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- PDB-6l1q: Crystal structure of AfCbbQ2, a MoxR AAA+-ATPase and CbbQO-type R... -

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Basic information

Entry
Database: PDB / ID: 6l1q
TitleCrystal structure of AfCbbQ2, a MoxR AAA+-ATPase and CbbQO-type Rubisco activase from Acidithiobacillus ferrooxidans
ComponentsCbbQ protein
KeywordsCHAPERONE / AAA+ ATPase / cbbQO-type rubisco activase / MoxR family / Molecular chaperone
Function / homology
Function and homology information


ATP hydrolysis activity / ATP binding
Similarity search - Function
CbbQ/NirQ/NorQ, C-terminal / CbbQ/NirQ/NorQ C-terminal / ATPase, dynein-related, AAA domain / AAA domain (dynein-related subfamily) / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
ADENOSINE-5'-DIPHOSPHATE / PHOSPHATE ION / CbbQ protein
Similarity search - Component
Biological speciesAcidithiobacillus ferrooxidans ATCC 23270 (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.2 Å
AuthorsYe, F.Z. / Tsai, Y.C.C. / Mueller-Cajar, O. / Gao, Y.G.
Funding support Singapore, 1items
OrganizationGrant numberCountry
Ministry of Education (Singapore)MOE2016-T2-02-088 Singapore
CitationJournal: Proc Natl Acad Sci U S A / Year: 2020
Title: Insights into the mechanism and regulation of the CbbQO-type Rubisco activase, a MoxR AAA+ ATPase.
Authors: Yi-Chin Candace Tsai / Fuzhou Ye / Lynette Liew / Di Liu / Shashi Bhushan / Yong-Gui Gao / Oliver Mueller-Cajar /
Abstract: The vast majority of biological carbon dioxide fixation relies on the function of ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco). In most cases the enzyme exhibits a tendency to become ...The vast majority of biological carbon dioxide fixation relies on the function of ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco). In most cases the enzyme exhibits a tendency to become inhibited by its substrate RuBP and other sugar phosphates. The inhibition is counteracted by diverse molecular chaperones known as Rubisco activases (Rcas). In some chemoautotrophic bacteria, the CbbQO-type Rca Q2O2 repairs inhibited active sites of hexameric form II Rubisco. The 2.2-Å crystal structure of the MoxR AAA+ protein CbbQ2 from reveals the helix 2 insert (H2I) that is critical for Rca function and forms the axial pore of the CbbQ hexamer. Negative-stain electron microscopy shows that the essential CbbO adaptor protein binds to the conserved, concave side of the CbbQ2 hexamer. Site-directed mutagenesis supports a model in which adenosine 5'-triphosphate (ATP)-powered movements of the H2I are transmitted to CbbO via the concave residue L85. The basal ATPase activity of Q2O2 Rca is repressed but strongly stimulated by inhibited Rubisco. The characterization of multiple variants where this repression is released indicates that binding of inhibited Rubisco to the C-terminal CbbO VWA domain initiates a signal toward the CbbQ active site that is propagated via elements that include the CbbQ α4-β4 loop, pore loop 1, and the presensor 1-β hairpin (PS1-βH). Detailed mechanistic insights into the enzyme repair chaperones of the highly diverse CO fixation machinery of Proteobacteria will facilitate their successful implementation in synthetic biology ventures.
History
DepositionSep 30, 2019Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Dec 18, 2019Provider: repository / Type: Initial release
Revision 1.1Jan 1, 2020Group: Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_PubMed / _citation.title ..._citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _citation_author.name
Revision 1.2Jan 22, 2020Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.year
Revision 1.3Nov 22, 2023Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
C: CbbQ protein
A: CbbQ protein
B: CbbQ protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)127,4969
Polymers125,9303
Non-polymers1,5676
Water3,243180
1
C: CbbQ protein
hetero molecules
x 6


Theoretical massNumber of molelcules
Total (without water)254,99318
Polymers251,8606
Non-polymers3,13312
Water1086
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation2_555-y,x-y,z1
crystal symmetry operation3_555-x+y,-x,z1
crystal symmetry operation4_555-x,-y,z1
crystal symmetry operation5_555y,-x+y,z1
crystal symmetry operation6_555x-y,x,z1
Buried area27440 Å2
ΔGint-131 kcal/mol
Surface area58780 Å2
MethodPISA
2
A: CbbQ protein
B: CbbQ protein
hetero molecules

A: CbbQ protein
B: CbbQ protein
hetero molecules

A: CbbQ protein
B: CbbQ protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)254,99318
Polymers251,8606
Non-polymers3,13312
Water1086
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation2_665-y+1,x-y+1,z1
crystal symmetry operation3_565-x+y,-x+1,z1
Buried area29960 Å2
ΔGint-120 kcal/mol
Surface area56780 Å2
MethodPISA
Unit cell
Length a, b, c (Å)167.753, 167.753, 48.291
Angle α, β, γ (deg.)90.000, 90.000, 120.000
Int Tables number168
Space group name H-MP6
Symmetry operation#1: x,y,z
#2: x-y,x,z
#3: y,-x+y,z
#4: -y,x-y,z
#5: -x+y,-x,z
#6: -x,-y,z

