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Open data
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Basic information
Entry | ![]() | |||||||||
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Title | bacteriophage T4 stalled primosome with mutant gp41-E227Q | |||||||||
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Function / homology | ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() Similarity search - Function | |||||||||
Biological species | ![]() ![]() | |||||||||
Method | ![]() ![]() | |||||||||
![]() | Feng X / Li H | |||||||||
Funding support | ![]()
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![]() | Journal: bioRxiv / Year: 2023 Title: Structural basis of the T4 bacteriophage primosome assembly and primer synthesis. Authors: Xiang Feng / Michelle M Spiering / Ruda de Luna Almeida Santos / Stephen J Benkovic / Huilin Li / ![]() Abstract: The T4 bacteriophage gp41 helicase and gp61 primase assemble into a primosome complex to couple DNA unwinding with RNA primer synthesis for DNA replication. How a primosome is assembled and how the ...The T4 bacteriophage gp41 helicase and gp61 primase assemble into a primosome complex to couple DNA unwinding with RNA primer synthesis for DNA replication. How a primosome is assembled and how the length of the RNA primer is defined in the T4 bacteriophage, or in any model system, are unclear. Here we report a series of cryo-EM structures of T4 primosome assembly intermediates at resolutions up to 2.7 Å. We show that the gp41 helicase is an open spiral in the absence of ssDNA, and ssDNA binding triggers a large-scale scissor-like conformational change that drives the open spiral to a closed ring that activates the helicase. We found that the activation of the gp41 helicase exposes a cryptic hydrophobic primase-binding surface allowing for the recruitment of the gp61 primase. The primase binds the gp41 helicase in a bipartite mode in which the N-terminal Zn-binding domain (ZBD) and the C-terminal RNA polymerase domain (RPD) each contain a helicase-interacting motif (HIM1 and HIM2, respectively) that bind to separate gp41 N-terminal hairpin dimers, leading to the assembly of one primase on the helicase hexamer. Based on two observed primosome conformations - one in a DNA-scanning mode and the other in a post RNA primer-synthesis mode - we suggest that the linker loop between the gp61 ZBD and RPD contributes to the T4 pentaribonucleotide primer. Our study reveals T4 primosome assembly process and sheds light on RNA primer synthesis mechanism. | |||||||||
History |
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Structure visualization
Supplemental images |
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Downloads & links
-EMDB archive
Map data | ![]() | 883.2 KB | ![]() | |
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Header (meta data) | ![]() ![]() | 19.1 KB 19.1 KB | Display Display | ![]() |
Images | ![]() | 59.6 KB | ||
Archive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Related structure data | ![]() 8gaoMC ![]() 8dtpC ![]() 8dueC ![]() 8duoC ![]() 8dvfC ![]() 8dviC ![]() 8dw6C ![]() 8dwjC ![]() 8g0zC C: citing same article ( M: atomic model generated by this map |
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Similar structure data | Similarity search - Function & homology ![]() |
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Links
EMDB pages | ![]() ![]() |
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Related items in Molecule of the Month |
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Map
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Voxel size | X=Y=Z: 1.029 Å | ||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||
Details | EMDB XML:
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-Supplemental data
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Sample components
-Entire : complex of T4 primosome with mutant helicase gp41
Entire | Name: complex of T4 primosome with mutant helicase gp41 |
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Components |
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-Supramolecule #1: complex of T4 primosome with mutant helicase gp41
Supramolecule | Name: complex of T4 primosome with mutant helicase gp41 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#5 |
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Source (natural) | Organism: ![]() ![]() |
-Macromolecule #1: DnaB-like replicative helicase
Macromolecule | Name: DnaB-like replicative helicase / type: protein_or_peptide / ID: 1 / Number of copies: 6 / Enantiomer: LEVO EC number: ![]() |
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Source (natural) | Organism: ![]() ![]() |
Molecular weight | Theoretical: 48.796711 KDa |
Recombinant expression | Organism: ![]() ![]() ![]() |
Sequence | String: MVEIILSHLI FDQAYFSKVW PYMDSEYFES GPAKNTFKLI KSHVNEYHSV PSINALNVAL ENSSFTETEY SGVKTLISKL ADSPEDHSW LVKETEKYVQ QRAMFNATSK IIEIQTNAEL PPEKRNKKMP DVGAIPDIMR QALSISFDSY VGHDWMDDYE A RWLSYMNK ...String: MVEIILSHLI FDQAYFSKVW PYMDSEYFES GPAKNTFKLI KSHVNEYHSV PSINALNVAL ENSSFTETEY SGVKTLISKL ADSPEDHSW LVKETEKYVQ QRAMFNATSK IIEIQTNAEL PPEKRNKKMP DVGAIPDIMR QALSISFDSY VGHDWMDDYE A RWLSYMNK ARKVPFKLRI LNKITKGGAE TGTLNVLMAG VNVGKSLGLC SLAADYLQLG HNVLYISMQM AEEVCAKRID AN MLDVSLD DIDDGHISYA EYKGKMEKWR EKSTLGRLIV KQYPTGGADA NTFRSLLNEL KLKKNFVPTI IIVDYLGICK SCR IRVYSE NSYTTVKAIA EELRALAVET ETVLWTAAQV GKQAWDSSDV NMSDIAESAG LPATADFMLA VIETEELAAA EQQL IKQIK SRYGDKNKWN KFLMGVQKGN QKWVEIE UniProtKB: DnaB-like replicative helicase |
