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- PDB-7qt1: Non-obligately L8S8-complex forming RubisCO derived from ancestra... -

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Basic information

Entry
Database: PDB / ID: 7qt1
TitleNon-obligately L8S8-complex forming RubisCO derived from ancestral sequence reconstruction and rational engineering in L8S8 complex with substitution e170N
Components
  • RubisCO large subunit
  • RubisCO small subunit
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: 2.1 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 small subunit
C: RubisCO large subunit
D: RubisCO small subunit
E: RubisCO large subunit
F: RubisCO small subunit
G: RubisCO large subunit
H: RubisCO small subunit
hetero molecules


Theoretical massNumber of molelcules
Total (without water)255,77216
Polymers254,2518
Non-polymers1,5228
Water28,8421601
1
A: RubisCO large subunit
B: RubisCO small subunit
C: RubisCO large subunit
D: RubisCO small subunit
E: RubisCO large subunit
F: RubisCO small subunit
G: RubisCO large subunit
H: RubisCO small subunit
hetero molecules

A: RubisCO large subunit
B: RubisCO small subunit
C: RubisCO large subunit
D: RubisCO small subunit
E: RubisCO large subunit
F: RubisCO small subunit
G: RubisCO large subunit
H: RubisCO small subunit
hetero molecules


Theoretical massNumber of molelcules
Total (without water)511,54532
Polymers508,50116
Non-polymers3,04316
Water28816
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation2_555-x,y,-z1
Buried area83590 Å2
ΔGint-325 kcal/mol
Surface area119880 Å2
MethodPISA
Unit cell
Length a, b, c (Å)108.942, 106.604, 191.968
Angle α, β, γ (deg.)90.000, 98.780, 90.000
Int Tables number5
Space group name H-MI121
Components on special symmetry positions
IDModelComponents
11G-822-

HOH

21G-911-

HOH

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Components

#1: Protein
RubisCO large subunit


Mass: 51082.785 Da / Num. of mol.: 4
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)
#2: Protein
RubisCO small subunit


Mass: 12479.868 Da / Num. of mol.: 4
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)
#3: Sugar
ChemComp-CAP / 2-CARBOXYARABINITOL-1,5-DIPHOSPHATE


Type: saccharide / Mass: 356.115 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C6H14O13P2 / Feature type: SUBJECT OF INVESTIGATION
#4: Chemical
ChemComp-MG / MAGNESIUM ION


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


Mass: 18.015 Da / Num. of mol.: 1601 / 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: 2.17 Å3/Da / Density % sol: 43.23 %
Crystal growTemperature: 288 K / Method: vapor diffusion, sitting drop / pH: 9.1
Details: Purified enzyme (3.25 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 0.182 mM CABP and 2.9 mM MgCl2. The ...Details: Purified enzyme (3.25 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 0.182 mM CABP and 2.9 mM MgCl2. The enzyme was then mixed in a 1:1 ratio with 0.2 M Bis-Tris propane, 20 % (w/v) polyethylene glycol 4000, pH 9.1. Before flash freezing the crystals in liquid nitrogen PEG200 was added to the mother liquor as cryoprotectant to a final concentration of 33 % (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: Dec 2, 2021
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.873128 Å / Relative weight: 1
ReflectionResolution: 2.1→29.29 Å / Num. obs: 126281 / % possible obs: 99.9 % / Redundancy: 6 % / Rpim(I) all: 0.096 / Rrim(I) all: 0.239 / Rsym value: 0.218 / Net I/av σ(I): 3.3 / Net I/σ(I): 7.1
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
2.1-2.216.10.711.1112922183990.3110.7770.712.4100
2.21-2.356.10.5471.4107000174250.240.5990.5473.2100
2.35-2.516.10.4381.799667163400.1930.480.4383.9100
2.51-2.716.10.3442.192674152770.1520.3770.3444.9100
2.71-2.9760.272.784063140450.120.2960.276.1100
2.97-3.326.10.1953.777183127100.0860.2130.1958.4100
3.32-3.8360.1315.567789112170.0580.1440.13112.1100
3.83-4.75.80.0937.55475894820.0420.1030.09315.899.9
4.7-6.645.60.0977.14165173820.0450.1070.09713.899.8
6.64-29.2865.70.0689.92298740040.0310.0750.06817.797.3

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Processing

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

6ura


Resolution: 2.1→29.29 Å / SU ML: 0.2 / Cross valid method: THROUGHOUT / σ(F): 1 / Phase error: 17.52 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.1857 2000 1.58 %
Rwork0.1629 124265 -
obs0.1633 126265 99.9 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso max: 81.35 Å2 / Biso mean: 17.7926 Å2 / Biso min: 9.53 Å2
Refinement stepCycle: final / Resolution: 2.1→29.29 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms17541 0 88 1601 19230
Biso mean--11.42 25.34 -
Num. residues----2204
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Rfactor Rfree error: 0 / Total num. of bins used: 14

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkNum. reflection all% reflection obs (%)
2.1-2.150.22861430.217588639006100
2.15-2.210.23211420.201488478989100
2.21-2.280.22731430.195588649007100
2.28-2.350.2391420.18488218963100
2.35-2.430.20651420.182488488990100
2.43-2.530.20671430.177188759018100
2.53-2.650.21521430.17388488991100
2.65-2.780.18471420.162988619003100
2.78-2.960.19941430.165288659008100
2.96-3.190.17871430.162388699012100
3.19-3.510.17321430.14989039046100
3.51-4.010.15091430.134489159058100
4.01-5.050.13351430.129189129055100
5.05-29.290.18131450.16848974911999
Refinement TLS params.Method: refined / Origin x: -16.7404 Å / Origin y: -5.4243 Å / Origin z: 3.9278 Å
111213212223313233
T0.1094 Å2-0.0031 Å20.0042 Å2-0.1358 Å20.007 Å2--0.1265 Å2
L0.0106 °2-0.0164 °20.0095 °2-0.1306 °20.0017 °2--0.0423 °2
S-0.001 Å °-0.0066 Å °-0.0082 Å °0.014 Å °0.001 Å °0.0382 Å °-0.0019 Å °-0.0153 Å °-0.0001 Å °
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection detailsAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1allA6 - 453
2X-RAY DIFFRACTION1allA500 - 510
3X-RAY DIFFRACTION1allB1 - 99
4X-RAY DIFFRACTION1allC6 - 510
5X-RAY DIFFRACTION1allD1 - 105
6X-RAY DIFFRACTION1allE6 - 510
7X-RAY DIFFRACTION1allF1 - 99
8X-RAY DIFFRACTION1allG6 - 510
9X-RAY DIFFRACTION1allH1 - 99
10X-RAY DIFFRACTION1allS1 - 1602

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