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- EMDB-11028: CryoEM structure of Rubisco Activase with its substrate Rubisco f... -

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Basic information

Entry
Database: EMDB / ID: EMD-11028
TitleCryoEM structure of Rubisco Activase with its substrate Rubisco from Nostoc sp. (strain PCC7120)
Map data
Sample
  • Complex: NosRca-deltaC:Rubisco complex
    • Complex: AAA-core of the Rca hexamer from Nostoc sp. PCC7120 in complex with ATP/ATPgammaS
      • Protein or peptide: Ribulose bisphosphate carboxylase/oxygenase activase
    • Complex: Rubisco complex from Nostoc sp. PCC7120 in complex with inhibitor CABP
      • Protein or peptide: Ribulose bisphosphate carboxylase large chain
      • Protein or peptide: Ribulose bisphosphate carboxylase small chain
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER
  • Ligand: MAGNESIUM ION
  • Ligand: 2-CARBOXYARABINITOL-1,5-DIPHOSPHATE
Function / homology
Function and homology information


photorespiration / carboxysome / ribulose-bisphosphate carboxylase / ribulose-bisphosphate carboxylase activity / reductive pentose-phosphate cycle / monooxygenase activity / magnesium ion binding / ATP hydrolysis activity / ATP binding
Similarity search - Function
Ribulose bisphosphate carboxylase/oxygenase activase-like / : / Ribulose bisphosphate carboxylase/oxygenase activase, AAA, helical / Ribulose bisphosphate carboxylase, small subunit / Ribulose bisphosphate carboxylase small subunit, domain / Ribulose bisphosphate carboxylase, small subunit superfamily / Ribulose bisphosphate carboxylase, small chain / Ribulose bisphosphate carboxylase, small chain / Ribulose bisphosphate carboxylase large subunit, type I / Ribulose bisphosphate carboxylase, large chain, active site ...Ribulose bisphosphate carboxylase/oxygenase activase-like / : / Ribulose bisphosphate carboxylase/oxygenase activase, AAA, helical / Ribulose bisphosphate carboxylase, small subunit / Ribulose bisphosphate carboxylase small subunit, domain / Ribulose bisphosphate carboxylase, small subunit superfamily / Ribulose bisphosphate carboxylase, small chain / Ribulose bisphosphate carboxylase, small chain / Ribulose bisphosphate carboxylase large subunit, type I / Ribulose bisphosphate carboxylase, large chain, active site / Ribulose bisphosphate carboxylase large chain active site. / Ribulose bisphosphate carboxylase, large subunit, ferrodoxin-like N-terminal / Ribulose bisphosphate carboxylase large chain, N-terminal domain / Ribulose bisphosphate carboxylase, large subunit, C-terminal / RuBisCO / Ribulose bisphosphate carboxylase, large subunit, C-terminal domain superfamily / RuBisCO large subunit, N-terminal domain superfamily / Ribulose bisphosphate carboxylase large chain, catalytic domain / ATPase family associated with various cellular activities (AAA) / ATPase, AAA-type, core / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Ribulose bisphosphate carboxylase large chain / Ribulose bisphosphate carboxylase small subunit / Ribulose bisphosphate carboxylase/oxygenase activase
Similarity search - Component
Biological speciesNostoc sp. PCC 7120 = FACHB-418 (bacteria) / Nostoc sp. (strain PCC 7120 / SAG 25.82 / UTEX 2576) (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.86 Å
AuthorsWang H / Bracher A / Flecken M / Popilka L / Hartl FU / Hayer-Hartl M
CitationJournal: Cell / Year: 2020
Title: Dual Functions of a Rubisco Activase in Metabolic Repair and Recruitment to Carboxysomes.
Authors: Mirkko Flecken / Huping Wang / Leonhard Popilka / F Ulrich Hartl / Andreas Bracher / Manajit Hayer-Hartl /
Abstract: Rubisco, the key enzyme of CO fixation in photosynthesis, is prone to inactivation by inhibitory sugar phosphates. Inhibited Rubisco undergoes conformational repair by the hexameric AAA+ chaperone ...Rubisco, the key enzyme of CO fixation in photosynthesis, is prone to inactivation by inhibitory sugar phosphates. Inhibited Rubisco undergoes conformational repair by the hexameric AAA+ chaperone Rubisco activase (Rca) in a process that is not well understood. Here, we performed a structural and mechanistic analysis of cyanobacterial Rca, a close homolog of plant Rca. In the Rca:Rubisco complex, Rca is positioned over the Rubisco catalytic site under repair and pulls the N-terminal tail of the large Rubisco subunit (RbcL) into the hexamer pore. Simultaneous displacement of the C terminus of the adjacent RbcL opens the catalytic site for inhibitor release. An alternative interaction of Rca with Rubisco is mediated by C-terminal domains that resemble the small Rubisco subunit. These domains, together with the N-terminal AAA+ hexamer, ensure that Rca is packaged with Rubisco into carboxysomes. The cyanobacterial Rca is a dual-purpose protein with functions in Rubisco repair and carboxysome organization.
History
DepositionMay 13, 2020-
Header (metadata) releaseSep 23, 2020-
Map releaseSep 23, 2020-
UpdateApr 7, 2021-
Current statusApr 7, 2021Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.015
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.015
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6z1f
  • Surface level: 0.015
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_11028.png