-Macromolecule #2: DNA primase
Macromolecule | Name: DNA primase / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO EC number: ![]() |
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Source (natural) | Organism: ![]() ![]() |
Molecular weight | Theoretical: 39.510613 KDa |
Recombinant expression | Organism: ![]() ![]() ![]() |
Sequence | String: SIPWIDNEFA YRALAHLPKF TQVNNSSTFK LRFRCPVCGD SKTDQNKARG WYYGDNNEGN IHCYNCNYHA PIGIYLKEFE PDLYREYIF EIRKEKGKSR PIEKPKELPK QPEKKIIKSL PSCVRLDKLA EDHPIIKYVK ARCIPKDKWK YLWFTTEWPK L VNSIAPGT ...String: SIPWIDNEFA YRALAHLPKF TQVNNSSTFK LRFRCPVCGD SKTDQNKARG WYYGDNNEGN IHCYNCNYHA PIGIYLKEFE PDLYREYIF EIRKEKGKSR PIEKPKELPK QPEKKIIKSL PSCVRLDKLA EDHPIIKYVK ARCIPKDKWK YLWFTTEWPK L VNSIAPGT YKKEISEPRL VIPIYNANGK AESFQGRALK KDAPQKYITI EAYPEATKIY GVERVKDGDV YVLEGPIDSL FI ENGIAIT GGQLDLEVVP FKDRRVWVLD NEPRHPDTIK RMTKLVDAGE RVMFWDKSPW KSKDVNDMIR KEGATPEQIM EYM KNNIAQ GLMAKMRLSK YAK UniProtKB: ![]() |
-Macromolecule #3: DNA (70-mer)
Macromolecule | Name: DNA (70-mer) / type: dna / ID: 3 / Number of copies: 1 / Classification: DNA |
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Source (natural) | Organism: synthetic construct (others) |
Molecular weight | Theoretical: 3.605356 KDa |
Sequence | String: (DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT) (DT)(DT) |
-Macromolecule #5: DNA (70-mer)
Macromolecule | Name: DNA (70-mer) / type: dna / ID: 5 / Number of copies: 1 / Classification: DNA |
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Source (natural) | Organism: synthetic construct (others) |
Molecular weight | Theoretical: 21.900025 KDa |
Sequence | String: (DG)(DA)(DA)(DT)(DG)(DA)(DG)(DG)(DA)(DG) (DT)(DA)(DG)(DT)(DA)(DG)(DT)(DG)(DA)(DA) (DT)(DG)(DT)(DA)(DG)(DT)(DG)(DA)(DG) (DG)(DT)(DA)(DA)(DT)(DA)(DT)(DC)(DG)(DG) (DC) (DT)(DG)(DG)(DT)(DC)(DT) ...String: (DG)(DA)(DA)(DT)(DG)(DA)(DG)(DG)(DA)(DG) (DT)(DA)(DG)(DT)(DA)(DG)(DT)(DG)(DA)(DA) (DT)(DG)(DT)(DA)(DG)(DT)(DG)(DA)(DG) (DG)(DT)(DA)(DA)(DT)(DA)(DT)(DC)(DG)(DG) (DC) (DT)(DG)(DG)(DT)(DC)(DT)(DG)(DG) (DT)(DC)(DT)(DG)(DT)(DG)(DC)(DC)(DA)(DA) (DG)(DT) (DT)(DG)(DC)(DT)(DG)(DC)(DA) (DA)(DA)(DA) |
-Macromolecule #4: RNA (5'-D(*(GTP))-R(P*CP*CP*GP*A)-3')
Macromolecule | Name: RNA (5'-D(*(GTP))-R(P*CP*CP*GP*A)-3') / type: rna / ID: 4 / Number of copies: 1 |
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Source (natural) | Organism: synthetic construct (others) |
Molecular weight | Theoretical: 1.744983 KDa |
Sequence | String: (GTP)CCGA |
-Macromolecule #6: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER
Macromolecule | Name: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / type: ligand / ID: 6 / Number of copies: 5 / Formula: AGS |
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Molecular weight | Theoretical: 523.247 Da |
Chemical component information | ![]() ChemComp-AGS: |
-Macromolecule #7: MAGNESIUM ION
Macromolecule | Name: MAGNESIUM ION / type: ligand / ID: 7 / Number of copies: 5 / Formula: MG |
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Molecular weight | Theoretical: 24.305 Da |
-Macromolecule #8: ZINC ION
Macromolecule | Name: ZINC ION / type: ligand / ID: 8 / Number of copies: 1 / Formula: ZN |
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Molecular weight | Theoretical: 65.409 Da |
-Experimental details
-Structure determination
Method | ![]() |
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Aggregation state | particle |
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Sample preparation
Buffer | pH: 7.8 Component:
Details: 20 mM HEPES pH 7.8, 100 mM NaCl, 10 mM MgCl2 and 2 mM DTT | |||||||||||||||
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Vitrification | Cryogen name: ETHANE |
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Electron microscopy
Microscope | FEI TITAN KRIOS |
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Electron beam | Acceleration voltage: 300 kV / Electron source: ![]() |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD![]() |
Image recording | Film or detector model: GATAN K3 (6k x 4k) / Average electron dose: 66.0 e/Å2 |
Experimental equipment | ![]() Model: Titan Krios / Image courtesy: FEI Company |
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Image processing
Startup model | Type of model: PDB ENTRY PDB model - PDB ID: |
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Initial angle assignment | Type: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.3.1) |
Final 3D classification | Number classes: 3 / Avg.num./class: 800000 / Software - Name: RELION (ver. 3.1) |
Final angle assignment | Type: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1) |
Final reconstruction | Resolution.type: BY AUTHOR / Resolution: 4.1 Å / Resolution method: OTHER / Software - Name: RELION (ver. 3.1) Details: lowest resolution in two combined focus refined maps Number images used: 272868 |
-Atomic model buiding 1
Refinement | Space: REAL / Protocol: OTHER |
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Output model | ![]() PDB-8gao: |