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- EMDB-63358: Cryo-EM structure of the Klebsiella pneumoniae CitS (citrate-boun... -

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Basic information

Entry
Database: EMDB / ID: EMD-63358
TitleCryo-EM structure of the Klebsiella pneumoniae CitS (citrate-bound occluded state)
Map data
Sample
  • Complex: CitS
    • Protein or peptide: Citrate/sodium symporter
  • Ligand: CITRIC ACID
  • Ligand: PALMITIC ACID
KeywordsDisulfide-bridged diabody / cryo-electron microscopy (cryo-EM) / small protein imaging / structural marker / antibody engineering / protein nanotechnology / MEMBRANE PROTEIN
Function / homology
Function and homology information


citrate metabolic process / organic anion transmembrane transporter activity / 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
Citrate/sodium symporter
Similarity search - Component
Biological speciesKlebsiella pneumoniae (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.9 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-
UpdateApr 16, 2025-
Current statusApr 16, 2025Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_63358.png

Downloads & links

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Map

FileDownload / File: emd_63358.map.gz / Format: CCP4 / Size: 59.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
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AxesZ (Sec.)Y (Row.)X (Col.)
0.82 Å/pix.
x 250 pix.
= 206.2 Å		0.82 Å/pix.
x 250 pix.
= 206.2 Å		0.82 Å/pix.
x 250 pix.
= 206.2 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.8248 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0596
Minimum - Maximum-0.20902531 - 0.43008196
Average (Standard dev.)0.0018264703 (±0.015080544)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions250250250
Spacing250250250
CellA=B=C: 206.2 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_63358_half_map_1.map
Projections & Slices
AxesZYX

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Histogram (log scale)

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Half map: #2

Fileemd_63358_half_map_2.map
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Sample components

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Entire : CitS

EntireName: CitS
Components
  • Complex: CitS
    • Protein or peptide: Citrate/sodium symporter
  • Ligand: CITRIC ACID
  • Ligand: PALMITIC ACID

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Supramolecule #1: CitS

SupramoleculeName: CitS / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Klebsiella pneumoniae (bacteria)

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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: CITRIC ACID

MacromoleculeName: CITRIC ACID / type: ligand / ID: 2 / Number of copies: 2 / Formula: CIT
Molecular weightTheoretical: 192.124 Da
Chemical component information

ChemComp-CIT:
CITRIC ACID

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Macromolecule #3: PALMITIC ACID

MacromoleculeName: PALMITIC ACID / type: ligand / ID: 3 / Number of copies: 1 / Formula: PLM
Molecular weightTheoretical: 256.424 Da
Chemical component information

ChemComp-PLM:
PALMITIC ACID

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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: 1.8 µm / Nominal defocus min: 0.8 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 235752
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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