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- PDB-7b5p: AcrB in cycloalkane amphipol -

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Basic information

Entry
Database: PDB / ID: 7b5p
TitleAcrB in cycloalkane amphipol
ComponentsEfflux pump membrane transporter
KeywordsMEMBRANE PROTEIN / drug exporter / amphipol
Function / homologyHydrophobe/amphiphile efflux-1 HAE1 / Acriflavin resistance protein / Multidrug efflux transporter AcrB TolC docking domain, DN/DC subdomains / AcrB/AcrD/AcrF family / efflux transmembrane transporter activity / xenobiotic transmembrane transporter activity / plasma membrane / Efflux pump membrane transporter
Function and homology information
Biological speciesEscherichia coli (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsHiggins, A.J. / Flynn, A.J. / Muench, S.P.
Funding support United Kingdom, 1items
OrganizationGrant numberCountry
Biotechnology and Biological Sciences Research Council (BBSRC)BB/R018561/1/ United Kingdom
CitationJournal: Commun Biol / Year: 2021
Title: Cycloalkane-modified amphiphilic polymers provide direct extraction of membrane proteins for CryoEM analysis.
Authors: Anna J Higgins / Alex J Flynn / Anaïs Marconnet / Laura J Musgrove / Vincent L G Postis / Jonathan D Lippiat / Chun-Wa Chung / Tom Ceska / Manuela Zoonens / Frank Sobott / Stephen P Muench /
Abstract: Membrane proteins are essential for cellular growth, signalling and homeostasis, making up a large proportion of therapeutic targets. However, the necessity for a solubilising agent to extract them ...Membrane proteins are essential for cellular growth, signalling and homeostasis, making up a large proportion of therapeutic targets. However, the necessity for a solubilising agent to extract them from the membrane creates challenges in their structural and functional study. Although amphipols have been very effective for single-particle electron cryo-microscopy (cryoEM) and mass spectrometry, they rely on initial detergent extraction before exchange into the amphipol environment. Therefore, circumventing this pre-requirement would be a big advantage. Here we use an alternative type of amphipol: a cycloalkane-modified amphiphile polymer (CyclAPol) to extract Escherichia coli AcrB directly from the membrane and demonstrate that the protein can be isolated in a one-step purification with the resultant cryoEM structure achieving 3.2 Å resolution. Together this work shows that cycloalkane amphipols provide a powerful approach for the study of membrane proteins, allowing native extraction and high-resolution structure determination by cryoEM.
History
DepositionDec 5, 2020Deposition site: PDBE / Processing site: PDBE
Revision 1.0Dec 8, 2021Provider: repository / Type: Initial release
Revision 1.1Jul 10, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond / em_admin / Item: _em_admin.last_update

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Structure visualization

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Assembly

Deposited unit
B: Efflux pump membrane transporter
C: Efflux pump membrane transporter
A: Efflux pump membrane transporter


Theoretical massNumber of molelcules
Total (without water)341,7583
Polymers341,7583
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area17280 Å2
ΔGint-90 kcal/mol
Surface area119280 Å2
MethodPISA

