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- EMDB-4039: Maltose binding protein genetically fused to dodecameric glutamin... -
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Open data
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Basic information
Entry | Database: EMDB / ID: EMD-4039 | |||||||||
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Title | Maltose binding protein genetically fused to dodecameric glutamine synthetase | |||||||||
![]() | Maltose-binding protein fused to dodecameric glutamine synthetase | |||||||||
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Function / homology | ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() Similarity search - Function | |||||||||
Biological species | ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() | |||||||||
Method | ![]() ![]() | |||||||||
![]() | Coscia F / Petosa C | |||||||||
Funding support | ![]()
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![]() | ![]() Title: Fusion to a homo-oligomeric scaffold allows cryo-EM analysis of a small protein. Authors: Francesca Coscia / Leandro F Estrozi / Fabienne Hans / Hélène Malet / Marjolaine Noirclerc-Savoye / Guy Schoehn / Carlo Petosa / ![]() Abstract: Recent technical advances have revolutionized the field of cryo-electron microscopy (cryo-EM). However, most monomeric proteins remain too small (<100 kDa) for cryo-EM analysis. To overcome this limitation, we explored a strategy whereby a monomeric target protein is genetically fused to a homo-oligomeric scaffold protein and the junction optimized to allow the target to adopt the scaffold symmetry, thereby generating a chimeric particle suitable for cryo-EM. To demonstrate the concept, we fused maltose-binding protein (MBP), a 40 kDa monomer, to glutamine synthetase, a dodecamer formed by two hexameric rings. Chimeric constructs with different junction lengths were screened by biophysical analysis and negative-stain EM. The optimal construct yielded a cryo-EM reconstruction that revealed the MBP structure at sub-nanometre resolution. These findings illustrate the feasibility of using homo-oligomeric scaffolds to enable cryo-EM analysis of monomeric proteins, paving the way for applying this strategy to challenging structures resistant to crystallographic and NMR analysis. | |||||||||
History |
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Structure visualization
Movie |
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Structure viewer | EM map: ![]() ![]() ![]() |
Supplemental images |
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Downloads & links
-EMDB archive
Map data | ![]() | 191.9 MB | ![]() | |
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Header (meta data) | ![]() ![]() | 14.8 KB 14.8 KB | Display Display | ![]() |
Images | ![]() | 216 KB | ||
Filedesc metadata | ![]() | 6.4 KB | ||
Archive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Related structure data | ![]() 5ldfMC M: atomic model generated by this map C: citing same article ( |
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Similar structure data |
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Links
EMDB pages | ![]() ![]() |
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Related items in Molecule of the Month |
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Map
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Annotation | Maltose-binding protein fused to dodecameric glutamine synthetase | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 0.8155 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
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Sample components
-Entire : Maltose-binding protein genetically fused to glutamine synthetase
Entire | Name: Maltose-binding protein genetically fused to glutamine synthetase |
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Components |
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-Supramolecule #1: Maltose-binding protein genetically fused to glutamine synthetase
Supramolecule | Name: Maltose-binding protein genetically fused to glutamine synthetase type: complex / ID: 1 / Parent: 0 / Macromolecule list: all |
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Source (natural) | Organism: ![]() ![]() ![]() |
Molecular weight | Theoretical: 1.11 MDa |
-Macromolecule #1: Glutamine synthetase
Macromolecule | Name: Glutamine synthetase / type: protein_or_peptide / ID: 1 / Number of copies: 12 / Enantiomer: LEVO / EC number: ![]() |
