+Open data
-Basic information
Entry | Database: PDB / ID: 8dmm | ||||||
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Title | Structure of the vanadate-trapped MsbA bound to KDL | ||||||
Components | ATP-binding transport protein MsbA | ||||||
Keywords | TRANSPORT PROTEIN / ABC transporter | ||||||
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 / membrane Similarity search - Function | ||||||
Biological species | Escherichia coli (E. coli) | ||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.47 Å | ||||||
Authors | Liu, C. / Lyu, J. / Laganowsky, A.D. / Zhao, M. | ||||||
Funding support | United States, 1items
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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
Structure viewer | Molecule: MolmilJmol/JSmol |
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-Downloads & links
-Download
PDBx/mmCIF format | 8dmm.cif.gz | 256.4 KB | Display | PDBx/mmCIF format |
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PDB format | pdb8dmm.ent.gz | 171 KB | Display | PDB format |
PDBx/mmJSON format | 8dmm.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/dm/8dmm ftp://data.pdbj.org/pub/pdb/validation_reports/dm/8dmm | HTTPS FTP |
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-Related structure data
Related structure data | 27544MC 8dhyC 8dmoC C: citing same article (ref.) M: map data used to model this data |
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Similar structure data | Similarity search - Function & homologyF&H Search |
-Links
-Assembly
Deposited unit |
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Noncrystallographic symmetry (NCS) | NCS domain:
NCS domain segments:
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-Components
#1: Protein | Mass: 64543.473 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) References: UniProt: C3TGA2, Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to catalyse transmembrane movement of substances #2: Chemical | #3: Chemical | Has ligand of interest | Y | |
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-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
-Sample preparation
Component | Name: Vanadate-trapped MsbA KDL and ADP Orthovanadate (AOV) / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT |
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Source (natural) | Organism: Escherichia coli (E. coli) |
Source (recombinant) | Organism: Escherichia coli (E. coli) |
Buffer solution | pH: 7.4 |
Specimen | Conc.: 8 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES |
Vitrification | Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 281 K |
-Electron microscopy imaging
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
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Microscopy | Model: FEI TITAN KRIOS |
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM |
Electron lens | Mode: BRIGHT FIELD / Nominal defocus max: 2500 nm / Nominal defocus min: 1000 nm |
Image recording | Electron dose: 50 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) |
-Processing
Software |
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EM software |
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CTF correction | Type: PHASE FLIPPING ONLY | ||||||||||||||||||||||||
Symmetry | Point symmetry: C2 (2 fold cyclic) | ||||||||||||||||||||||||
3D reconstruction | Resolution: 3.47 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 340615 / Symmetry type: POINT | ||||||||||||||||||||||||
Atomic model building | B value: 153 / Protocol: AB INITIO MODEL / Space: REAL | ||||||||||||||||||||||||
Refinement | Cross valid method: NONE Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2 | ||||||||||||||||||||||||
Displacement parameters | Biso mean: 71.06 Å2 | ||||||||||||||||||||||||
Refine LS restraints |
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Refine LS restraints NCS | Type: NCS constraints / Rms dev position: 3.09285176042E-13 Å |