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Components

#1: Protein Efflux pump membrane transporter


Mass: 113919.477 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli)
Gene: acrB_1, acrB, acrB_2, acrB_3, acrB_4, acrB_5, acrB_6, A8C65_09270, A9819_02265, A9X72_18695, AC789_1c04620, ACN002_0478, ACN68_10695, ACN81_24630, ACU57_11680, ACU90_20590, AM270_01980, AM464_ ...Gene: acrB_1, acrB, acrB_2, acrB_3, acrB_4, acrB_5, acrB_6, A8C65_09270, A9819_02265, A9X72_18695, AC789_1c04620, ACN002_0478, ACN68_10695, ACN81_24630, ACU57_11680, ACU90_20590, AM270_01980, AM464_19305, AMK83_09190, AML35_17310, AUQ13_18190, AW059_13640, AWB10_19050, AWG78_005565, B6V57_02435, BANRA_00889, BANRA_00999, BB545_15910, BEN53_04520, BHF03_18080, BHF46_21335, BHS81_02950, BHS87_02495, BIQ87_02540, BIZ41_13825, BJJ90_19940, BK248_01725, BK292_14280, BK296_16050, BK334_05580, BK373_00965, BK383_14595, BMA87_04530, BMT49_03610, BMT91_04935, BN17_02651, BOH76_16460, BON63_11900, BON65_19130, BON66_08715, BON69_11225, BON71_10040, BON72_14720, BON75_11085, BON76_24190, BON86_26060, BON94_15210, BON95_06075, BON98_18530, BTQ06_00925, BUE81_06435, BvCms12BK_04863, BvCms2454_01835, BvCms28BK_00632, BvCmsC61A_03075, BvCmsHHP019_03148, BvCmsHHP056_01479, BvCmsKKP061_00163, BvCmsKSNP073_04604, BvCmsKSNP081_04683, BvCmsKSNP120_04201, BvCmsKSP011_05014, BvCmsKSP024_04861, BvCmsKSP026_02627, BvCmsKSP045_04703, BvCmsKSP058_04933, BvCmsKSP067_05122, BvCmsKSP076_04688, BvCmsNSNP036_04234, BvCmsNSP006_01210, BvCmsNSP007_00776, BvCmsNSP047_01693, BvCmsNSP072_02397, BvCmsOUP014_01037, BvCmsSINP011_02642, BvCmsSINP022_01605, BvCmsSIP019_03696, BvCmsSIP044_00930, BW690_06940, BWI89_12175, BXT93_07785, BZL31_19295, C2U48_07740, C4K41_07725, C4M78_18085, C5F73_09745, C5N07_13110, C5P01_15940, C5P44_16955, C6669_02375, C6B13_10325, C7B02_04170, C7B06_13000, C7B07_14810, C9114_13235, C9141_12200, C9160_00185, C9162_13005, C9201_04165, C9E25_04215, C9Z03_06210, C9Z23_00255, C9Z28_08635, C9Z37_05005, C9Z39_05195, C9Z43_06095, C9Z69_16095, C9Z70_09245, CA593_00820, CDC27_01240, CDL37_08810, CF006_06195, CG692_04680, CI693_16095, CI694_14685, CIG45_11530, CJU63_02500, CJU64_02490, CMR93_03420, CO706_24730, COD46_04485, CR538_19130, CR539_06565, CRD98_02045, CRJUMX01_650090, CRM83_13415, CT146_16100, CV83915_01104, CWS33_13665, D0X26_07790, D2184_11155, D2185_12425, D3C88_31425, D3O91_18365, D4023_06520, D4628_13155, D4636_06025, D4638_05215, D4718_02720, D4V08_10150, D6T60_09670, D6T98_07735, D6W00_12590, D6X36_16210, D7W70_07875, D7Z75_05690, D9C99_07900, D9D20_11125, D9D44_10685, D9E13_00485, D9E19_02400, D9E35_07135, D9E73_21575, D9F17_03155, D9F32_03530, D9F87_10685, D9G11_17580, D9G29_00450, D9G42_13565, D9G69_10585, D9G95_10390, D9H36_07220, D9H68_03680, D9H70_06065, D9H94_07380, D9I18_05035, D9I87_03680, D9I97_02625, D9J11_02570, D9J44_06200, D9J52_11760, D9J63_03815, D9K02_03900, D9K54_20470, D9Z28_24640, DAH18_15350, DAH30_15550, DAH34_12755, DAH37_04875, DB359_14850, DBQ99_19255, DEN89_14865, DEN97_16890, DEO04_04920, DEO19_17525, DEO20_17510, DJ487_10315, DJ503_01450, DK132_02395, DL292_07005, DL326_11785, DL479_02540, DL545_18765, DL705_13145, DLU50_07900, DLU67_10935, DLU82_01970, DLW88_19215, DLX40_09005, DLY41_06715, DLY44_14465, DM129_08550, DM267_01255, DM296_06955, DM382_09705, DM820_06155, DM973_09850, DMY83_13165, DN627_04140, DN660_06030, DN700_07110, DN808_01530, DNC98_06145, DND79_04450, DNI21_09935, DNR35_00855, DNW42_12745, DNX19_03500, DOY61_02460, DOY67_09480, DP258_04725, DP265_04400, DP277_07910, DQE91_07055, DQF36_01200, DQF57_08040, DQF72_04240, DQO13_05880, DQP61_17770, DRW19_06780, DS732_07285, DT034_10130, DTL43_07030, DTL90_18880, DTM10_06440, DU333_14630, DW236_16180, DWB25_18810, DXT69_07740, DXT71_02600, DXT73_02225, E0I42_07045, E0K84_10260, E2112_11465, E2114_15395, E2115_11930, E2119_01370, E2127_09005, E2128_08320, E2129_12030, E2134_12580, E2135_12650, E2148_08850, E4A44_12425, E4K51_12425, E4K55_09900, E4K60_11595, E5P22_01620, E5P24_11315, E5P28_18315, E5P37_02890, E5S42_13425, E5S56_04760, EA184_13145, EA189_17430, EA218_08750, EA223_15405, EA231_12325, EA239_15375, EA250_11755, EA429_15685, EAI46_01