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- EMDB-26126: Structure of the L. blandensis dGTPase in the apo form -

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Basic information

Entry
Database: EMDB / ID: EMD-26126
TitleStructure of the L. blandensis dGTPase in the apo form
Map dataDeepEMhancer post-processed map
Sample
  • Complex: dGTP triphosphohydrolaseDGTPase
    • Protein or peptide: dGTP triphosphohydrolaseDGTPase
  • Ligand: MAGNESIUM ION
  • Ligand: water
KeywordsdGTPase / nucleotide binding / HYDROLASE
Function / homology
Function and homology information


triphosphoric monoester hydrolase activity
Similarity search - Function
Deoxyguanosinetriphosphate triphosphohydrolase, C-terminal / Deoxyguanosinetriphosphate triphosphohydrolase, central domain superfamily / dNTP triphosphohydrolase / HD domain profile. / HD domain / HD domain / Metal dependent phosphohydrolases with conserved 'HD' motif. / HD/PDEase domain
Similarity search - Domain/homology
dGTP triphosphohydrolase
Similarity search - Component
Biological speciesLeeuwenhoekiella blandensis MED217 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.1 Å
AuthorsKlemm BP / Sikkema AP
Funding support United States, 3 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of Environmental Health Sciences (NIH/NIEHS)1ZIAES050165 United States
National Institutes of Health/National Institute of Environmental Health Sciences (NIH/NIEHS)1ZICES103326 United States
National Institutes of Health/National Institute of Environmental Health Sciences (NIH/NIEHS)1ZIAES101905 United States
CitationJournal: J Biol Chem / Year: 2022
Title: High-resolution structures of the SAMHD1 dGTPase homolog from Leeuwenhoekiella blandensis reveal a novel mechanism of allosteric activation by dATP.
Authors: Bradley P Klemm / Andrew P Sikkema / Allen L Hsu / James C Horng / Traci M Tanaka Hall / Mario J Borgnia / Roel M Schaaper /
Abstract: Deoxynucleoside triphosphate (dNTP) triphosphohydrolases (dNTPases) are important enzymes that may perform multiple functions in the cell, including regulating the dNTP pools and contributing to ...Deoxynucleoside triphosphate (dNTP) triphosphohydrolases (dNTPases) are important enzymes that may perform multiple functions in the cell, including regulating the dNTP pools and contributing to innate immunity against viruses. Among the homologs that are best studied are human sterile alpha motif and HD domain-containing protein 1 (SAMHD1), a tetrameric dNTPase, and the hexameric Escherichia coli dGTPase; however, it is unclear whether these are representative of all dNTPases given their wide distribution throughout life. Here, we investigated a hexameric homolog from the marine bacterium Leeuwenhoekiella blandensis, revealing that it is a dGTPase that is subject to allosteric activation by dATP, specifically. Allosteric regulation mediated solely by dATP represents a novel regulatory feature among dNTPases that may facilitate maintenance of cellular dNTP pools in L. blandensis. We present high-resolution X-ray crystallographic structures (1.80-2.26 Å) in catalytically important conformations as well as cryo-EM structures (2.1-2.7 Å) of the enzyme bound to dGTP and dATP ligands. The structures, the highest resolution cryo-EM structures of any SAMHD1-like dNTPase to date, reveal an intact metal-binding site with the dGTP substrate coordinated to three metal ions. These structural and biochemical data yield insights into the catalytic mechanism and support a conserved catalytic mechanism for the tetrameric and hexameric dNTPase homologs. We conclude that the allosteric activation by dATP appears to rely on structural connectivity between the allosteric and active sites, as opposed to the changes in oligomeric state upon ligand binding used by SAMHD1.
History
DepositionFeb 2, 2022-
Header (metadata) releaseJun 1, 2022-
Map releaseJun 1, 2022-
UpdateFeb 21, 2024-
Current statusFeb 21, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_26126.png

Downloads & links

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Map

FileDownload / File: emd_26126.map.gz / Format: CCP4 / Size: 190.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationDeepEMhancer post-processed map
Voxel sizeX=Y=Z: 0.646 Å
Density
Contour LevelBy AUTHOR: 0.0295
Minimum - Maximum-0.096104555 - 1.6289883
Average (Standard dev.)0.0027564785 (±0.03676543)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions368368368
Spacing368368368
CellA=B=C: 237.72801 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_26126_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Full-map from RELION refinement

Fileemd_26126_additional_1.map
AnnotationFull-map from RELION refinement
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: RELION post-processed map

