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- PDB-7qsx: Non-obligately L8S8-complex forming RubisCO derived from ancestra... -

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Basic information

Entry
Database: PDB / ID: 7qsx
TitleNon-obligately L8S8-complex forming RubisCO derived from ancestral sequence reconstruction and rational engineering in L8 complex
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)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.7 Å
AuthorsZarzycki, J. / Schulz, L. / Erb, T.J. / Hochberg, G.K.A.
Funding support Germany, 1items
OrganizationGrant numberCountry
Max Planck Society 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 14, 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
Revision 1.2Jan 31, 2024Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond / pdbx_initial_refinement_model

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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
hetero molecules


Theoretical massNumber of molelcules
Total (without water)411,82624
Polymers408,7828
Non-polymers3,04316
Water12,701705
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: light scattering, 424 kDa measured by mass photometry
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area50780 Å2
ΔGint-271 kcal/mol
Surface area102770 Å2
MethodPISA
Unit cell
Length a, b, c (Å)113.599, 159.197, 127.910
Angle α, β, γ (deg.)90.000, 107.900, 90.000
Int Tables number4
Space group name H-MP1211

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Components

#1: Protein
RubisCO large subunit / RuBisCO


Mass: 51097.797 Da / Num. of mol.: 8
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) synthetic construct (others)
Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
Variant (production host): ArcticExpress (DE3) / References: ribulose-bisphosphate carboxylase
#2: Sugar
ChemComp-CAP / 2-CARBOXYARABINITOL-1,5-DIPHOSPHATE


Type: saccharideCarbohydrate / Mass: 356.115 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: C6H14O13P2 / Feature type: SUBJECT OF INVESTIGATION
#3: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
#4: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 705 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 2.69 Å3/Da / Density % sol: 54.35 %
Crystal growTemperature: 288 K / Method: vapor diffusion, sitting drop / pH: 6.5
Details: Purified enzyme (5 mg/mL) in 25 mM Tricine-NaOH, 75 mM NaCl, pH 8.0 was incubated at 3% (v/v) CO2 in air and 30 degrees C for 1 h in the presence of 0.2 mM CABP and 3.2 mM MgCl2. The enzyme ...Details: Purified enzyme (5 mg/mL) in 25 mM Tricine-NaOH, 75 mM NaCl, pH 8.0 was incubated at 3% (v/v) CO2 in air and 30 degrees C for 1 h in the presence of 0.2 mM CABP and 3.2 mM MgCl2. The enzyme was then mixed in a 1:1 ratio with 0.1 M ammonium sulfate, 0.3 M sodium formate, 0.1 M sodium cacodylate , 3% (w/v) gamma-PGA, and 2% (w/v) PEG 3350, pH 6.5. Before flash freezing the crystals in liquid nitrogen PEG200 was added to the mother liquor as cryoprotectant to a final concentration of 40 % (w/v).

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: ESRF / Beamline: ID23-2 / Wavelength: 0.873128 Å
DetectorType: DECTRIS PILATUS3 2M / Detector: PIXEL / Date: Jul 9, 2021
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.873128 Å / Relative weight: 1
ReflectionResolution: 2.7→29.889 Å / Num. all: 118081 / Num. obs: 118081 / % possible obs: 99.6 % / Redundancy: 6 % / Rpim(I) all: 0.119 / Rrim(I) all: 0.298 / Rsym value: 0.272 / Net I/av σ(I): 2.6 / Net I/σ(I): 5.8 / Num. measured all: 708705
Reflection shell

Diffraction-ID: 1

Resolution (Å)Redundancy (%)Rmerge(I) obsMean I/σ(I) obsNum. measured allNum. unique obsRpim(I) allRrim(I) allRsym valueNet I/σ(I) obs% possible all
2.7-2.856.20.8210.9107444172910.3530.8950.8212.2100
2.85-3.026.20.6611.1100882163130.2850.7210.6612.7100
3.02-3.236.10.5051.493809153420.2190.5510.5053.6100
3.23-3.4960.363286587143160.1570.3960.3635100
3.49-3.8260.2562.878747131460.1120.280.2566.8100
3.82-4.2760.183.971977119400.0780.1970.189.199.9
4.27-4.935.90.1474.862173105050.0650.1610.14710.399.8
4.93-6.045.60.1773.94992688580.080.1950.1778.299.4
6.04-8.545.70.1484.73941269020.0660.1630.148999.6
8.54-29.8895.10.09271774834680.0430.1010.09211.391.2

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Processing

Software
NameVersionClassification
PHENIX1.18.2_3874refinement
SCALA3.3.22data scaling
PDB_EXTRACT3.27data extraction
XDS20210323data reduction
PHENIX1.18.2_3874phasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 6URA

6ura


Resolution: 2.7→29.889 Å / SU ML: 0.39 / Cross valid method: THROUGHOUT / σ(F): 0.76 / Phase error: 27.09 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.2603 1999 1.69 %
Rwork0.2182 116026 -
obs0.2189 118025 99.6 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso max: 135.36 Å2 / Biso mean: 32.3019 Å2 / Biso min: 16.01 Å2
Refinement stepCycle: final / Resolution: 2.7→29.889 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms28347 0 176 705 29228
Biso mean--27.8 29.61 -
Num. residues----3597
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Rfactor Rfree error: 0

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection Rwork% reflection obs (%)
2.7-2.770.3651430.2788289100
2.77-2.840.30311430.2648310100
2.84-2.930.31231420.25818255100
2.93-3.020.31191430.25978281100
3.02-3.130.29621430.25818276100
3.13-3.250.28051430.23828354100
3.25-3.40.2461430.23268264100
3.4-3.580.2691430.22698305100
3.58-3.80.27581430.20458322100
3.8-4.10.22171420.19438309100
4.1-4.510.231440.19138293100
4.51-5.160.21691430.18858341100
5.16-6.490.26421430.2152829299
6.49-29.8890.23951410.1906813596
Refinement TLS params.Method: refined / Origin x: 22.029 Å / Origin y: -80.8588 Å / Origin z: 89.0077 Å
111213212223313233
T0.2319 Å20.0095 Å2-0.003 Å2-0.1782 Å20.0079 Å2--0.1748 Å2
L0.2061 °20.0686 °2-0.0239 °2-0.2358 °20.0061 °2--0.1912 °2
S-0.0096 Å °-0.0036 Å °0.0207 Å °0.0063 Å °0.0191 Å °0.0222 Å °-0.0125 Å °-0.0046 Å °-0.0086 Å °
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection detailsAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1allA6 - 455
2X-RAY DIFFRACTION1allA500 - 510
3X-RAY DIFFRACTION1allB6 - 455
4X-RAY DIFFRACTION1allB500 - 510
5X-RAY DIFFRACTION1allC6 - 455
6X-RAY DIFFRACTION1allC500 - 510
7X-RAY DIFFRACTION1allD6 - 455
8X-RAY DIFFRACTION1allD500 - 510
9X-RAY DIFFRACTION1allE6 - 510
10X-RAY DIFFRACTION1allF6 - 510
11X-RAY DIFFRACTION1allG6 - 455
12X-RAY DIFFRACTION1allG500 - 510
13X-RAY DIFFRACTION1allH6 - 510
14X-RAY DIFFRACTION1allS1 - 705

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