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- EMDB-11029: CryoEM structure of the interaction between Rubisco Activase smal... -

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Basic information

Entry
Database: EMDB / ID: EMD-11029
TitleCryoEM structure of the interaction between Rubisco Activase small-subunit-like (SSUL) domain with Rubisco from Nostoc sp. (strain PCC7120)
Map data
Sample
  • Complex: Complex between Rubisco Activase small-subunit-like domain with Rubisco from Nostoc sp. (strain PCC7120)
    • Complex: SSUL domain of Rca, part of the (Rca)6 hexamer
      • Protein or peptide: Ribulose bisphosphate carboxylase/oxygenase activase
    • Complex: (RbcL)2(RbcS)2 unit of the (RbcL)8(RbcS)8 Rubisco complex
      • Protein or peptide: Ribulose bisphosphate carboxylase large chain
      • Protein or peptide: Ribulose bisphosphate carboxylase small chain
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: 8.2 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: 0.03
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.03
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6z1g
  • Surface level: 0.03
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-6z1g
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_11029.png

Downloads & links

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Map

FileDownload / File: emd_11029.map.gz / Format: CCP4 / Size: 30.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.885 Å
Density
Contour LevelBy AUTHOR: 0.03 / Movie #1: 0.03
Minimum - Maximum-0.059503756 - 0.11776942
Average (Standard dev.)0.00010899712 (±0.0022778183)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions200200200
Spacing200200200
CellA=B=C: 377.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.8851.8851.885
M x/y/z200200200
origin x/y/z0.0000.0000.000
length x/y/z377.000377.000377.000
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ138139230
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS200200200
D min/max/mean-0.0600.1180.000

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Supplemental data

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Sample components

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Entire : Complex between Rubisco Activase small-subunit-like domain with R...

EntireName: Complex between Rubisco Activase small-subunit-like domain with Rubisco from Nostoc sp. (strain PCC7120)
Components
  • Complex: Complex between Rubisco Activase small-subunit-like domain with Rubisco from Nostoc sp. (strain PCC7120)
    • Complex: SSUL domain of Rca, part of the (Rca)6 hexamer
      • Protein or peptide: Ribulose bisphosphate carboxylase/oxygenase activase
    • Complex: (RbcL)2(RbcS)2 unit of the (RbcL)8(RbcS)8 Rubisco complex
      • Protein or peptide: Ribulose bisphosphate carboxylase large chain
      • Protein or peptide: Ribulose bisphosphate carboxylase small chain

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Supramolecule #1: Complex between Rubisco Activase small-subunit-like domain with R...

SupramoleculeName: Complex between Rubisco Activase small-subunit-like domain with Rubisco from Nostoc sp. (strain PCC7120)
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Nostoc sp. PCC 7120 = FACHB-418 (bacteria)
Recombinant expressionOrganism: Escherichia coli BL21 (bacteria)

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Supramolecule #2: SSUL domain of Rca, part of the (Rca)6 hexamer

SupramoleculeName: SSUL domain of Rca, part of the (Rca)6 hexamer / 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 pBAD33-EcGroSEL / Recombinant plasmid: pHUE

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Supramolecule #3: (RbcL)2(RbcS)2 unit of the (RbcL)8(RbcS)8 Rubisco complex

SupramoleculeName: (RbcL)2(RbcS)2 unit of the (RbcL)8(RbcS)8 Rubisco complex
type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2-#3
Details: The (RbcL)8(RbcS)8 Rubisco complex has D4 symmetry. The (RbcL)2(RbcS)2 unit has two-fold roational symmetry.
Source (natural)Organism: Nostoc sp. PCC 7120 = FACHB-418 (bacteria)
Recombinant expressionOrganism: Escherichia coli BL21 (bacteria) / Recombinant strain: BL21 STAR / Recombinant plasmid: pET11a-NosGroSEL-H6ubi-NosLS

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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: 1 / Enantiomer: LEVO
Source (natural)Organism: Nostoc sp. (strain PCC 7120 / SAG 25.82 / UTEX 2576) (bacteria)
Molecular weightTheoretical: 10.266549 KDa
Recombinant expressionOrganism: Escherichia coli BL21 (bacteria)
SequenceString:
HLSLETQEQI RQILSQGHKI TFEHVDARRF RTGSWQSCGT LHIDAESDAI STLEACLVDY DGEYVRMVGI DPKGKRRVVE TIIQRPNGK N

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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: 2 / Enantiomer: LEVO / EC number: ribulose-bisphosphate carboxylase
Source (natural)Organism: Nostoc sp. (strain PCC 7120 / SAG 25.82 / UTEX 2576) (bacteria)
Molecular weightTheoretical: 53.112125 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 DFTKDDENIN SAPFQRWRDR FLFVADAITK AQAETGEIKG HY LNVTAPT CEEMLKRAEY AKELKQPIIM HDYLTAGFTA NTTLARWCRD NGVLLHIHRA MHAVIDRQKN HGIHFRVLAK ALR LSGGDH IHTGTVVGKL EGERGITMGF VDLLRENYVE QDKSRGIYFT QDWASLPGVM AVASGGIHVW HMPALVEIFG DDSV LQFGG 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: 2 / 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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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration4.9 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 mMC4H10O2S2Dithiothreitol
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: 95 % / Chamber temperature: 298 K / Instrument: FEI VITROBOT MARK IV
DetailsFor Preparing the NosRca:Rubisco complex, NosRubisco (1.25 uM) was mixed with NosRca (15 uM).

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Electron microscopy

MicroscopeTFS GLACIOS
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELDBright-field microscopy
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Number grids imaged: 1 / Number real images: 1570 / Average exposure time: 12.0 sec. / Average electron dose: 47.0 e/Å2 / Details: 40 frames per image

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Image processing

Particle selectionNumber selected: 298336
CTF correctionSoftware - Name: CTFFIND (ver. 4.1)
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: 8.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Number images used: 32128
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementProtocol: RIGID BODY FIT
Output model

PDB-6z1g:
CryoEM structure of the interaction between Rubisco Activase small-subunit-like (SSUL) domain with Rubisco from Nostoc sp. (strain PCC7120)

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