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- PDB-9lsj: Cryo-EM structure of the G15C-R66C and T83C-T83C diabody complex ... -

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Basic information

Entry
Database: PDB / ID: 9lsj
TitleCryo-EM structure of the G15C-R66C and T83C-T83C diabody complex (CitS-diabody #7-TLR3)
Components
  • Citrate/sodium symporter
  • Diabody (CitS VH-TLR3 VL)
  • Diabody (TLR3 VH-CitS VL)
  • Toll-like receptor 3
KeywordsSTRUCTURAL PROTEIN/IMMUNE SYSTEM / Disulfide-bridged diabody / cryo-electron microscopy (cryo-EM) / small protein imaging / structural marker / antibody engineering / protein nanotechnology / STRUCTURAL PROTEIN / STRUCTURAL PROTEIN-IMMUNE SYSTEM complex
Function / homology
Function and homology information


TLR3 deficiency - HSE / response to dsRNA / UNC93B1 deficiency - HSE / type III interferon production / positive regulation of type III interferon production / TICAM1 deficiency - HSE / TRAF3 deficiency - HSE / regulation of dendritic cell cytokine production / Toll Like Receptor 3 (TLR3) Cascade / citrate metabolic process ...TLR3 deficiency - HSE / response to dsRNA / UNC93B1 deficiency - HSE / type III interferon production / positive regulation of type III interferon production / TICAM1 deficiency - HSE / TRAF3 deficiency - HSE / regulation of dendritic cell cytokine production / Toll Like Receptor 3 (TLR3) Cascade / citrate metabolic process / inflammatory response to wounding / TLR3-mediated TICAM1-dependent programmed cell death / activation of NF-kappaB-inducing kinase activity / toll-like receptor 3 signaling pathway / detection of virus / necroptotic signaling pathway / symporter activity / : / RIP-mediated NFkB activation via ZBP1 / positive regulation of cytokine production involved in inflammatory response / endolysosome membrane / Trafficking and processing of endosomal TLR / hyperosmotic response / positive regulation of macrophage cytokine production / toll-like receptor signaling pathway / sodium ion transport / pattern recognition receptor activity / cellular response to exogenous dsRNA / RSV-host interactions / response to exogenous dsRNA / negative regulation of osteoclast differentiation / ubiquitin-like protein ligase binding / positive regulation of interferon-alpha production / cellular response to interferon-beta / positive regulation of chemokine production / JNK cascade / extrinsic apoptotic signaling pathway / positive regulation of interleukin-12 production / Regulation of TBK1, IKKε-mediated activation of IRF3, IRF7 upon TLR3 ligation / extracellular matrix / TICAM1,TRAF6-dependent induction of TAK1 complex / TICAM1-dependent activation of IRF3/IRF7 / positive regulation of interferon-beta production / TICAM1, RIP1-mediated IKK complex recruitment / positive regulation of interleukin-8 production / positive regulation of JNK cascade / microglial cell activation / cellular response to mechanical stimulus / cellular response to virus / cellular response to type II interferon / positive regulation of interleukin-6 production / male gonad development / cellular response to xenobiotic stimulus / positive regulation of type II interferon production / positive regulation of angiogenesis / positive regulation of inflammatory response / positive regulation of tumor necrosis factor production / transmembrane signaling receptor activity / signaling receptor activity / double-stranded RNA binding / defense response to virus / early endosome / positive regulation of canonical NF-kappaB signal transduction / endosome membrane / defense response to bacterium / positive regulation of apoptotic process / Golgi membrane / lysosomal membrane / innate immune response / positive regulation of gene expression / endoplasmic reticulum membrane / signal transduction / positive regulation of transcription by RNA polymerase II / extracellular space / metal ion binding / identical protein binding / membrane / plasma membrane / cytoplasm
Similarity search - Function
