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- EMDB-0071: GAPDH-CP12-PRK complex -

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Basic information

Entry
Database: EMDB / ID: EMD-0071
TitleGAPDH-CP12-PRK complex
Map dataNone
Sample
  • Complex: GAPDH-CP12-PRK complex
    • Complex: GAPDH-CP12-PRK complex
      • Protein or peptide: CP12 polypeptide
    • Complex: GAPDH-CP12-PRK complex
      • Protein or peptide: Glyceraldehyde-3-phosphate dehydrogenaseGlyceraldehyde 3-phosphate dehydrogenase
      • Protein or peptide: Phosphoribulokinase
  • Ligand: NICOTINAMIDE-ADENINE-DINUCLEOTIDE
Function / homology
Function and homology information


phosphoribulokinase / phosphoribulokinase activity / 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 / nucleotide binding / ATP binding
Similarity search - Function
Phosphoribulokinase signature. / Phosphoribulokinase / Calvin cycle protein CP12-like / CP12 domain / CP12 domain / CP12 / Phosphoribulokinase/uridine kinase / Phosphoribulokinase / Uridine kinase family / Glyceraldehyde-3-phosphate dehydrogenase, type I / Glyceraldehyde 3-phosphate dehydrogenase, active site ...Phosphoribulokinase signature. / Phosphoribulokinase / Calvin cycle protein CP12-like / CP12 domain / CP12 domain / CP12 / Phosphoribulokinase/uridine kinase / Phosphoribulokinase / Uridine kinase family / 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 / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Phosphoribulokinase / CP12 polypeptide / Glyceraldehyde-3-phosphate dehydrogenase
Similarity search - Component
Biological speciesThermosynechococcus elongatus BP-1 (bacteria) / Thermosynechococcus elongatus (strain BP-1) (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.9 Å
AuthorsMcFarlane CR / Shah N / Bubeck D / Murray JW
Funding support United Kingdom, 1 items
OrganizationGrant numberCountry
Biotechnology and Biological Sciences Research CouncilBB/J014575/1 United Kingdom
CitationJournal: 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
DepositionJun 20, 2018-
Header (metadata) releaseAug 22, 2018-
Map releaseJul 3, 2019-
UpdateNov 25, 2020-
Current statusNov 25, 2020Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.014
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.014
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6gve
  • Surface level: 0.014
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_0071.png

Downloads & links

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Map

FileDownload / File: emd_0071.map.gz / Format: CCP4 / Size: 163.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationNone
Voxel sizeX=Y=Z: 1.047 Å
Density
Contour LevelBy AUTHOR: 0.00952 / Movie #1: 0.014
Minimum - Maximum-0.04345707 - 0.07975341
Average (Standard dev.)0.00000546325 (±0.0021943953)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions350350350
Spacing350350350
CellA=B=C: 366.45 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0471.0471.047
M x/y/z350350350
origin x/y/z0.0000.0000.000
length x/y/z366.450366.450366.450
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS350350350
D min/max/mean-0.0430.0800.000

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Supplemental data

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Half map: half map 1

Fileemd_0071_half_map_1.map
Annotationhalf map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half map 2

Fileemd_0071_half_map_2.map
Annotationhalf map 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : GAPDH-CP12-PRK complex

EntireName: GAPDH-CP12-PRK complex
Components
  • Complex: GAPDH-CP12-PRK complex
    • Complex: GAPDH-CP12-PRK complex
      • Protein or peptide: CP12 polypeptide
    • Complex: GAPDH-CP12-PRK complex
      • Protein or peptide: Glyceraldehyde-3-phosphate dehydrogenaseGlyceraldehyde 3-phosphate dehydrogenase
      • Protein or peptide: Phosphoribulokinase
  • Ligand: NICOTINAMIDE-ADENINE-DINUCLEOTIDE

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Supramolecule #1: GAPDH-CP12-PRK complex

SupramoleculeName: GAPDH-CP12-PRK complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Molecular weightExperimental: 480 KDa

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Supramolecule #2: GAPDH-CP12-PRK complex

SupramoleculeName: GAPDH-CP12-PRK complex / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Thermosynechococcus elongatus BP-1 (bacteria)

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Supramolecule #3: GAPDH-CP12-PRK complex

SupramoleculeName: GAPDH-CP12-PRK complex / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2-#3
Source (natural)Organism: Thermosynechococcus elongatus BP-1 (bacteria)
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Macromolecule #1: CP12 polypeptide

MacromoleculeName: CP12 polypeptide / type: protein_or_peptide / ID: 1 / Number of copies: 4 / Enantiomer: LEVO
Source (natural)Organism: Thermosynechococcus elongatus (strain BP-1) (bacteria)
Strain: BP-1
Molecular weightTheoretical: 8.611127 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
GSMSNLEKQI EQAREEAHKI CDTEGATSGQ CAAAWDALEE LQAEAAHQRA EQQDHKTSFQ QYCDDNPDAA ECRIYDD

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Macromolecule #2: Glyceraldehyde-3-phosphate dehydrogenase

