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- EMDB-47824: T4 bacteriophage replicative holoenzyme, DNA in left Polymerase(S... -

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Basic information

Entry
Database: EMDB / ID: EMD-47824
TitleT4 bacteriophage replicative holoenzyme, DNA in left Polymerase(State 2)
Map dataT4 bacteriophage replicative holoenzyme, DNA in left Polymerase(State 2)
Sample
  • Complex: T4 bacteriophage replication holoenzyme complex
    • Protein or peptide: DNA-directed DNA polymerase
    • Protein or peptide: Sliding clamp
    • DNA: DNA (5'-AGC TAT GAC CAT GAT TAC GAA TTG ddC-3')
    • DNA: DNA (38-MER)
Keywordsreplication / T4 / holoenzyme / replication-DNA complex
Function / homology
Function and homology information


bidirectional double-stranded viral DNA replication / viral DNA genome replication / Hydrolases; Acting on ester bonds; Exodeoxyribonucleases producing 5'-phosphomonoesters / DNA polymerase processivity factor activity / viral transcription / 3'-5' exonuclease activity / DNA-templated DNA replication / DNA-directed DNA polymerase / DNA-directed DNA polymerase activity / DNA replication ...bidirectional double-stranded viral DNA replication / viral DNA genome replication / Hydrolases; Acting on ester bonds; Exodeoxyribonucleases producing 5'-phosphomonoesters / DNA polymerase processivity factor activity / viral transcription / 3'-5' exonuclease activity / DNA-templated DNA replication / DNA-directed DNA polymerase / DNA-directed DNA polymerase activity / DNA replication / nucleotide binding / DNA binding / metal ion binding
Similarity search - Function
Sliding clamp, C-terminal / Sliding clamp / gp45 sliding clamp, C terminal / DNA polymerase processivity factor / DNA polymerase processivity factor / DNA-directed DNA polymerase T4 type / : / DNA-directed DNA polymerase, family B, multifunctional domain / DNA-directed DNA polymerase, family B, conserved site / DNA polymerase family B signature. ...Sliding clamp, C-terminal / Sliding clamp / gp45 sliding clamp, C terminal / DNA polymerase processivity factor / DNA polymerase processivity factor / DNA-directed DNA polymerase T4 type / : / DNA-directed DNA polymerase, family B, multifunctional domain / DNA-directed DNA polymerase, family B, conserved site / DNA polymerase family B signature. / DNA polymerase family B / : / DNA polymerase family B, exonuclease domain / DNA-directed DNA polymerase, family B, exonuclease domain / DNA polymerase, palm domain superfamily / DNA polymerase type-B family / DNA-directed DNA polymerase, family B / Ribonuclease H superfamily / Ribonuclease H-like superfamily / DNA/RNA polymerase superfamily
Similarity search - Domain/homology
DNA-directed DNA polymerase / Sliding clamp
Similarity search - Component
Biological speciesEscherichia phage T4 (virus) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.7 Å
AuthorsLi H / Feng X
Funding support United States, 2 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM013306 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM131754 United States
CitationJournal: Nucleic Acids Res / Year: 2025
Title: Structural insights into the exchange mechanism of a replicative DNA polymerase.
Authors: Xiang Feng / Michelle M Spiering / Almeida Ruda de Luna Santos / Stephen J Benkovic / Huilin Li /
Abstract: Replicative DNA polymerases are distinguished by their speed, processivity, and fidelity. While speed and fidelity arise from the polymerase's intrinsic catalytic and proofreading activities, ...Replicative DNA polymerases are distinguished by their speed, processivity, and fidelity. While speed and fidelity arise from the polymerase's intrinsic catalytic and proofreading activities, processivity is typically attributed to the DNA sliding clamp that tethers the polymerase to DNA. However, additional mechanisms may also contribute. The T4 bacteriophage polymerase can exchange on-the-fly, a process likely contributing to its ∼10-fold higher synthesis rate compared with human polymerases. Here, we reconstituted the T4 holoenzyme and polymerase exchange complexes using purified gp43 polymerase, gp45 sliding clamp, and a primer-template DNA substrate. Cryo-electron microscopy (cryo-EM) analysis revealed either one or two polymerases bound to the clamp and DNA. In the one-polymerase complex, the DNA threads perpendicularly through the clamp, supporting processive synthesis. In contrast, the two-polymerase complex displays a markedly tilted DNA orientation, impeding sliding and representing exchange intermediates. Three distinct conformational states of the two-polymerase complex define a multistep exchange mechanism. To our knowledge, these findings provide the first molecular-level view of replicative polymerase exchange.
History
DepositionNov 10, 2024-
Header (metadata) releaseNov 19, 2025-
Map releaseNov 19, 2025-
UpdateJan 14, 2026-
Current statusJan 14, 2026Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_47824.png

