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- EMDB-36797: mycobacterial efflux pump, AMPPNP bound state -

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Basic information

Entry
Database: EMDB / ID: EMD-36797
Titlemycobacterial efflux pump, AMPPNP bound state
Map dataEM map of mycobacterial efflux pump, AMPPNP bound state
Sample
  • Complex: ternary complex of an ABC transporter Rv1217c-1218c
    • Protein or peptide: Multidrug efflux system permease protein Rv1217c
    • Protein or peptide: Multidrug efflux system ATP-binding protein Rv1218c
  • Ligand: CARDIOLIPIN
  • Ligand: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER
  • Ligand: MAGNESIUM ION
KeywordsABC transporter / efflux pump / TRANSPORT PROTEIN
Function / homology
Function and homology information


Translocases; Catalysing the translocation of other compounds; Linked to the hydrolysis of a nucleoside triphosphate / response to antibiotic / ATP hydrolysis activity / ATP binding / plasma membrane
Similarity search - Function
: / Domain of unknown function DUF4162 / ATP-binding protein DrrA1-3-like, C-terminal domain / 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
Multidrug efflux system permease protein Rv1217c / Multidrug efflux system ATP-binding protein Rv1218c
Similarity search - Component
Biological speciesMycobacterium tuberculosis (strain ATCC 25618 / H37Rv) (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.9 Å
AuthorsWang Y / Wu F / Zhang L / Rao Z
Funding support China, 1 items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC)32170703 China
CitationJournal: Proc Natl Acad Sci U S A / Year: 2024
Title: Cryo-EM structures of a mycobacterial ABC transporter that mediates rifampicin resistance.
Authors: Yinan Wang / Shan Gao / Fangyu Wu / Yicheng Gong / Nengjiang Mu / Chuancun Wei / Chengyao Wu / Jun Wang / Ning Yan / Huifang Yang / Yifan Zhang / Jiayi Liu / Zeyu Wang / Xiuna Yang / Sin Man ...Authors: Yinan Wang / Shan Gao / Fangyu Wu / Yicheng Gong / Nengjiang Mu / Chuancun Wei / Chengyao Wu / Jun Wang / Ning Yan / Huifang Yang / Yifan Zhang / Jiayi Liu / Zeyu Wang / Xiuna Yang / Sin Man Lam / Guanghou Shui / Siyuan Li / Lintai Da / Luke W Guddat / Zihe Rao / Lu Zhang /
Abstract: Drug-resistant Tuberculosis (TB) is a global public health problem. Resistance to rifampicin, the most effective drug for TB treatment, is a major growing concern. The etiological agent, (), has a ...Drug-resistant Tuberculosis (TB) is a global public health problem. Resistance to rifampicin, the most effective drug for TB treatment, is a major growing concern. The etiological agent, (), has a cluster of ATP-binding cassette (ABC) transporters which are responsible for drug resistance through active export. Here, we describe studies characterizing Rv1217c-1218c as an ABC transporter that can mediate mycobacterial resistance to rifampicin and have determined the cryo-electron microscopy structures of Rv1217c-1218c. The structures show Rv1217c-1218c has a type V exporter fold. In the absence of ATP, Rv1217c-1218c forms a periplasmic gate by two juxtaposed-membrane helices from each transmembrane domain (TMD), while the nucleotide-binding domains (NBDs) form a partially closed dimer which is held together by four salt-bridges. Adenylyl-imidodiphosphate (AMPPNP) binding induces a structural change where the NBDs become further closed to each other, which downstream translates to a closed conformation for the TMDs. AMPPNP binding results in the collapse of the outer leaflet cavity and the opening of the periplasmic gate, which was proposed to play a role in substrate export. The rifampicin-bound structure shows a hydrophobic and periplasm-facing cavity is involved in rifampicin binding. Phospholipid molecules are observed in all determined structures and form an integral part of the Rv1217c-1218c transporter system. Our results provide a structural basis for a mycobacterial ABC exporter that mediates rifampicin resistance, which can lead to different insights into combating rifampicin resistance.
History
DepositionJul 11, 2023-
Header (metadata) releaseJul 17, 2024-
Map releaseJul 17, 2024-
UpdateJan 29, 2025-
Current statusJan 29, 2025Processing site: PDBc / Status: Released

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Structure visualization

Supplemental images

emd_36797.png

Downloads & links

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Map

FileDownload / File: emd_36797.map.gz / Format: CCP4 / Size: 216 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationEM map of mycobacterial efflux pump, AMPPNP bound state
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.82 Å/pix.
x 384 pix.
= 314.88 Å		0.82 Å/pix.
x 384 pix.
= 314.88 Å		0.82 Å/pix.
x 384 pix.
= 314.88 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.82 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.5
Minimum - Maximum-3.4402783 - 6.8974166
Average (Standard dev.)-0.0011862626 (±0.089036316)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions384384384
Spacing384384384
CellA=B=C: 314.88 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: half map B of mycobacterial efflux pump, AMPPNP bound state

Fileemd_36797_half_map_1.map
Annotationhalf map B of mycobacterial efflux pump, AMPPNP bound state
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half map A of mycobacterial efflux pump, AMPPNP bound state

Fileemd_36797_half_map_2.map
Annotationhalf map A of mycobacterial efflux pump, AMPPNP bound state
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : ternary complex of an ABC transporter Rv1217c-1218c

EntireName: ternary complex of an ABC transporter Rv1217c-1218c
Components
  • Complex: ternary complex of an ABC transporter Rv1217c-1218c
    • Protein or peptide: Multidrug efflux system permease protein Rv1217c
    • Protein or peptide: Multidrug efflux system ATP-binding protein Rv1218c
  • Ligand: CARDIOLIPIN
  • Ligand: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER
  • Ligand: MAGNESIUM ION

