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- EMDB-63357: Cryo-EM structure of the G15C-R66C and T83C-T83C diabody complex ... -

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Entry
Database: EMDB / ID: EMD-63357
TitleCryo-EM structure of the G15C-R66C and T83C-T83C diabody complex (CitS-diabody #7-TLR3)
Map data
Sample
  • Complex: CitS-diabody #1-TLR3 complex
    • Complex: CitS
      • Protein or peptide: Citrate/sodium symporter
    • Complex: TLR3
      • Protein or peptide: Toll-like receptor 3
    • Complex: Diabody
      • Protein or peptide: Diabody (CitS VH-TLR3 VL)
      • Protein or peptide: Diabody (TLR3 VH-CitS VL)
  • Ligand: CITRIC ACID
  • Ligand: PALMITIC ACID
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
KeywordsDisulfide-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 / TICAM1 deficiency - HSE / type III interferon production / positive regulation of type III interferon production / TRAF3 deficiency - HSE / regulation of dendritic cell cytokine production / I-kappaB phosphorylation / Toll Like Receptor 3 (TLR3) Cascade ...TLR3 deficiency - HSE / response to dsRNA / UNC93B1 deficiency - HSE / TICAM1 deficiency - HSE / type III interferon production / positive regulation of type III interferon production / TRAF3 deficiency - HSE / regulation of dendritic cell cytokine production / I-kappaB phosphorylation / Toll Like Receptor 3 (TLR3) Cascade / TLR3-mediated TICAM1-dependent programmed cell death / citrate metabolic process / inflammatory response to wounding / activation of NF-kappaB-inducing kinase activity / toll-like receptor 3 signaling pathway / detection of virus / necroptotic signaling pathway / symporter activity / organic anion transmembrane transporter activity / RIP-mediated NFkB activation via ZBP1 / hyperosmotic response / endolysosome membrane / positive regulation of cytokine production involved in inflammatory response / Trafficking and processing of endosomal TLR / positive regulation of macrophage cytokine production / sodium ion transport / toll-like receptor signaling pathway / 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 / extrinsic apoptotic signaling pathway / JNK cascade / 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 / positive regulation of non-canonical NF-kappaB signal transduction / cellular response to virus / cellular response to type II interferon / positive regulation of interleukin-6 production / positive regulation of type II interferon production / cellular response to mechanical stimulus / positive regulation of inflammatory response / male gonad development / positive regulation of angiogenesis / positive regulation of tumor necrosis factor production / transmembrane signaling receptor activity / cellular response to xenobiotic stimulus / double-stranded RNA binding / signaling receptor activity / defense response to virus / positive regulation of canonical NF-kappaB signal transduction / early endosome / 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
Toll-like receptor 3 / Citrate/sodium symporter
Similarity search - Component
Biological speciesKlebsiella pneumoniae (bacteria) / Homo sapiens (human) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.1 Å
AuthorsKim S / Kim JW / Park JG / Lee SS / Choi SH / Lee J-O / Jin MS
Funding support Korea, Republic Of, 3 items
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, 2025-
Header (metadata) releaseMar 12, 2025-
Map releaseMar 12, 2025-
UpdateJun 18, 2025-
Current statusJun 18, 2025Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_63357.png

Downloads & links

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Map

FileDownload / File: emd_63357.map.gz / Format: CCP4 / Size: 83.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

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AxesZ (Sec.)Y (Row.)X (Col.)
1.05 Å/pix.
x 280 pix.
= 294. Å		1.05 Å/pix.
x 280 pix.
= 294. Å		1.05 Å/pix.
x 280 pix.
= 294. Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.05 Å
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Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0934
Minimum - Maximum-0.27441087 - 0.63435227
Average (Standard dev.)0.0013727502 (±0.020890389)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions280280280
Spacing280280280
CellA=B=C: 294.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_63357_half_map_1.map
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Half map: #1

Fileemd_63357_half_map_2.map
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Sample components

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Entire : CitS-diabody #1-TLR3 complex

EntireName: CitS-diabody #1-TLR3 complex
Components
  • Complex: CitS-diabody #1-TLR3 complex
    • Complex: CitS
      • Protein or peptide: Citrate/sodium symporter
    • Complex: TLR3
      • Protein or peptide: Toll-like receptor 3
    • Complex: Diabody
      • Protein or peptide: Diabody (CitS VH-TLR3 VL)
      • Protein or peptide: Diabody (TLR3 VH-CitS VL)
  • Ligand: CITRIC ACID
  • Ligand: PALMITIC ACID
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose

