+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-3458 | |||||||||
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Title | negative-stain volume of Sso DNA PolB1 | |||||||||
Map data | Final map from iteration 10 onto which FSC has been calculated and displayed (Figure S4C, right). Contour level in Chimera | |||||||||
Sample |
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Biological species | Archaea (unknown) | |||||||||
Method | single particle reconstruction / negative staining / Resolution: 22.8 Å | |||||||||
Authors | Abrescia NGA / Bell SD | |||||||||
Citation | Journal: Nat Commun / Year: 2017 Title: Identification and characterization of a heterotrimeric archaeal DNA polymerase holoenzyme. Authors: Jiangyu Yan / Thomas R Beattie / Adriana L Rojas / Kelly Schermerhorn / Tamzin Gristwood / Jonathan C Trinidad / Sonja V Albers / Pietro Roversi / Andrew F Gardner / Nicola G A Abrescia / Stephen D Bell / Abstract: Since their initial characterization over 30 years ago, it has been believed that the archaeal B-family DNA polymerases are single-subunit enzymes. This contrasts with the multi-subunit B-family ...Since their initial characterization over 30 years ago, it has been believed that the archaeal B-family DNA polymerases are single-subunit enzymes. This contrasts with the multi-subunit B-family replicative polymerases of eukaryotes. Here we reveal that the highly studied PolB1 from Sulfolobus solfataricus exists as a heterotrimeric complex in cell extracts. Two small subunits, PBP1 and PBP2, associate with distinct surfaces of the larger catalytic subunit and influence the enzymatic properties of the DNA polymerase. Thus, multi-subunit replicative DNA polymerase holoenzymes are present in all three domains of life. We reveal the architecture of the assembly by a combination of cross-linking coupled with mass spectrometry, X-ray crystallography and single-particle electron microscopy. The small subunits stabilize the holoenzyme assembly and the acidic tail of one small subunit mitigates the ability of the enzyme to perform strand-displacement synthesis, with important implications for lagging strand DNA synthesis. | |||||||||
History |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | EM map: SurfViewMolmilJmol/JSmol |
Supplemental images |
-Downloads & links
-EMDB archive
Map data | emd_3458.map.gz | 6.1 MB | EMDB map data format | |
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Header (meta data) | emd-3458-v30.xml emd-3458.xml | 21.5 KB 21.5 KB | Display Display | EMDB header |
Images | emd_3458.png | 111.1 KB | ||
Masks | emd_3458_msk_1.map | 8 MB | Mask map | |
Others | emd_3458_additional.map.gz emd_3458_half_map_1.map.gz emd_3458_half_map_2.map.gz | 874.8 KB 6.1 MB 6.1 MB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-3458 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-3458 | HTTPS FTP |
-Validation report
Summary document | emd_3458_validation.pdf.gz | 303.9 KB | Display | EMDB validaton report |
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Full document | emd_3458_full_validation.pdf.gz | 303.1 KB | Display | |
Data in XML | emd_3458_validation.xml.gz | 8.1 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-3458 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-3458 | HTTPS FTP |
-Related structure data
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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-Map
File | Download / File: emd_3458.map.gz / Format: CCP4 / Size: 8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | Final map from iteration 10 onto which FSC has been calculated and displayed (Figure S4C, right). Contour level in Chimera | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 1.66 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Mask #1
File | emd_3458_msk_1.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Additional map: This is the masked map used in Figure...
