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- EMDB-4039: Maltose binding protein genetically fused to dodecameric glutamin... -

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Basic information

Entry
Database: EMDB / ID: EMD-4039
TitleMaltose binding protein genetically fused to dodecameric glutamine synthetase
Map dataMaltose-binding protein fused to dodecameric glutamine synthetase
Sample
  • Complex: Maltose-binding protein genetically fused to glutamine synthetase
    • Protein or peptide: Glutamine synthetase
    • Protein or peptide: Maltose-binding periplasmic protein
Function / homology
Function and homology information


glutamine synthetase / glutamine biosynthetic process / glutamine synthetase activity / carbohydrate transmembrane transporter activity / outer membrane-bounded periplasmic space / ATP binding / metal ion binding / cytoplasm
Similarity search - Function
Glutamine synthetase class-I, adenylation site / Glutamine synthetase class-I adenylation site. / Glutamine synthetase type I / Glutamine synthetase, N-terminal conserved site / Glutamine synthetase signature 1. / Glutamine synthetase, beta-Grasp domain / Glutamine synthetase, glycine-rich site / Glutamine synthetase putative ATP-binding region signature. / Glutamine synthetase, N-terminal domain / Glutamine synthetase, N-terminal domain superfamily ...Glutamine synthetase class-I, adenylation site / Glutamine synthetase class-I adenylation site. / Glutamine synthetase type I / Glutamine synthetase, N-terminal conserved site / Glutamine synthetase signature 1. / Glutamine synthetase, beta-Grasp domain / Glutamine synthetase, glycine-rich site / Glutamine synthetase putative ATP-binding region signature. / Glutamine synthetase, N-terminal domain / Glutamine synthetase, N-terminal domain superfamily / Glutamine synthetase, catalytic domain / Glutamine synthetase, catalytic domain / Glutamine synthetase, catalytic domain / Glutamine synthetase/guanido kinase, catalytic domain / Maltose/Cyclodextrin ABC transporter, substrate-binding protein / Solute-binding family 1, conserved site / Bacterial extracellular solute-binding proteins, family 1 signature. / Bacterial extracellular solute-binding protein / Bacterial extracellular solute-binding protein
Similarity search - Domain/homology
Glutamine synthetase / Maltose/maltodextrin-binding periplasmic protein
Similarity search - Component
Biological speciesEscherichia coli (E. coli) / Salmonella typhi (bacteria) / Escherichia coli O157:H7 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 6.2 Å
AuthorsCoscia F / Petosa C / Schoehn G
Funding support France, 1 items
OrganizationGrant numberCountry
Nanosciences Foundation France
CitationJournal: Sci Rep / Year: 2016
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
DepositionJun 25, 2016-
Header (metadata) releaseAug 10, 2016-
Map releaseAug 10, 2016-
UpdateJul 29, 2020-
Current statusJul 29, 2020Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0015
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.0015
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-5ldf
  • Surface level: 0.0015
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_4039.png

Downloads & links

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Map

FileDownload / File: emd_4039.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationMaltose-binding protein fused to dodecameric glutamine synthetase
Voxel sizeX=Y=Z: 0.8155 Å
Density
Contour LevelBy AUTHOR: 0.0015 / Movie #1: 0.0015
Minimum - Maximum-0.0026587392 - 0.008522521
Average (Standard dev.)-0.00003801997 (±0.0005987295)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions400400400
Spacing400400400
CellA=B=C: 326.2 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.81550.81550.8155
M x/y/z400400400
origin x/y/z0.0000.0000.000
length x/y/z326.200326.200326.200
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS400400400
D min/max/mean-0.0030.009-0.000

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Supplemental data

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Sample components

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Entire : Maltose-binding protein genetically fused to glutamine synthetase

EntireName: Maltose-binding protein genetically fused to glutamine synthetase
Components
  • Complex: Maltose-binding protein genetically fused to glutamine synthetase
    • Protein or peptide: Glutamine synthetase
    • Protein or peptide: Maltose-binding periplasmic protein

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Supramolecule #1: Maltose-binding protein genetically fused to glutamine synthetase

SupramoleculeName: Maltose-binding protein genetically fused to glutamine synthetase
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Escherichia coli (E. coli)
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant plasmid: pETM-11
Molecular weightTheoretical: 1.11 MDa

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Macromolecule #1: Glutamine synthetase

MacromoleculeName: Glutamine synthetase / type: protein_or_peptide / ID: 1 / Number of copies: 12 / Enantiomer: LEVO / EC number: glutamine synthetase
Source (natural)Organism: Salmonella typhi (bacteria)
Molecular weightTheoretical: 51.586266 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: 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

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Macromolecule #2: Maltose-binding periplasmic protein

MacromoleculeName: Maltose-binding periplasmic protein / type: protein_or_peptide / ID: 2 / Number of copies: 12 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli O157:H7 (bacteria)
Molecular weightTheoretical: 40.753152 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: 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

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.5 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
50.0 mMC4H11NO3TRIS pH 8
150.0 mMNaClSodium chloridesodium chloride
5.0 mMMgCl2magnesium chloride
10.0 mMC12H22O11maltose
GridModel: Quantifoil / Material: COPPER/RHODIUM / Mesh: 400 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 290 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TECNAI F30
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 3.5 µm / Nominal defocus min: 1.0 µm
Sample stageCooling holder cryogen: NITROGEN
DetailsPolara Top entry
Image recordingFilm 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 selectionNumber selected: 39167
CTF correctionSoftware: (Name: CTFFIND (ver. 3), Bsoft)
Startup modelType 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 assignmentType: ANGULAR RECONSTITUTION / Software - Name: IMAGIC
Final 3D classificationSoftware - Name: RELION
Final angle assignmentType: PROJECTION MATCHING
Final reconstructionApplied symmetry - Point group: D6 (2x6 fold dihedral) / Resolution.type: BY AUTHOR / Resolution: 6.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Number images used: 13847

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-5ldf:
Maltose binding protein genetically fused to dodecameric glutamine synthetase

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