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- EMDB-13654: DNA polymerase from M. tuberculosis -

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Basic information

Entry
Database: EMDB / ID: EMD-13654
TitleDNA polymerase from M. tuberculosis
Map data
Sample
  • Complex: DNA polymerase from M. tuberculosis
    • Protein or peptide: DNA polymerase III subunit alphaDNA polymerase III holoenzyme
    • Other: Template
    • Other: primer
  • Ligand: ZINC ION
  • Ligand: [(1~{S},3~{R},4~{R},5~{R},6~{R},7~{S},8~{R},11~{S},13~{S},16~{S},17~{R},18~{E})-13-methoxy-5,17,19-trimethyl-6-oxidanyl-16-[(1~{R})-1-oxidanylethyl]-14-oxidanylidene-2,15-dioxatetracyclo[9.8.0.0^{1,7}.0^{3,8}]nonadeca-9,18-dien-4-yl] 1~{H}-pyrrole-2-carboxylate
  • Ligand: water
Function / homology
Function and homology information


3'-5' exonuclease activity / DNA replication / DNA-directed DNA polymerase / DNA-directed DNA polymerase activity / cytoplasm
Similarity search - Function
Bacterial DNA polymerase III alpha subunit, thumb domain / DNA polymerase III, alpha subunit / Bacterial DNA polymerase III, alpha subunit, NTPase domain / DNA polymerase, helix-hairpin-helix motif / DNA polymerase III alpha subunit finger domain / Bacterial DNA polymerase III alpha NTPase domain / Helix-hairpin-helix motif / Bacterial DNA polymerase III alpha subunit finger domain / PHP domain / PHP domain ...Bacterial DNA polymerase III alpha subunit, thumb domain / DNA polymerase III, alpha subunit / Bacterial DNA polymerase III, alpha subunit, NTPase domain / DNA polymerase, helix-hairpin-helix motif / DNA polymerase III alpha subunit finger domain / Bacterial DNA polymerase III alpha NTPase domain / Helix-hairpin-helix motif / Bacterial DNA polymerase III alpha subunit finger domain / PHP domain / PHP domain / Polymerase/histidinol phosphatase, N-terminal / DNA polymerase alpha chain like domain / Polymerase/histidinol phosphatase-like
Similarity search - Domain/homology
DNA-directed DNA polymerase
Similarity search - Component
Biological speciesMycobacterium tuberculosis (bacteria) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.9 Å
AuthorsBorsellini A / Lamers MH
Funding support United States, 1 items
OrganizationGrant numberCountry
Bill & Melinda Gates Foundation United States
CitationJournal: ACS Infect Dis / Year: 2022
Title: DNA-Dependent Binding of Nargenicin to DnaE1 Inhibits Replication in .
Authors: Melissa D Chengalroyen / Mandy K Mason / Alessandro Borsellini / Raffaella Tassoni / Garth L Abrahams / Sasha Lynch / Yong-Mo Ahn / Jon Ambler / Katherine Young / Brendan M Crowley / David B ...Authors: Melissa D Chengalroyen / Mandy K Mason / Alessandro Borsellini / Raffaella Tassoni / Garth L Abrahams / Sasha Lynch / Yong-Mo Ahn / Jon Ambler / Katherine Young / Brendan M Crowley / David B Olsen / Digby F Warner / Clifton E Barry Iii / Helena I M Boshoff / Meindert H Lamers / Valerie Mizrahi /
Abstract: Natural products provide a rich source of potential antimicrobials for treating infectious diseases for which drug resistance has emerged. Foremost among these diseases is tuberculosis. Assessment of ...Natural products provide a rich source of potential antimicrobials for treating infectious diseases for which drug resistance has emerged. Foremost among these diseases is tuberculosis. Assessment of the antimycobacterial activity of nargenicin, a natural product that targets the replicative DNA polymerase of , revealed that it is a bactericidal genotoxin that induces a DNA damage response in () and inhibits growth by blocking the replicative DNA polymerase, DnaE1. Cryo-electron microscopy revealed that binding of nargenicin to DnaE1 requires the DNA substrate such that nargenicin is wedged between the terminal base pair and the polymerase and occupies the position of both the incoming nucleotide and templating base. Comparative analysis across three bacterial species suggests that the activity of nargenicin is partly attributable to the DNA binding affinity of the replicative polymerase. This work has laid the foundation for target-led drug discovery efforts focused on DnaE1.
History
DepositionSep 28, 2021-
Header (metadata) releaseFeb 23, 2022-
Map releaseFeb 23, 2022-
UpdateApr 13, 2022-
Current statusApr 13, 2022Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.019
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.019
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7pu7
  • Surface level: 0.019
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_13654.png

