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Yorodumi- PDB-8w9m: Cryo-EM structure of the cyanobacterial nitrate transporter NrtBC... -
+Open data
-Basic information
Entry | Database: PDB / ID: 8w9m | ||||||
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Title | Cryo-EM structure of the cyanobacterial nitrate transporter NrtBCD in complex with ATP | ||||||
Components |
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Keywords | MEMBRANE PROTEIN / ATP-dependent transporter / ABC transporter / nitrate/nitrite transporter | ||||||
Function / homology | Function and homology information nitrate transmembrane transporter activity / monoatomic ion transport / ATP hydrolysis activity / ATP binding / membrane / plasma membrane Similarity search - Function | ||||||
Biological species | Nostoc sp. PCC 7120 = FACHB-418 (bacteria) | ||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.1 Å | ||||||
Authors | Li, B. / Zhou, C.Z. / Chen, Y.X. / Jiang, Y.L. | ||||||
Funding support | China, 1items
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Citation | Journal: Proc Natl Acad Sci U S A / Year: 2024 Title: Allosteric regulation of nitrate transporter NRT via the signaling protein PII. Authors: Bo Li / Xiao-Qian Wang / Qin-Yao Li / Da Xu / Jing Li / Wen-Tao Hou / Yuxing Chen / Yong-Liang Jiang / Cong-Zhao Zhou / Abstract: Coordinated carbon and nitrogen metabolism is crucial for bacteria living in the fluctuating environments. Intracellular carbon and nitrogen homeostasis is maintained by a sophisticated network, in ...Coordinated carbon and nitrogen metabolism is crucial for bacteria living in the fluctuating environments. Intracellular carbon and nitrogen homeostasis is maintained by a sophisticated network, in which the widespread signaling protein PII acts as a major regulatory hub. In cyanobacteria, PII was proposed to regulate the nitrate uptake by an ABC (ATP-binding cassette)-type nitrate transporter NrtABCD, in which the nucleotide-binding domain of NrtC is fused with a C-terminal regulatory domain (CRD). Here, we solved three cryoelectron microscopy structures of NrtBCD, bound to nitrate, ATP, and PII, respectively. Structural and biochemical analyses enable us to identify the key residues that form a hydrophobic and a hydrophilic cavity along the substrate translocation channel. The core structure of PII, but not the canonical T-loop, binds to NrtC and stabilizes the CRD, making it visible in the complex structure, narrows the substrate translocation channel in NrtB, and ultimately locks NrtBCD at an inhibited inward-facing conformation. Based on these results and previous reports, we propose a putative transport cycle driven by NrtABCD, which is allosterically inhibited by PII in response to the cellular level of 2-oxoglutarate. Our findings provide a distinct regulatory mechanism of ABC transporter via asymmetrically binding to a signaling protein. | ||||||
History |
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-Structure visualization
Structure viewer | Molecule: MolmilJmol/JSmol |
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-Downloads & links
-Download
PDBx/mmCIF format | 8w9m.cif.gz | 202 KB | Display | PDBx/mmCIF format |
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PDB format | pdb8w9m.ent.gz | 149.1 KB | Display | PDB format |
PDBx/mmJSON format | 8w9m.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/w9/8w9m ftp://data.pdbj.org/pub/pdb/validation_reports/w9/8w9m | HTTPS FTP |
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-Related structure data
Related structure data | 37375MC 8wm7C 8wm8C M: map data used to model this data C: citing same article (ref.) |
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Similar structure data | Similarity search - Function & homologyF&H Search |
-Links
-Assembly
Deposited unit |
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1 |
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-Components
#1: Protein | Mass: 30483.053 Da / Num. of mol.: 2 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Nostoc sp. PCC 7120 = FACHB-418 (bacteria) Gene: nrtB / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): C43 / References: UniProt: Q8YZ77 #2: Protein | | Mass: 76325.156 Da / Num. of mol.: 1 / Mutation: E164Q Source method: isolated from a genetically manipulated source Source: (gene. exp.) Nostoc sp. PCC 7120 = FACHB-418 (bacteria) Gene: nrtC / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): C43 / References: UniProt: Q8YZ76 #3: Protein | | Mass: 31398.152 Da / Num. of mol.: 1 / Mutation: E179Q Source method: isolated from a genetically manipulated source Source: (gene. exp.) Nostoc sp. PCC 7120 = FACHB-418 (bacteria) Gene: nrtD / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): C43 / References: UniProt: Q8YZ75 #4: Chemical | #5: 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: nitrate transporter NrtBCD complexed with ATP / Type: COMPLEX / Entity ID: #1-#3 / Source: RECOMBINANT |
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Source (natural) | Organism: Nostoc sp. PCC 7120 = FACHB-418 (bacteria) |
Source (recombinant) | Organism: Escherichia coli BL21(DE3) (bacteria) / Strain: C43(DE3) |
Buffer solution | pH: 7.5 |
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 |
-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 FIELDBright-field microscopy / Nominal defocus max: 2300 nm / Nominal defocus min: 1500 nm |
Image recording | Electron dose: 60 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) |
-Processing
EM software | Name: PHENIX / Version: 1.19.2-4158 / Category: model refinement | ||||||||||||||||||||||||
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||
3D reconstruction | Resolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 1222929 / Symmetry type: POINT | ||||||||||||||||||||||||
Refinement | Cross valid method: NONE Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2 | ||||||||||||||||||||||||
Displacement parameters | Biso mean: 47.69 Å2 | ||||||||||||||||||||||||
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