2-hydroxycarboxylate transporter / 2-hydroxycarboxylate transporter, Proteobacteria/Firmicutes / 2-hydroxycarboxylate transporter family / Toll-like receptor 3 trans-membrane domain / Toll-like receptor 3 trans-membrane domain / TIR domain / Leucine Rich repeat / Cysteine-rich flanking region, C-terminal / Leucine rich repeat C-terminal domain / Toll - interleukin 1 - resistance ...2-hydroxycarboxylate transporter / 2-hydroxycarboxylate transporter, Proteobacteria/Firmicutes / 2-hydroxycarboxylate transporter family / Toll-like receptor 3 trans-membrane domain / Toll-like receptor 3 trans-membrane domain / TIR domain / Leucine Rich repeat / Cysteine-rich flanking region, C-terminal / Leucine rich repeat C-terminal domain / Toll - interleukin 1 - resistance / TIR domain profile. / Leucine-rich repeat, SDS22-like subfamily / Toll/interleukin-1 receptor homology (TIR) domain / Toll/interleukin-1 receptor homology (TIR) domain superfamily / Leucine rich repeat / Leucine-rich repeat, typical subtype / Leucine-rich repeats, typical (most populated) subfamily / Leucine-rich repeat profile. / Leucine-rich repeat / Leucine-rich repeat domain superfamily
Similarity search - Domain/homology
CITRIC ACID / PALMITIC ACID / Toll-like receptor 3 / Citrate/sodium symporter
Similarity search - Component
Biological speciesKlebsiella pneumoniae (bacteria)
Homo sapiens (human)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.1 Å
AuthorsKim, S. / Kim, J.W. / Park, J.G. / Lee, S.S. / Choi, S.H. / Lee, J.-O. / Jin, M.S.
Funding support Korea, Republic Of, 3items
OrganizationGrant numberCountry
Samsung Science and Technology FoundationSSTF-BA1702-14 Korea, Republic Of
National Research Foundation (NRF, Korea)RS-2024-00344154 Korea, Republic Of
National Research Foundation (NRF, Korea)RS-2024-00440614 Korea, Republic Of
CitationJournal: Structure / Year: 2025
Title: Disulfide-stabilized diabodies enable near-atomic cryo-EM imaging of small proteins: A case study of the bacterial Na/citrate symporter CitS.
Authors: Subin Kim / Ji Won Kim / Jun Gyou Park / Sang Soo Lee / Seung Hun Choi / Jie-Oh Lee / Mi Sun Jin /
Abstract: Diabodies are engineered antibody fragments with two antigen-binding Fv domains. Previously, we demonstrated that they are often highly flexible but can be rigidified by introducing a disulfide bond ...Diabodies are engineered antibody fragments with two antigen-binding Fv domains. Previously, we demonstrated that they are often highly flexible but can be rigidified by introducing a disulfide bond at the Fv interface. In this study, we explored the potential of disulfide-bridged, bispecific diabodies for near-atomic cryoelectron microscopy (cryo-EM) imaging of small proteins because they can predictably link target proteins to "structural marker" proteins. As a case study, we used the bacterial citrate transporter CitS as the target protein, and the horseshoe-shaped ectodomain of human Toll-like receptor 3 (TLR3) as the marker. We show that diabodies containing one or two disulfide bonds enabled the 3D reconstruction of CitS at resolutions of 3.3 Å and 3.1 Å, respectively. This resolution surpassed previous crystallographic results and allowed us to visualize the high-resolution structural features of the transporter. Our work expands the application of diabodies in structural biology to address a key limitation in the field.
History
DepositionFeb 4, 2025Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Mar 12, 2025Provider: repository / Type: Initial release
Revision 1.0Mar 12, 2025Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Mar 12, 2025Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
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Revision 1.0Mar 12, 2025Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Citrate/sodium symporter
B: Citrate/sodium symporter
C: Toll-like receptor 3
D: Diabody (CitS VH-TLR3 VL)
E: Diabody (TLR3 VH-CitS VL)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)257,89215
Polymers254,6995
Non-polymers3,19310
Water00
1


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

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Components

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Protein , 2 types, 3 molecules ABC

#1: Protein Citrate/sodium symporter / Citrate transporter CitS / Na(+)-dependent citrate carrier / Sodium-dependent citrate transport system


