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- EMDB-19338: SPNS2:sfGFP hetero dimer assembled by Di-Gluebody -

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Basic information

Entry
Database: EMDB / ID: EMD-19338
TitleSPNS2:sfGFP hetero dimer assembled by Di-Gluebody
Map dataSPNS2:sfGFP hetero dimer assembled by Di-Gluebody
Sample
  • Complex: SPNS2:sfGFP heterodimer assembled by Di-Gluebody
    • Complex: Green fluorescent protein
      • Protein or peptide: Green fluorescent protein
    • Complex: Di-Gluebody
      • Protein or peptide: Gluebody GbEnhancer
      • Protein or peptide: Gluebody GbC4
    • Complex: Sphingosine-1-phosphate transporter SPNS2
      • Protein or peptide: Sphingosine-1-phosphate transporter SPNS2
  • Ligand: DODECYL-BETA-D-MALTOSIDE
KeywordsSPNS2 / Di-Gluebody / sfGFP / MEMBRANE PROTEIN
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)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.9 Å
AuthorsYi G / Ye M / Mamalis D / Li H / Sauer DB / von Delft F / Davis BG / Gilbert RJC
Funding support United Kingdom, 1 items
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 DB / von Delft F / Davis BG / Gilbert RJC
History
DepositionJan 2, 2024-
Header (metadata) releaseJan 15, 2025-
Map releaseJan 15, 2025-
UpdateJan 15, 2025-
Current statusJan 15, 2025Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_19338.png

Downloads & links

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Map

FileDownload / File: emd_19338.map.gz / Format: CCP4 / Size: 163.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSPNS2:sfGFP hetero dimer assembled by Di-Gluebody
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.93 Å/pix.
x 350 pix.
= 326.2 Å		0.93 Å/pix.
x 350 pix.
= 326.2 Å		0.93 Å/pix.
x 350 pix.
= 326.2 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.932 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.188
Minimum - Maximum-0.82444066 - 1.2261571
Average (Standard dev.)-0.00016261855 (±0.0166484)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions350350350
Spacing350350350
CellA=B=C: 326.19998 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: half map B

Fileemd_19338_half_map_1.map
Annotationhalf map B
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half map A

Fileemd_19338_half_map_2.map
Annotationhalf map A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : SPNS2:sfGFP heterodimer assembled by Di-Gluebody

EntireName: SPNS2:sfGFP heterodimer assembled by Di-Gluebody
Components
  • Complex: SPNS2:sfGFP heterodimer assembled by Di-Gluebody
    • Complex: Green fluorescent protein
      • Protein or peptide: Green fluorescent protein
    • Complex: Di-Gluebody
      • Protein or peptide: Gluebody GbEnhancer
      • Protein or peptide: Gluebody GbC4
    • Complex: Sphingosine-1-phosphate transporter SPNS2
      • Protein or peptide: Sphingosine-1-phosphate transporter SPNS2
  • Ligand: DODECYL-BETA-D-MALTOSIDE

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Supramolecule #1: SPNS2:sfGFP heterodimer assembled by Di-Gluebody

SupramoleculeName: SPNS2:sfGFP heterodimer assembled by Di-Gluebody / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4
Details: GbEnhancer and GbC4 were assembled via a disulfide to form a hetero Di-Gluebody
Molecular weightTheoretical: 112 KDa

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Supramolecule #2: Green fluorescent protein

SupramoleculeName: Green fluorescent protein / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1 / Details: Green fluorescent protein
Source (natural)Organism: Aequorea victoria (jellyfish)

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Supramolecule #3: Di-Gluebody

SupramoleculeName: Di-Gluebody / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2-#3 / Details: Gluebody GbEnhancer and Gluebody GbC4
Source (natural)Organism: Lama glama (llama)

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Supramolecule #4: Sphingosine-1-phosphate transporter SPNS2

SupramoleculeName: Sphingosine-1-phosphate transporter SPNS2 / type: complex / ID: 4 / Parent: 1 / Macromolecule list: #4 / Details: Sphingosine-1-phosphate transporter SPNS2
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: Green fluorescent protein

MacromoleculeName: Green fluorescent protein / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Aequorea victoria (jellyfish)
Molecular weightTheoretical: 26.81923 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MSKGEELFTG VVPILVELDG DVNGHKFSVR GEGEGDATNG KLTLKFICTT GKLPVPWPTL VTTLTYGVQC FSRYPDHMKR HDFFKSAMP EGYVQERTIS FKDDGTYKTR AEVKFEGDTL VNRIELKGID FKEDGNILGH KLEYNFNSHN VYITADKQKN G IKANFKIR ...String:
MSKGEELFTG VVPILVELDG DVNGHKFSVR GEGEGDATNG KLTLKFICTT GKLPVPWPTL VTTLTYGVQC FSRYPDHMKR HDFFKSAMP EGYVQERTIS FKDDGTYKTR AEVKFEGDTL VNRIELKGID FKEDGNILGH KLEYNFNSHN VYITADKQKN G IKANFKIR HNVEDGSVQL ADHYQQNTPI GDGPVLLPDN HYLSTQSVLS KDPNEKRDHM VLLEFVTAAG ITHGMDELYK

