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

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Basic information

Entry
Database: PDB / ID: 7qsz
TitleNon-obligately L8S8-complex forming RubisCO derived from ancestral sequence reconstruction and rational engineering in L8 complex with substitution e170N
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: 2.25 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
hetero molecules


Theoretical massNumber of molelcules
Total (without water)205,85312
Polymers204,3314
Non-polymers1,5228
Water19,0781059
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)411,70624
Polymers408,6628
Non-polymers3,04316
Water1448
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation3_655-x+1,y,-z+1/21
Unit cell
Length a, b, c (Å)122.434, 204.691, 148.457
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number20
Space group name H-MC2221
Components on special symmetry positions
IDModelComponents
11A-867-

HOH

21C-651-

HOH

31C-799-

HOH

41C-845-

HOH

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Components

#1: Protein
RubisCO large subunit / RuBisCO


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: Sugar
ChemComp-CAP / 2-CARBOXYARABINITOL-1,5-DIPHOSPHATE


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


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


Mass: 18.015 Da / Num. of mol.: 1059 / 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: 2.28 Å3/Da / Density % sol: 45.96 %
Crystal growTemperature: 288 K / Method: vapor diffusion, sitting drop / pH: 7.5
Details: Purified enzyme (2.25 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 (2.25 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 0.1 M Hepes, 0.2 M MgCl2, 30 % (w/v) polyethylene glycol 400, pH 7.5. Crystals were flash frozen in liquid nitrogen (no additional cryoprotectant added).

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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: 2.25→29.33 Å / Num. obs: 88310 / % possible obs: 99.9 % / Redundancy: 15 % / Rpim(I) all: 0.069 / Rrim(I) all: 0.269 / Rsym value: 0.26 / Net I/av σ(I): 2.9 / Net I/σ(I): 10.6
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
2.25-2.3715.41.1760.7196536127720.3091.2161.1762.6100
2.37-2.5215.40.9050.8185703120920.2380.9370.9053.4100
2.52-2.6915.20.6991.1173671114020.1840.7230.6994.4100
2.69-2.915.10.4991.5160783106330.1320.5170.4996.1100
2.9-3.1814.90.3492.214614697970.0930.3610.3498.4100
3.18-3.5615.10.2143.513445688890.0570.2220.21413.2100
3.56-4.11150.1315.711786078780.0350.1350.13120.3100
4.11-5.0314.20.0957.59500166810.0260.0990.09525.7100
5.03-7.1213.80.1056.97249752430.0290.1090.10522.2100
7.12-29.32513.50.06113937329230.0170.0620.0633.398.3

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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: 2.25→29.33 Å / SU ML: 0.23 / Cross valid method: THROUGHOUT / σ(F): 0.63 / Phase error: 18.32 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.1939 2003 2.27 %
Rwork0.1608 86270 -
obs0.1616 88273 99.97 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso max: 91.64 Å2 / Biso mean: 27.047 Å2 / Biso min: 15 Å2
Refinement stepCycle: final / Resolution: 2.25→29.33 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms14148 0 88 1059 15295
Biso mean--20.66 32.58 -
Num. residues----1796
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Rfactor Rfree error: 0 / Total num. of bins used: 14 / % reflection obs: 100 %

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkNum. reflection all
2.25-2.310.27471340.21460816215
2.31-2.370.25871490.194260826231
2.37-2.440.2381340.193361606294
2.44-2.520.23831480.189960736221
2.52-2.610.22241470.181661416288
2.61-2.710.25651420.179561356277
2.71-2.830.20661440.17461126256
2.83-2.980.21821420.182161536295
2.98-3.170.18211420.174161206262
3.17-3.410.1951440.162961736317
3.41-3.760.18891370.150461666303
3.76-4.30.1641460.129661996345
4.3-5.410.14341440.131262736417
5.41-29.330.16451500.145664026552
Refinement TLS params.Method: refined / Origin x: 53.0729 Å / Origin y: 58.2375 Å / Origin z: 45.8227 Å
111213212223313233
T0.1702 Å2-0.0049 Å20.0041 Å2-0.1865 Å20.0063 Å2--0.1968 Å2
L0.0726 °20.0055 °20.0367 °2-0.0852 °20.0083 °2--0.2184 °2
S0.007 Å °-0.0113 Å °-0.0151 Å °0.0141 Å °0.0088 Å °0.0134 Å °0.0079 Å °-0.0384 Å °-0.0156 Å °
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection detailsAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1allA6 - 453
2X-RAY DIFFRACTION1allA500 - 510
3X-RAY DIFFRACTION1allB6 - 455
4X-RAY DIFFRACTION1allB500 - 510
5X-RAY DIFFRACTION1allC6 - 453
6X-RAY DIFFRACTION1allC500 - 510
7X-RAY DIFFRACTION1allD6 - 510
8X-RAY DIFFRACTION1allS1 - 1065

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