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Supramolecule #1: CitS-diabody #1-TLR3 complex

SupramoleculeName: CitS-diabody #1-TLR3 complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4

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Supramolecule #2: CitS

SupramoleculeName: CitS / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Klebsiella pneumoniae (bacteria)

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Supramolecule #3: TLR3

SupramoleculeName: TLR3 / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2
Source (natural)Organism: Homo sapiens (human)

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Supramolecule #4: Diabody

SupramoleculeName: Diabody / type: complex / ID: 4 / Parent: 1 / Macromolecule list: #3-#4
Source (natural)Organism: synthetic construct (others)

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Macromolecule #1: Citrate/sodium symporter

MacromoleculeName: Citrate/sodium symporter / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Klebsiella pneumoniae (bacteria)
Molecular weightTheoretical: 47.592492 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MTNMSQPPAT EKKGVSDLLG FKIFGMPLPL YAFALITLLL SHFYNALPTD IVGGFAIMFI IGAIFGEIGK RLPIFNKYIG GAPVMIFLV AAYFVYAGIF TQKEIDAISN VMDKSNFLNL FIAVLITGAI LSVNRRLLLK SLLGYIPTIL MGIVGASIFG I AIGLVFGI ...String:
MTNMSQPPAT EKKGVSDLLG FKIFGMPLPL YAFALITLLL SHFYNALPTD IVGGFAIMFI IGAIFGEIGK RLPIFNKYIG GAPVMIFLV AAYFVYAGIF TQKEIDAISN VMDKSNFLNL FIAVLITGAI LSVNRRLLLK SLLGYIPTIL MGIVGASIFG I AIGLVFGI PVDRIMMLYV LPIMGGGNGA GAVPLSEIYH SVTGRSREEY YSTAIAILTI ANIFAIVFAA VLDIIGKKHT WL SGEGELV RKASFKVEED EKTGQITHRE TAVGLVLSTT CFLLAYVVAK KILPSIGGVA IHYFAWMVLI VAALNASGLC SPE IKAGAK RLSDFFSKQL LWVLMVGVGV CYTDLQEIIN AITFANVVIA AIIVIGAVLG AAIGGWLMGF FPIESAITAG LCMA NRGGS GDLEVLSACN RMNLISYAQI SSRLGGGIVL VIASIVFGMM I

UniProtKB: Citrate/sodium symporter

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Macromolecule #2: Toll-like receptor 3

MacromoleculeName: Toll-like receptor 3 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 103.94632 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: MRQTLPCIYF WGGLLPFGML CASSTTKCTV SHEVADCSHL KLTQVPDDLP TNITVLNLTH NQLRRLPAAN FTRYSQLTSL DVGFNTISK LEPELCQKLP MLKVLNLQHN ELSQLSDKTF AFCTNLTELH LMSNSIQKIK NNPFVKQKNL ITLDLSHNGL S STKLGTQV ...String:
MRQTLPCIYF WGGLLPFGML CASSTTKCTV SHEVADCSHL KLTQVPDDLP TNITVLNLTH NQLRRLPAAN FTRYSQLTSL DVGFNTISK LEPELCQKLP MLKVLNLQHN ELSQLSDKTF AFCTNLTELH LMSNSIQKIK NNPFVKQKNL ITLDLSHNGL S STKLGTQV QLENLQELLL SNNKIQALKS EELDIFANSS LKKLELSSNQ IKEFSPGCFH AIGRLFGLFL NNVQLGPSLT EK LCLELAN TSIRNLSLSN SQLSTTSNTT FLGLKWTNLT MLDLSYNNLN VVGNDSFAWL PQLEYFFLEY NNIQHLFSHS LHG LFNVRY LNLKRSFTKQ SISLASLPKI DDFSFQWLKC LEHLNMEDND IPGIKSNMFT GLINLKYLSL SNSFTSLRTL TNET FVSLA HSPLHILNLT KNKISKIESD AFSWLGHLEV LDLGLNEIGQ ELTGQEWRGL ENIFEIYLSY NKYLQLTRNS FALVP SLQR LMLRRVALKN VDSSPSPFQP LRNLTILDLS NNNIANINDD MLEGLEKLEI LDLQHNNLAR LWKHANPGGP IYFLKG LSH LHILNLESNG FDEIPVEVFK DLFELKIIDL GLNNLNTLPA SVFNNQVSLK SLNLQKNLIT SVEKKVFGPA FRNLTEL DM RFNPFDCTCE SIAWFVNWIN ETHTNIPELS SHYLCNTPPH YHGFPVRLFD TSSCKDSAPF ELFFMINTSI LLIFIFIV L LIHFEGWRIS FYWNVSVHRV LGFKEIDRQT EQFEYAAYII HAYKDKDWVW EHFSSMEKED QSLKFCLEER DFEAGVFEL EAIVNSIKRS RKIIFVITHH LLKDPLCKRF KVHHAVQQAI EQNLDSIILV FLEEIPDYKL NHALCLRRGM FKSHCILNWP VQKERIGAF RHKLQVALGS KNSVH

