+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-26126 | ||||||||||||
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Title | Structure of the L. blandensis dGTPase in the apo form | ||||||||||||
Map data | DeepEMhancer post-processed map | ||||||||||||
Sample |
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Keywords | dGTPase / nucleotide binding / HYDROLASE | ||||||||||||
Function / homology | Function and homology information | ||||||||||||
Biological species | Leeuwenhoekiella blandensis MED217 (bacteria) | ||||||||||||
Method | single particle reconstruction / cryo EM / Resolution: 2.1 Å | ||||||||||||
Authors | Klemm BP / Sikkema AP | ||||||||||||
Funding support | United States, 3 items
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Citation | Journal: 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 |
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-Structure visualization
Supplemental images |
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-Downloads & links
-EMDB archive
Map data | emd_26126.map.gz | 159.1 MB | EMDB map data format | |
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Header (meta data) | emd-26126-v30.xml emd-26126.xml | 27.3 KB 27.3 KB | Display Display | EMDB header |
FSC (resolution estimation) | emd_26126_fsc.xml | 13 KB | Display | FSC data file |
Images | emd_26126.png | 135.4 KB | ||
Masks | emd_26126_msk_1.map | 190.1 MB | Mask map | |
Filedesc metadata | emd-26126.cif.gz | 6.9 KB | ||
Others | emd_26126_additional_1.map.gz emd_26126_additional_2.map.gz emd_26126_additional_3.map.gz emd_26126_half_map_1.map.gz emd_26126_half_map_2.map.gz | 149.4 MB 19.6 MB 176.2 MB 150.8 MB 150.8 MB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-26126 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-26126 | HTTPS FTP |
-Validation report
Summary document | emd_26126_validation.pdf.gz | 844.8 KB | Display | EMDB validaton report |
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Full document | emd_26126_full_validation.pdf.gz | 844.4 KB | Display | |
Data in XML | emd_26126_validation.xml.gz | 20.7 KB | Display | |
Data in CIF | emd_26126_validation.cif.gz | 27.1 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-26126 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-26126 | HTTPS FTP |
-Related structure data
Related structure data | 7tu5MC 7tu0C 7tu1C 7tu2C 7tu3C 7tu4C 7tu6C 7tu7C 7tu8C C: citing same article (ref.) M: atomic model generated by this map |
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Similar structure data | Similarity search - Function & homologyF&H Search |
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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Related items in Molecule of the Month |
-Map
File | Download / File: emd_26126.map.gz / Format: CCP4 / Size: 190.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||
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Annotation | DeepEMhancer post-processed map | ||||||||||||||||||||||||||||||||||||
Projections & slices | Image control
Images are generated by Spider. | ||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 0.646 Å | ||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
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-Supplemental data
-Mask #1
File | emd_26126_msk_1.map | ||||||||||||
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Density Histograms |
-Additional map: Full-map from RELION refinement
File | emd_26126_additional_1.map | ||||||||||||
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Annotation | Full-map from RELION refinement | ||||||||||||
Projections & Slices |
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Density Histograms |
-Additional map: RELION post-processed map
File | emd_26126_additional_2.map | ||||||||||||
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Annotation | RELION post-processed map | ||||||||||||
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Density Histograms |
-Additional map: PHENIX auto-sharpened map
File | emd_26126_additional_3.map | ||||||||||||
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Annotation | PHENIX auto-sharpened map | ||||||||||||
Projections & Slices |
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Density Histograms |
-Half map: Half-map 1
File | emd_26126_half_map_1.map | ||||||||||||
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Annotation | Half-map 1 | ||||||||||||
Projections & Slices |
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Density Histograms |
-Half map: Half-map 2
File | emd_26126_half_map_2.map | ||||||||||||
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Annotation | Half-map 2 | ||||||||||||
Projections & Slices |
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Density Histograms |
-Sample components
-Entire : dGTP triphosphohydrolase
Entire | Name: dGTP triphosphohydrolase |
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Components |
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-Supramolecule #1: dGTP triphosphohydrolase
Supramolecule | Name: dGTP triphosphohydrolase / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1 |
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Source (natural) | Organism: Leeuwenhoekiella blandensis MED217 (bacteria) |
-Macromolecule #1: dGTP triphosphohydrolase
Macromolecule | Name: dGTP triphosphohydrolase / type: protein_or_peptide / ID: 1 / Number of copies: 6 / Enantiomer: LEVO / EC number: dGTPase |
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Source (natural) | Organism: Leeuwenhoekiella blandensis MED217 (bacteria) / Strain: CECT 7118 / CCUG 51940 / MED217 |
Molecular weight | Theoretical: 52.723242 KDa |
Recombinant expression | Organism: Escherichia coli BL21(DE3) (bacteria) |
Sequence | String: 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 |
-Macromolecule #2: MAGNESIUM ION
Macromolecule | Name: MAGNESIUM ION / type: ligand / ID: 2 / Number of copies: 6 / Formula: MG |
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Molecular weight | Theoretical: 24.305 Da |
-Macromolecule #3: water
Macromolecule | Name: water / type: ligand / ID: 3 / Number of copies: 6 / Formula: HOH |
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Molecular weight | Theoretical: 18.015 Da |
Chemical component information | ChemComp-HOH: |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 0.2 mg/mL | ||||||||||||
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Buffer | pH: 7.5 Component:
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Grid | Model: UltrAuFoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE | ||||||||||||
Vitrification | Cryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 290 K / Instrument: LEICA EM GP / Details: 3.5 second blot time (front). | ||||||||||||
Details | Hexamer concentration is listed. |
-Electron microscopy
Microscope | FEI TITAN KRIOS |
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Specialist optics | Energy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV |
Details | Defocus values as determined by CTFFIND-4.1 |
Image recording | Film 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 |
Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 215000 |
Sample stage | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN |
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
+Image processing
-Atomic model buiding 1
Initial model | PDB ID: Chain - Source name: PDB / Chain - Initial model type: experimental model |
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Details | The hexameric biological assembly of PDB ID: 3BG2 was used as the initial input model to fit into the EM map. |
Refinement | Space: REAL / Protocol: RIGID BODY FIT |
Output model | PDB-7tu5: |