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- PDB-5nc5: Crystal structure of AcrBZ in complex with antibiotic puromycin -

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Basic information

Entry
Database: PDB / ID: 5nc5
TitleCrystal structure of AcrBZ in complex with antibiotic puromycin
Components
  • (Multidrug efflux pump ...) x 2
  • DARPin
KeywordsTRANSPORT PROTEIN / Membrane transporter / Multidrug efflux pump
Function / homology
Function and homology information


alkane transmembrane transporter activity / alkane transport / xenobiotic detoxification by transmembrane export across the cell outer membrane / efflux pump complex / enterobactin transport / enterobactin transmembrane transporter activity / periplasmic side of plasma membrane / bile acid transmembrane transporter activity / xenobiotic detoxification by transmembrane export across the plasma membrane / bile acid and bile salt transport ...alkane transmembrane transporter activity / alkane transport / xenobiotic detoxification by transmembrane export across the cell outer membrane / efflux pump complex / enterobactin transport / enterobactin transmembrane transporter activity / periplasmic side of plasma membrane / bile acid transmembrane transporter activity / xenobiotic detoxification by transmembrane export across the plasma membrane / bile acid and bile salt transport / xenobiotic transport / efflux transmembrane transporter activity / xenobiotic transmembrane transporter activity / fatty acid transport / response to toxic substance / response to xenobiotic stimulus / response to antibiotic / identical protein binding / membrane / plasma membrane
Similarity search - Function
: / Multidrug efflux pump accessory protein AcrZ / Multidrug efflux pump-associated protein AcrZ / Multidrug efflux transporter AcrB transmembrane fold / Multidrug efflux transporter AcrB transmembrane domain / Multidrug efflux transporter AcrB TolC docking domain; DN and DC subdomains / Multidrug efflux transporter AcrB TolC docking domain; DN and DC subdomains / Multidrug efflux transporter AcrB pore domain / Multidrug efflux transporter AcrB pore domain like / Multidrug efflux transporter AcrB pore domain ...: / Multidrug efflux pump accessory protein AcrZ / Multidrug efflux pump-associated protein AcrZ / Multidrug efflux transporter AcrB transmembrane fold / Multidrug efflux transporter AcrB transmembrane domain / Multidrug efflux transporter AcrB TolC docking domain; DN and DC subdomains / Multidrug efflux transporter AcrB TolC docking domain; DN and DC subdomains / Multidrug efflux transporter AcrB pore domain / Multidrug efflux transporter AcrB pore domain like / Multidrug efflux transporter AcrB pore domain / Hydrophobe/amphiphile efflux-1 HAE1 / Acriflavin resistance protein / Multidrug efflux transporter AcrB TolC docking domain, DN/DC subdomains / AcrB/AcrD/AcrF family / Ankyrin repeat-containing domain / Serine Threonine Protein Phosphatase 5, Tetratricopeptide repeat / Alpha Horseshoe / Alpha-Beta Plaits / Up-down Bundle / 2-Layer Sandwich / Mainly Alpha / Alpha Beta
Similarity search - Domain/homology
DECANE / DODECANE / nonane / HEXANE / PUROMYCIN / Multidrug efflux pump accessory protein AcrZ / Multidrug efflux pump subunit AcrB
Similarity search - Component
Biological speciesEscherichia coli K-12 (bacteria)
Escherichia coli (E. coli)
Escherichia coli O157:H7 (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 3.2 Å
AuthorsDu, D. / Luisi, B.
Funding support United Kingdom, 1items
OrganizationGrant numberCountry
Wellcome TrustRG61065 United Kingdom
CitationJournal: Elife / Year: 2017
Title: An allosteric transport mechanism for the AcrAB-TolC multidrug efflux pump.
Authors: Zhao Wang / Guizhen Fan / Corey F Hryc / James N Blaza / Irina I Serysheva / Michael F Schmid / Wah Chiu / Ben F Luisi / Dijun Du /
Abstract: Bacterial efflux pumps confer multidrug resistance by transporting diverse antibiotics from the cell. In Gram-negative bacteria, some of these pumps form multi-protein assemblies that span the cell ...Bacterial efflux pumps confer multidrug resistance by transporting diverse antibiotics from the cell. In Gram-negative bacteria, some of these pumps form multi-protein assemblies that span the cell envelope. Here, we report the near-atomic resolution cryoEM structures of the AcrAB-TolC multidrug efflux pump in resting and drug transport states, revealing a quaternary structural switch that allosterically couples and synchronizes initial ligand binding with channel opening. Within the transport-activated state, the channel remains open even though the pump cycles through three distinct conformations. Collectively, our data provide a dynamic mechanism for the assembly and operation of the AcrAB-TolC pump.
History
DepositionMar 3, 2017Deposition site: PDBE / Processing site: PDBE
Revision 1.0Apr 12, 2017Provider: repository / Type: Initial release
Revision 1.1Apr 19, 2017Group: Atomic model
Revision 1.2Oct 16, 2019Group: Data collection / Category: reflns_shell
Revision 1.3Jan 17, 2024Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Multidrug efflux pump subunit AcrB
B: Multidrug efflux pump subunit AcrB
C: Multidrug efflux pump subunit AcrB
D: DARPin
E: DARPin
F: Multidrug efflux pump accessory protein AcrZ
G: Multidrug efflux pump accessory protein AcrZ
H: Multidrug efflux pump accessory protein AcrZ
hetero molecules


