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- EMDB-23641: The structure of Bacillus subtilis BmrCD in the inward-facing con... -

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Entry
Database: EMDB / ID: EMD-23641
TitleThe structure of Bacillus subtilis BmrCD in the inward-facing conformation bound to Hoechst-33342 and ATP
Map dataPostprocessed maps used for model building and model refinement in Phenix.
Sample
  • Complex: BmrCD
    • Protein or peptide: Probable multidrug resistance ABC transporter ATP-binding/permease protein YheI
    • Protein or peptide: Probable multidrug resistance ABC transporter ATP-binding/permease protein YheH
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE
  • Ligand: 2'-(4-ETHOXYPHENYL)-5-(4-METHYL-1-PIPERAZINYL)-2,5'-BI-BENZIMIDAZOLE
KeywordsABC transporter / multi-drug efflux transporter / ABC exporter / TRANSPORT PROTEIN
Function / homology
Function and homology information


ATPase-coupled lipid transmembrane transporter activity / Translocases; Catalysing the translocation of other compounds; Linked to the hydrolysis of a nucleoside triphosphate / ATPase-coupled transmembrane transporter activity / ABC-type transporter activity / transmembrane transport / response to antibiotic / ATP hydrolysis activity / ATP binding / plasma 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. / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Probable multidrug resistance ABC transporter ATP-binding/permease protein YheH / Probable multidrug resistance ABC transporter ATP-binding/permease protein YheI
Similarity search - Component
Biological speciesBacillus subtilis (bacteria) / Bacillus subtilis subsp. subtilis str. 168 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.55 Å
AuthorsThaker TM / Tomasiak TM
Funding support United States, 2 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM114245 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM128087 United States
CitationJournal: Nat Chem Biol / Year: 2022
Title: Asymmetric drug binding in an ATP-loaded inward-facing state of an ABC transporter.
Authors: Tarjani M Thaker / Smriti Mishra / Wenchang Zhou / Michael Mohan / Qingyu Tang / José D Faraldo-Goméz / Hassane S Mchaourab / Thomas M Tomasiak /
Abstract: Substrate efflux by ATP-binding cassette (ABC) transporters, which play a major role in multidrug resistance, entails the ATP-powered interconversion between transporter intermediates. Despite recent ...Substrate efflux by ATP-binding cassette (ABC) transporters, which play a major role in multidrug resistance, entails the ATP-powered interconversion between transporter intermediates. Despite recent progress in structure elucidation, a number of intermediates have yet to be visualized and mechanistically interpreted. Here, we combine cryogenic-electron microscopy (cryo-EM), double electron-electron resonance spectroscopy and molecular dynamics simulations to profile a previously unobserved intermediate of BmrCD, a heterodimeric multidrug ABC exporter from Bacillus subtilis. In our cryo-EM structure, ATP-bound BmrCD adopts an inward-facing architecture featuring two molecules of the substrate Hoechst-33342 in a striking asymmetric head-to-tail arrangement. Deletion of the extracellular domain capping the substrate-binding chamber or mutation of Hoechst-coordinating residues abrogates cooperative stimulation of ATP hydrolysis. Together, our findings support a mechanistic role for symmetry mismatch between the nucleotide binding and the transmembrane domains in the conformational cycle of ABC transporters and is of notable importance for rational design of molecules for targeted ABC transporter inhibition.
History
DepositionMar 18, 2021-
Header (metadata) releaseJan 5, 2022-
Map releaseJan 5, 2022-
UpdateMay 29, 2024-
Current statusMay 29, 2024Processing site: RCSB / 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
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  • Surface view colored by cylindrical radius
  • Surface level: 0.025
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7m33
  • Surface level: 0.025
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_23641.png

Downloads & links

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Map

FileDownload / File: emd_23641.map.gz / Format: CCP4 / Size: 91.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationPostprocessed maps used for model building and model refinement in Phenix.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.06 Å/pix.
x 288 pix.
= 304.992 Å		1.06 Å/pix.
x 288 pix.
= 304.992 Å		1.06 Å/pix.
x 288 pix.
= 304.992 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.059 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.023 / Movie #1: 0.025
Minimum - Maximum-0.16292481 - 0.2624507
Average (Standard dev.)-0.00009320474 (±0.0042563616)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions288288288
Spacing288288288
CellA=B=C: 304.992 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0591.0591.059
M x/y/z288288288
origin x/y/z0.0000.0000.000
length x/y/z304.992304.992304.992
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS288288288
D min/max/mean-0.1630.262-0.000

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Supplemental data

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Mask #1

Fileemd_23641_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Masked postprocessed maps used for model building and...

