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- PDB-9lsk: Cryo-EM structure of the Klebsiella pneumoniae CitS (citrate-boun... -

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Basic information

Entry
Database: PDB / ID: 9lsk
TitleCryo-EM structure of the Klebsiella pneumoniae CitS (citrate-bound occluded state)
ComponentsCitrate/sodium symporter
KeywordsMEMBRANE PROTEIN / Disulfide-bridged diabody / cryo-electron microscopy (cryo-EM) / small protein imaging / structural marker / antibody engineering / protein nanotechnology
Function / homology
Function and homology information


citrate metabolic process / symporter activity / : / sodium ion transport / metal ion binding / plasma membrane
Similarity search - Function
2-hydroxycarboxylate transporter / 2-hydroxycarboxylate transporter, Proteobacteria/Firmicutes / 2-hydroxycarboxylate transporter family
Similarity search - Domain/homology
CITRIC ACID / PALMITIC ACID / Citrate/sodium symporter
Similarity search - Component
Biological speciesKlebsiella pneumoniae (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.9 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
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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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Revision 1.1Apr 16, 2025Group: Data collection / Database references / Category: citation / citation_author / em_admin
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Citrate/sodium symporter
B: Citrate/sodium symporter
hetero molecules


Theoretical massNumber of molelcules
Total (without water)95,8265
Polymers95,1852
Non-polymers6413
Water00
1


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

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Components

#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: Chemical ChemComp-CIT / CITRIC ACID


Mass: 192.124 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C6H8O7 / Feature type: SUBJECT OF INVESTIGATION
#3: Chemical ChemComp-PLM / PALMITIC ACID


Mass: 256.424 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C16H32O2
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: CitS / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Source (natural)Organism: Klebsiella pneumoniae (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
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: 1800 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: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 235752 / Symmetry type: POINT
RefinementStereochemistry target values: REAL-SPACE (WEIGHTED MAP SUM AT ATOM CENTERS)
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0046545
ELECTRON MICROSCOPYf_angle_d0.6028883
ELECTRON MICROSCOPYf_dihedral_angle_d10.681932
ELECTRON MICROSCOPYf_chiral_restr0.0421080
ELECTRON MICROSCOPYf_plane_restr0.0031085

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