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Source (natural) | Organism: ![]() ![]() |
Molecular weight | Theoretical: 51.586266 KDa |
Recombinant expression | Organism: ![]() ![]() ![]() |
Sequence | String: EHVLTMLNEH EVKFVDLRFT DTKGKEQHVT IPAHQVNAEF FEEGKMFDGS SIGGWKGINE SDMVLMPDAS TAVIDPFFAD STLIIRCDI LEPGTLQGYD RDPRSIAKRA EDYLRATGIA DTVLFGPEPE FFLFDDIRFG ASISGSHVAI DDIEGAWNSS T KYEGGNKG ...String: EHVLTMLNEH EVKFVDLRFT DTKGKEQHVT IPAHQVNAEF FEEGKMFDGS SIGGWKGINE SDMVLMPDAS TAVIDPFFAD STLIIRCDI LEPGTLQGYD RDPRSIAKRA EDYLRATGIA DTVLFGPEPE FFLFDDIRFG ASISGSHVAI DDIEGAWNSS T KYEGGNKG HRPGVKGGYF PVPPVDSAQD IRSEMCLVME QMGLVVEAHH HEVATAGQNE VATRFNTMTK KADEIQIYKY VV HNVAHRF GKTATFMPKP MFGDNGSGMH CHMSLAKNGT NLFSGDKYAG LSEQALYYIG GVIKHAKAIN ALANPTTNSY KRL VPGYEA PVMLAYSARN RSASIRIPVV ASPKARRIEV RFPDPAANPY LCFAALLMAG LDGIKNKIHP GEPMDKNLYD LPPE EAKEI PQVAGSLEEA LNALDLDREF LKAGGVFTDE AIDAYIALRR EEDDRVRMTP HPVEFELYYS V UniProtKB: ![]() |
-Macromolecule #2: Maltose-binding periplasmic protein
Macromolecule | Name: Maltose-binding periplasmic protein / type: protein_or_peptide / ID: 2 / Number of copies: 12 / Enantiomer: LEVO |
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Source (natural) | Organism: ![]() ![]() ![]() |
Molecular weight | Theoretical: 40.753152 KDa |
Recombinant expression | Organism: ![]() ![]() ![]() |
Sequence | String: KIEEGKLVIW INGDKGYNGL AEVGKKFEKD TGIKVTVEHP DKLEEKFPQV AATGDGPDII FWAHDRFGGY AQSGLLAEIT PDKAFQDKL YPFTWDAVRY NGKLIAYPIA VEALSLIYNK DLLPNPPKTW EEIPALDKEL KAKGKSALMF NLQEPYFTWP L IAADGGYA ...String: KIEEGKLVIW INGDKGYNGL AEVGKKFEKD TGIKVTVEHP DKLEEKFPQV AATGDGPDII FWAHDRFGGY AQSGLLAEIT PDKAFQDKL YPFTWDAVRY NGKLIAYPIA VEALSLIYNK DLLPNPPKTW EEIPALDKEL KAKGKSALMF NLQEPYFTWP L IAADGGYA FKYENGKYDI KDVGVDNAGA KAGLTFLVDL IKNKHMNADT DYSIAEAAFN KGETAMTING PWAWSNIDTS KV NYGVTVL PTFKGQPSKP FVGVLSAGIN AASPNKELAK EFLENYLLTD EGLEAVNKDK PLGAVALKSY EEELAKDPRI AAT MENAQK GEIMPNIPQM SAFWYAVRTA VINAASGRQT VDEALKDAQT RITK UniProtKB: Maltose/maltodextrin-binding periplasmic protein |
-Experimental details
-Structure determination
Method | ![]() |
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Aggregation state | particle |
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Sample preparation
Concentration | 0.5 mg/mL | |||||||||||||||
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Buffer | pH: 8 Component:
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Grid | Model: Quantifoil / Material: COPPER/RHODIUM / Mesh: 400 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec. | |||||||||||||||
Vitrification | Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 290 K / Instrument: FEI VITROBOT MARK IV |
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Electron microscopy
Microscope | FEI TECNAI F30 |
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Electron beam | Acceleration voltage: 300 kV / Electron source: ![]() |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD![]() |
Sample stage | Cooling holder cryogen: NITROGEN |
Details | Polara Top entry |
Image recording | Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Number real images: 165 / Average exposure time: 6.0 sec. / Average electron dose: 25.0 e/Å2 |
Experimental equipment | ![]() Model: Tecnai F30 / Image courtesy: FEI Company |
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Image processing
Particle selection | Number selected: 39167 |
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Startup model | Type of model: OTHER Details: Reference models were generated by angular reconstitution using IMAGIC and volumes refined by projection matching using SPIDER. As a control to check for possible model bias, we generated an ...Details: Reference models were generated by angular reconstitution using IMAGIC and volumes refined by projection matching using SPIDER. As a control to check for possible model bias, we generated an alternative ab initio reference model using RIco, which uses symmetry adapted functions. |
Initial angle assignment | Type: ANGULAR RECONSTITUTION / Software - Name: IMAGIC |
Final 3D classification | Software - Name: RELION |
Final angle assignment | Type: PROJECTION MATCHING |
Final reconstruction | Applied symmetry - Point group: D6 (2x6 fold dihedral![]() |
-Atomic model buiding 1
Refinement | Space: REAL / Protocol: RIGID BODY FIT |
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Output model | ![]() PDB-5ldf: |