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- EMDB-11785: OpuA inward-facing in the presence of glycine betaine -

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Basic information

Entry
Database: EMDB / ID: EMD-11785
TitleOpuA inward-facing in the presence of glycine betaine
Map data
Sample
  • Complex: OpuA inward facing in the presence of glycine betaine
    • Protein or peptide: OpuABC
    • Protein or peptide: OpuAA
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)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.5 Å
AuthorsSikkema HR / Rheinberger J / Paulino C / Poolman B
Funding support Netherlands, 3 items
OrganizationGrant numberCountry
European Research Council (ERC)670578 Netherlands
Netherlands Organisation for Scientific Research (NWO)740.018.016 Netherlands
Netherlands Organisation for Scientific Research (NWO)722.017.001 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-
UpdateDec 2, 2020-
Current statusDec 2, 2020Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.025
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.025
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_11785.png

Downloads & links

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Map

FileDownload / File: emd_11785.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: 0.025 / Movie #1: 0.025
Minimum - Maximum-0.031224249 - 0.06116654
Average (Standard dev.)0.00004696392 (±0.0028408004)
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
start NX/NY/NZ79740
NX/NY/NZ93103213
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-0.0310.0610.000

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Supplemental data

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Mask #1

Fileemd_11785_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_11785_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_11785_half_map_2.map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : OpuA inward facing in the presence of glycine betaine

EntireName: OpuA inward facing in the presence of glycine betaine
Components
  • Complex: OpuA inward facing in the presence of glycine betaine
    • Protein or peptide: OpuABC
    • Protein or peptide: OpuAA

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Supramolecule #1: OpuA inward facing in the presence of glycine betaine

SupramoleculeName: OpuA inward facing in the presence of glycine betaine / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Lactococcus lactis subsp. lactis (lactic acid bacteria)
Recombinant expressionOrganism: Lactococcus lactis subsp. lactis (lactic acid bacteria)

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Macromolecule #1: OpuABC

MacromoleculeName: OpuABC / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Lactococcus lactis subsp. lactis (lactic acid bacteria)
Recombinant expressionOrganism: Lactococcus lactis subsp. lactis (lactic acid bacteria)
SequenceString: MIDLAIGQV PIANWVSSAT DWITSTFSSG FDVIQKSGTV LMNGITGALT AVPFWLMIAV VTILAILVSG KKIAFPLFTF IGLSLIANQG LWSDLMSTIT LVLLSSLLSI IIGVPLGIWM AKSDLVAKIV QPILDFMQTM PGFVYLIPAV AFFGIGVVPG VFASVIFALP ...String:
MIDLAIGQV PIANWVSSAT DWITSTFSSG FDVIQKSGTV LMNGITGALT AVPFWLMIAV VTILAILVSG KKIAFPLFTF IGLSLIANQG LWSDLMSTIT LVLLSSLLSI IIGVPLGIWM AKSDLVAKIV QPILDFMQTM PGFVYLIPAV AFFGIGVVPG VFASVIFALP PTVRMTNLGI RQVSTELVEA ADSFGSTARQ KLFKLEFPLA KGTIMAGVNQ TIMLALSMVV IASMIGAPGL GRGVLAAVQS ADIGKGFVSG ISLVILAIII DRFTQKLNVS PLEKQGNPTV KKWKRGIALV SLLALIIGAF SGMSFGKTAS DKKVDLVYMN WDSEVASINV LTQAMKEHGF DVKTTALDNA VAWQTVANGQ ADGMVSAWLP NTHKTQWQKY GKSVDLLGPN LKGAKVGFVV PSYMNVNSIE DLTNQANKTI TGIEPGAGVM AASEKTLNSY DNLKDWKLVP SSSGAMTVAL GEAIKQHKDI VITGWSPHWM FNKYDLKYLA DPKGTMGTSE NINTIVRKGL KKENPEAYKV LDKFNWTTKD MEAVMLDIQN GKTPEEAAKN WIKDHQKEVD KWFKGSIEGR HHHHHH

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Macromolecule #2: OpuAA

MacromoleculeName: OpuAA / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO
Source (natural)Organism: Lactococcus lactis subsp. lactis (lactic acid bacteria)
Recombinant expressionOrganism: Lactococcus lactis subsp. lactis (lactic acid bacteria)
SequenceString: MAVKIKIEHL TKIFGKRIKT ALTMVEKGEP KNEILKKTGA TVGVYDTNFE INEGEIFVIM GLSGSGKSTL LRLLNRLIEP TSGKIFIDNQ DVATLNKEDL LQVRRKTMSM VFQNFGLFPH RTILENTEYG LEVQNVPKEE RRKRAEKALD NANLLDFKDQ YPKQLSGGMQ ...String:
MAVKIKIEHL TKIFGKRIKT ALTMVEKGEP KNEILKKTGA TVGVYDTNFE INEGEIFVIM GLSGSGKSTL LRLLNRLIEP TSGKIFIDNQ DVATLNKEDL LQVRRKTMSM VFQNFGLFPH RTILENTEYG LEVQNVPKEE RRKRAEKALD NANLLDFKDQ YPKQLSGGMQ QRVGLARALA NDPEILLMDE AFSALDPLIR REMQDELLEL QAKFQKTIIF VSHDLNEALR IGDRIAIMKD GKIMQIGTGE EILTNPANDY VKTFVEDVDR AKVITAENIM IPALTTNIDV DGPSVALKKM KTEEVSSLMA VDKKRQFRGV VTSEQAIAAR KNNQPLKDVM TTDVGTVSKE MLVRDILPII YDAPTPLAVV DDNGFLKGVL IRGSVLEALA DIPDEDEVEE IEKEEENK

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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 / 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
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 FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 49407
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
TemperatureMin: 90.0 K / Max: 105.0 K
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: 901 / Average exposure time: 9.0 sec. / Average electron dose: 53.0 e/Å2
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 615299
CTF correctionSoftware - Name: CTFFIND
Initial angle assignmentType: ANGULAR RECONSTITUTION
Final angle assignmentType: ANGULAR RECONSTITUTION
Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 4.5 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Number images used: 30077
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL

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