MacromoleculeName: Glyceraldehyde-3-phosphate dehydrogenase / type: protein_or_peptide / ID: 2 / Number of copies: 8 / Enantiomer: LEVO
EC number: Oxidoreductases; Acting on the aldehyde or oxo group of donors; With NAD+ or NADP+ as acceptor
Source (natural)Organism: Thermosynechococcus elongatus (strain BP-1) (bacteria)
Strain: BP-1
Molecular weightTheoretical: 36.792734 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: GSMVRVAING FGRIGRNFMR CWLQRKANSK LEIVGINDTS DPRTNAHLLK YDSMLGIFQD AEITADDDCI YAGGHAVKCV SDRNPENLP WSAWGIDLVI EATGVFTSRE GASKHLSAGA KKVLITAPGK GNIPTYVVGV NHHTYDPSED IVSNASCTTN C LAPIVKVL ...String:
GSMVRVAING FGRIGRNFMR CWLQRKANSK LEIVGINDTS DPRTNAHLLK YDSMLGIFQD AEITADDDCI YAGGHAVKCV SDRNPENLP WSAWGIDLVI EATGVFTSRE GASKHLSAGA KKVLITAPGK GNIPTYVVGV NHHTYDPSED IVSNASCTTN C LAPIVKVL HEAFGIQQGM MTTTHSYTGD QRLLDASHRD LRRARAAAMN IVPTSTGAAK AVGLVIPELQ GKLNGIALRV PT PNVSVVD FVAQVEKPTI AEQVNQVIKE ASETTMKGII HYSELELVSS DYRGHNASSI LDASLTMVLG GNLVKVVAWY DNE WGYSQR VLDLAEHMAA HWA

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Macromolecule #3: Phosphoribulokinase

MacromoleculeName: Phosphoribulokinase / type: protein_or_peptide / ID: 3 / Number of copies: 4 / Enantiomer: LEVO / EC number: phosphoribulokinase
Source (natural)Organism: Thermosynechococcus elongatus (strain BP-1) (bacteria)
Strain: BP-1
Molecular weightTheoretical: 38.038234 KDa
SequenceString: MSSKPDRVVL IGVAGDSGCG KSTFLRRLAD LFGEDFMTVI CLDDYHSLDR KQRKEMGITA LDPRANNFDL MYEQIKALKN GESIMKPIY NHETGTIDPP EKVDPNHVIV IEGLHPLYDE RVRSLIDFSV YLDISDDVKI AWKIKRDMAE RGHSYEDVIA S INARRPDF ...String:
MSSKPDRVVL IGVAGDSGCG KSTFLRRLAD LFGEDFMTVI CLDDYHSLDR KQRKEMGITA LDPRANNFDL MYEQIKALKN GESIMKPIY NHETGTIDPP EKVDPNHVIV IEGLHPLYDE RVRSLIDFSV YLDISDDVKI AWKIKRDMAE RGHSYEDVIA S INARRPDF MAYIDPQKQY ADVVLQILPS QLAKEEKVGN ILRVRMLQRE GIPGFEPVYL FDEGSTITWI PCGRKLTCSY PG IRLSYGP DEYYGHPVSV LEVDGRFEKL DELIYIESHL SNTSTKHYGE VTELLLKHRD YPGSDNGSGL FQVLTGLKMR ATY ERLTSR DAATVTNR

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Macromolecule #4: NICOTINAMIDE-ADENINE-DINUCLEOTIDE

MacromoleculeName: NICOTINAMIDE-ADENINE-DINUCLEOTIDE / type: ligand / ID: 4 / Number of copies: 8 / Formula: NAD
Molecular weightTheoretical: 663.425 Da
Chemical component information

ChemComp-NAD:
NICOTINAMIDE-ADENINE-DINUCLEOTIDE / NAD*YM / Nicotinamide adenine dinucleotide

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7.9
Component:
ConcentrationFormulaName
1.0 mMC21H27N7O14P2Nicotinamide adenine dinucleotide
1.0 mMC10H15N5O10P2Adenosine diphosphate
1.0 mMC4H8O2S2trans-4,5-Dihydroxy-1,2-dithiane
50.0 mMNaClSodium chloridesoidium chloride
10.0 mMMgCl2magnesium chloride
50.0 mMC4H11NO3Tris-HClTris
GridModel: Quantifoil R2/2 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Support film - Film thickness: 3.0 nm / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 85 % / Chamber temperature: 283 K / Instrument: FEI VITROBOT MARK IV
Details: Blot force 3. Wait time 60 seconds, then blotted for 3.5 seconds before plunging. 2.5 ul of sample..

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Average electron dose: 44.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: Correction of CTF was implemented within the Bayesian framework of Relion 2.1
Startup modelType of model: INSILICO MODEL
In silico model: Generated in Relion by the Initial Model generation tool
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionApplied symmetry - Point group: D2 (2x2 fold dihedral) / Resolution.type: BY AUTHOR / Resolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 197212
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-6gve:
GAPDH-CP12-PRK complex

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