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- PDB-7q53: Single Particle Cryo-EM structure of photosynthetic A2B2 glyceral... -

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Basic information

Entry
Database: PDB / ID: 7q53
TitleSingle Particle Cryo-EM structure of photosynthetic A2B2 glyceraldehyde 3-phosphate dehydrogenase from Spinacia oleracia
Components
  • Glyceraldehyde-3-phosphate dehydrogenase A, chloroplastic,Glyceraldehyde-3-phosphate dehydrogenase A, chloroplastic,Glyceraldehyde-3-phosphate dehydrogenase A, chloroplastic,Glyceraldehyde-3-phosphate dehydrogenase A, chloroplastic
  • Glyceraldehyde-3-phosphate dehydrogenase B, chloroplastic
KeywordsOXIDOREDUCTASE / Photosynthesis / Calvin-Benson cycle / redox regulation / glyceraldehyde-3-phosphate dehydrogenase
Function / homology
Function and homology information


glyceraldehyde-3-phosphate dehydrogenase (NADP+) (phosphorylating) / glyceraldehyde-3-phosphate dehydrogenase (NADP+) (phosphorylating) activity / reductive pentose-phosphate cycle / chloroplast / glucose metabolic process / NAD binding / NADP binding
Similarity search - Function
Glyceraldehyde-3-phosphate dehydrogenase, type I / Glyceraldehyde 3-phosphate dehydrogenase, active site / Glyceraldehyde 3-phosphate dehydrogenase active site. / Glyceraldehyde 3-phosphate dehydrogenase, NAD binding domain / Glyceraldehyde 3-phosphate dehydrogenase, NAD(P) binding domain / Glyceraldehyde 3-phosphate dehydrogenase, catalytic domain / Glyceraldehyde/Erythrose phosphate dehydrogenase family / Glyceraldehyde 3-phosphate dehydrogenase, C-terminal domain / Glyceraldehyde 3-phosphate dehydrogenase, NAD binding domain / NAD(P)-binding domain superfamily
Similarity search - Domain/homology
NICOTINAMIDE-ADENINE-DINUCLEOTIDE / Glyceraldehyde-3-phosphate dehydrogenase B, chloroplastic / Glyceraldehyde-3-phosphate dehydrogenase A, chloroplastic
Similarity search - Component
Biological speciesSpinacia oleracea (spinach)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 6.3 Å
AuthorsMarotta, R. / Fermani, S. / Sparla, F. / Trost, P. / Del Giudice, A.
Funding support France, 1items
OrganizationGrant numberCountry
Grenoble Instruct-ERIC Center (ISBG)PID 1829 France
CitationJournal: Acta Crystallogr D Struct Biol / Year: 2022
Title: Unravelling the regulation pathway of photosynthetic AB-GAPDH.
Authors: Roberto Marotta / Alessandra Del Giudice / Libero Gurrieri / Silvia Fanti / Paolo Swuec / Luciano Galantini / Giuseppe Falini / Paolo Trost / Simona Fermani / Francesca Sparla /
Abstract: Oxygenic phototrophs perform carbon fixation through the Calvin-Benson cycle. Different mechanisms adjust the cycle and the light-harvesting reactions to rapid environmental changes. Photosynthetic ...Oxygenic phototrophs perform carbon fixation through the Calvin-Benson cycle. Different mechanisms adjust the cycle and the light-harvesting reactions to rapid environmental changes. Photosynthetic glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is a key enzyme in the cycle. In land plants, different photosynthetic GAPDHs exist: the most abundant isoform is formed by AB heterotetramers and the least abundant by A homotetramers. Regardless of the subunit composition, GAPDH is the major consumer of photosynthetic NADPH and its activity is strictly regulated. While A-GAPDH is regulated by CP12, AB-GAPDH is autonomously regulated through the C-terminal extension (CTE) of its B subunits. Reversible inhibition of AB-GAPDH occurs via the oxidation of a cysteine pair located in the CTE and the substitution of NADP(H) with NAD(H) in the cofactor-binding site. These combined conditions lead to a change in the oligomerization state and enzyme inhibition. SEC-SAXS and single-particle cryo-EM analysis were applied to reveal the structural basis of this regulatory mechanism. Both approaches revealed that spinach (AB)-GAPDH oligomers with n = 1, 2, 4 and 5 co-exist in a dynamic system. B subunits mediate the contacts between adjacent tetramers in AB and AB oligomers. The CTE of each B subunit penetrates into the active site of a B subunit of the adjacent tetramer, which in turn moves its CTE in the opposite direction, effectively preventing the binding of the substrate 1,3-bisphosphoglycerate in the B subunits. The whole mechanism is made possible, and eventually controlled, by pyridine nucleotides. In fact, NAD(H), by removing NADP(H) from A subunits, allows the entrance of the CTE into the active site of the B subunit, hence stabilizing inhibited oligomers.
History
DepositionNov 2, 2021Deposition site: PDBE / Processing site: PDBE
Revision 1.0Nov 16, 2022Provider: repository / Type: Initial release
Revision 1.1Jul 17, 2024Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_3d_fitting_list / em_admin / pdbx_initial_refinement_model
Item: _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id ..._em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
O: Glyceraldehyde-3-phosphate dehydrogenase B, chloroplastic
P: Glyceraldehyde-3-phosphate dehydrogenase A, chloroplastic,Glyceraldehyde-3-phosphate dehydrogenase A, chloroplastic,Glyceraldehyde-3-phosphate dehydrogenase A, chloroplastic,Glyceraldehyde-3-phosphate dehydrogenase A, chloroplastic
Q: Glyceraldehyde-3-phosphate dehydrogenase B, chloroplastic
R: Glyceraldehyde-3-phosphate dehydrogenase A, chloroplastic,Glyceraldehyde-3-phosphate dehydrogenase A, chloroplastic,Glyceraldehyde-3-phosphate dehydrogenase A, chloroplastic,Glyceraldehyde-3-phosphate dehydrogenase A, chloroplastic
hetero molecules