Downloads & links

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Map

FileDownload / File: emd_11028.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.8512 Å
Density
Contour LevelBy AUTHOR: 0.015 / Movie #1: 0.015
Minimum - Maximum-0.038927805 - 0.071099915
Average (Standard dev.)0.0002686747 (±0.0026960569)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions360360360
Spacing360360360
CellA=B=C: 306.432 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.85120.85120.8512
M x/y/z360360360
origin x/y/z0.0000.0000.000
length x/y/z306.432306.432306.432
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ138139230
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS360360360
D min/max/mean-0.0390.0710.000

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Supplemental data

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Additional map: #1

Fileemd_11028_additional.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : NosRca-deltaC:Rubisco complex

EntireName: NosRca-deltaC:Rubisco complex
Components
  • Complex: NosRca-deltaC:Rubisco complex
    • Complex: AAA-core of the Rca hexamer from Nostoc sp. PCC7120 in complex with ATP/ATPgammaS
      • Protein or peptide: Ribulose bisphosphate carboxylase/oxygenase activase
    • Complex: Rubisco complex from Nostoc sp. PCC7120 in complex with inhibitor CABP
      • Protein or peptide: Ribulose bisphosphate carboxylase large chain
      • Protein or peptide: Ribulose bisphosphate carboxylase small chain
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER
  • Ligand: MAGNESIUM ION
  • Ligand: 2-CARBOXYARABINITOL-1,5-DIPHOSPHATE

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Supramolecule #1: NosRca-deltaC:Rubisco complex

SupramoleculeName: NosRca-deltaC:Rubisco complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Details: The Rca hexamer captured in the complex with its substrate Rubisco from Nostoc sp. PCC 7120.
Source (natural)Organism: Nostoc sp. PCC 7120 = FACHB-418 (bacteria)
Recombinant expressionOrganism: Escherichia coli BL21 (bacteria) / Recombinant strain: BL21 sTAR

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Supramolecule #2: AAA-core of the Rca hexamer from Nostoc sp. PCC7120 in complex wi...

SupramoleculeName: AAA-core of the Rca hexamer from Nostoc sp. PCC7120 in complex with ATP/ATPgammaS
type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Nostoc sp. PCC 7120 = FACHB-418 (bacteria)
Recombinant expressionOrganism: Escherichia coli BL21 (bacteria) / Recombinant strain: BL21 sTAR

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Supramolecule #3: Rubisco complex from Nostoc sp. PCC7120 in complex with inhibitor CABP