035, EAI52_03305, EAN77_07570, EAX79_06460, EB476_04025, EB525_RS00175, EC1094V2_3386, EC3426_01300, EC382_09135, ECONIH1_02500, ECTO6_03638, ED178_10910, ED307_06795, ED648_02095, EEP23_01410, EG075_08750, EG796_01740, EH186_13990, EHD79_03845, EI021_20720, EI028_12150, EI032_07905, EI041_08500, EIA08_09625, EIA21_12180, EJC75_24510, EL75_3290, EL79_3385, EL80_3339, ELT20_07985, ELT58_12170, ELV08_01140, ELY05_12005, EO241_14205, EPT01_06710, EPU41_02295, EQ825_17705, ERS085365_02371, ERS085366_03391, ERS085374_02303, ERS085379_01141, ERS139211_01948, ERS150873_01787, ExPECSC019_00381, ExPECSC038_03243, EXX06_12030, EXX13_09300, EXX23_10330, EXX24_02235, EXX53_07265, EXX55_11505, EXX71_19130, EXX78_05470, EXX87_11195, EYD11_17095, EYX82_01500, EYX99_02255, EYY27_15910, EYY78_02330, F1E13_02930, F1E19_02810, F7F11_07205, F7F18_02645, F7F29_13450, F7F56_10820, F9040_08845, F9225_13995, F9X20_11065, F9Z53_11120, F9Z74_08955, FORC82_3586, FQ022_06350, FQ915_11580, FQR64_17285, FQU83_20500, FQZ46_14155, FRV13_04215, FV293_05765, FV438_08525, FZ043_05490, FZN06_02310, FZN30_08470, G3V64_03945, G5603_08575, G5608_10915, G5616_17800, G5632_14455, G5686_06550, G5688_07040, GHR40_21640, GII67_09125, GII91_03825, GKF21_12125, GKF28_11295, GKF34_11195, GKF39_01530, GKF47_11385, GKF52_13635, GKF74_01825, GKF86_04555, GKF89_04410, GKG12_03295, GNZ03_09590, GNZ05_01310, GP650_17935, GP654_09140, GP666_11010, GP671_13130, GP678_16470, GP689_14290, GP698_14015, GP700_10545, GP720_06095, GP727_13610, GP892_11190, GP912_14175, GP935_15175, GP945_16670, GP946_00315, GP950_13345, GQA06_04590, GQA23_11080, GQA63_01155, GQA64_12425, GQE22_08970, GQE30_17545, GQE33_07470, GQE34_01120, GQE42_04545, GQE51_10380, GQE58_13150, GQE64_19110, GQE87_01345, GQE93_05675, GQF59_01155, GQL64_11830, GQM10_04910, GQM17_15105, GQM18_02835, GQM28_04965, GQN16_06365, GQN24_05120, GQY14_07730, GRW05_06170, GRW12_18865, GRW27_17010, GRW30_01790, GRW42_02940, GRW80_13135, GRW81_04465, GUB85_11530, GUB91_14860, GUB95_14530, GUC01_07830, GUC12_14650, GZS12_17565, HmCms184_01943, HmCmsJML079_00721, HmCmsJML146_03063, HmCmsJML204_02413, HW43_05935, MJ49_04125, MS6198_04670, MS8345_00466, NCTC10418_05566, NCTC10766_05800, NCTC10865_04672, NCTC10963_03279, NCTC11126_00116, NCTC11181_01060, NCTC12650_04129, NCTC13148_06601, NCTC13216_02965, NCTC13846_03608, NCTC9007_00190, NCTC9036_03767, NCTC9045_04279, NCTC9055_00632, NCTC9058_03192, NCTC9062_04550, NCTC9111_03904, NCTC9117_04708, NCTC9119_03922, NCTC9434_02883, NCTC9706_01042, NCTC9969_03956, PGD_02849, PU06_12600, RG28_02520, RK56_026290, RX35_02133, SAMEA3472043_02753, SAMEA3472044_00392, SAMEA3472047_02966, SAMEA3472055_02089, SAMEA3472056_03557, SAMEA3472070_02210, SAMEA3472080_01739, SAMEA3472090_01741, SAMEA3472108_01933, SAMEA3472114_01186, SAMEA3484427_03472, SAMEA3484429_01960, SAMEA3485101_03912, SAMEA3752553_00212, SAMEA3752557_00298, SAMEA3752559_02826, SAMEA3752620_00857, SAMEA3753064_01307, SAMEA3753097_00439, SAMEA3753164_00290, SAMEA3753290_01796, SAMEA3753300_00529, SK85_00486, UN86_09270, UN91_18355, WQ89_01050, WR15_04005
Production host: Escherichia coli (E. coli) / References: UniProt: E2QH56

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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: AcrB / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Escherichia coli (E. coli)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 8
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid type: Quantifoil
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 279 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: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 130000 X
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 10 sec. / Electron dose: 58 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.18_3861: / Classification: refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 100932 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0123541
ELECTRON MICROSCOPYf_angle_d0.90331980
ELECTRON MICROSCOPYf_dihedral_angle_d13.1373225
ELECTRON MICROSCOPYf_chiral_restr0.0553787
ELECTRON MICROSCOPYf_plane_restr0.0054086

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