File | emd_3458_additional.map | ||||||||||||
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Annotation | This is the masked map used in Figure 4A and in submitted figure. Contour level in Chimera. | ||||||||||||
Projections & Slices |
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Density Histograms |
-Half map: half2 map from iteration 10. Contour level in Chimera
File | emd_3458_half_map_1.map | ||||||||||||
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Annotation | half2 map from iteration 10. Contour level in Chimera | ||||||||||||
Projections & Slices |
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Density Histograms |
-Half map: half1 map from iteration 10. Contour level in Chimera
File | emd_3458_half_map_2.map | ||||||||||||
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Annotation | half1 map from iteration 10. Contour level in Chimera | ||||||||||||
Projections & Slices |
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Density Histograms |
-Sample components
-Entire : archaeal DNA polymerase B1
Entire | Name: archaeal DNA polymerase B1 |
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Components |
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-Supramolecule #1: archaeal DNA polymerase B1
Supramolecule | Name: archaeal DNA polymerase B1 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all / Details: This is the apo enzyme. |
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Source (natural) | Organism: Archaea (unknown) |
Recombinant expression | Organism: Escherichia coli (E. coli) / Recombinant plasmid: pET33b |
Molecular weight | Theoretical: 101 KDa |
-Macromolecule #1: DNA polymerase B1
Macromolecule | Name: DNA polymerase B1 / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO |
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Source (natural) | Organism: Archaea (unknown) |
Recombinant expression | Organism: Escherichia coli (E. coli) |
Sequence | String: MAKQLTLFDI PSSKPAKSEQ NTQQSQQSAP VEEKKVVRRE WLEEAQENKI YFLLQVDYDG KKGKAVCKLF DKETQKIYAL YDNTGHKPYF LVDLEPDKVG KIPKIVRDPS FDHIETVSKI DPYTWNKFKL TKIVVRDPLA VRRLRNDVPK AYEAHIKYFN NYMYDIGLIP ...String: MAKQLTLFDI PSSKPAKSEQ NTQQSQQSAP VEEKKVVRRE WLEEAQENKI YFLLQVDYDG KKGKAVCKLF DKETQKIYAL YDNTGHKPYF LVDLEPDKVG KIPKIVRDPS FDHIETVSKI DPYTWNKFKL TKIVVRDPLA VRRLRNDVPK AYEAHIKYFN NYMYDIGLIP GMPYVVKNGK LESVYLSLDE KDVEEIKKAF ADSDEMTRQM AVDWLPIFET EIPKIKRVAI DIEVYTPVKG RIPDSQKAEF PIISIALAGS DGLKKVLVLN RNDVNEGSVK LDGISVERFN TEYELLGRFF DILLEYPIVL TFNGDDFDLP YIYFRALKLG YFPEEIPIDV AGKDEAKYLA GLHIDLYKFF FNKAVRNYAF EGKYNEYNLD AVAKALLGTS KVKVDTLISF LDVEKLIEYN FRDAEITLQL TTFNNDLTMK LIVLFSRISR LGIEELTRTE ISTWVKNLYY WEHRKRNWLI PLKEEILAKS SNIRTSALIK GKGYKGAVVI DPPAGIFFNI TVLDFASLYP SIIRTWNLSY ETVDIQQCKK PYEVKDETGE VLHIVCMDRP GITAVITGLL RDFRVKIYKK KAKNPNNSEE QKLLYDVVQR AMKVFINATY GVFGAETFPL YAPAVAESVT ALGRYVITST VKKAREEGLT VLYGDTDSLF LLNPPKNSLE NIIKWVKTTF NLDLEVDKTY KFVAFSGLKK NYFGVYQDGK VDIKGMLVKK RNTPEFVKKV FNEVKELMIS INSPNDVKEI KRKIVDVVKG SYEKLKNKGY NLDELAFKVM LSKPLDAYKK NTPQHVKAAL QLRPFGVNVL PRDIIYYVKV RSKDGVKPVQ LAKVTEIDAE KYLEALRSTF EQILRAFGVS WDEIAATMSI DSFFSYPSKG NS |
-Experimental details
-Structure determination
Method | negative staining |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 0.01 mg/mL | ||||||||||||
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Buffer | pH: 7.5 Component:
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Staining | Type: NEGATIVE / Material: uranyl formate Details: Negatively stained EM specimens were prepared using carbon-coated grids and stained with 2% of uranyl formate solution. | ||||||||||||
Grid | Model: EMS / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.02 kPa |
-Electron microscopy
Microscope | JEOL 2200FSC |
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Specialist optics | Energy filter - Name: In-column Omega Filter / Energy filter - Lower energy threshold: 0 eV / Energy filter - Upper energy threshold: 20 eV |
Image recording | Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Number real images: 108 / Average exposure time: 0.5 sec. / Average electron dose: 30.0 e/Å2 |
Electron beam | Acceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Calibrated magnification: 90201 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.0 mm / Nominal defocus max: 1.7 µm / Nominal defocus min: 1.3 µm / Nominal magnification: 60000 |
Sample stage | Specimen holder model: JEOL |
+Image processing
-Atomic model buiding 1
Refinement | Space: REAL / Protocol: RIGID BODY FIT / Target criteria: Cross-correlation coefficient |
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