SupramoleculeName: Rubisco complex from Nostoc sp. PCC7120 in complex with inhibitor CABP
type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2-#3
Source (natural)Organism: Nostoc sp. PCC 7120 = FACHB-418 (bacteria)
Recombinant expressionOrganism: Escherichia coli BL21 (bacteria) / Recombinant strain: BL21 sTAR

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Macromolecule #1: Ribulose bisphosphate carboxylase/oxygenase activase

MacromoleculeName: Ribulose bisphosphate carboxylase/oxygenase activase / type: protein_or_peptide / ID: 1 / Number of copies: 6 / Enantiomer: LEVO
Source (natural)Organism: Nostoc sp. (strain PCC 7120 / SAG 25.82 / UTEX 2576) (bacteria)
Molecular weightTheoretical: 32.773316 KDa
Recombinant expressionOrganism: Escherichia coli BL21 (bacteria)
SequenceString: SYYIAPRFLD KLAVHITKNF LNIPGVRVPL ILGIHGRKGE GKTFQCELAF EKMGIEVTLI SGGELESPDA GDPARLIRLR YRETAELIK VRGKMCVLMI NDLDAGAGRF DEGTQYTVNT QLVNATLMNI ADNPTDVQLP GSYDSNPIRR VPIIVTGNDF S TLYAPLIR ...String:
SYYIAPRFLD KLAVHITKNF LNIPGVRVPL ILGIHGRKGE GKTFQCELAF EKMGIEVTLI SGGELESPDA GDPARLIRLR YRETAELIK VRGKMCVLMI NDLDAGAGRF DEGTQYTVNT QLVNATLMNI ADNPTDVQLP GSYDSNPIRR VPIIVTGNDF S TLYAPLIR DGRMEKFYWE PNRDDKVGIV GGIFAEDGLS QREIEQLVDT FPKQSIDFFS ALRSRIYDIQ IRDFIHKVGF ER ISLRVVN SLEAPPEFKK PDFSLAHLIE SGNLVLGEQQ RVDNSQLVDE YNR

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Macromolecule #2: Ribulose bisphosphate carboxylase large chain

MacromoleculeName: Ribulose bisphosphate carboxylase large chain / type: protein_or_peptide / ID: 2 / Number of copies: 8 / Enantiomer: LEVO / EC number: ribulose-bisphosphate carboxylase
Source (natural)Organism: Nostoc sp. (strain PCC 7120 / SAG 25.82 / UTEX 2576) (bacteria)
Molecular weightTheoretical: 53.155125 KDa
Recombinant expressionOrganism: Escherichia coli BL21 (bacteria)
SequenceString: MSYAQTKTQT KSGYKAGVQD YRLTYYTPDY TPKDTDILAA FRVTPQPGVP FEEAAAAVAA ESSTGTWTTV WTDLLTDLDR YKGRCYDIE PVPGEDNQFI AYIAYPLDLF EEGSITNVLT SIVGNVFGFK ALRALRLEDI RFPVAYIKTF QGPPHGIQVE R DKLNKYGR ...String:
MSYAQTKTQT KSGYKAGVQD YRLTYYTPDY TPKDTDILAA FRVTPQPGVP FEEAAAAVAA ESSTGTWTTV WTDLLTDLDR YKGRCYDIE PVPGEDNQFI AYIAYPLDLF EEGSITNVLT SIVGNVFGFK ALRALRLEDI RFPVAYIKTF QGPPHGIQVE R DKLNKYGR PLLGCTIKPK LGLSAKNYGR AVYECLRGGL DFT(KCX)DDENIN SAPFQRWRDR FLFVADAITK AQAETGEI K GHYLNVTAPT CEEMLKRAEY AKELKQPIIM HDYLTAGFTA NTTLARWCRD NGVLLHIHRA MHAVIDRQKN HGIHFRVLA KALRLSGGDH IHTGTVVGKL EGERGITMGF VDLLRENYVE QDKSRGIYFT QDWASLPGVM AVASGGIHVW HMPALVEIFG DDSVLQFGG GTLGHPWGNA PGATANRVAL EACVQARNEG RNLAREGNDV IREAAKWSPE LAVACELWKE IKFEFEAMDT V