Theoretical massNumber of molelcules
Total (without water)402,49161
Polymers393,5448
Non-polymers8,94753
Water1,76598
1


  • Idetical with deposited unit
  • defined by software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area47620 Å2
ΔGint68 kcal/mol
Surface area128690 Å2
MethodPISA
Unit cell
Length a, b, c (Å)147.245, 167.646, 249.980
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number19
Space group name H-MP212121

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Components

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Multidrug efflux pump ... , 2 types, 6 molecules ABCFGH

#1: Protein Multidrug efflux pump subunit AcrB / AcrAB-TolC multidrug efflux pump subunit AcrB / Acridine resistance protein B


Mass: 113665.180 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli K-12 (bacteria) / Gene: acrB, acrE, b0462, JW0451 / Production host: Escherichia coli (E. coli) / References: UniProt: P31224
#3: Protein/peptide Multidrug efflux pump accessory protein AcrZ / AcrAB-TolC multidrug efflux pump accessory protein AcrZ / Acridine resistance protein Z


Mass: 5304.423 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli O157:H7 (bacteria) / Gene: acrZ, Z0932, ECs0790 / Production host: Escherichia coli (E. coli) / References: UniProt: P0AAX1

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Protein / Sugars , 2 types, 5 molecules DE

#2: Protein DARPin


Mass: 18317.566 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Production host: Escherichia coli (E. coli)
#4: Sugar ChemComp-LMT / DODECYL-BETA-D-MALTOSIDE


Type: D-saccharide / Mass: 510.615 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C24H46O11 / Comment: detergent*YM

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Non-polymers , 6 types, 148 molecules

#5: Chemical
ChemComp-D12 / DODECANE


Mass: 170.335 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: C12H26
#6: Chemical...
ChemComp-D10 / DECANE


Mass: 142.282 Da / Num. of mol.: 26 / Source method: obtained synthetically / Formula: C10H22
#7: Chemical
ChemComp-DD9 / nonane


Mass: 128.255 Da / Num. of mol.: 14 / Source method: obtained synthetically / Formula: C9H20
#8: Chemical ChemComp-PUY / PUROMYCIN


Type: RNA linking / Mass: 471.510 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C22H29N7O5 / Comment: antibiotic, inhibitor*YM
#9: Chemical ChemComp-HEX / HEXANE


Mass: 86.175 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C6H14
#10: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 98 / Source method: isolated from a natural source / Formula: H2O

