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Yorodumi- EMDB-3699: Structure of Rubisco from Rhodobacter sphaeroides in complex with CABP -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-3699 | |||||||||
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Title | Structure of Rubisco from Rhodobacter sphaeroides in complex with CABP | |||||||||
Map data | postprocessed Rubisco map; D4 symmetry | |||||||||
Sample |
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Function / homology | Function and homology information ribulose-bisphosphate carboxylase / ribulose-bisphosphate carboxylase activity / reductive pentose-phosphate cycle / monooxygenase activity / magnesium ion binding Similarity search - Function | |||||||||
Biological species | Rhodobacter sphaeroides (bacteria) | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 3.39 Å | |||||||||
Authors | Bracher A / Milicic G / Ciniawsky S / Wendler P / Hayer-Hartl M / Hartl FU | |||||||||
Citation | Journal: Mol Cell / Year: 2017 Title: Mechanism of Enzyme Repair by the AAA Chaperone Rubisco Activase. Authors: Javaid Y Bhat / Goran Miličić / Gabriel Thieulin-Pardo / Andreas Bracher / Andrew Maxwell / Susanne Ciniawsky / Oliver Mueller-Cajar / John R Engen / F Ulrich Hartl / Petra Wendler / Manajit Hayer-Hartl / Abstract: How AAA+ chaperones conformationally remodel specific target proteins in an ATP-dependent manner is not well understood. Here, we investigated the mechanism of the AAA+ protein Rubisco activase (Rca) ...How AAA+ chaperones conformationally remodel specific target proteins in an ATP-dependent manner is not well understood. Here, we investigated the mechanism of the AAA+ protein Rubisco activase (Rca) in metabolic repair of the photosynthetic enzyme Rubisco, a complex of eight large (RbcL) and eight small (RbcS) subunits containing eight catalytic sites. Rubisco is prone to inhibition by tight-binding sugar phosphates, whose removal is catalyzed by Rca. We engineered a stable Rca hexamer ring and analyzed its functional interaction with Rubisco. Hydrogen/deuterium exchange and chemical crosslinking showed that Rca structurally destabilizes elements of the Rubisco active site with remarkable selectivity. Cryo-electron microscopy revealed that Rca docks onto Rubisco over one active site at a time, positioning the C-terminal strand of RbcL, which stabilizes the catalytic center, for access to the Rca hexamer pore. The pulling force of Rca is fine-tuned to avoid global destabilization and allow for precise enzyme repair. | |||||||||
History |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | EM map: SurfViewMolmilJmol/JSmol |
Supplemental images |
-Downloads & links
-EMDB archive
Map data | emd_3699.map.gz | 10.7 MB | EMDB map data format | |
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Header (meta data) | emd-3699-v30.xml emd-3699.xml | 17.4 KB 17.4 KB | Display Display | EMDB header |
Images | emd_3699.png | 296.7 KB | ||
Others | emd_3699_additional.map.gz | 94.9 MB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-3699 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-3699 | HTTPS FTP |
-Validation report
Summary document | emd_3699_validation.pdf.gz | 261.8 KB | Display | EMDB validaton report |
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Full document | emd_3699_full_validation.pdf.gz | 261 KB | Display | |
Data in XML | emd_3699_validation.xml.gz | 6.5 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-3699 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-3699 | HTTPS FTP |
-Related structure data
Related structure data | 5nv3MC 3700C 3701C 3702C M: atomic model generated by this map C: citing same article (ref.) |
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Similar structure data |
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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Related items in Molecule of the Month |
-Map
File | Download / File: emd_3699.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | postprocessed Rubisco map; D4 symmetry | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Projections & slices | Image control
Images are generated by Spider. | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 1.04 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Additional map: unfiltered Rubisco map; D4 symmetry
File | emd_3699_additional.map | ||||||||||||
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Annotation | unfiltered Rubisco map; D4 symmetry | ||||||||||||
Projections & Slices |
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Density Histograms |
-Sample components
-Entire : Rubisco
