+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-12043 | |||||||||
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Title | AcrB in cycloalkane amphipol | |||||||||
Map data | ||||||||||
Sample |
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Keywords | drug exporter / membrane protein / amphipol | |||||||||
Function / homology | : Function and homology information | |||||||||
Biological species | Escherichia coli (E. coli) | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 3.2 Å | |||||||||
Authors | Higgins AJ / Flynn AJ | |||||||||
Funding support | United Kingdom, 1 items
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Citation | Journal: 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 |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | EM map: SurfViewMolmilJmol/JSmol |
Supplemental images |
-Downloads & links
-EMDB archive
Map data | emd_12043.map.gz | 59.9 MB | EMDB map data format | |
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Header (meta data) | emd-12043-v30.xml emd-12043.xml | 11.5 KB 11.5 KB | Display Display | EMDB header |
Images | emd_12043.png | 92 KB | ||
Filedesc metadata | emd-12043.cif.gz | 9.2 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-12043 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-12043 | HTTPS FTP |
-Validation report
Summary document | emd_12043_validation.pdf.gz | 623 KB | Display | EMDB validaton report |
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Full document | emd_12043_full_validation.pdf.gz | 622.6 KB | Display | |
Data in XML | emd_12043_validation.xml.gz | 6.1 KB | Display | |
Data in CIF | emd_12043_validation.cif.gz | 6.9 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-12043 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-12043 | HTTPS FTP |
-Related structure data
Related structure data | 7b5pMC M: atomic model generated by this map C: citing same article (ref.) |
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Similar structure data |
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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-Map
File | Download / File: emd_12043.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Projections & slices | Image control
Images are generated by Spider. | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 1.07 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Sample components
-Entire : AcrB
Entire | Name: AcrB |
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Components |
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-Supramolecule #1: AcrB
Supramolecule | Name: AcrB / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all |
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Source (natural) | Organism: Escherichia coli (E. coli) |
-Macromolecule #1: Efflux pump membrane transporter
Macromolecule | Name: Efflux pump membrane transporter / type: protein_or_peptide / ID: 1 / Number of copies: 3 / Enantiomer: LEVO |
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Source (natural) | Organism: Escherichia coli (E. coli) |
Molecular weight | Theoretical: 113.919477 KDa |
Recombinant expression | Organism: Escherichia coli (E. coli) |
Sequence | String: MPRPNFFIDR PIFAWVIAII IMLAGGLAIL KLPVAQYPTI APPAVTISAS YPGADAKTVQ DTVTQVIEQN MNGIDNLMYM SSNSDSTGT VQITLTFESG TDADIAQVQV QNKLQLAMPL LPQEVQQQGV SVEKSSSSFL MVVGVINTDG TMTQEDISDY V AANMKDAI ...String: MPRPNFFIDR PIFAWVIAII IMLAGGLAIL KLPVAQYPTI APPAVTISAS YPGADAKTVQ DTVTQVIEQN MNGIDNLMYM SSNSDSTGT VQITLTFESG TDADIAQVQV QNKLQLAMPL LPQEVQQQGV SVEKSSSSFL MVVGVINTDG TMTQEDISDY V AANMKDAI SRTSGVGDVQ LFGSQYAMRI WMNPNELNKF QLTPVDVITA IKAQNAQVAA GQLGGTPPVK GQQLNASIIA QT RLTSTEE FGKILLKVNQ DGSRVLLRDV AKIELGGENY DIIAEFNGQP ASGLGIKLAT GANALDTAAA IRAELAKMEP FFP SGLKIV YPYDTTPFVK ISIHEVVKTL VEAIILVFLV MYLFLQNFRA TLIPTIAVPV VLLGTFAVLA AFGFSINTLT MFGM VLAIG LLVDDAIVVV ENVERVMAEE GLPPKEATRK SMGQIQGALV GIAMVLSAVF VPMAFFGGST GAIYRQFSIT IVSAM ALSV LVALILTPAL CATMLKPIAK GDHGEGKKGF FGWFNRMFEK STHHYTDSVG GILRSTGRYL VLYLIIVVGM AYLFVR LPS SFLPDEDQGV FMTMVQLPAG ATQERTQKVL NEVTHYYLTK EKNNVESVFA VNGFGFAGRG QNTGIAFVSL KDWADRP GE ENKVEAITMR ATRAFSQIKD AMVFAFNLPA IVELGTATGF DFELIDQAGL GHEKLTQARN QLLAEAAKHP DMLTSVRP N GLEDTPQFKI DIDQEKAQAL GVSINDINTT LGAAWGGSYV NDFIDRGRVK KVYVMSEAKY RMLPDDIGDW YVRAADGQM VPFSAFSSSR WEYGSPRLER YNGLPSMEIL GQAAPGKSTG EAMELMEQLA SKLPTGVGYD WTGMSYQERL SGNQAPSLYA ISLIVVFLC LAALYESWSI PFSVMLVVPL GVIGALLAAT FRGLTNDVYF QVGLLTTIGL SAKNAILIVE FAKDLMDKEG K GLIEATLD AVRMRLRPIL MTSLAFILGV MPLVISTGAG SGAQNAVGTG VMGGMVTATV LAIFFVPVFF VVVRRRFSRK NE DIEHSHT VDHH UniProtKB: UNIPROTKB: E2QH56 |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 1 mg/mL |
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Buffer | pH: 8 |
Grid | Model: Quantifoil / Material: COPPER |
Vitrification | Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 279 K / Instrument: FEI VITROBOT MARK IV |
-Electron microscopy
Microscope | FEI TITAN KRIOS |
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Image recording | Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Average exposure time: 10.0 sec. / Average electron dose: 58.0 e/Å2 |
Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal magnification: 130000 |
Sample stage | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN |
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
-Image processing
Startup model | Type of model: PDB ENTRY PDB model - PDB ID: |
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Final reconstruction | Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 100932 |
Initial angle assignment | Type: MAXIMUM LIKELIHOOD |
Final angle assignment | Type: MAXIMUM LIKELIHOOD |