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 3.92 Å3/Da / Density % sol: 68.62 %
Crystal growTemperature: 293 K / Method: vapor diffusion, hanging drop
Details: 80 mM Bis-Tris, pH 6.0, 50 mM sodium citrate, 120 mM KCl, 10% PEG 4000, 0.5% N,N-dimethyldodecylamine N-oxide

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: Diamond / Beamline: I24 / Wavelength: 0.9794 Å
DetectorType: DECTRIS PILATUS3 6M / Detector: PIXEL / Date: Sep 1, 2014
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9794 Å / Relative weight: 1
ReflectionResolution: 3→50 Å / Num. obs: 94271 / % possible obs: 92.5 % / Redundancy: 3 % / Rmerge(I) obs: 0.175 / Net I/σ(I): 5.2

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Processing

Software
NameVersionClassification
PHENIX(1.10.1_2155: ???)refinement
iMOSFLMdata reduction
SCALEPACKdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 4DX5

4dx5


Resolution: 3.2→34.928 Å / SU ML: 0.46 / Cross valid method: FREE R-VALUE / σ(F): 1.35 / Phase error: 24.08
RfactorNum. reflection% reflection
Rfree0.2448 4695 4.98 %
Rwork0.1827 --
obs0.1859 94200 91.97 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å
Refinement stepCycle: LAST / Resolution: 3.2→34.928 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms26769 0 530 98 27397
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.01927753
X-RAY DIFFRACTIONf_angle_d1.9137490
X-RAY DIFFRACTIONf_dihedral_angle_d17.20416589
X-RAY DIFFRACTIONf_chiral_restr0.1124385
X-RAY DIFFRACTIONf_plane_restr0.0154742
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
3.2-3.23640.3741480.31452864X-RAY DIFFRACTION89
3.2364-3.27440.35431380.29962844X-RAY DIFFRACTION89
3.2744-3.31430.36321460.28072874X-RAY DIFFRACTION90
3.3143-3.35620.331650.25752994X-RAY DIFFRACTION93
3.3562-3.40040.2841420.24033042X-RAY DIFFRACTION95
3.4004-3.44690.28641450.24483066X-RAY DIFFRACTION95
3.4469-3.49610.33151540.24343044X-RAY DIFFRACTION94
3.4961-3.54820.3061820.23532974X-RAY DIFFRACTION94
3.5482-3.60360.30051500.23193033X-RAY DIFFRACTION94
3.6036-3.66260.30731760.22672952X-RAY DIFFRACTION93
3.6626-3.72570.30471610.2213039X-RAY DIFFRACTION94
3.7257-3.79340.27941520.20113013X-RAY DIFFRACTION94
3.7934-3.86630.28211440.19463024X-RAY DIFFRACTION94
3.8663-3.94510.27231500.18383014X-RAY DIFFRACTION93
3.9451-4.03070.26341530.17673007X-RAY DIFFRACTION92
4.0307-4.12440.25631660.17262886X-RAY DIFFRACTION90
4.1244-4.22730.23931450.16782842X-RAY DIFFRACTION88
4.2273-4.34140.20251360.15132965X-RAY DIFFRACTION91
4.3414-4.46890.19661720.14313031X-RAY DIFFRACTION94
4.4689-4.61290.19071660.13942989X-RAY DIFFRACTION93
4.6129-4.77730.21811820.13752988X-RAY DIFFRACTION93
4.7773-4.96810.19691530.13692999X-RAY DIFFRACTION93
4.9681-5.19350.20771510.14353022X-RAY DIFFRACTION93
5.1935-5.46640.22931500.15613037X-RAY DIFFRACTION93
5.4664-5.80740.23991440.16272972X-RAY DIFFRACTION91
5.8074-6.25340.23831520.17812870X-RAY DIFFRACTION88
6.2534-6.87840.2431550.1733025X-RAY DIFFRACTION92
6.8784-7.86380.20121620.14963006X-RAY DIFFRACTION91
7.8638-9.87040.16931800.12533035X-RAY DIFFRACTION91
9.8704-34.92980.22411750.20633054X-RAY DIFFRACTION88

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