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Components

#1: Protein CbbQ protein / cbbQ2-type rubisco activase


Mass: 41976.648 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Acidithiobacillus ferrooxidans ATCC 23270 (bacteria)
Strain: ATCC 23270 / Gene: cbbQ-2, AFE_2156 / Plasmid: pOPThisLipo / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / References: UniProt: B7J5E4
#2: Chemical ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE


Mass: 427.201 Da / Num. of mol.: 3 / Source method: isolated from a natural source / Formula: C10H15N5O10P2 / Feature type: SUBJECT OF INVESTIGATION / Comment: ADP, energy-carrying molecule*YM
#3: Chemical ChemComp-PO4 / PHOSPHATE ION


Mass: 94.971 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: PO4
#4: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 180 / 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: 1.56 Å3/Da / Density % sol: 21.03 %
Crystal growTemperature: 293 K / Method: vapor diffusion / pH: 4.2
Details: 25% (v/v) 1,2-propanediol, phosphate-citrate pH4.2, 5% (v/v) PEG3000, 10% (v/v) glycerol

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: NSRRC / Beamline: BL13B1 / Wavelength: 1.05 Å
DetectorType: ADSC QUANTUM 315r / Detector: CCD / Date: Aug 15, 2015
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.05 Å / Relative weight: 1
ReflectionResolution: 2.2→30 Å / Num. obs: 56135 / % possible obs: 99.9 % / Redundancy: 10.5 % / Biso Wilson estimate: 28 Å2 / CC1/2: 0.99 / Rmerge(I) obs: 0.09 / Rpim(I) all: 0.04 / Net I/σ(I): 16.1
Reflection shellResolution: 2.2→2.27 Å / Redundancy: 9.7 % / Rmerge(I) obs: 0.36 / Mean I/σ(I) obs: 5.1 / Num. unique obs: 8038 / CC1/2: 0.95 / Rpim(I) all: 0.18 / % possible all: 99.5

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Processing

Software
NameVersionClassification
PHENIX1.9_1692refinement
XDSdata reduction
SCALAdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 5c3c

5c3c


Resolution: 2.2→29.056 Å / SU ML: 0.25 / Cross valid method: THROUGHOUT / σ(F): 1.34 / Phase error: 28.6 / Stereochemistry target values: ML
Details: the entry contains Friedel pairs in F_Plus/Minus columns
RfactorNum. reflection% reflection
Rfree0.238 3496 6.23 %
Rwork0.1943 52639 -
obs0.197 56135 72.8 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso max: 134.71 Å2 / Biso mean: 37.7988 Å2 / Biso min: 13.83 Å2
Refinement stepCycle: final / Resolution: 2.2→29.056 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms6151 0 96 180 6427
Biso mean--32.39 34.94 -
Num. residues----801
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0056369
X-RAY DIFFRACTIONf_angle_d0.9458682
X-RAY DIFFRACTIONf_chiral_restr0.034991
X-RAY DIFFRACTIONf_plane_restr0.0041113
X-RAY DIFFRACTIONf_dihedral_angle_d14.672317
LS refinement shell

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

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection Rwork% reflection obs (%)
2.2002-2.23030.303100.2791305
2.2303-2.26210.3886120.27991937
2.2621-2.29590.3052180.272533011
2.2959-2.33180.2863370.297352819
2.3318-2.370.244460.287970425
2.37-2.41080.262660.298599934
2.4108-2.45460.3017800.2706130945
2.4546-2.50180.23581090.27158255
2.5018-2.55290.32331190.2559187464
2.5529-2.60830.31121540.2408226479
2.6083-2.6690.33641720.2362246285
2.669-2.73570.2421770.2362274695
2.7357-2.80960.32481910.22852877100
2.8096-2.89220.25971880.23722866100
2.8922-2.98540.28791850.24272895100
2.9854-3.0920.23631970.23812881100
3.092-3.21570.25241900.21722925100
3.2157-3.36180.25421980.19992872100
3.3618-3.53880.25971840.19092872100
3.5388-3.76010.26761980.18342931100
3.7601-4.04970.16452000.15742890100
4.0497-4.45590.21861800.14972895100
4.4559-5.09770.17792010.14062871100
5.0977-6.41110.26531890.1849288099
6.4111-100.16591950.1516286399
Refinement TLS params.Method: refined / Origin x: 6.8442 Å / Origin y: 57.6726 Å / Origin z: 4.3962 Å
111213212223313233
T0.1961 Å2-0.0187 Å20.0136 Å2-0.1819 Å20.0099 Å2--0.153 Å2
L0.4225 °20.0209 °20.0209 °2--0.007 °2-0.014 °2--0.0516 °2
S-0.0122 Å °-0.0525 Å °-0.0781 Å °0.0143 Å °-0.0102 Å °0.0095 Å °0.0046 Å °-0.0167 Å °0.0215 Å °
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection detailsAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1allC6 - 272
2X-RAY DIFFRACTION1allC1001 - 2001
3X-RAY DIFFRACTION1allA6 - 272
4X-RAY DIFFRACTION1allA1002 - 2002
5X-RAY DIFFRACTION1allB6 - 272
6X-RAY DIFFRACTION1allB1003 - 2003
7X-RAY DIFFRACTION1allS1 - 80
8X-RAY DIFFRACTION1allS81 - 180

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