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- PDB-7qsv: L8-complex forming RubisCO derived from ancestral sequence recons... -

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Basic information

Entry
Database: PDB / ID: 7qsv
TitleL8-complex forming RubisCO derived from ancestral sequence reconstruction of the last common ancestor of Form I'' and Form I RubisCOs
ComponentsRubisCO large 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 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)410,92024
Polymers407,8778
Non-polymers3,04316
Water42,6782369
1
A: RubisCO large subunit
B: RubisCO large subunit
C: RubisCO large subunit
D: RubisCO large subunit
hetero molecules

A: RubisCO large subunit
B: RubisCO large subunit
C: RubisCO large subunit
D: RubisCO large subunit
hetero molecules


Theoretical massNumber of molelcules
Total (without water)410,92024
Polymers407,8778
Non-polymers3,04316
Water1448
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation2_556-x,y,-z+11
2
E: RubisCO large subunit
F: RubisCO large subunit
G: RubisCO large subunit
H: RubisCO large subunit
hetero molecules

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)410,92024
Polymers407,8778
Non-polymers3,04316
Water1448
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation2_655-x+1,y,-z1
Unit cell
Length a, b, c (Å)114.007, 203.445, 174.974
Angle α, β, γ (deg.)90.000, 100.430, 90.000
Int Tables number5
Space group name H-MC121
Components on special symmetry positions
IDModelComponents
11A-2295-

HOH

21B-931-

HOH

31C-947-

HOH

41E-839-

HOH

51F-869-

HOH

61G-915-

HOH

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Components

#1: Protein
RubisCO large subunit


Mass: 50984.617 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: saccharide / 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


Mass: 18.015 Da / Num. of mol.: 2369 / 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: 2.45 Å3/Da / Density % sol: 49.72 %
Crystal growTemperature: 288 K / Method: vapor diffusion, sitting drop / pH: 8
Details: Purified enzyme (7.5 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 0.25 mM CABP and 4 mM MgCl2. The enzyme ...Details: Purified enzyme (7.5 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 0.25 mM CABP and 4 mM MgCl2. The enzyme was then mixed in a 1:1 ratio with 40 % v/v Glycerol ethoxylate. Crystals were flash frozen in liquid nitrogen.

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: PETRA III, EMBL c/o DESY / Beamline: P14 (MX2) / Wavelength: 0.97626 Å
DetectorType: DECTRIS EIGER2 X 16M / Detector: PIXEL / Date: Aug 20, 2021
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.97626 Å / Relative weight: 1
ReflectionResolution: 2.1→29.863 Å / Num. all: 225493 / Num. obs: 225493 / % possible obs: 99.2 % / Redundancy: 7.2 % / Rpim(I) all: 0.054 / Rrim(I) all: 0.146 / Rsym value: 0.135 / Net I/av σ(I): 4.9 / Net I/σ(I): 8.9 / Num. measured all: 1613528
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
2.1-2.217.20.9110.8235574329270.3650.9830.9112.199.3
2.21-2.357.10.6371.2221167310390.2560.6870.6372.899.1
2.35-2.517.10.4611.7208713292520.1850.4980.4613.899.2
2.51-2.717.20.3252.4197003272820.130.350.3255.299.3
2.71-2.977.20.2223.4180044249810.0890.2390.2227.299.1
2.97-3.327.10.1425.1161312227170.0570.1540.14210.599.3
3.32-3.8370.0877.8139733199560.0350.0940.08715.699.1
3.83-4.77.30.05710.5124336170060.0230.0610.05722.899.7
4.7-6.647.10.05810.293769131350.0230.0630.05822.199.5
6.64-29.8637.20.044115187771980.0170.0470.04427.298.6

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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→29.86 Å / SU ML: 0.24 / Cross valid method: THROUGHOUT / σ(F): 1.03 / Phase error: 27.99 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.223 1994 89 %
Rwork0.1887 222940 -
obs0.189 224934 98.94 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso max: 124.17 Å2 / Biso mean: 32.9975 Å2 / Biso min: 16.79 Å2
Refinement stepCycle: final / Resolution: 2.1→29.86 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms28309 0 176 2369 30854
Biso mean--27.24 37.48 -
Num. residues----3599
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 (%)
2.1-2.150.32471430.2907159291607299
2.15-2.210.32581420.2721159011604399
2.21-2.280.31911430.252158981604199
2.28-2.350.29721420.2378158211596399
2.35-2.430.26951430.224159651610899
2.43-2.530.23091420.2117159191606199
2.53-2.650.22781430.207159061604999
2.65-2.780.25281430.1983158961603999
2.78-2.960.24021420.1998158761601899
2.96-3.190.21621430.1903159781612199
3.19-3.510.19991420.1833158871602999
3.51-4.010.2271440.1581160151615999
4.01-5.050.15451420.1409160651620799
5.05-29.860.18221400.1618158841602498
Refinement TLS params.Method: refined / Origin x: 23.9881 Å / Origin y: -55.669 Å / Origin z: 54.4461 Å
111213212223313233
T0.1849 Å20.0072 Å2-0.0341 Å2-0.2389 Å20.0056 Å2--0.2747 Å2
L0.0854 °20.0021 °2-0.0422 °2-0.0231 °20.0048 °2--0.0705 °2
S-0.0103 Å °0.0364 Å °-0.0233 Å °-0.0054 Å °0.0021 Å °-0.03 Å °0.021 Å °0.038 Å °0.0095 Å °
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection detailsAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1allA5 - 454
2X-RAY DIFFRACTION1allA500 - 520
3X-RAY DIFFRACTION1allB5 - 455
4X-RAY DIFFRACTION1allB500 - 520
5X-RAY DIFFRACTION1allC5 - 454
6X-RAY DIFFRACTION1allC500 - 520
7X-RAY DIFFRACTION1allD5 - 454
8X-RAY DIFFRACTION1allD500 - 520
9X-RAY DIFFRACTION1allE6 - 454
10X-RAY DIFFRACTION1allE500 - 520
11X-RAY DIFFRACTION1allF5 - 454
12X-RAY DIFFRACTION1allF500 - 520
13X-RAY DIFFRACTION1allG5 - 454
14X-RAY DIFFRACTION1allG500 - 520
15X-RAY DIFFRACTION1allH6 - 454
16X-RAY DIFFRACTION1allH500 - 520
17X-RAY DIFFRACTION1allS1 - 2369

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