|Entry||Database: PDB / ID: 5ng5|
|Title||multi-drug efflux; membrane transport; RND superfamily; Drug resistance|
|Keywords||MEMBRANE PROTEIN / multi-drug efflux / membrane transport / RND superfamily / Drug resistance / membrane protein|
|Specimen source||Escherichia coli / bacteria / /|
|Method||Electron microscopy (6.5 Å resolution / Particle / Single particle) / Transmission electron microscopy|
|Authors||Wang, Z. / Fan, G. / Hryc, C.F. / Blaza, J.N. / Serysheva, I.I. / Schmid, M.F. / Chiu, W. / Luisi, B.F. / Du, D.|
|Citation||Elife, 2017, 6|
SummaryFull reportAbout validation report
|Date||Deposition: Mar 16, 2017 / Release: Apr 19, 2017|
Downloads & links
E: Multidrug efflux pump subunit AcrA
D: Multidrug efflux pump subunit AcrA
G: Multidrug efflux pump subunit AcrA
H: Multidrug efflux pump subunit AcrA
A: Multidrug efflux pump subunit AcrA
B: Multidrug efflux pump subunit AcrA
F: Outer membrane protein TolC
I: Outer membrane protein TolC
C: Outer membrane protein TolC
K: Multidrug efflux pump subunit AcrB
L: Multidrug efflux pump subunit AcrB
M: Multidrug efflux pump accessory protein AcrZ
N: Multidrug efflux pump accessory protein AcrZ
O: Multidrug efflux pump accessory protein AcrZ
J: Multidrug efflux pump subunit AcrB
Mass: 39800.660 Da / Num. of mol.: 6 / Source: (gene. exp.) Escherichia coli / bacteria / / / Gene: acrA, lir, mtcA, b0463, JW0452 / Production host: Escherichia coli / References: UniProt: P0AE06
Mass: 53783.355 Da / Num. of mol.: 3 / Source: (gene. exp.) Escherichia coli / bacteria / /
Gene: tolC, colE1-i, mtcB, mukA, refI, toc, weeA, b3035, JW5503
Production host: Escherichia coli / References: UniProt: P02930
Mass: 113665.180 Da / Num. of mol.: 3 / Source: (gene. exp.) Escherichia coli / bacteria / / / Gene: acrB, acrE, b0462, JW0451 / Production host: Escherichia coli / References: UniProt: P31224
Mass: 5995.157 Da / Num. of mol.: 3 / Source: (gene. exp.) Escherichia coli / bacteria / / / Gene: acrZ, ybhT, b0762, JW5102 / Production host: Escherichia coli / References: UniProt: P0AAW9
|Experiment||Method: ELECTRON MICROSCOPY|
|EM experiment||Aggregation state: PARTICLE / Reconstruction method: SINGLE PARTICLE|
|Component||Name: AcrABTolC in apo state / Type: COMPLEX / Entity ID: 1,||Source (natural)||Organism: Escherichia coli||Source (recombinant)||Organism: Escherichia coli||Buffer solution||pH: 8||Specimen||Conc.: 2 mg/ml / Details: AcrABTolC complex in apo state / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES||Specimen support||Grid material: GOLD / Grid mesh size: 300 / Grid type: Quantifoil||Vitrification||Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 20 kelvins|
Details: a 3ul aliquot at a concentration of 2 mg per ml was applied onto glow-discharged holey carbon grid (Quantifoil Au R1.21.3, 300 mesh)
-Electron microscopy imaging
Model: Titan Krios / Image courtesy: FEI Company
|Microscopy||Microscope model: FEI TITAN KRIOS|
|Electron gun||Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM|
|Electron lens||Mode: BRIGHT FIELD|
|Image recording||Electron dose: 1 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k)|
|Image scans||Movie frames/image: 45|
|CTF correction||Type: PHASE FLIPPING ONLY|
|Particle selection||Details: auto box by relion / Number of particles selected: 95410|
|3D reconstruction||Resolution: 6.5 Å / Resolution method: FSC 0.143 CUT-OFF / Number of particles: 13544 / Algorithm: BACK PROJECTION / Number of class averages: 1 / Symmetry type: POINT|
|Atomic model building||Details: Crystal structures were rigid-body fit into the density map and model optimization was then carried out with Phenix real-space refine. Due to the weaker resolution, stronger stereochemical and secondary structure restraints were used to ensure that alpha-helices and beta-sheets did not deviate far from their expected geometry. Manual adjustments were kept to a minimum to reduce human bias in the modeling procedure, with Coot only being used to fix obvious errors such as C-beta deviations. A final check of MolProbity and cross correlation was done to ensure model quality.|
Ref protocol: FLEXIBLE FIT / Ref space: REAL / Target criteria: Cross-correlation coefficient
-Oct 4, 2017. Three pioneers of this field were awarded Nobel Prize in Chemistry 2017
Three pioneers of this field were awarded Nobel Prize in Chemistry 2017
- Jacques Dubochet (University of Lausanne, Switzerland) is a pioneer of ice-embedding method of EM specimen (as known as cryo-EM), Most of 3DEM structures in EMDB and PDB are obtained using his method.
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External links: The 2017 Nobel Prize in Chemistry - Press Release
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