Theoretical massNumber of molelcules
Total (without water)147,7448
Polymers145,0904
Non-polymers2,6544
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: SAXS, electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Glyceraldehyde-3-phosphate dehydrogenase B, chloroplastic / NADP-dependent glyceraldehydephosphate dehydrogenase subunit B


Mass: 36288.660 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Spinacia oleracea (spinach) / Organ: chloroplast
References: UniProt: P12860, glyceraldehyde-3-phosphate dehydrogenase (NADP+) (phosphorylating)
#2: Protein Glyceraldehyde-3-phosphate dehydrogenase A, chloroplastic,Glyceraldehyde-3-phosphate dehydrogenase A, chloroplastic,Glyceraldehyde-3-phosphate dehydrogenase A, chloroplastic,Glyceraldehyde-3-phosphate dehydrogenase A, chloroplastic / NADP-dependent glyceraldehydephosphate dehydrogenase subunit A


Mass: 36256.391 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Spinacia oleracea (spinach) / Organ: chloroplast
References: UniProt: P19866, glyceraldehyde-3-phosphate dehydrogenase (NADP+) (phosphorylating)
#3: Chemical
ChemComp-NAD / NICOTINAMIDE-ADENINE-DINUCLEOTIDE


Mass: 663.425 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C21H27N7O14P2 / Feature type: SUBJECT OF INVESTIGATION / Comment: NAD*YM
Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Photosynthetic A2B2 glyceraldehyde-3-phosphate dehydrogenase hetero-tetramer complexed with NAD.
Type: COMPLEX / Entity ID: #1-#2 / Source: NATURAL
Source (natural)Organism: Spinacia oleracea (spinach)
Buffer solutionpH: 7.4
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: C-flat-1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company
MicroscopyModel: FEI POLARA 300
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 3500 nm / Nominal defocus min: 1500 nm
Image recordingElectron dose: 42 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.17.1_3660: / Classification: refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: D2 (2x2 fold dihedral)
3D reconstructionResolution: 6.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 19636 / Symmetry type: POINT
Atomic model buildingPDB-ID: 2PKQ

2pkq


Accession code: 2PKQ / Source name: PDB / Type: experimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00610674
ELECTRON MICROSCOPYf_angle_d0.82314527
ELECTRON MICROSCOPYf_dihedral_angle_d7.4811468
ELECTRON MICROSCOPYf_chiral_restr0.0481723
ELECTRON MICROSCOPYf_plane_restr0.0051836

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