Entire | Name: Rubisco |
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Components |
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-Supramolecule #1: Rubisco
Supramolecule | Name: Rubisco / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2 / Details: Rubisco was treated with the inhibitor CABP. |
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Source (natural) | Organism: Rhodobacter sphaeroides (bacteria) |
Recombinant expression | Organism: Escherichia coli BL21(DE3) (bacteria) |
-Macromolecule #1: Ribulose bisphosphate carboxylase large chain
Macromolecule | Name: Ribulose bisphosphate carboxylase large chain / type: protein_or_peptide / ID: 1 / Number of copies: 8 / Enantiomer: LEVO / EC number: ribulose-bisphosphate carboxylase |
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Source (natural) | Organism: Rhodobacter sphaeroides (bacteria) |
Molecular weight | Theoretical: 51.768852 KDa |
Recombinant expression | Organism: Escherichia coli BL21(DE3) (bacteria) |
Sequence | String: RYKAGVLKYA QMGYWDGDYV PKDTDVLALF RITPQEGVDP VEAAAAVAGE SSTATWTVVW TDRLTACDSY RAKAYRVEPV PGTPGQYFC YVAYDLILFE EGSIANLTAS IIGNVFSFKP LKAARLEDMR FPVAYVKTYK GPPTGIVGER ERLDKFGKPL L GATTKPKL ...String: RYKAGVLKYA QMGYWDGDYV PKDTDVLALF RITPQEGVDP VEAAAAVAGE SSTATWTVVW TDRLTACDSY RAKAYRVEPV PGTPGQYFC YVAYDLILFE EGSIANLTAS IIGNVFSFKP LKAARLEDMR FPVAYVKTYK GPPTGIVGER ERLDKFGKPL L GATTKPKL GLSGKNYGRV VYEGLKGGLD FM(KCX)DDENINS QPFMHWRDRF LYVMEAVNLA SAQTGEVKGH YLNITAGT M EEMYRRAEFA KSLGSVIVMV DLIIGYTAIQ SISEWCRQND MILHMHRAGH GTYTRQKNHG ISFRVIAKWL RLAGVDHLH CGTAVGKLEG DPLTVQGYYN VCREPFNTVD LPRGIFFEQD WADLRKVMPV ASGGIHAGQM HQLLSLFGDD VVLQFGGGTI GHPMGIQAG ATANRVALEA MVLARNEGRN IDVEGPEILR AAAKWCKPLE AALDTWGNIT FNYTSTDTSD FV |
-Macromolecule #2: Ribulose bisphosphate carboxylase small chain 1
Macromolecule | Name: Ribulose bisphosphate carboxylase small chain 1 / type: protein_or_peptide / ID: 2 / Number of copies: 8 / Enantiomer: LEVO / EC number: ribulose-bisphosphate carboxylase |
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Source (natural) | Organism: Rhodobacter sphaeroides (bacteria) |
Molecular weight | Theoretical: 15.183234 KDa |
Recombinant expression | Organism: Escherichia coli BL21(DE3) (bacteria) |
Sequence | String: MRITQGCFSF LPDLTDEQIS AQVDYCLGRG WAVSLEHTDD PHPRNTYWEM WGMPMFDLRD PKGVMIELDE CRKAWPGRYI RINAFDSTR GFETVTMSFI VNRPEVEPSL RMERTEVDGR SIRYTHSIVR |
-Macromolecule #3: 2-CARBOXYARABINITOL-1,5-DIPHOSPHATE
Macromolecule | Name: 2-CARBOXYARABINITOL-1,5-DIPHOSPHATE / type: ligand / ID: 3 / Number of copies: 8 / Formula: CAP |
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Molecular weight | Theoretical: 356.115 Da |
Chemical component information | ChemComp-CAP: |
-Macromolecule #4: MAGNESIUM ION
Macromolecule | Name: MAGNESIUM ION / type: ligand / ID: 4 / Number of copies: 8 / Formula: MG |
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Molecular weight | Theoretical: 24.305 Da |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 0.3 mg/mL | ||||||||||||||||||
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Buffer | pH: 8 Component:
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Vitrification | Cryogen name: ETHANE / Instrument: FEI VITROBOT MARK IV | ||||||||||||||||||
Details | RcaCC hexamers (20 micromolar monomer) were mixed with E.C.M-CABP octamers (10 micromolar monomer) in a reaction containing 20 mM HEPES pH 7.5, 50 mM NaCl, 10 mM MgCl2, 10 mM ATP and 1mM RuBP, for 1 min at 25oC prior to addition of 0.125 % of glutaraldehyde (GA). After 10 min the reaction was quenched by addition of 0.1M Tris HCl pH 8 followed by gel filtration on a Superdex 200 PC 3.2/30 column (GE Healthcare).The fractions were eluted in buffer A and analyzed on a 6 % native gel. Fraction 13 containing HMW complexes with the least amount of free Rubisco were chosen for cryo-EM. The crosslinked E.C.M.-CABP-RcaCC complexes were diluted to 0.0030-0.0035 g ml-1 in 20 mM Tris-HCl pH 8.0, 50 mM NaCl, 1 mM ATP, 1 mM ATP-gammaS and 1 mM RuBP |
-Electron microscopy
Microscope | FEI TITAN KRIOS |
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Details | Cs corrected Krios 1 at NeCEN (June 2016) |
Image recording | Film or detector model: FEI FALCON II (4k x 4k) / Average exposure time: 1.25 sec. / Average electron dose: 50.0 e/Å2 |
Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD |
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
+Image processing
-Atomic model buiding 1
Refinement | Space: RECIPROCAL / Protocol: OTHER / Target criteria: Maximum likelihood |
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Output model | PDB-5nv3: |