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- PDB-8rl8: SPNS2 in complex with homo Di-Gluebody GbD12 - SPNS2 local refinement -

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Basic information

Entry
Database: PDB / ID: 8rl8
TitleSPNS2 in complex with homo Di-Gluebody GbD12 - SPNS2 local refinement
ComponentsSphingosine-1-phosphate transporter SPNS2
KeywordsMEMBRANE PROTEIN / SPNS2 / Di-Gluebody
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 / 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
Similarity search - Domain/homology
Sphingosine-1-phosphate transporter SPNS2
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.79 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: Nat Chem Biol / Year: 2025
Title: Covalently constrained 'Di-Gembodies' enable parallel structure solutions by cryo-EM.
Authors: Gangshun Yi / Dimitrios Mamalis / Mingda Ye / Loic Carrique / Michael Fairhead / Huanyu Li / Katharina L Duerr / Peijun Zhang / David B Sauer / Frank von Delft / Benjamin G Davis / Robert J C Gilbert /
Abstract: Whilst cryo-electron microscopy(cryo-EM) has become a routine methodology in structural biology, obtaining high-resolution cryo-EM structures of small proteins (<100 kDa) and increasing overall throughput remain challenging. One approach to augment protein size and improve particle alignment involves the use of binding proteins or protein-based scaffolds. However, a given imaging scaffold or linking module may prove inadequate for structure solution and availability of such scaffolds remains limited. Here, we describe a strategy that exploits covalent dimerization of nanobodies to trap an engineered, predisposed nanobody-to-nanobody interface, giving Di-Gembodies as modular constructs created in homomeric and heteromeric forms. By exploiting side-chain-to-side-chain assembly, they can simultaneously display two copies of the same or two distinct proteins through a subunit interface that provides sufficient constraint required for cryo-EM structure determination. We validate this method with multiple soluble and membrane structural targets, down to 14 kDa, demonstrating a flexible and scalable platform for expanded protein structure determination.
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
A: Sphingosine-1-phosphate transporter SPNS2
hetero molecules


Theoretical massNumber of molelcules
Total (without water)59,4732
Polymers58,9621
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 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
#2: 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 modificationN

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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: Local refinement of the SPNS2 part of SPNS2 homodimer assembled by GbD12 homo Di-Gluebody
Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.146 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMHEPESC8H18N2O4S1
2150 mMsodium chlorideNaCl1
30.025 %DDMC24H46O111
SpecimenConc.: 9 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: 12013

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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
13cryoSPARC4classification
14cryoSPARC43D reconstruction
CTF correctionDetails: patch-CTF correction in cryoSPARC / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 871094
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.79 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 570074 / Num. of class averages: 6 / Symmetry type: POINT
Atomic model buildingB value: 100.8 / Space: REAL
Atomic model buildingPDB-ID: 8QV6

8qv6


Accession code: 8QV6 / Source name: PDB / Type: experimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0023291
ELECTRON MICROSCOPYf_angle_d0.5014471
ELECTRON MICROSCOPYf_dihedral_angle_d5.188461
ELECTRON MICROSCOPYf_chiral_restr0.035531
ELECTRON MICROSCOPYf_plane_restr0.004544

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