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- PDB-7qvi: Fiber-forming RubisCO derived from ancestral sequence reconstruct... -

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Basic information

Entry
Database: PDB / ID: 7qvi
TitleFiber-forming RubisCO derived from ancestral sequence reconstruction and rational engineering
ComponentsRubisCO large subunitRuBisCO
KeywordsLYASE / Ribulose 1 / 5-bisphosphate carboxylase/oxydase / RubisCO
Function / homology2-CARBOXYARABINITOL-1,5-DIPHOSPHATE
Function and homology information
Biological speciessynthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3 Å
AuthorsSchulz, L. / Zarzycki, J. / Prinz, S. / Schuller, J.M. / Erb, T.J. / Hochberg, G.K.A.
Funding support Germany, 2items
OrganizationGrant numberCountry
Max Planck Society Germany
German Research Foundation (DFG)SCHU 3364/1-1 Germany
CitationJournal: Science / Year: 2022
Title: Evolution of increased complexity and specificity at the dawn of form I Rubiscos.
Authors: Luca Schulz / Zhijun Guo / Jan Zarzycki / Wieland Steinchen / Jan M Schuller / Thomas Heimerl / Simone Prinz / Oliver Mueller-Cajar / Tobias J Erb / Georg K A Hochberg /
Abstract: The evolution of ribulose-1,5-bisphosphate carboxylase/oxygenases (Rubiscos) that discriminate strongly between their substrate carbon dioxide and the undesired side substrate dioxygen was an ...The evolution of ribulose-1,5-bisphosphate carboxylase/oxygenases (Rubiscos) that discriminate strongly between their substrate carbon dioxide and the undesired side substrate dioxygen was an important event for photosynthetic organisms adapting to an oxygenated environment. We use ancestral sequence reconstruction to recapitulate this event. We show that Rubisco increased its specificity and carboxylation efficiency through the gain of an accessory subunit before atmospheric oxygen was present. Using structural and biochemical approaches, we retrace how this subunit was gained and became essential. Our work illuminates the emergence of an adaptation to rising ambient oxygen levels, provides a template for investigating the function of interactions that have remained elusive because of their essentiality, and sheds light on the determinants of specificity in Rubisco.
History
DepositionJan 21, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0Oct 12, 2022Provider: repository / Type: Initial release
Revision 1.1Oct 26, 2022Group: Database references / Category: citation
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: RubisCO large subunit
B: RubisCO large subunit
C: RubisCO large subunit
D: RubisCO large subunit
E: RubisCO large subunit
F: RubisCO large subunit
G: RubisCO large subunit
H: RubisCO large subunit
I: RubisCO large subunit
J: RubisCO large subunit
K: RubisCO large subunit
L: RubisCO large subunit
M: RubisCO large subunit
N: RubisCO large subunit
O: RubisCO large subunit
P: RubisCO large subunit
hetero molecules


Theoretical massNumber of molelcules
Total (without water)825,23848
Polymers819,15116
Non-polymers6,08732
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: light scattering, 873 kDa measured by mass photometry
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
RubisCO large subunit / RuBisCO


Mass: 51196.930 Da / Num. of mol.: 16
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) synthetic construct (others)
Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
Strain (production host): ArcticExpress (DE3)
#2: Sugar
ChemComp-CAP / 2-CARBOXYARABINITOL-1,5-DIPHOSPHATE


Type: saccharideCarbohydrate / Mass: 356.115 Da / Num. of mol.: 16 / Source method: obtained synthetically / Formula: C6H14O13P2 / Feature type: SUBJECT OF INVESTIGATION
#3: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 16 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: 2D ARRAY / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: ancestral sequence reconstruction of RubisCO large subunit in complex with CABP and Magnesium
Type: COMPLEX / Details: complex of two octamers (2x L8) / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.873 MDa / Experimental value: YES
Source (natural)Organism: synthetic construct (others)
Source (recombinant)Organism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
Strain: ArtricExpress (DE3)
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
125 mMTricineC6H13NO51
275 mMsodium chlorideNaClSodium chloride1
30.07 mMCABPFrozen state pensionC6H14O13P21
41.33 mMmagnesium chlorideMgCl21
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid type: Quantifoil R2/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2500 nm / Nominal defocus min: 500 nm
Image recordingElectron dose: 40 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

Software
NameVersionClassificationNB
phenix.real_space_refine1.18.2_3874refinement
PHENIX1.18.2_3874refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 171572 / Symmetry type: POINT
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 65.59 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.003657992
ELECTRON MICROSCOPYf_angle_d0.543278648
ELECTRON MICROSCOPYf_chiral_restr0.04078248
ELECTRON MICROSCOPYf_plane_restr0.003910360
ELECTRON MICROSCOPYf_dihedral_angle_d28.60688112

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