Fileemd_23641_additional_1.map
AnnotationMasked postprocessed maps used for model building and model refinement in Phenix.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half-map 1 out of Refine3D in Relion 3.1.

Fileemd_23641_half_map_1.map
AnnotationHalf-map 1 out of Refine3D in Relion 3.1.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half-map 2 out of Refine3D in Relion 3.1.

Fileemd_23641_half_map_2.map
AnnotationHalf-map 2 out of Refine3D in Relion 3.1.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : BmrCD

EntireName: BmrCD
Components
  • Complex: BmrCD
    • Protein or peptide: Probable multidrug resistance ABC transporter ATP-binding/permease protein YheI
    • Protein or peptide: Probable multidrug resistance ABC transporter ATP-binding/permease protein YheH
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE
  • Ligand: 2'-(4-ETHOXYPHENYL)-5-(4-METHYL-1-PIPERAZINYL)-2,5'-BI-BENZIMIDAZOLE

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Supramolecule #1: BmrCD

SupramoleculeName: BmrCD / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Details: heterodimeric multi-drug ABC exporter from Bacillus subtilis
Source (natural)Organism: Bacillus subtilis (bacteria)
Molecular weightTheoretical: 141.39865 KDa

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Macromolecule #1: Probable multidrug resistance ABC transporter ATP-binding/permeas...

MacromoleculeName: Probable multidrug resistance ABC transporter ATP-binding/permease protein YheI
type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
EC number: Translocases; Catalysing the translocation of other compounds; Linked to the hydrolysis of a nucleoside triphosphate
Source (natural)Organism: Bacillus subtilis subsp. subtilis str. 168 (bacteria)
Molecular weightTheoretical: 67.602961 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MGSSHHHHHH SSGLVPRGSH MLEFSVLKKL GWFFKAYWLR YTIAIVLLLA VNVIEMFPPK LLGNAIDDMK AGAFTAEGLL FYIGIFFVL TAAVYIMSYF WMHQLFGGAN LMEKILRTKL MGHLLTMSPP FYEKNRTGDL MARGTNDLQA VSLTTGFGIL T LVDSTMFM ...String:
MGSSHHHHHH SSGLVPRGSH MLEFSVLKKL GWFFKAYWLR YTIAIVLLLA VNVIEMFPPK LLGNAIDDMK AGAFTAEGLL FYIGIFFVL TAAVYIMSYF WMHQLFGGAN LMEKILRTKL MGHLLTMSPP FYEKNRTGDL MARGTNDLQA VSLTTGFGIL T LVDSTMFM MTIFLTMGFL ISWKLTFAAI IPLPVMAIAI SLYGSKIHER FTEAQNAFGA LNDRVLESVS GVRVIRAYVQ ET NDVRRFN EMTADVYQKN MKVAFIDSLF EPTVKLLVGA SYLIGLGYGA FLVFRNELTL GELVSFNVYL GMMIWPMFAI GEL INVMQR GNASLDRVNE TLSYETDVTD PKQPADLKEP GDIVFSHVSF TYPSSTSDNL QDISFTVRKG QTVGIAGKTG SGKT TIIKQ LLRQYPPGEG SITFSGVPIQ QIPLDRLRGW IGYVPQDHLL FSRTVKENIL YGKQDATDKE VQQAIAEAHF EKDLH MLPS GLETMVGEKG VALSGGQKQR ISIARALMAN PEILILDQSL SAVDAKTEAA IIKNIRENRK GKTTFILTHR LSAVEH ADL ILVMDGGVIA ERGTHQELLA NNGWYREQYE RQQLFTAEEG GAGA

UniProtKB: Probable multidrug resistance ABC transporter ATP-binding/permease protein YheI

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Macromolecule #2: Probable multidrug resistance ABC transporter ATP-binding/permeas...