UniProtKB: Toll-like receptor 3

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

MacromoleculeName: Diabody (CitS VH-TLR3 VL) / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 27.183098 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: EVQLVESGGG LVQPCGSLRL SCAASGFTFR NSAMHWVRQA PGKGLEWVSS IWYSGSNTYY ADSVKGRFTI SRDNSKNTLY LQMNSLCAE DTAVYYCARF AGGWGAYDVW GQGTLVTVSS GGGGSDIQMT QSPSSLSASV GDRVTITCRA SQSIGLYLAW Y QQKPGKAP ...String:
EVQLVESGGG LVQPCGSLRL SCAASGFTFR NSAMHWVRQA PGKGLEWVSS IWYSGSNTYY ADSVKGRFTI SRDNSKNTLY LQMNSLCAE DTAVYYCARF AGGWGAYDVW GQGTLVTVSS GGGGSDIQMT QSPSSLSASV GDRVTITCRA SQSIGLYLAW Y QQKPGKAP KLLIYAASSL QSGVPSRFSG SGSGTDFTLT ISSLQPEDFA TYYCQQGNTL SYTFGQGTKV EIKPWLVPRG SH HHHHHSG GGGSGGGGS

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Macromolecule #4: Diabody (TLR3 VH-CitS VL)

MacromoleculeName: Diabody (TLR3 VH-CitS VL) / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 28.384414 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: GGGGSGGGGL VPRGSPGEVQ LVQSGAEVKK PGESLKISCK GSGYSFTNYW VGWVRQMPGK GLEWMGFIDP SDSYTNYADS VKGCFTISA DKSISTAYLQ MNSLCASDTA MYYCARELYQ GYMDTFDSWG QGTLVTVSSG GGGSDIVLTQ SPATLSLSPG E RATLSCRA ...String:
GGGGSGGGGL VPRGSPGEVQ LVQSGAEVKK PGESLKISCK GSGYSFTNYW VGWVRQMPGK GLEWMGFIDP SDSYTNYADS VKGCFTISA DKSISTAYLQ MNSLCASDTA MYYCARELYQ GYMDTFDSWG QGTLVTVSSG GGGSDIVLTQ SPATLSLSPG E RATLSCRA SQSVSSNYLA WYQQKPGQAP RLLIYDSSSR ATGVPARFSG SGSGTDFTLT ISSLEPEDFA VYYCHQYSDI SP TFGQGTK VEIKSGRLVP RGSHHHHHH

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Macromolecule #7: CITRIC ACID

MacromoleculeName: CITRIC ACID / type: ligand / ID: 7 / Number of copies: 1 / Formula: CIT
Molecular weightTheoretical: 192.124 Da
Chemical component information

ChemComp-CIT:
CITRIC ACID

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Macromolecule #8: PALMITIC ACID

MacromoleculeName: PALMITIC ACID / type: ligand / ID: 8 / Number of copies: 1 / Formula: PLM
Molecular weightTheoretical: 256.424 Da
Chemical component information

ChemComp-PLM:
PALMITIC ACID

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Macromolecule #9: 2-acetamido-2-deoxy-beta-D-glucopyranose

MacromoleculeName: 2-acetamido-2-deoxy-beta-D-glucopyranose / type: ligand / ID: 9 / Number of copies: 4 / Formula: NAG
Molecular weightTheoretical: 221.208 Da
Chemical component information

ChemComp-NAG:
2-acetamido-2-deoxy-beta-D-glucopyranose

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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

BufferpH: 7.5
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 60.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.8 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 259962
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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