Fileemd_26126_additional_2.map
AnnotationRELION post-processed map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: PHENIX auto-sharpened map

Fileemd_26126_additional_3.map
AnnotationPHENIX auto-sharpened map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half-map 1

Fileemd_26126_half_map_1.map
AnnotationHalf-map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half-map 2

Fileemd_26126_half_map_2.map
AnnotationHalf-map 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : dGTP triphosphohydrolase

EntireName: dGTP triphosphohydrolaseDGTPase
Components
  • Complex: dGTP triphosphohydrolaseDGTPase
    • Protein or peptide: dGTP triphosphohydrolaseDGTPase
  • Ligand: MAGNESIUM ION
  • Ligand: water

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Supramolecule #1: dGTP triphosphohydrolase

SupramoleculeName: dGTP triphosphohydrolase / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Leeuwenhoekiella blandensis MED217 (bacteria)

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Macromolecule #1: dGTP triphosphohydrolase

MacromoleculeName: dGTP triphosphohydrolase / type: protein_or_peptide / ID: 1 / Number of copies: 6 / Enantiomer: LEVO / EC number: dGTPase
Source (natural)Organism: Leeuwenhoekiella blandensis MED217 (bacteria) / Strain: CECT 7118 / CCUG 51940 / MED217
Molecular weightTheoretical: 52.723242 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MHHHHHHSSG VDLGTENLYF QSNANWEHLL SLKRQGDTAK RLRIEQDDTR LGFEVDYDRI IFSAPFRSLQ DKTQVIPLSK TDFVHTRLT HSLEVSVVGR SLGRMVGKKL LEKYPHLEQV YGYKFNDFGA IVAAAALAHD IGNPPFGHSG EKAIGEFFKN G YGKRYKDS ...String:
MHHHHHHSSG VDLGTENLYF QSNANWEHLL SLKRQGDTAK RLRIEQDDTR LGFEVDYDRI IFSAPFRSLQ DKTQVIPLSK TDFVHTRLT HSLEVSVVGR SLGRMVGKKL LEKYPHLEQV YGYKFNDFGA IVAAAALAHD IGNPPFGHSG EKAIGEFFKN G YGKRYKDS LTAKEYQDLI KFEGNANGFK VLSQSKPGAQ GGLRLSYATL GAFMKYPKES LPHKPSDHIA DKKYGFFQSE RA LFEDVAQ ELGLLKRSTT DDVSWSRHPL AYLVEAADDI CYTIIDFEDG INLGLIPEEY ALEYMVKLVG QTIDRNKYNA LQE TSDRVS YLRALAIGTL INESVDTFMK YEEEILAGTF DQSLIDKSNY QAQITDIINL SIERIYNSRE VIEKEIAGYE ILST LLEAR CRALDNNDTH YNQLIQQLLA PNDHSEKSLY ENLIQICAEV STMTDGKALR NYKKIKGLD

UniProtKB: dGTP triphosphohydrolase

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Macromolecule #2: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 2 / Number of copies: 6 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #3: water

MacromoleculeName: water / type: ligand / ID: 3 / Number of copies: 6 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER / Water

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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

Concentration0.2 mg/mL
BufferpH: 7.5
Component:
ConcentrationNameFormula
20.0 mMTris
100.0 mMsodium chlorideNaClSodium chloride
5.0 mMmagnesium chlorideMgCl2
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 290 K / Instrument: LEICA EM GP / Details: 3.5 second blot time (front).
DetailsHexamer concentration is listed.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 215000
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
DetailsDefocus values as determined by CTFFIND-4.1
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 3838 pixel / Digitization - Dimensions - Height: 3710 pixel / Number grids imaged: 1 / Number real images: 3780 / Average exposure time: 4.0 sec. / Average electron dose: 58.6 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 996971 / Details: Laplacian-of-Gaussian auto-picking
Startup modelType of model: INSILICO MODEL / In silico model: RELION-generated initial model / Details: Low-pass filtered to ~60 Angstrom
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
Final 3D classificationNumber classes: 5 / Avg.num./class: 119275 / Software - Name: RELION
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: D3 (2x3 fold dihedral) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 2.1 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Number images used: 299926
Details3239 micrographs selected using a maximum resolution cutoff of 4 Angstrom based on CTF fitting.
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

3bg2


Chain - Source name: PDB / Chain - Initial model type: experimental model
DetailsThe hexameric biological assembly of PDB ID: 3BG2 was used as the initial input model to fit into the EM map.
RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-7tu5:
Structure of the L. blandensis dGTPase in the apo form

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