UniProtKB: Green fluorescent protein

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Macromolecule #2: Gluebody GbEnhancer

MacromoleculeName: Gluebody GbEnhancer / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Lama glama (llama)
Molecular weightTheoretical: 12.602062 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
QVQLVENGGA CVKPGGSLRL SCAASGFPVN RYSMRWYRQA PGKEREWVAG MSSAGDRSSY EDSVKGRFTI SRDDARNTVY LQMNSLKPE DTAVYYCNVN VGFEYWGQGT QVMV

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Macromolecule #3: Gluebody GbC4

MacromoleculeName: Gluebody GbC4 / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Lama glama (llama)
Molecular weightTheoretical: 12.982609 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
SQGQLVENGG GCVKAGGSLR LSCAASQGTL SNLVTGWFRR APGKEREFVA NIGRDGLTVY SNSVKGRFTI SRDRAKNTVY LQMDSLKPE DTAVYYCAGR LSRFPGEYDY WSKGTPVMVS

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Macromolecule #4: Sphingosine-1-phosphate transporter SPNS2

MacromoleculeName: Sphingosine-1-phosphate transporter SPNS2 / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 58.962211 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MMCLECASAA AGGAEEEEAD AERRRRRRGA QRGAGGSGCC GARGAGGAGV SAAGDEVQTL SGSVRRAPTG PPGTPGTPGC AATAKGPGA QQPKPASLGR GRGAAAAILS LGNVLNYLDR YTVAGVLLDI QQHFGVKDRG AGLLQSVFIC SFMVAAPIFG Y LGDRFNRK ...String:
MMCLECASAA AGGAEEEEAD AERRRRRRGA QRGAGGSGCC GARGAGGAGV SAAGDEVQTL SGSVRRAPTG PPGTPGTPGC AATAKGPGA QQPKPASLGR GRGAAAAILS LGNVLNYLDR YTVAGVLLDI QQHFGVKDRG AGLLQSVFIC SFMVAAPIFG Y LGDRFNRK VILSCGIFFW SAVTFSSSFI PQQYFWLLVL SRGLVGIGEA SYSTIAPTII GDLFTKNTRT LMLSVFYFAI PL GSGLGYI TGSSVKQAAG DWHWALRVSP VLGMITGTLI LILVPATKRG HADQLGDQLK ARTSWLRDMK ALIRNRSYVF SSL ATSAVS FATGALGMWI PLYLHRAQVV QKTAETCNSP PCGAKDSLIF GAITCFTGFL GVVTGAGATR WCRLKTQRAD PLVC AVGML GSAIFICLIF VAAKSSIVGA YICIFVGETL LFSNWAITAD ILMYVVIPTR RATAVALQSF TSHLLGDAGS PYLIG FISD LIRQSTKDSP LWEFLSLGYA LMLCPFVVVL GGMFFLATAL FFVSDRARAE QQVNQLAMPP ASVKVAENLY FQ

UniProtKB: Sphingosine-1-phosphate transporter SPNS2

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Macromolecule #5: DODECYL-BETA-D-MALTOSIDE

MacromoleculeName: DODECYL-BETA-D-MALTOSIDE / type: ligand / ID: 5 / Number of copies: 1 / Formula: LMT
Molecular weightTheoretical: 510.615 Da
Chemical component information

ChemComp-LMT:
DODECYL-BETA-D-MALTOSIDE / detergent*YM

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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

Concentration4.2 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
20.0 mMC8H18N2O4SHEPES
150.0 mMNaClsodium chloride
0.025 %C24H46O11DDM
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number real images: 8674 / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.5 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 1127724
Startup modelType of model: INSILICO MODEL / In silico model: Ab-initio reconstruction in cryosparc
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.0) / Number images used: 321178
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.0) / Details: non-uniform refinement in cryoSPARC
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.0) / Details: non-uniform refinement in cryoSPARC
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model
PDB IDChain

8qv6

source_name: PDB, initial_model_type: experimental model

3k1k

source_name: PDB, initial_model_type: experimental model
RefinementSpace: REAL / Overall B value: 131.4
Output model

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

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