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- PDB-8rle: SPNS2:sfGFP hetero dimer assembled by Di-Gluebody - sfGFP local r... -

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Basic information

Entry
Database: PDB / ID: 8rle
TitleSPNS2:sfGFP hetero dimer assembled by Di-Gluebody - sfGFP local refinement
Components
  • Gluebody GbC4
  • Gluebody GbEnhancer
  • Green fluorescent protein
KeywordsHYDROLASE / DNA helicase / Di-Gluebody
Function / homologyGreen fluorescent protein, GFP / Green fluorescent protein-related / Green fluorescent protein / Green fluorescent protein / bioluminescence / generation of precursor metabolites and energy / Green fluorescent protein
Function and homology information
Biological speciesAequorea victoria (jellyfish)
Lama glama (llama)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.75 Å
AuthorsYi, G. / Ye, M. / Mamalis, D. / Sauer, D.B. / von Delft, F. / Davis, B.G. / Gilbert, R.J.C.
Funding support United Kingdom, 1items
OrganizationGrant numberCountry
Other private United Kingdom
CitationJournal: To Be Published
Title: Di-Gluebodies: Rigid modular nanobody protein assemblies enabling simultaneous determination of high-resolution cryo-EM structures
Authors: Yi, G. / Mamalis, D. / Ye, M. / Carrique, L. / Fairhead, M. / Li, H. / Duerr, K. / Zhang, P. / Sauer, D.B. / von Delft, F. / Davis, B.G. / Gilbert, R.J.C.
History
DepositionJan 2, 2024Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jan 15, 2025Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
B: Green fluorescent protein
D: Gluebody GbEnhancer
C: Gluebody GbC4


Theoretical massNumber of molelcules
Total (without water)52,4913
Polymers52,4913
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Green fluorescent protein


Mass: 26819.230 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Aequorea victoria (jellyfish) / Gene: gfp / Production host: Escherichia coli (E. coli) / References: UniProt: A0A059PIQ0
#2: Antibody Gluebody GbEnhancer


Mass: 12689.139 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Lama glama (llama) / Production host: Escherichia coli (E. coli)
#3: Antibody Gluebody GbC4


Mass: 12982.609 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Lama glama (llama) / Production host: Escherichia coli (E. coli)
Has protein modificationY

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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

Component
IDNameTypeDetailsEntity IDParent-IDSource
1Local refinement of GFP part of the SPNS2:sfGFP heterodimer assembled by Di-GluebodyCOMPLEXGbEnhancer and GbC4 were assembled via a disulfide to form a hetero Di-Gluebodyall0MULTIPLE SOURCES
2Green fluorescent proteinCOMPLEXGreen fluorescent protein1RECOMBINANT
3Di-GluebodyCOMPLEXGluebody GbEnhancer and Gluebody GbC41RECOMBINANT
Molecular weightValue: 0.112 MDa / Experimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Aequorea victoria (jellyfish)6100
33Lama glama (llama)9844
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
22Escherichia coli (E. coli)562
33Escherichia coli (E. coli)562
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMHEPESC8H18N2O4S1
2150 mMsodium chlorideNaCl1
30.025 %DDMC24H46O111
SpecimenConc.: 4.2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2500 nm / Nominal defocus min: 1500 nm
Image recordingElectron dose: 50 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k) / Num. of real images: 8674

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Processing

EM software
IDNameVersionCategory
1cryoSPARC4particle selection
2EPUimage acquisition
4cryoSPARC4CTF correction
7UCSF ChimeraXmodel fitting
9PHENIXmodel refinement
10Cootmodel refinement
11cryoSPARC4initial Euler assignment
12cryoSPARC4final Euler assignment
14cryoSPARC43D reconstruction
CTF correctionDetails: patch-ctf correction in cryoSPARC / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1127724
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.75 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 321178 / Symmetry type: POINT
Atomic model buildingB value: 154.9 / Space: REAL
Atomic model buildingPDB-ID: 3K1K
Accession code: 3K1K / Source name: PDB / Type: experimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0023502
ELECTRON MICROSCOPYf_angle_d0.5114752
ELECTRON MICROSCOPYf_dihedral_angle_d4.116493
ELECTRON MICROSCOPYf_chiral_restr0.044512
ELECTRON MICROSCOPYf_plane_restr0.004630

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