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- EMDB-0789: Negatively stained reconstruction of a rubisco activase -

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Basic information

Entry
Database: EMDB / ID: EMD-0789
TitleNegatively stained reconstruction of a rubisco activase
Map dataNegative stained 3D map of the rubisco activase
Sample
  • Complex: CBBQO type Rubisco activase
    • Protein or peptide: Rubisco activase
Biological speciesAcidithiobacillus ferrooxidans (bacteria)
Methodsingle particle reconstruction / negative staining / Resolution: 16.0 Å
AuthorsTsai Y / Liu D / Bhushan S / Mueller-Cajar O
Funding support Singapore, 1 items
OrganizationGrant numberCountry
Ministry of Education (Czech Republic)MOE2016-T2-02-088, MOE2015-T2-1-078, MOE2017-T2-2-089 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 24, 2019-
Header (metadata) releaseDec 18, 2019-
Map releaseDec 18, 2019-
UpdateDec 18, 2019-
Current statusDec 18, 2019Processing site: PDBj / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0452
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.0452
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_0789.png

Downloads & links

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Map

FileDownload / File: emd_0789.map.gz / Format: CCP4 / Size: 489.3 KB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationNegative stained 3D map of the rubisco activase
Voxel sizeX=Y=Z: 4.19 Å
Density
Contour LevelBy AUTHOR: 0.0452 / Movie #1: 0.0452
Minimum - Maximum-0.07980231 - 0.36390454
Average (Standard dev.)0.004813181 (±0.02760975)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions505050
Spacing505050
CellA=B=C: 209.5 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z4.194.194.19
M x/y/z505050
origin x/y/z0.0000.0000.000
length x/y/z209.500209.500209.500
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS505050
D min/max/mean-0.0800.3640.005

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Supplemental data

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Sample components

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Entire : CBBQO type Rubisco activase

EntireName: CBBQO type Rubisco activase
Components
  • Complex: CBBQO type Rubisco activase
    • Protein or peptide: Rubisco activase

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Supramolecule #1: CBBQO type Rubisco activase

SupramoleculeName: CBBQO type Rubisco activase / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Acidithiobacillus ferrooxidans (bacteria)
Recombinant expressionOrganism: Escherichia coli (E. coli)
Molecular weightExperimental: 0.27 kDa/nm

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Macromolecule #1: Rubisco activase

MacromoleculeName: Rubisco activase / type: protein_or_peptide / ID: 1 / Enantiomer: DEXTRO
Source (natural)Organism: Acidithiobacillus ferrooxidans (bacteria)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MTATDSSILN QYLVGKEPFY QPQHDEVALF EAAYRKRLPV MVKGPTGCGK SRFVEFMAWR LGKPLVTVAC NEDMTAADLV GRWLLDKDGT RWQDGPLTVA ARYGAICYLD EIVEARQDTT VVIHPLTDHR RTLPLDKKGE LIRAHPDFQL VISYNPGYQS LMKDLKQSTK ...String:
MTATDSSILN QYLVGKEPFY QPQHDEVALF EAAYRKRLPV MVKGPTGCGK SRFVEFMAWR LGKPLVTVAC NEDMTAADLV GRWLLDKDGT RWQDGPLTVA ARYGAICYLD EIVEARQDTT VVIHPLTDHR RTLPLDKKGE LIRAHPDFQL VISYNPGYQS LMKDLKQSTK QRFTGFEFDY PNAELEAGIL VQETGVAPSI AAQLVTVAAT ARRLKGHGLD EGISTRLLVY AAMLMDDGVA PRAACRMALV QPITDDADIR ATLEHAIDMT FA MTDHPRD ASLPSRLAAY RKQLDCRFPR VGEVFSDCIA KASARLGPAG VSAYVDAARA LCKLGRGEEP VLIFLEEWPD VGAALGDGTL EMVMQMVQFM QRTPNGNAIG GFLQSLAPVS RALLSREQLG HYLNTLRDMM ERTTGSIHGH HQTHPSPGLP ELLRQAPTLL QSLTVDGLRN WVDYGVRNYL HHPERQKDFF SLQSADSRAV LQRERHGTLL ADVERKLDLY LRGLWQDSEV LVPYSTAFAT LRTPQPYYDA LGMRLPDVLD DLPGIGALDH YRAILAHMVG HRRWSTPQIA DNWSPFQRLA VEFFEDARID TLLIRTYPGL RTLFLALHPK PGEDACDPET TSCLRHRLAM LSRALLDPEH GYRNPVLHDF VDRFHGELAG GNADTATMAR LALDYVTTTR RQSDQFAKVH FADTEISYRD DNRGLWRFIE SGDEEEAFDA EQRKAPDLET QGLPPRHYPE WDYQSQSYRP DWVSLYEGLH ASAPAATIDQ LLLKHAALAK HLKRLLDLLK PQDKVRIRYQ EEGSELDLDV ALRSWIDFKS GSTPDPRINM SHRTAGRDIA VTLLLDLSES LNESVKTGGG DGQTVLQLSQ EAVSLLAWSI EQLGDPLAIA GFNSNTRHEV RYQHIKGFSE PWGDVVKGRL AALQAGYSTR MGAAMRHAGH YLATRKADKK LMLVLTDGRP SDVDVQDDRL LIEDARQAVN ELDRDGIFTY CISLDPHADA YVADIFGRQY TVIDHIARLP EKLPELFIAL TR

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Experimental details

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Structure determination

Methodnegative staining
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.05 mg/mL
BufferpH: 8 / Details: 20 mM Tris-HCl pH8.0, 50 mM NaCl
StainingType: NEGATIVE / Material: Uranyl Acetate
Details: The sample was applied to a carbon-coated TEM grid and stained with 2% (w/v) uranyl acetate
GridModel: Homemade / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Support film - Film thickness: 6.0 nm / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR
DetailsMonodisperse

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Electron microscopy

MicroscopeFEI TECNAI 12
Image recordingFilm or detector model: FEI EAGLE (4k x 4k) / Number grids imaged: 1 / Number real images: 60 / Average exposure time: 1.0 sec. / Average electron dose: 25.0 e/Å2
Electron beamAcceleration voltage: 120 kV / Electron source: LAB6
Electron opticsCalibrated defocus max: 1.5 µm / Calibrated defocus min: 1.25 µm / Calibrated magnification: 49000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 6.3 mm / Nominal defocus max: 1.5 µm / Nominal defocus min: 1.25 µm
Sample stageSpecimen holder model: GATAN 910 MULTI-SPECIMEN SINGLE TILT CRYO TRANSFER HOLDER

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Image processing

Particle selectionNumber selected: 32879
CTF correctionSoftware - Name: EMAN2
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 16.0 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 15386
Initial angle assignmentType: OTHER / Software - Name: RELION
Final angle assignmentType: PROJECTION MATCHING / Software - Name: RELION

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