Mass: 47592.492 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Klebsiella pneumoniae (bacteria) / Gene: citS / Production host: Escherichia coli (E. coli) / References: UniProt: P31602
#2: Protein Toll-like receptor 3


Mass: 103946.320 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: TLR3 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: O15455

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Antibody , 2 types, 2 molecules DE

#3: Antibody Diabody (CitS VH-TLR3 VL)


Mass: 27183.098 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) synthetic construct (others) / Production host: Trichoplusia ni (cabbage looper)
#4: Antibody Diabody (TLR3 VH-CitS VL)


Mass: 28384.414 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) synthetic construct (others) / Production host: Trichoplusia ni (cabbage looper)

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Sugars , 3 types, 8 molecules

#5: Polysaccharide 2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 424.401 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DGlcpNAcb1-4DGlcpNAcb1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/1,2,1/[a2122h-1b_1-5_2*NCC/3=O]/1-1/a4-b1WURCSPDB2Glycan 1.1.0
[][D-1-deoxy-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{}}LINUCSPDB-CARE
#6: Polysaccharide beta-D-mannopyranose-(1-3)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta- ...beta-D-mannopyranose-(1-3)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 586.542 Da / Num. of mol.: 1 / Source method: obtained synthetically
DescriptorTypeProgram
DManpb1-3DGlcpNAcb1-4DGlcpNAcb1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/2,3,2/[a2122h-1b_1-5_2*NCC/3=O][a1122h-1b_1-5]/1-1-2/a4-b1_b3-c1WURCSPDB2Glycan 1.1.0
[][D-1-deoxy-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{[(3+1)][b-D-Manp]{}}}LINUCSPDB-CARE
#9: Sugar
ChemComp-NAG / 2-acetamido-2-deoxy-beta-D-glucopyranose / N-acetyl-beta-D-glucosamine / 2-acetamido-2-deoxy-beta-D-glucose / 2-acetamido-2-deoxy-D-glucose / 2-acetamido-2-deoxy-glucose / N-ACETYL-D-GLUCOSAMINE


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Formula: C8H15NO6
IdentifierTypeProgram
DGlcpNAcbCONDENSED IUPAC CARBOHYDRATE SYMBOLGMML 1.0
N-acetyl-b-D-glucopyranosamineCOMMON NAMEGMML 1.0
b-D-GlcpNAcIUPAC CARBOHYDRATE SYMBOLPDB-CARE 1.0
GlcNAcSNFG CARBOHYDRATE SYMBOLGMML 1.0

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Non-polymers , 2 types, 2 molecules

#7: Chemical ChemComp-CIT / CITRIC ACID


Mass: 192.124 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C6H8O7 / Feature type: SUBJECT OF INVESTIGATION
#8: Chemical ChemComp-PLM / PALMITIC ACID


Mass: 256.424 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C16H32O2

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Details

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
IDNameTypeEntity IDParent-IDSource
1CitS-diabody #1-TLR3 complexCOMPLEX#1-#40MULTIPLE SOURCES
2CitSCOMPLEX#11RECOMBINANT
3TLR3COMPLEX#21RECOMBINANT
4DiabodyCOMPLEX#3-#41RECOMBINANT
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Klebsiella pneumoniae (bacteria)573
33Homo sapiens (human)9606
44synthetic construct (others)32630
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
22Escherichia coli (E. coli)562
33Trichoplusia ni (cabbage looper)7111
44Trichoplusia ni (cabbage looper)7111
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2000 nm / Nominal defocus min: 800 nm
Image recordingElectron dose: 60 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

EM softwareName: PHENIX / Version: 1.18.2_3874 / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 259962 / Symmetry type: POINT
RefinementStereochemistry target values: REAL-SPACE (WEIGHTED MAP SUM AT ATOM CENTERS)
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00615780
ELECTRON MICROSCOPYf_angle_d0.71121420
ELECTRON MICROSCOPYf_dihedral_angle_d9.7022218
ELECTRON MICROSCOPYf_chiral_restr0.0492535
ELECTRON MICROSCOPYf_plane_restr0.0042672

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