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- PDB-8k1m: mycobacterial efflux pump, apo state -

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Basic information

Entry
Database: PDB / ID: 8k1m
Titlemycobacterial efflux pump, apo state
Components
  • Multidrug efflux system ATP-binding protein Rv1218c
  • Multidrug efflux system permease protein Rv1217c
KeywordsTRANSPORT PROTEIN / ABC transporter / efflux pump
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
CARDIOLIPIN / Chem-L9Q / Multidrug efflux system permease protein Rv1217c / Multidrug efflux system ATP-binding protein Rv1218c
Similarity search - Component
Biological speciesMycobacterium tuberculosis (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.9 Å
AuthorsWang, Y. / Wu, F. / Zhang, L. / Rao, Z.
Funding support China, 1items
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, 2023Deposition site: PDBJ / Processing site: PDBC
Revision 1.0Jul 17, 2024Provider: repository / Type: Initial release
Revision 1.1Jan 29, 2025Group: Data collection / Database references / Structure summary
Category: citation / citation_author ...citation / citation_author / em_admin / pdbx_entry_details
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _em_admin.last_update / _pdbx_entry_details.has_protein_modification

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
B: Multidrug efflux system permease protein Rv1217c
C: Multidrug efflux system ATP-binding protein Rv1218c
D: Multidrug efflux system ATP-binding protein Rv1218c
hetero molecules


Theoretical massNumber of molelcules
Total (without water)121,3635
Polymers119,1533
Non-polymers2,2102
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Multidrug efflux system permease protein Rv1217c


Mass: 56698.039 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: transmembrane domain
Source: (gene. exp.) Mycobacterium tuberculosis (strain ATCC 25618 / H37Rv) (bacteria)
Gene: Rv1217c / Plasmid: pMV261
Production host: Mycolicibacterium smegmatis MC2 155 (bacteria)
References: UniProt: O05318
#2: Protein Multidrug efflux system ATP-binding protein Rv1218c


Mass: 31227.666 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Details: nucleotide binding domain
Source: (gene. exp.) Mycobacterium tuberculosis (strain ATCC 25618 / H37Rv) (bacteria)
Gene: Rv1218c / Plasmid: pMV261
Production host: Mycolicibacterium smegmatis MC2 155 (bacteria)
References: UniProt: O86311, Translocases; Catalysing the translocation of other compounds; Linked to the hydrolysis of a nucleoside triphosphate
#3: Chemical ChemComp-L9Q / (1S)-2-{[(S)-(2-aminoethoxy)(hydroxy)phosphoryl]oxy}-1-[(octadecanoyloxy)methyl]ethyl (9Z)-octadec-9-enoate / 1-stearoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine


Mass: 746.050 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C41H80NO8P / Feature type: SUBJECT OF INVESTIGATION
#4: Chemical ChemComp-CDL / CARDIOLIPIN / DIPHOSPHATIDYL GLYCEROL / BIS-(1,2-DIACYL-SN-GLYCERO-3-PHOSPHO)-1',3'-SN-GLYCEROL


Mass: 1464.043 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C81H156O17P2 / Comment: phospholipid*YM
Has ligand of interestY
Has protein modificationN

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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: ternary complex of an ABC transporter Rv1217c-1218c / Type: COMPLEX / Entity ID: #1-#2 / Source: MULTIPLE SOURCES
Molecular weightValue: 0.12 MDa / Experimental value: YES
Source (natural)Organism: Mycobacterium tuberculosis (strain ATCC 25618 / H37Rv) (bacteria)
Source (recombinant)Organism: Mycolicibacterium smegmatis MC2 155 (bacteria) / Plasmid: pMV261
Buffer solutionpH: 7.4 / Details: 150mM NaCl, 20mM Tris, detergent
SpecimenConc.: 8 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: sample was monodisperse
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in.
VitrificationInstrument: FEI VITROBOT MARK II / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 281 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: SPOT SCAN
Electron lensMode: BRIGHT FIELD / Nominal magnification: 29000 X / Nominal defocus max: 2200 nm / Nominal defocus min: 1200 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm / Alignment procedure: BASIC
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 2.4 sec. / Electron dose: 60 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 5900

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Processing

EM software
IDNameCategory
1cryoSPARCparticle selection
2SerialEMimage acquisition
4cryoSPARCCTF correction
12cryoSPARCclassification
13cryoSPARC3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1616966
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 270648 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0048372
ELECTRON MICROSCOPYf_angle_d0.72711379
ELECTRON MICROSCOPYf_dihedral_angle_d25.6221198
ELECTRON MICROSCOPYf_chiral_restr0.0461368
ELECTRON MICROSCOPYf_plane_restr0.0051448

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