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- EMDB-11575: CryoEM Local map of Rubisco Activase from the complex with its su... -

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Basic information

Entry
Database: EMDB / ID: EMD-11575
TitleCryoEM Local map of Rubisco Activase from the complex with its substrate Rubisco from Nostoc sp. (strain PCC7120)
Map dataCryoEM local map of Rubisco Activase from the complex with its substrate Rubisco from Nostoc sp. (strain PCC7120)
Sample
  • Complex: NosRca-deltaC:Rubisco complex
    • Complex: AAA-core of the Rca hexamer from Nostoc sp. PCC7120 in complex with ATP/ATPgammaS
    • Complex: Rubisco complex from Nostoc sp. PCC7120 in complex with inhibitor CABP
Function / homology
Function and homology information


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, domain / Ribulose bisphosphate carboxylase, small subunit superfamily / Ribulose bisphosphate carboxylase, small chain / Ribulose bisphosphate carboxylase, small chain / 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/oxygenase activase
Similarity search - Component
Biological speciesNostoc sp. PCC 7120 = FACHB-418 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.29 Å
AuthorsWang H / Bracher A
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
DepositionAug 3, 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: 0.012
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.012
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_11575.png

Downloads & links

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Map

FileDownload / File: emd_11575.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryoEM local map of Rubisco Activase from the complex with its substrate Rubisco from Nostoc sp. (strain PCC7120)
Voxel sizeX=Y=Z: 0.8512 Å
Density
Contour LevelBy AUTHOR: 0.012 / Movie #1: 0.012
Minimum - Maximum-0.031081885 - 0.05226625
Average (Standard dev.)2.3150458e-05 (±0.0011149904)
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
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS360360360
D min/max/mean-0.0310.0520.000

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Supplemental data

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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
    • Complex: Rubisco complex from Nostoc sp. PCC7120 in complex with inhibitor CABP

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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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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 / Support film - Material: CARBON / Support film - topology: HOLEY ARRAY
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 reconstructionResolution.type: BY AUTHOR / Resolution: 3.29 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

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