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- PDB-5ldf: Maltose binding protein genetically fused to dodecameric glutamin... -

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Basic information

Entry
Database: PDB / ID: 5ldf
TitleMaltose binding protein genetically fused to dodecameric glutamine synthetase
Components
  • Glutamine synthetase
  • Maltose-binding periplasmic protein
KeywordsLIGASE / Fusion protein / chimera / dodecamer / symmetrized construct
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
alpha-maltose / Glutamine synthetase / Maltose/maltodextrin-binding periplasmic protein
Similarity search - Component
Biological speciesSalmonella typhi (bacteria)
Escherichia coli O157:H7 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 6.2 Å
AuthorsCoscia, F. / Petosa, C. / Schoehn, G.
Funding support France, 1items
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, 2016Deposition site: PDBE / Processing site: PDBE
Revision 1.0Aug 10, 2016Provider: repository / Type: Initial release
Revision 1.1Aug 2, 2017Group: Data collection / Derived calculations / Category: em_software / struct_conf / Item: _em_software.name / _em_software.version
Revision 1.2Nov 21, 2018Group: Advisory / Data collection / Derived calculations / Category: pdbx_validate_close_contact / struct_conn
Revision 1.3Oct 2, 2019Group: Data collection / Other / Category: atom_sites / cell
Item: _atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] ..._atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] / _atom_sites.fract_transf_matrix[3][3] / _cell.length_a / _cell.length_b / _cell.length_c
Revision 1.4Oct 23, 2019Group: Data collection / Other / Category: cell / Item: _cell.Z_PDB
Revision 2.0Jul 29, 2020Group: Atomic model / Data collection ...Atomic model / Data collection / Derived calculations / Non-polymer description / Source and taxonomy / Structure summary
Category: atom_site / chem_comp ...atom_site / chem_comp / em_entity_assembly / entity / entity_name_com / entity_src_gen / pdbx_branch_scheme / pdbx_chem_comp_identifier / pdbx_entity_branch / pdbx_entity_branch_descriptor / pdbx_entity_branch_link / pdbx_entity_branch_list / pdbx_entity_nonpoly / pdbx_molecule_features / pdbx_nonpoly_scheme / struct_conn / struct_site / struct_site_gen
Item: _atom_site.Cartn_x / _atom_site.Cartn_y ..._atom_site.Cartn_x / _atom_site.Cartn_y / _atom_site.Cartn_z / _atom_site.auth_asym_id / _atom_site.auth_atom_id / _atom_site.auth_comp_id / _atom_site.auth_seq_id / _atom_site.label_atom_id / _atom_site.label_comp_id / _chem_comp.formula / _chem_comp.formula_weight / _chem_comp.id / _chem_comp.mon_nstd_flag / _chem_comp.name / _chem_comp.type / _em_entity_assembly.entity_id_list / _entity.formula_weight / _entity.pdbx_description / _entity.type
Description: Carbohydrate remediation / Provider: repository / Type: Remediation

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Structure visualization

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Structure viewerMolecule:
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Assembly

Deposited unit
A: Glutamine synthetase
B: Glutamine synthetase
C: Glutamine synthetase
D: Glutamine synthetase
E: Glutamine synthetase
F: Glutamine synthetase
G: Glutamine synthetase
H: Glutamine synthetase
I: Glutamine synthetase
J: Glutamine synthetase
K: Glutamine synthetase
L: Glutamine synthetase
M: Maltose-binding periplasmic protein
N: Maltose-binding periplasmic protein
O: Maltose-binding periplasmic protein
P: Maltose-binding periplasmic protein
Q: Maltose-binding periplasmic protein
R: Maltose-binding periplasmic protein
S: Maltose-binding periplasmic protein
T: Maltose-binding periplasmic protein
U: Maltose-binding periplasmic protein
V: Maltose-binding periplasmic protein
W: Maltose-binding periplasmic protein
X: Maltose-binding periplasmic protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)1,112,18136
Polymers1,108,07324
Non-polymers4,10812
Water0
1


  • Idetical with deposited unit
  • defined by software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area93370 Å2
ΔGint-420 kcal/mol
Surface area347990 Å2
MethodPISA

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Components

#1: Protein
Glutamine synthetase / / Glutamate--ammonia ligase


Mass: 51586.266 Da / Num. of mol.: 12 / Mutation: Deletion of residues 1-2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Salmonella typhi (bacteria) / Gene: glnA, STY3874, t3614 / Production host: Escherichia coli (E. coli) / References: UniProt: P0A1P7, glutamine synthetase
#2: Protein
Maltose-binding periplasmic protein / MBP / MMBP / Maltodextrin-binding protein


Mass: 40753.152 Da / Num. of mol.: 12
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli O157:H7 (bacteria) / Gene: malE, Z5632, ECs5017 / Production host: Escherichia coli (E. coli) / References: UniProt: P0AEY0
#3: Polysaccharide
alpha-D-glucopyranose-(1-4)-alpha-D-glucopyranose / alpha-maltose


Type: Oligosaccharide / Class: Nutrient / Mass: 342.297 Da / Num. of mol.: 12
Source method: isolated from a genetically manipulated source
Details: oligosaccharide / References: alpha-maltose
DescriptorTypeProgram
DGlcpa1-4DGlcpa1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/1,2,1/[a2122h-1a_1-5]/1-1/a4-b1WURCSPDB2Glycan 1.1.0
[][a-D-Glcp]{[(4+1)][a-D-Glcp]{}}LINUCSPDB-CARE

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Maltose-binding protein genetically fused to glutamine synthetase
Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT
Molecular weightValue: 1.11 MDa / Experimental value: NO
Source (natural)Organism: Escherichia coli (E. coli)
Source (recombinant)Organism: Escherichia coli (E. coli) / Plasmid: pETM-11
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
150 mMTRIS pH 8C4H11NO31
2150 mMsodium chlorideNaClSodium chloride1
35 mMmagnesium chlorideMgCl21
410 mMmaltoseC12H22O111
SpecimenConc.: 0.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER/RHODIUM / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 290 K

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Electron microscopy imaging

Experimental equipment
Model: Tecnai F30 / Image courtesy: FEI Company
MicroscopyModel: FEI TECNAI F30 / Details: Polara Top entry
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 3500 nm / Nominal defocus min: 1000 nm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN
Image recordingAverage exposure time: 6 sec. / Electron dose: 25 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of real images: 165
Image scansMovie frames/image: 40

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Processing

EM software
IDNameVersionCategory
1EMANparticle selection
2RELIONparticle selection
5CTFFIND3CTF correction
6BsoftCTF correction
9UCSF Chimera1.10.2model fitting
11IMAGICinitial Euler assignment
13RELIONclassification
14RELION3D reconstruction
CTF correctionType: PHASE FLIPPING ONLY
Particle selectionNum. of particles selected: 39167
SymmetryPoint symmetry: D6 (2x6 fold dihedral)
3D reconstructionResolution: 6.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 13847 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL

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