Downloads & links

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Map

FileDownload / File: emd_13654.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.87 Å/pix.
x 256 pix.
= 221.696 Å		0.87 Å/pix.
x 256 pix.
= 221.696 Å		0.87 Å/pix.
x 256 pix.
= 221.696 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.866 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0216 / Movie #1: 0.019
Minimum - Maximum-0.046119027 - 0.110750996
Average (Standard dev.)0.00023045097 (±0.0024507917)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 221.696 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.8660.8660.866
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z221.696221.696221.696
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-0.0460.1110.000

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Supplemental data

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Sample components

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Entire : DNA polymerase from M. tuberculosis

EntireName: DNA polymerase from M. tuberculosis
Components
  • Complex: DNA polymerase from M. tuberculosis
    • Protein or peptide: DNA polymerase III subunit alphaDNA polymerase III holoenzyme
    • Other: Template
    • Other: primer
  • Ligand: ZINC ION
  • Ligand: [(1~{S},3~{R},4~{R},5~{R},6~{R},7~{S},8~{R},11~{S},13~{S},16~{S},17~{R},18~{E})-13-methoxy-5,17,19-trimethyl-6-oxidanyl-16-[(1~{R})-1-oxidanylethyl]-14-oxidanylidene-2,15-dioxatetracyclo[9.8.0.0^{1,7}.0^{3,8}]nonadeca-9,18-dien-4-yl] 1~{H}-pyrrole-2-carboxylate
  • Ligand: water

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Supramolecule #1: DNA polymerase from M. tuberculosis

SupramoleculeName: DNA polymerase from M. tuberculosis / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Source (natural)Organism: Mycobacterium tuberculosis (bacteria)
Recombinant expressionOrganism: Mycolicibacterium smegmatis (bacteria)
Molecular weightTheoretical: 129.3 kDa/nm

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Macromolecule #1: DNA polymerase III subunit alpha

MacromoleculeName: DNA polymerase III subunit alpha / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: DNA-directed DNA polymerase
Source (natural)Organism: Mycobacterium tuberculosis (bacteria)
Molecular weightTheoretical: 129.479734 KDa
Recombinant expressionOrganism: Mycolicibacterium smegmatis (bacteria)
SequenceString: MSGSSAGSSF VHLHNHTEYS MLDGAAKITP MLAEVERLGM PAVGMTDHGN MFGASEFYNS ATKAGIKPII GVEAYIAPGS RFDTRRILW GDPSQKADDV SGSGSYTHLT MMAENATGLR NLFKLSSHAS FEGQLSKWSR MDAELIAEHA EGIIITTGCP S GEVQTRLR ...String:
MSGSSAGSSF VHLHNHTEYS MLDGAAKITP MLAEVERLGM PAVGMTDHGN MFGASEFYNS ATKAGIKPII GVEAYIAPGS RFDTRRILW GDPSQKADDV SGSGSYTHLT MMAENATGLR NLFKLSSHAS FEGQLSKWSR MDAELIAEHA EGIIITTGCP S GEVQTRLR LGQDREALEA AAKWREIVGP DNYFLELMDH GLTIERRVRD GLLEIGRALN IPPLATNDCH YVTRDAAHNH EA LLCVQTG KTLSDPNRFK FDGDGYYLKS AAEMRQIWDD EVPGACDSTL LIAERVQSYA DVWTPRDRMP VFPVPDGHDQ ASW LRHEVD AGLRRRFPAG PPDGYRERAA YEIDVICSKG FPSYFLIVAD LISYARSAGI RVGPGRGSAA GSLVAYALGI TDID PIPHG LLFERFLNPE RTSMPDIDID FDDRRRGEMV RYAADKWGHD RVAQVITFGT IKTKAALKDS ARIHYGQPGF AIADR ITKA LPPAIMAKDI PLSGITDPSH ERYKEAAEVR GLIETDPDVR TIYQTARGLE GLIRNAGVHA CAVIMSSEPL TEAIPL WKR PQDGAIITGW DYPACEAIGL LKMDFLGLRN LTIIGDAIDN VRANRGIDLD LESVPLDDKA TYELLGRGDT LGVFQLD GG PMRDLLRRMQ PTGFEDVVAV IALYRPGPMG MNAHNDYADR KNNRQAIKPI HPELEEPLRE ILAETYGLIV YQEQIMRI A QKVASYSLAR ADILRKAMGK KKREVLEKEF EGFSDGMQAN GFSPAAIKAL WDTILPFADY AFNKSHAAGY GMVSYWTAY LKANYPAEYM AGLLTSVGDD KDKAAVYLAD CRKLGITVLP PDVNESGLNF ASVGQDIRYG LGAVRNVGAN VVGSLLQTRN DKGKFTDFS DYLNKIDISA CNKKVTESLI KAGAFDSLGH ARKGLFLVHS DAVDSVLGTK KAEALGQFDL FGSNDDGTGT A DPVFTIKV PDDEWEDKHK LALEREMLGL YVSGHPLNGV AHLLAAQVDT AIPAILDGDV PNDAQVRVGG ILASVNRRVN KN GMPWASA QLEDLTGGIE VMFFPHTYSS YGADIVDDAV VLVNAKVAVR DDRIALIAND LTVPDFSNAE VERPLAVSLP TRQ CTFDKV SALKQVLARH PGTSQVHLRL ISGDRITTLA LDQSLRVTPS PALMGDLKEL LGPGCLGS