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Supramolecule #1: ternary complex of an ABC transporter Rv1217c-1218c

SupramoleculeName: ternary complex of an ABC transporter Rv1217c-1218c / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Mycobacterium tuberculosis (strain ATCC 25618 / H37Rv) (bacteria)
Molecular weightTheoretical: 120 KDa

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Macromolecule #1: Multidrug efflux system permease protein Rv1217c

MacromoleculeName: Multidrug efflux system permease protein Rv1217c / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Mycobacterium tuberculosis (strain ATCC 25618 / H37Rv) (bacteria)
Molecular weightTheoretical: 56.698039 KDa
Recombinant expressionOrganism: Mycolicibacterium smegmatis MC2 155 (bacteria)
SequenceString: MSSTVIDRAR PAGHRAPHRG SGFTGTLGLL RLYLRRDRVS LPLWVLLLSV PLATVYIASV ETVYPDRSAR AAAAAAIMAS PAQRALYGP VYNDSLGAVG IWKAGMFHTL IAVAVILTVI RHTRADEESG RAELIDSTVV GRYANLTGAL LLSFGASIAT G AIGALGLL ...String:
MSSTVIDRAR PAGHRAPHRG SGFTGTLGLL RLYLRRDRVS LPLWVLLLSV PLATVYIASV ETVYPDRSAR AAAAAAIMAS PAQRALYGP VYNDSLGAVG IWKAGMFHTL IAVAVILTVI RHTRADEESG RAELIDSTVV GRYANLTGAL LLSFGASIAT G AIGALGLL ATDVAPAGSV AFGVALAASG MVFTAVAAVA AQLSPSARFT RAVAFAVLGT AFALRAIGDA GSGTLSWCSP LG WSLQVRP YAGERWWVLL LSLATAAVLT VLAYRLRAGR DVGAGLIAER PGAGTAGPML SEPFGLAWRL NRGSLLLWTV GLC LYGLVM GSVVHGIGDQ LGDNTAVRDI VTRMGGTGAL EQAFLALAFT MIGMVAAAFA VSLTLRLHQE ETGLRAETLL AGAV SRTHW LASHLAMALA GSAVATLISG VAAGLAYGMT VGDVGGKLPT VVGTAAVQLP AVWLLSAVTV GLFGLAPRFT PVAWG VLVG FIALYLLGSL AGFPQMLLNL EPFAHIPRVG GGDFTAVPLL WLLAIDAALI TLGAMAFRRR DVRC

UniProtKB: Multidrug efflux system permease protein Rv1217c

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Macromolecule #2: Multidrug efflux system ATP-binding protein Rv1218c

MacromoleculeName: Multidrug efflux system ATP-binding protein Rv1218c / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO
EC number: Translocases; Catalysing the translocation of other compounds; Linked to the hydrolysis of a nucleoside triphosphate
Source (natural)Organism: Mycobacterium tuberculosis (strain ATCC 25618 / H37Rv) (bacteria)
Molecular weightTheoretical: 33.491082 KDa
Recombinant expressionOrganism: Mycolicibacterium smegmatis MC2 155 (bacteria)
SequenceString: MSADNHQVPI EIRGLTKHFG SVRALDGLDL TVREGEVHGF LGPNGAGKST TLRILLGLVK ADGGSVRLLG GDPWTDAVDL HRHIAYVPG DVTLWPSLTG GETIDLLARM RGGIDNARRA ELIERFGLDP TKKARTYSKG NRQKVSLISA LSSHATLLLL D EPSSGLDP ...String:
MSADNHQVPI EIRGLTKHFG SVRALDGLDL TVREGEVHGF LGPNGAGKST TLRILLGLVK ADGGSVRLLG GDPWTDAVDL HRHIAYVPG DVTLWPSLTG GETIDLLARM RGGIDNARRA ELIERFGLDP TKKARTYSKG NRQKVSLISA LSSHATLLLL D EPSSGLDP LMENVFQQCI GEARQRGVTV LLSSHILAET EALCEKVTII RAGKTVESGS LDALRHLSRT SIKAEMIGDP GD LSQIKGV EDISIEGTTV RAQVDSESLR ELIQVLGHAG VRSLVSQPPT LEELFLRHYS LGPEVAAEQQ VATP

UniProtKB: Multidrug efflux system ATP-binding protein Rv1218c

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Macromolecule #3: CARDIOLIPIN

MacromoleculeName: CARDIOLIPIN / type: ligand / ID: 3 / Number of copies: 2 / Formula: CDL
Molecular weightTheoretical: 1.464043 KDa
Chemical component information

ChemComp-CDL:
CARDIOLIPIN / phospholipid*YM

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Macromolecule #4: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER

MacromoleculeName: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER / type: ligand / ID: 4 / Number of copies: 2 / Formula: ANP
Molecular weightTheoretical: 506.196 Da
Chemical component information

ChemComp-ANP:
PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER / AMP-PNP, energy-carrying molecule analogue*YM

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Macromolecule #5: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 5 / Number of copies: 2 / Formula: MG
Molecular weightTheoretical: 24.305 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

Concentration8 mg/mL
BufferpH: 7.4 / Details: 150mM NaCl, 20mM Tris, detergent
GridMaterial: COPPER / Mesh: 300 / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Time: 30 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 281 K / Instrument: FEI VITROBOT MARK II
Detailssample mono disperse

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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: 3629 / Average exposure time: 2.4 sec. / Average electron dose: 60.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.2 µm / Nominal defocus min: 1.2 µm / Nominal magnification: 29000
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

Particle selectionNumber selected: 1859573
Startup modelType of model: NONE
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 157931
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final 3D classificationNumber classes: 4 / Software - Name: cryoSPARC

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