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- PDB-8rlc: SPNS2:sfGFP hetero dimer assembled by Di-Gluebody -

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Basic information

Entry
Database: PDB / ID: 8rlc
TitleSPNS2:sfGFP hetero dimer assembled by Di-Gluebody
Components
  • Gluebody GbC4
  • Gluebody GbEnhancer
  • Green fluorescent protein
  • Sphingosine-1-phosphate transporter SPNS2
KeywordsMEMBRANE PROTEIN / SPNS2 / Di-Gluebody / sfGFP
Function / homology
Function and homology information


regulation of eye pigmentation / regulation of humoral immune response / regulation of T cell migration / sphingolipid transporter activity / lymphocyte migration / sphingolipid biosynthetic process / Sphingolipid de novo biosynthesis / sphingosine-1-phosphate receptor signaling pathway / lipid transport / T cell homeostasis ...regulation of eye pigmentation / regulation of humoral immune response / regulation of T cell migration / sphingolipid transporter activity / lymphocyte migration / sphingolipid biosynthetic process / Sphingolipid de novo biosynthesis / sphingosine-1-phosphate receptor signaling pathway / lipid transport / T cell homeostasis / B cell homeostasis / transmembrane transporter activity / lymph node development / bioluminescence / generation of precursor metabolites and energy / sensory perception of sound / bone development / endosome membrane / membrane / plasma membrane
Similarity search - Function
Protein spinster-like / Major facilitator superfamily / Major Facilitator Superfamily / Major facilitator superfamily domain / Major facilitator superfamily (MFS) profile. / MFS transporter superfamily / Green fluorescent protein, GFP / Green fluorescent protein-related / Green fluorescent protein / Green fluorescent protein
Similarity search - Domain/homology
Green fluorescent protein / Sphingosine-1-phosphate transporter SPNS2
Similarity search - Component
Biological speciesAequorea victoria (jellyfish)
Lama glama (llama)
Homo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.9 Å
AuthorsYi, G. / Ye, M. / Mamalis, D. / Li, H. / 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

Downloads & links

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Assembly

Deposited unit
B: Green fluorescent protein
D: Gluebody GbEnhancer
C: Gluebody GbC4
A: Sphingosine-1-phosphate transporter SPNS2
hetero molecules


Theoretical massNumber of molelcules
Total (without water)111,8775
Polymers111,3664
Non-polymers5111
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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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: 12602.062 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)
#4: Protein Sphingosine-1-phosphate transporter SPNS2


Mass: 58962.211 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: SPNS2 / Production host: Homo sapiens (human) / References: UniProt: Q8IVW8
#5: Sugar ChemComp-LMT / DODECYL-BETA-D-MALTOSIDE


Type: D-saccharide / Mass: 510.615 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C24H46O11 / Feature type: SUBJECT OF INVESTIGATION / Comment: detergent*YM
Has ligand of interestY
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
1SPNS2:sfGFP heterodimer assembled by Di-GluebodyCOMPLEXGbEnhancer and GbC4 were assembled via a disulfide to form a hetero Di-Gluebody#1-#40MULTIPLE SOURCES
2Green fluorescent proteinCOMPLEXGreen fluorescent protein#11RECOMBINANT
3Di-GluebodyCOMPLEXGluebody GbEnhancer and Gluebody GbC4#2-#31RECOMBINANT
4Sphingosine-1-phosphate transporter SPNS2COMPLEXSphingosine-1-phosphate transporter SPNS2#41RECOMBINANT
Molecular weightValue: 0.112 MDa / Experimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Aequorea victoria (jellyfish)6100
33Lama glama (llama)9844
44Homo sapiens (human)9606
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
22Escherichia coli (E. coli)562
33Escherichia coli (E. coli)562
44Homo sapiens (human)9606
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: GATAN K3 (6k 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: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 321178 / Symmetry type: POINT
Atomic model buildingB value: 131.4 / Space: REAL
Atomic model building
IDPDB-ID 3D fitting-IDAccession codeInitial refinement model-IDSource nameType
18QV6

8qv6

18QV61PDBexperimental model
23K1K

3k1k

13K1K2PDBexperimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0026744
ELECTRON MICROSCOPYf_angle_d0.4449160
ELECTRON MICROSCOPYf_dihedral_angle_d4.598949
ELECTRON MICROSCOPYf_chiral_restr0.0381037
ELECTRON MICROSCOPYf_plane_restr0.0021167

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