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Macromolecule #2: Template

MacromoleculeName: Template / type: other / ID: 2 / Number of copies: 1
Classification: polydeoxyribonucleotide/polyribonucleotide hybrid
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 5.944902 KDa
SequenceString:
(DA)(DA)(DA)(DA)(DG)(DA)(DA)(DG)(DG)(DA) (DC)(DG)(DA)(DA)(DG)(DG)(DA)(DC)(DT)

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Macromolecule #3: primer

MacromoleculeName: primer / type: other / ID: 3 / Number of copies: 1
Classification: polydeoxyribonucleotide/polyribonucleotide hybrid
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 4.197727 KDa
SequenceString:
(DA)(DG)(DT)(DC)(DC)(DT)(DT)(DC)(DG)(DT) (DC)(DC)(DT)(DT)

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Macromolecule #4: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 4 / Number of copies: 3 / Formula: ZN
Molecular weightTheoretical: 65.409 Da

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Macromolecule #5: [(1~{S},3~{R},4~{R},5~{R},6~{R},7~{S},8~{R},11~{S},13~{S},16~{S},...

MacromoleculeName: [(1~{S},3~{R},4~{R},5~{R},6~{R},7~{S},8~{R},11~{S},13~{S},16~{S},17~{R},18~{E})-13-methoxy-5,17,19-trimethyl-6-oxidanyl-16-[(1~{R})-1-oxidanylethyl]-14-oxidanylidene-2,15-dioxatetracyclo[9.8.0. ...Name: [(1~{S},3~{R},4~{R},5~{R},6~{R},7~{S},8~{R},11~{S},13~{S},16~{S},17~{R},18~{E})-13-methoxy-5,17,19-trimethyl-6-oxidanyl-16-[(1~{R})-1-oxidanylethyl]-14-oxidanylidene-2,15-dioxatetracyclo[9.8.0.0^{1,7}.0^{3,8}]nonadeca-9,18-dien-4-yl] 1~{H}-pyrrole-2-carboxylate
type: ligand / ID: 5 / Number of copies: 1 / Formula: 82W
Molecular weightTheoretical: 515.595 Da
Chemical component information

ChemComp-82W:
[(1~{S},3~{R},4~{R},5~{R},6~{R},7~{S},8~{R},11~{S},13~{S},16~{S},17~{R},18~{E})-13-methoxy-5,17,19-trimethyl-6-oxidanyl-16-[(1~{R})-1-oxidanylethyl]-14-oxidanylidene-2,15-dioxatetracyclo[9.8.0.0^{1,7}.0^{3,8}]nonadeca-9,18-dien-4-yl] 1~{H}-pyrrole-2-carboxylate

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Macromolecule #6: water

MacromoleculeName: water / type: ligand / ID: 6 / Number of copies: 3 / 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

BufferpH: 7
Component:
ConcentrationFormulaName
20.0 mMC8H18N2O6S2PIPES
50.0 mMC5H10KNO4potassium glutammate
2.0 mMC4H10O2S2DTT
0.01 %C58H114O26tween20
GridModel: Quantifoil R2/1 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.0002 kPa
VitrificationCryogen name: ETHANE / Chamber humidity: 76 % / Chamber temperature: 277 K / Instrument: LEICA EM GP

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 0.8 µm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 2 / Average electron dose: 50.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 196709
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-7pu7:
DNA polymerase from M. tuberculosis

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