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- EMDB-35056: Cryo-EM Structure of D5 ADP-ssDNA form -

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Basic information

Entry
Database: EMDB / ID: EMD-35056
TitleCryo-EM Structure of D5 ADP-ssDNA form
Map data
Sample
  • Complex: D5 ADP-ssDNA form
    • Protein or peptide: Primase D5
    • DNA: DNA (5'-D(P*TP*TP*TP*TP*TP*T)-3')
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: MAGNESIUM ION
KeywordsMPVX / VIRAL PROTEIN
Function / homologyDNA primase/nucleoside triphosphatase, C-terminal / Poxvirus D5 protein-like / Bacteriophage/plasmid primase, P4, C-terminal / D5 N terminal like / Helicase, superfamily 3, DNA virus / Superfamily 3 helicase of DNA viruses domain profile. / hydrolase activity / P-loop containing nucleoside triphosphate hydrolase / Uncoating factor OPG117
Function and homology information
Biological speciesMonkeypox virus / Homo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsLi YN / Zhu J / Guo YY / Yan RH
Funding support China, 1 items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC)82202517 China
CitationJournal: Nat Struct Mol Biol / Year: 2024
Title: Structural insight into the assembly and working mechanism of helicase-primase D5 from Mpox virus.
Authors: Yaning Li / Jing Zhu / Yingying Guo / Renhong Yan /
Abstract: The Mpox pandemic, caused by the Mpox virus (or monkeypox virus, MPXV), has gained global attention. The D5 protein, a putative helicase-primase found in MPXV, plays a vital role in viral replication ...The Mpox pandemic, caused by the Mpox virus (or monkeypox virus, MPXV), has gained global attention. The D5 protein, a putative helicase-primase found in MPXV, plays a vital role in viral replication and genome uncoating. Here we determined multiple cryo-EM structures of full-length hexameric D5 in diverse states. These states were captured during ATP hydrolysis while moving along the single-stranded DNA (ssDNA) track. Through comprehensive structural analysis combined with the helicase activity system, we revealed that when the primase domain is truncated or the interaction between the primase and helicase domains is disrupted, the double-stranded DNA (dsDNA) unwinds into ssDNA, suggesting a critical regulatory role of the primase domain. Two transition states bound with ssDNA substrate during unwinding reveals that two ATP molecules were consumed to drive DNA moving forward two nucleotides. Collectively, our findings shed light on the molecular mechanism that links ATP hydrolysis to the DNA unwinding in poxviruses.
History
DepositionDec 29, 2022-
Header (metadata) releaseJan 10, 2024-
Map releaseJan 10, 2024-
UpdateJan 31, 2024-
Current statusJan 31, 2024Processing site: PDBc / Status: Released

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Structure visualization

Supplemental images

emd_35056.png

Downloads & links

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Map

FileDownload / File: emd_35056.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
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AxesZ (Sec.)Y (Row.)X (Col.)
1.07 Å/pix.
x 256 pix.
= 274.432 Å		1.07 Å/pix.
x 256 pix.
= 274.432 Å		1.07 Å/pix.
x 256 pix.
= 274.432 Å

Surface

Projections

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.072 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.02
Minimum - Maximum-0.06213218 - 0.12450667
Average (Standard dev.)0.00013444283 (±0.0040892838)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 274.432 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_35056_half_map_1.map
Projections & Slices
AxesZYX

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Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_35056_half_map_2.map
Projections & Slices
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Histogram (log scale)

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Sample components

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Entire : D5 ADP-ssDNA form

EntireName: D5 ADP-ssDNA form
Components
  • Complex: D5 ADP-ssDNA form
    • Protein or peptide: Primase D5
    • DNA: DNA (5'-D(P*TP*TP*TP*TP*TP*T)-3')
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: MAGNESIUM ION

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Supramolecule #1: D5 ADP-ssDNA form

SupramoleculeName: D5 ADP-ssDNA form / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Monkeypox virus

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Macromolecule #1: Primase D5

MacromoleculeName: Primase D5 / type: protein_or_peptide / ID: 1 / Number of copies: 6 / Enantiomer: LEVO
Source (natural)Organism: Monkeypox virus
Molecular weightTheoretical: 90.476344 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MDAAIRGNDV IFVLKTIGVP SACRQNEDPR FVEAFKCDEL ERYIDNNPEC TLFESLRDEE AYSIVRIFMD VDLDACLDEI DYLTAIQDF IIEVSNCVAR FAFTECGAIH ENVIKSMRSN FSLTKSTNRD KTSFHIIFLD TYTTMDTLIA MKRTLLELSR S SENPLTRS ...String:
MDAAIRGNDV IFVLKTIGVP SACRQNEDPR FVEAFKCDEL ERYIDNNPEC TLFESLRDEE AYSIVRIFMD VDLDACLDEI DYLTAIQDF IIEVSNCVAR FAFTECGAIH ENVIKSMRSN FSLTKSTNRD KTSFHIIFLD TYTTMDTLIA MKRTLLELSR S SENPLTRS IDTAVYRRKT TLRVVGTRKN PNCDTIHVMQ PPHDNIEDYL FTYVDMNNNS YYFSLQRRLE DLVPDKLWEP GF ISFEDAI KRVSKIFINS IINFNDLDEN NFTTVPLVID YVTPCALCKK RSHKHPHQLS LENGAIRIYK TGNPHSCKVK IVP LDGNKL FNIAQRILDT NSVLLTERGD HIVWINNSWK FNSEEPLITK LILSIRHQLP KEYSSELLCP RKRKTVEANI RDML VDSVE TDTYPDKLPF KNGVLDLVDG MFYSGDDAKK YTCTVSTGFK FDDTKFVEDS PEMEELMNII NDIQPLTDEN KKNRE LYEK TLSSCLCGAT KGCLTFFFGE TATGKSTTKR LLKSAIGDLF VETGQTILTD VLDKGPNPFI ANMHLKRSVF CSELPD FAC SGSKKIRSDN IKKLTEPCVI GRPCFSNKIN NRNHATIIID TNYKPVFDRI DNALMRRIAV VRFRTHFSQP SGREAAE NN DAYDKVKLLD EGLDGKIQNN RYRFAFLYLL VKWYKKYHIP IMKLYPTPEE IPDFAFYLKI GTLLVSSSVK HIPLMTDL S KKGYILYDNV VTLPLTTFQQ KISKYFNSRL FGHDIESFIN RHKKFANVSD EYLQYIFIED ISSP

UniProtKB: Uncoating factor OPG117

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Macromolecule #2: DNA (5'-D(P*TP*TP*TP*TP*TP*T)-3')

MacromoleculeName: DNA (5'-D(P*TP*TP*TP*TP*TP*T)-3') / type: dna / ID: 2 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 1.780199 KDa
SequenceString:
(DT)(DT)(DT)(DT)(DT)(DT)

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Macromolecule #3: ADENOSINE-5'-DIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-DIPHOSPHATE / type: ligand / ID: 3 / Number of copies: 6 / Formula: ADP
Molecular weightTheoretical: 427.201 Da
Chemical component information

ChemComp-ADP:
ADENOSINE-5'-DIPHOSPHATE / ADP, energy-carrying molecule*YM

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Macromolecule #4: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 4 / Number of copies: 3 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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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: 7.5
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.2 µm / Nominal defocus min: 1.2 µm
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: OTHER
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0.6) / Number images used: 60551
Initial angle assignmentType: ANGULAR RECONSTITUTION
Final angle assignmentType: MAXIMUM LIKELIHOOD

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