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TitleDisulfide-stabilized diabodies enable near-atomic cryo-EM imaging of small proteins: A case study of the bacterial Na/citrate symporter CitS.
Journal, issue, pagesStructure, Vol. 33, Issue 6, Page 1088-11100.e3, Year 2025
Publish dateJun 5, 2025
AuthorsSubin Kim / Ji Won Kim / Jun Gyou Park / Sang Soo Lee / Seung Hun Choi / Jie-Oh Lee / Mi Sun Jin /
PubMed AbstractDiabodies 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.
External linksStructure / PubMed:40169000
MethodsEM (single particle)
Resolution2.9 - 3.8 Å
Structure data

63355

9lsh

EMDB-63355, PDB-9lsh:
Cryo-EM structure of the wild-type diabody complex (CitS-diabody #1-TLR3)
Method: EM (single particle) / Resolution: 3.8 Å

63356

9lsi

EMDB-63356, PDB-9lsi:
Cryo-EM structure of the S82C-S82C diabody complex (CitS-diabody #2-TLR3)
Method: EM (single particle) / Resolution: 3.3 Å

63357

9lsj

EMDB-63357, PDB-9lsj:
Cryo-EM structure of the G15C-R66C and T83C-T83C diabody complex (CitS-diabody #7-TLR3)
Method: EM (single particle) / Resolution: 3.1 Å

63358

9lsk

EMDB-63358, PDB-9lsk:
Cryo-EM structure of the Klebsiella pneumoniae CitS (citrate-bound occluded state)
Method: EM (single particle) / Resolution: 2.9 Å

Chemicals

ChemComp-CIT:
CITRIC ACID

ChemComp-PLM:
PALMITIC ACID

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

Source
  • klebsiella pneumoniae (bacteria)
  • homo sapiens (human)
  • synthetic construct (others)
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 / MEMBRANE PROTEIN

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