.412 Da

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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
Component:
ConcentrationFormulaName
50.0 mMKPipotassium phosphate
200.0 mMKClpotassium chloride
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 20 sec. / Details: at 5mA
VitrificationCryogen name: ETHANE-PROPANE / Chamber humidity: 100 % / Chamber temperature: 295 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
TemperatureMin: 90.0 K / Max: 105.0 K
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 3838 pixel / Digitization - Dimensions - Height: 3710 pixel / Digitization - Frames/image: 1-60 / Number grids imaged: 1 / Number real images: 1066 / Average exposure time: 9.0 sec. / Average electron dose: 53.0 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Calibrated defocus max: 2.0 µm / Calibrated defocus min: 0.5 µm / Calibrated magnification: 49407 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 49407
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 657116
Startup modelType of model: INSILICO MODEL / In silico model: Relion ab-initio model
Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 4.1 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Number images used: 13520
Initial angle assignmentType: ANGULAR RECONSTITUTION
Final angle assignmentType: ANGULAR RECONSTITUTION
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL
Output model

PDB-7ahe:
OpuA inhibited inward facing

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