Downloads & links

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Map

FileDownload / File: emd_47824.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationT4 bacteriophage replicative holoenzyme, DNA in left Polymerase(State 2)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 256 pix.
= 211.968 Å		0.83 Å/pix.
x 256 pix.
= 211.968 Å		0.83 Å/pix.
x 256 pix.
= 211.968 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.828 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.047
Minimum - Maximum-0.3630404 - 0.6360827
Average (Standard dev.)0.002513779 (±0.018769486)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 211.968 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Half Map B

Fileemd_47824_half_map_1.map
AnnotationHalf Map B
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half Map A

Fileemd_47824_half_map_2.map
AnnotationHalf Map A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : T4 bacteriophage replication holoenzyme complex

EntireName: T4 bacteriophage replication holoenzyme complex
Components
  • Complex: T4 bacteriophage replication holoenzyme complex
    • Protein or peptide: DNA-directed DNA polymerase
    • Protein or peptide: Sliding clamp
    • DNA: DNA (5'-AGC TAT GAC CAT GAT TAC GAA TTG ddC-3')
    • DNA: DNA (38-MER)

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Supramolecule #1: T4 bacteriophage replication holoenzyme complex

SupramoleculeName: T4 bacteriophage replication holoenzyme complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Escherichia phage T4 (virus)

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Macromolecule #1: DNA-directed DNA polymerase

MacromoleculeName: DNA-directed DNA polymerase / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO / EC number: DNA-directed DNA polymerase
Source (natural)Organism: Escherichia phage T4 (virus)
Molecular weightTheoretical: 103.702797 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MKEFYISIET VGNNIVERYI DENGKERTRE VEYLPTMFRH CKEESKYKDI YGKNCAPQKF PSMKDARDWM KRMEDIGLEA LGMNDFKLA YISDTYGSEI VYDRKFVRVA NCDIEVTGDK FPDPMKAEYE IDAITHYDSI DDRFYVFDLL NSMYGSVSKW D AKLAAKLD ...String:
MKEFYISIET VGNNIVERYI DENGKERTRE VEYLPTMFRH CKEESKYKDI YGKNCAPQKF PSMKDARDWM KRMEDIGLEA LGMNDFKLA YISDTYGSEI VYDRKFVRVA NCDIEVTGDK FPDPMKAEYE IDAITHYDSI DDRFYVFDLL NSMYGSVSKW D AKLAAKLD CEGGDEVPQE ILDRVIYMPF DNERDMLMEY INLWEQKRPA IFTGWNIEGF AVPYIMNRVK MILGERSMKR FS PIGRVKS KLIQNMYGSK EIYSIDGVSI LDYLDLYKKF AFTNLPSFSL ESVAQHETKK GKLPYDGPIN KLRETNHQRY ISY NIIDVE SVQAIDKIRG FIDLVLSMSY YAKMPFSGVM SPIKTWDAII FNSLKGEHKV IPQQGSHVKQ SFPGAFVFEP KPIA RRYIM SFDLTSLYPS IIRQVNISPE TIRGQFKVHP IHEYIAGTAP KPSDEYSCSP NGWMYDKHQE GIIPKEIAKV FFQRK DWKK KMFAEEMNAE AIKKIIMKGA GSCSTKPEVE RYVKFSDDFL NELSNYTESV LNSLIEECEK AATLANTNQL NRKILI NSL YGALGNIHFR YYDLRNATAI TIFGQVGIQW IARKINEYLN KVCGTNDEDF IAAGDTDSVY VCVDKVIEKV GLDRFKE QN DLVEFMNQFG KKKMEPMIDV AYRELCDYMN NREHLMHMDR EAISCPPLGS KGVGGFWKAK KRYALNVYDM EDKRFAEP H LKIMGMETQQ SSTPKAVQEA LEESIRRILQ EGEESVQEYY KNFEKEYRQL DYKVIAEVKT ANDIAKYDDK GWPGFKCPF HIRGVLTYRR AVSGLGVAPI LDGNKVMVLP LREGNPFGDK CIAWPSGTEL PKEIRSDVLS WIDHSTLFQK SFVKPLAGMC ESAGMDYEE KASLDFLFG