MacromoleculeName: Probable multidrug resistance ABC transporter ATP-binding/permease protein YheH
type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
EC number: Translocases; Catalysing the translocation of other compounds; Linked to the hydrolysis of a nucleoside triphosphate
Source (natural)Organism: Bacillus subtilis subsp. subtilis str. 168 (bacteria)
Molecular weightTheoretical: 77.369898 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MKIGKTLWRY ALLYRKLLIT AVLLLTVAVG AELTGPFIGK KMIDDHILGI EKTWYEAAEK DKNAVQFHGV SYVREDRLQE PVSKAKEAH IYQVGMAFYF VDQAVSFDGN RTVSDGKLTI TNGDKSRAYA AEKLTKQELF QFYQPEIKGM VLLIALYGGL L VFSVFFQY ...String:
MKIGKTLWRY ALLYRKLLIT AVLLLTVAVG AELTGPFIGK KMIDDHILGI EKTWYEAAEK DKNAVQFHGV SYVREDRLQE PVSKAKEAH IYQVGMAFYF VDQAVSFDGN RTVSDGKLTI TNGDKSRAYA AEKLTKQELF QFYQPEIKGM VLLIALYGGL L VFSVFFQY GQHYLLQMSA NRIIQKMRQD VFSHIQKMPI RYFDNLPAGK VVARITNDTE AIRDLYVTVL STFVTSGIYM FG IFTALFL LDVKLAFVAL AIVPIIWLWS VIYRRYASYY NQKIRSINSD INAKMNESIQ GMTIIQAFRH QKETMREFEE LNE SHFYFQ NRMLNLNSLM SHNLVNVIRN LAFVALIWHF GGASLNAAGI VSIGVLYAFV DYLNRLFQPI TGIVNQFSKL ELAR VSAGR VFELLEEKNT EEAGEPAKER ALGRVEFRDV SFAYQEGEEV LKHISFTAQK GETVALVGHT GSGKSSILNL LFRFY DAQK GDVLIDGKSI YNMSRQELRS HMGIVLQDPY LFSGTIGSNV SLDDERMTEE EIKNALRQVG AEPLLKKLPK GINEPV IEK GSTLSSGERQ LISFARALAF DPAILILDQA TAHIDTETEA VIQKALDVVK QGRTTFVIAH RLSTIRNADQ ILVLDKG EI VERGNHEELM ALEGQYYQMY ELQKGQKHSI ALEHHHHHH

UniProtKB: Probable multidrug resistance ABC transporter ATP-binding/permease protein YheH

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Macromolecule #3: ADENOSINE-5'-TRIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 3 / Number of copies: 2 / Formula: ATP
Molecular weightTheoretical: 507.181 Da
Chemical component information

ChemComp-ATP:
ADENOSINE-5'-TRIPHOSPHATE / ATP, energy-carrying molecule*YM

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Macromolecule #4: 2'-(4-ETHOXYPHENYL)-5-(4-METHYL-1-PIPERAZINYL)-2,5'-BI-BENZIMIDAZOLE

MacromoleculeName: 2'-(4-ETHOXYPHENYL)-5-(4-METHYL-1-PIPERAZINYL)-2,5'-BI-BENZIMIDAZOLE
type: ligand / ID: 4 / Number of copies: 2 / Formula: HT1
Molecular weightTheoretical: 452.551 Da
Chemical component information

ChemComp-HT1:
2'-(4-ETHOXYPHENYL)-5-(4-METHYL-1-PIPERAZINYL)-2,5'-BI-BENZIMIDAZOLE

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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

Concentration2.5 mg/mL
BufferpH: 7.4
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 85 % / Chamber temperature: 283 K / Instrument: LEICA EM GP / Details: blot for 4 seconds before plunging.
DetailsPurified by size exclusion chromatography using a Superose 6 column.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 1 / Number real images: 6919 / Average electron dose: 37.2 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.1 µm / Nominal defocus min: 0.8 µm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.55 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 157021
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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