9LSK

Cryo-EM structure of the Klebsiella pneumoniae CitS (citrate-bound occluded state)

Summary for 9LSK

• Entry DOI: 10.2210/pdb9lsk/pdb
• EMDB information: 63358
• Descriptor: Citrate/sodium symporter, CITRIC ACID, PALMITIC ACID (3 entities in total)
• Functional Keywords: disulfide-bridged diabody, cryo-electron microscopy (cryo-em), small protein imaging, structural marker, antibody engineering, protein nanotechnology, membrane protein
• Biological source: Klebsiella pneumoniae
• Total number of polymer chains: 2
• Total formula weight: 95825.66

Authors

Kim, S.,Kim, J.W.,Park, J.G.,Lee, S.S.,Choi, S.H.,Lee, J.-O.,Jin, M.S. (deposition date: 2025-02-04, release date: 2025-03-12, Last modification date: 2025-04-16)

Primary citation

Kim, S.,Kim, J.W.,Park, J.G.,Lee, S.S.,Choi, S.H.,Lee, J.O.,Jin, M.S.
Disulfide-stabilized diabodies enable near-atomic cryo-EM imaging of small proteins: A case study of the bacterial Na + /citrate symporter CitS.
Structure, 2025

PubMed 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 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.

PubMed: 40169000
DOI: 10.1016/j.str.2025.03.006

Experimental method

ELECTRON MICROSCOPY (2.9 Å)