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- EMDB-17948: Cryo-EM map of Giardia intestinalis deoxyadenosine kinase -

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Basic information

Entry
Database: EMDB / ID: EMD-17948
TitleCryo-EM map of Giardia intestinalis deoxyadenosine kinase
Map data
Sample
  • Organelle or cellular component: deoxyadenosine kinase
Keywordskinase / TRANSFERASE
Biological speciesGiardia intestinalis (eukaryote)
Methodsingle particle reconstruction / cryo EM / Resolution: 6.304 Å
AuthorsRanjbarian F / Rafie K / Shankar K / Krakovka S / Svaerd S / Carlson L-A / Hofer A
Funding support Sweden, 4 items
OrganizationGrant numberCountry
Swedish Research Council2018-05851 Sweden
Swedish Research Council2021-01145 Sweden
Swedish Research Council2022-005593 Sweden
Knut and Alice Wallenberg Foundation Sweden
CitationJournal: Nucleic Acids Res / Year: 2024
Title: Tetramerization of deoxyadenosine kinase meets the demands of a DNA replication substrate challenge in Giardia intestinalis.
Authors: Farahnaz Ranjbarian / Karim Rafie / Kasturika Shankar / Sascha Krakovka / Staffan G Svärd / Lars-Anders Carlson / Anders Hofer /
Abstract: The protozoan parasite Giardia intestinalis is one of only a few organisms lacking de novo synthesis of DNA building blocks (deoxyribonucleotides). Instead, the parasite relies exclusively on ...The protozoan parasite Giardia intestinalis is one of only a few organisms lacking de novo synthesis of DNA building blocks (deoxyribonucleotides). Instead, the parasite relies exclusively on salvaging deoxyadenosine and other deoxyribonucleosides from its host environment. Here, we report that G. intestinalis has a deoxyribonucleoside kinase with a 1000-fold higher catalytic efficiency (kcat/KM) for deoxyadenosine than the corresponding mammalian kinases and can thereby provide sufficient deoxyadenosine triphosphate levels for DNA synthesis despite the lack of de novo synthesis. Several deoxyadenosine analogs were also potent substrates and showed comparable EC50 values on cultured G. intestinalis cells as metronidazole, the current first-line treatment, with the additional advantage of being effective against metronidazole-resistant parasites. Structural analysis using cryo-EM and X-ray crystallography showed that the enzyme is unique within its family of deoxyribonucleoside kinases by forming a tetramer stabilized by extended N- and C-termini in a novel dimer-dimer interaction. Removal of the two termini resulted in lost ability to form tetramers and a markedly reduced affinity for the deoxyribonucleoside substrate. The development of highly efficient deoxyribonucleoside kinases via oligomerization may represent a critical evolutionary adaptation in organisms that rely solely on deoxyribonucleoside salvage.
History
DepositionJul 17, 2023-
Header (metadata) releaseJul 24, 2024-
Map releaseJul 24, 2024-
UpdateFeb 26, 2025-
Current statusFeb 26, 2025Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_17948.png

Downloads & links

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Map

FileDownload / File: emd_17948.map.gz / Format: CCP4 / Size: 59.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.58 Å/pix.
x 250 pix.
= 145. Å		0.58 Å/pix.
x 250 pix.
= 145. Å		0.58 Å/pix.
x 250 pix.
= 145. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.58 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.00306
Minimum - Maximum-0.004397238 - 0.012524658
Average (Standard dev.)0.000045005196 (±0.0010555568)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions250250250
Spacing250250250
CellA=B=C: 145.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_17948_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_17948_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : deoxyadenosine kinase

EntireName: deoxyadenosine kinase
Components
  • Organelle or cellular component: deoxyadenosine kinase

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Supramolecule #1: deoxyadenosine kinase

SupramoleculeName: deoxyadenosine kinase / type: organelle_or_cellular_component / ID: 1 / Parent: 0
Source (natural)Organism: Giardia intestinalis (eukaryote)
Molecular weightTheoretical: 117 kDa/nm

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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

Concentration1 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
150.0 mMNaClSodium chloride
20.0 mMC4H11NO3Tris
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Digitization - Dimensions - Width: 4000 pixel / Digitization - Dimensions - Height: 4000 pixel / Number real images: 3420 / 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 / Cs: 2.7 mm / Nominal defocus max: 3.5 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 215000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: INSILICO MODEL / In silico model: ab-initio model generation in relion
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 6.304 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Number images used: 105162
Initial angle assignmentType: OTHER / Software - Name: RELION
Final angle assignmentType: OTHER / Software - Name: RELION
Final 3D classificationNumber classes: 6 / Software - Name: RELION

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Atomic model buiding 1

RefinementProtocol: AB INITIO MODEL

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