+Open data
-Basic information
Entry | Database: PDB / ID: 6gve | ||||||
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Title | GAPDH-CP12-PRK complex | ||||||
Components |
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Keywords | PHOTOSYNTHESIS / complex / calvin cycle / redox regulation | ||||||
Function / homology | Function and homology information phosphoribulokinase / phosphoribulokinase activity / negative regulation of reductive pentose-phosphate cycle / Oxidoreductases; Acting on the aldehyde or oxo group of donors; With NAD+ or NADP+ as acceptor / oxidoreductase activity, acting on the aldehyde or oxo group of donors, NAD or NADP as acceptor / glucose metabolic process / NAD binding / NADP binding / carbohydrate metabolic process / ATP binding / metal ion binding Similarity search - Function | ||||||
Biological species | Thermosynechococcus elongatus (bacteria) | ||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.9 Å | ||||||
Authors | McFarlane, C.R. / Shah, N. / Bubeck, D. / Murray, J.W. | ||||||
Funding support | United Kingdom, 1items
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Citation | Journal: Proc Natl Acad Sci U S A / Year: 2019 Title: Structural basis of light-induced redox regulation in the Calvin-Benson cycle in cyanobacteria. Authors: Ciaran R McFarlane / Nita R Shah / Burak V Kabasakal / Blanca Echeverria / Charles A R Cotton / Doryen Bubeck / James W Murray / Abstract: Plants, algae, and cyanobacteria fix carbon dioxide to organic carbon with the Calvin-Benson (CB) cycle. Phosphoribulokinase (PRK) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) are essential ...Plants, algae, and cyanobacteria fix carbon dioxide to organic carbon with the Calvin-Benson (CB) cycle. Phosphoribulokinase (PRK) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) are essential CB-cycle enzymes that control substrate availability for the carboxylation enzyme Rubisco. PRK consumes ATP to produce the Rubisco substrate ribulose bisphosphate (RuBP). GAPDH catalyzes the reduction step of the CB cycle with NADPH to produce the sugar glyceraldehyde 3-phosphate (GAP), which is used for regeneration of RuBP and is the main exit point of the cycle. GAPDH and PRK are coregulated by the redox state of a conditionally disordered protein CP12, which forms a ternary complex with both enzymes. However, the structural basis of CB-cycle regulation by CP12 is unknown. Here, we show how CP12 modulates the activity of both GAPDH and PRK. Using thermophilic cyanobacterial homologs, we solve crystal structures of GAPDH with different cofactors and CP12 bound, and the ternary GAPDH-CP12-PRK complex by electron cryo-microscopy, we reveal that formation of the N-terminal disulfide preorders CP12 prior to binding the PRK active site, which is resolved in complex with CP12. We find that CP12 binding to GAPDH influences substrate accessibility of all GAPDH active sites in the binary and ternary inhibited complexes. Our structural and biochemical data explain how CP12 integrates responses from both redox state and nicotinamide dinucleotide availability to regulate carbon fixation. | ||||||
History |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | Molecule: MolmilJmol/JSmol |
-Downloads & links
-Download
PDBx/mmCIF format | 6gve.cif.gz | 711.3 KB | Display | PDBx/mmCIF format |
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PDB format | pdb6gve.ent.gz | 606.6 KB | Display | PDB format |
PDBx/mmJSON format | 6gve.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 6gve_validation.pdf.gz | 1.8 MB | Display | wwPDB validaton report |
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Full document | 6gve_full_validation.pdf.gz | 1.8 MB | Display | |
Data in XML | 6gve_validation.xml.gz | 122.2 KB | Display | |
Data in CIF | 6gve_validation.cif.gz | 166.2 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/gv/6gve ftp://data.pdbj.org/pub/pdb/validation_reports/gv/6gve | HTTPS FTP |
-Related structure data
Related structure data | 0071MC 6gfoC 6gfpC 6gfqC 6gfrC 6gg7C 6ghlC 6ghrC M: map data used to model this data C: citing same article (ref.) |
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Similar structure data |
-Links
-Assembly
Deposited unit |
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1 |
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-Components
#1: Protein | Mass: 8611.127 Da / Num. of mol.: 4 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Thermosynechococcus elongatus (strain BP-1) (bacteria) Strain: BP-1 / Gene: cp12 / Plasmid: pRSETA Details (production host): modified to include thrombin site Production host: Escherichia coli (E. coli) / Variant (production host): KRX / References: UniProt: Q8DHX3 #2: Protein | Mass: 36792.734 Da / Num. of mol.: 8 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Thermosynechococcus elongatus (strain BP-1) (bacteria) Strain: BP-1 / Gene: tll1466 / Plasmid: pRSETA / Details (production host): modified with thrombin site / Production host: Escherichia coli (E. coli) / Variant (production host): KRX References: UniProt: Q8DIW5, Oxidoreductases; Acting on the aldehyde or oxo group of donors; With NAD+ or NADP+ as acceptor #3: Protein | Mass: 38038.234 Da / Num. of mol.: 4 / Source method: isolated from a natural source Source: (natural) Thermosynechococcus elongatus (strain BP-1) (bacteria) Strain: BP-1 / References: UniProt: Q8DHN2, phosphoribulokinase #4: Chemical | ChemComp-NAD / |
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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
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Molecular weight | Value: 0.480 MDa / Experimental value: YES | |||||||||||||||||||||||||||||||||||
Source (natural) |
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Source (recombinant) | Organism: Escherichia coli (E. coli) | |||||||||||||||||||||||||||||||||||
Buffer solution | pH: 7.9 | |||||||||||||||||||||||||||||||||||
Buffer component |
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Specimen | Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES | |||||||||||||||||||||||||||||||||||
Specimen support | Grid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R2/2 | |||||||||||||||||||||||||||||||||||
Vitrification | Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 85 % / Chamber temperature: 283 K Details: Blot force 3. Wait time 60 seconds, then blotted for 3.5 seconds before plunging. 2.5 ul of sample. |
-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 |
Specimen holder | Cryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER |
Image recording | Electron dose: 44 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) |
-Processing
Software | Name: PHENIX / Version: 1.13_2998: / Classification: refinement | ||||||||||||||||||||
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EM software |
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CTF correction | Details: Correction of CTF was implemented within the Bayesian framework of Relion 2.1 Type: NONE | ||||||||||||||||||||
Symmetry | Point symmetry: D2 (2x2 fold dihedral) | ||||||||||||||||||||
3D reconstruction | Resolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 197212 / Symmetry type: POINT | ||||||||||||||||||||
Atomic model building | Protocol: FLEXIBLE FIT / Space: REAL |