UniProtKB: DNA-directed DNA polymerase

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Macromolecule #2: Sliding clamp

MacromoleculeName: Sliding clamp / type: protein_or_peptide / ID: 2 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Escherichia phage T4 (virus)
Molecular weightTheoretical: 24.881223 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MKLSKDTTAL LKNFATINSG IMLKSGQFIM TRAVNGTTYA EANISDVIDF DVAIYDLNGF LGILSLVNDD AEISQSEDGN IKIADARST IFWPAADPST VVAPNKPIPF PVASAVTEIK AEDLQQLLRV SRGLQIDTIA ITVKEGKIVI NGFNKVEDSA L TRVKYSLT ...String:
MKLSKDTTAL LKNFATINSG IMLKSGQFIM TRAVNGTTYA EANISDVIDF DVAIYDLNGF LGILSLVNDD AEISQSEDGN IKIADARST IFWPAADPST VVAPNKPIPF PVASAVTEIK AEDLQQLLRV SRGLQIDTIA ITVKEGKIVI NGFNKVEDSA L TRVKYSLT LGDYDGENTF NFIINMANMK MQPGNYKLLL WAKGKQGAAK FEGEHANYVV ALEADSTHDF

UniProtKB: Sliding clamp

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Macromolecule #3: DNA (5'-AGC TAT GAC CAT GAT TAC GAA TTG ddC-3')

MacromoleculeName: DNA (5'-AGC TAT GAC CAT GAT TAC GAA TTG ddC-3') / type: dna / ID: 3 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 7.681985 KDa
SequenceString:
(DA)(DG)(DC)(DT)(DA)(DT)(DG)(DA)(DC)(DC) (DA)(DT)(DG)(DA)(DT)(DT)(DA)(DC)(DG)(DA) (DA)(DT)(DT)(DG)(DC)

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Macromolecule #4: DNA (38-MER)

MacromoleculeName: DNA (38-MER) / type: dna / ID: 4 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 11.677539 KDa
SequenceString:
(DC)(DT)(DG)(DC)(DA)(DC)(DG)(DA)(DA)(DT) (DT)(DA)(DA)(DG)(DC)(DA)(DA)(DT)(DT)(DC) (DG)(DT)(DA)(DA)(DT)(DC)(DA)(DT)(DG) (DG)(DT)(DC)(DA)(DT)(DA)(DG)(DC)(DT)

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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
Component:
ConcentrationFormulaName
20.0 mMHEPESHEPES
150.0 mMKoACPotassium acetate
10.0 mMMgCl2Magnesium chloride
2.0 mMDTTDithiothreitol
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 283 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 51.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.0 µm / Nominal defocus min: 1.0 µm
Sample stageCooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionSoftware - Name: cryoSPARC (ver. 4.2.1) / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: OTHER
Details: Templates for Polymerase structures were generated with AF2. Templates for the sliding clamp are from PDB code 1ZCD.
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.7 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.2.1) / Number images used: 164340
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.2.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.2.1)
Final 3D classificationNumber classes: 3 / Avg.num./class: 215743 / Software - Name: cryoSPARC (ver. 4.2.1)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model
PDB IDChain

1czd

source_name: PDB, initial_model_type: experimental model
source_name: AlphaFold, initial_model_type: in silico model
RefinementSpace: REAL / Protocol: OTHER / Overall B value: 157.6
Output model

PDB-9ea2:
T4 Bacteriophage Replicative Polymerase Captured in Polymerase Exchange State 2

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