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Macromolecule #3: Ribulose bisphosphate carboxylase small chain

MacromoleculeName: Ribulose bisphosphate carboxylase small chain / type: protein_or_peptide / ID: 3 / Number of copies: 8 / Enantiomer: LEVO / EC number: ribulose-bisphosphate carboxylase
Source (natural)Organism: Nostoc sp. (strain PCC 7120 / SAG 25.82 / UTEX 2576) (bacteria)
Molecular weightTheoretical: 12.840725 KDa
Recombinant expressionOrganism: Escherichia coli BL21 (bacteria)
SequenceString:
MQTLPKERRY ETLSYLPPLT DVQIEKQVQY ILSQGYIPAV EFNEVSEPTE LYWTLWKLPL FGAKTSREVL AEVQSCRSQY PGHYIRVVG FDNIKQCQIL SFIVHKPSRY

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Macromolecule #4: ADENOSINE-5'-DIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-DIPHOSPHATE / type: ligand / ID: 4 / Number of copies: 1 / Formula: ADP
Molecular weightTheoretical: 427.201 Da
Chemical component information

ChemComp-ADP:
ADENOSINE-5'-DIPHOSPHATE / ADP, energy-carrying molecule*YM / Adenosine diphosphate

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Macromolecule #5: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER

MacromoleculeName: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / type: ligand / ID: 5 / Number of copies: 5 / Formula: AGS
Molecular weightTheoretical: 523.247 Da
Chemical component information

ChemComp-AGS:
PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / ATP-gamma-S, energy-carrying molecule analogue*YM

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Macromolecule #6: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 6 / Number of copies: 12 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #7: 2-CARBOXYARABINITOL-1,5-DIPHOSPHATE

MacromoleculeName: 2-CARBOXYARABINITOL-1,5-DIPHOSPHATE / type: ligand / ID: 7 / Number of copies: 7 / Formula: CAP
Molecular weightTheoretical: 356.115 Da
Chemical component information

ChemComp-CAP:
2-CARBOXYARABINITOL-1,5-DIPHOSPHATE

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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

Concentration2.2 mg/mL
BufferpH: 8.4
Component:
ConcentrationFormulaName
10.0 mMC4H6O5Malic acid
20.0 mMC6H13NO4S2-ethanesulfonic acid
20.0 mMC4H11NO32-Amino-2-hydroxymethyl-propane-1,3-diol
50.0 mMKClPotassium chloride
10.0 mMMgCl2Magnesium chloride
5.0 mMNaHCO3Sodium bicarbonate
0.004 mMC6H14O13P22-Carboxyarabinitol-1,5-diphosphate
2.0 mMC10H16N5O13P3Adenosine triphosphate sodium salt
2.0 mMC10H12Li4N5O12P3SAdenosine 5'-(3-thiotriphosphate) tetralithium salt
GridModel: Quantifoil R2/1 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY ARRAY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 298 K / Instrument: FEI VITROBOT MARK IV
DetailsNosRubisco (1 uM) was incubated with NaHCO3 (10 mM) at 298 K for 10 min followed by addition of CABP (8 uM). CABP-inhibited NosRubisco (0.5 uM) was then incubated with NosRcaDC (10 uM) in the presence of ATP (2 mM) for 10 s, followed by the addition of ATP-gammaS (2 mM), and incubated at 298 K for another 10 min before preparing the cryo-grids.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELDBright-field microscopy
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 1 / Number real images: 9042 / Average exposure time: 2.8 sec. / Average electron dose: 60.0 e/Å2 / Details: 31 frames per image
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 519151
CTF correctionSoftware - Name: CTFFIND (ver. 4.1)
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

1rbl

Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
Final 3D classificationSoftware - Name: RELION
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 2.86 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 21149
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: RECIPROCAL / Protocol: OTHER
Output model

PDB-6z1f:
CryoEM structure of Rubisco Activase with its substrate Rubisco from Nostoc sp. (strain PCC7120)

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