+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-27545 | |||||||||
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Title | Structure of open, inward-facing MsbA from E. coli | |||||||||
Map data | ||||||||||
Sample |
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Keywords | ABC transporter / TRANSPORT PROTEIN | |||||||||
Function / homology | Function and homology information Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to catalyse transmembrane movement of substances / ATPase-coupled lipid transmembrane transporter activity / ABC-type transporter activity / ATP hydrolysis activity / ATP binding / plasma membrane Similarity search - Function | |||||||||
Biological species | Escherichia coli (E. coli) | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 3.9 Å | |||||||||
Authors | Liu C / Lyu J / Laganowsky AD / Zhao M | |||||||||
Funding support | United States, 1 items
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Citation | Journal: Nat Commun / Year: 2022 Title: Structural basis for lipid and copper regulation of the ABC transporter MsbA. Authors: Jixing Lyu / Chang Liu / Tianqi Zhang / Samantha Schrecke / Nicklaus P Elam / Charles Packianathan / Georg K A Hochberg / David Russell / Minglei Zhao / Arthur Laganowsky / Abstract: A critical step in lipopolysaccharide (LPS) biogenesis involves flipping lipooligosaccharide, an LPS precursor, from the cytoplasmic to the periplasmic leaflet of the inner membrane, an operation ...A critical step in lipopolysaccharide (LPS) biogenesis involves flipping lipooligosaccharide, an LPS precursor, from the cytoplasmic to the periplasmic leaflet of the inner membrane, an operation carried out by the ATP-binding cassette transporter MsbA. Although LPS binding to the inner cavity of MsbA is well established, the selectivity of MsbA-lipid interactions at other site(s) remains poorly understood. Here we use native mass spectrometry (MS) to characterize MsbA-lipid interactions and guide structural studies. We show the transporter co-purifies with copper(II) and metal binding modulates protein-lipid interactions. A 2.15 Å resolution structure of an N-terminal region of MsbA in complex with copper(II) is presented, revealing a structure reminiscent of the GHK peptide, a high-affinity copper(II) chelator. Our results demonstrate conformation-dependent lipid binding affinities, particularly for the LPS-precursor, 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo)-lipid A (KDL). We report a 3.6 Å-resolution structure of MsbA trapped in an open, outward-facing conformation with adenosine 5'-diphosphate and vanadate, revealing a distinct KDL binding site, wherein the lipid forms extensive interactions with the transporter. Additional studies provide evidence that the exterior KDL binding site is conserved and a positive allosteric modulator of ATPase activity, serving as a feedforward activation mechanism to couple transporter activity with LPS biosynthesis. | |||||||||
History |
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-Structure visualization
Supplemental images |
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-Downloads & links
-EMDB archive
Map data | emd_27545.map.gz | 41.2 MB | EMDB map data format | |
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Header (meta data) | emd-27545-v30.xml emd-27545.xml | 16.8 KB 16.8 KB | Display Display | EMDB header |
Images | emd_27545.png | 120 KB | ||
Filedesc metadata | emd-27545.cif.gz | 5.5 KB | ||
Others | emd_27545_additional_1.map.gz emd_27545_half_map_1.map.gz emd_27545_half_map_2.map.gz | 78.9 MB 77.7 MB 77.7 MB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-27545 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-27545 | HTTPS FTP |
-Validation report
Summary document | emd_27545_validation.pdf.gz | 686.9 KB | Display | EMDB validaton report |
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Full document | emd_27545_full_validation.pdf.gz | 686.4 KB | Display | |
Data in XML | emd_27545_validation.xml.gz | 12.8 KB | Display | |
Data in CIF | emd_27545_validation.cif.gz | 15 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-27545 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-27545 | HTTPS FTP |
-Related structure data
Related structure data | 8dmoMC 8dhyC 8dmmC C: citing same article (ref.) M: atomic model generated by this map |
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Similar structure data | Similarity search - Function & homologyF&H Search |
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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Related items in Molecule of the Month |
-Map
File | Download / File: emd_27545.map.gz / Format: CCP4 / Size: 83.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||
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Voxel size | X=Y=Z: 1.065 Å | ||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||
Details | EMDB XML:
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-Supplemental data
-Additional map: #1
File | emd_27545_additional_1.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Half map: #2
File | emd_27545_half_map_1.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Half map: #1
File | emd_27545_half_map_2.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Sample components
-Entire : Copper-bound MsbA
Entire | Name: Copper-bound MsbA |
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Components |
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-Supramolecule #1: Copper-bound MsbA
Supramolecule | Name: Copper-bound MsbA / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all |
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Source (natural) | Organism: Escherichia coli (E. coli) |
-Macromolecule #1: ATP-binding transport protein MsbA
Macromolecule | Name: ATP-binding transport protein MsbA / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO EC number: Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to catalyse transmembrane movement of substances |
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Source (natural) | Organism: Escherichia coli (E. coli) |
Molecular weight | Theoretical: 64.543473 KDa |
Recombinant expression | Organism: Escherichia coli (E. coli) |
Sequence | String: GSHNDKDLST WQTFRRLWPT IAPFKAGLIV AGVALILNAA SDTFMLSLLK PLLDDGFGKT DRSVLVWMPL VVIGLMILRG ITSYVSSYC ISWVSGKVVM TMRRRLFGHM MGMPVSFFDK QSTGTLLSRI TYDSEQVASS SSGALITVVR EGASIIGLFI M MFYYSWQL ...String: GSHNDKDLST WQTFRRLWPT IAPFKAGLIV AGVALILNAA SDTFMLSLLK PLLDDGFGKT DRSVLVWMPL VVIGLMILRG ITSYVSSYC ISWVSGKVVM TMRRRLFGHM MGMPVSFFDK QSTGTLLSRI TYDSEQVASS SSGALITVVR EGASIIGLFI M MFYYSWQL SIILIVLAPI VSIAIRVVSK RFRNISKNMQ NTMGQVTTSA EQMLKGHKEV LIFGGQEVET KRFDKVSNRM RL QGMKMVS ASSISDPIIQ LIASLALAFV LYAASFPSVM DSLTAGTITV VFSSMIALMR PLKSLTNVNA QFQRGMAACQ TLF TILDSE QEKDEGKRVI ERATGDVEFR NVTFTYPGRD VPALRNINLK IPAGKTVALV GRSGSGKSTI ASLITRFYDI DEGE ILMDG HDLREYTLAS LRNQVALVSQ NVHLFNDTVA NNIAYARTEQ YSREQIEEAA RMAYAMDFIN KMDNGLDTVI GENGV LLSG GQRQRIAIAR ALLRDSPILI LDEATSALDT ESERAIQAAL DELQKNRTSL VIAHRLSTIE KADEIVVVED GVIVER GTH NDLLEHRGVY AQLHKMQFGQ UniProtKB: ATP-binding transport protein MsbA |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 8 mg/mL |
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Buffer | pH: 7.4 |
Vitrification | Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 281 K / Instrument: FEI VITROBOT MARK IV |
-Electron microscopy
Microscope | FEI TITAN KRIOS |
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Image recording | Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 50.0 e/Å2 |
Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.0 µm |
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
-Image processing
Startup model | Type of model: INSILICO MODEL / In silico model: Ab initio reconstruction in cryoSPARC |
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Final reconstruction | Applied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 421840 |
Initial angle assignment | Type: RANDOM ASSIGNMENT |
Final angle assignment | Type: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC |