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- EMDB-42402: Cryo-EM structure of T4 Bacteriophage Clamp Loader with Sliding Clamp -

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Basic information

Entry
Database: EMDB / ID: EMD-42402
TitleCryo-EM structure of T4 Bacteriophage Clamp Loader with Sliding Clamp
Map data
Sample
  • Complex: T4 Bacteriophage Clamp Loader with Sliding Clamp
    • Complex: T4 Bacteriophage Sliding Clamp (gp45)
      • Protein or peptide: Sliding clamp
    • Complex: T4 Bacteriophage Clamp Loader (gp44 + gp62)
      • Protein or peptide: Sliding-clamp-loader large subunit
      • Protein or peptide: Sliding-clamp-loader small subunit
  • Ligand: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER
  • Ligand: MAGNESIUM ION
Keywordsautoinhibited / DNA-free / catalytically inactive / stable / REPLICATION
Function / homology
Function and homology information


DNA replication factor C complex / DNA clamp loader activity / bidirectional double-stranded viral DNA replication / viral DNA genome replication / DNA polymerase processivity factor activity / viral transcription / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / DNA-templated DNA replication / DNA replication / DNA repair ...DNA replication factor C complex / DNA clamp loader activity / bidirectional double-stranded viral DNA replication / viral DNA genome replication / DNA polymerase processivity factor activity / viral transcription / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / DNA-templated DNA replication / DNA replication / DNA repair / ATP hydrolysis activity / DNA binding / ATP binding
Similarity search - Function
Sliding-clamp-loader small subunit gp62 / Sliding-clamp-loader large subunit / : / : / Bacteriophage clamp loader A subunit / Sliding-clamp-loader large subunit, AAA+ ATPase lid domain / Sliding-clamp-loader large subunit, C-terminal domain / Sliding clamp, C-terminal / Sliding clamp / gp45 sliding clamp, C terminal ...Sliding-clamp-loader small subunit gp62 / Sliding-clamp-loader large subunit / : / : / Bacteriophage clamp loader A subunit / Sliding-clamp-loader large subunit, AAA+ ATPase lid domain / Sliding-clamp-loader large subunit, C-terminal domain / Sliding clamp, C-terminal / Sliding clamp / gp45 sliding clamp, C terminal / DNA polymerase processivity factor / DNA polymerase processivity factor / : / : / ATPase family associated with various cellular activities (AAA) / ATPase, AAA-type, core / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Sliding clamp / Sliding-clamp-loader large subunit / Sliding-clamp-loader small subunit
Similarity search - Component
Biological speciesTequatrovirus T4
Methodsingle particle reconstruction / cryo EM / Resolution: 3.21 Å
AuthorsHuang Y / Marcus K / Subramanian S / Gee LC / Gorday K / Ghaffari-Kashani S / Luo X / Zhang L / O'Donnell M / Kuriyan J
Funding support United States, 1 items
OrganizationGrant numberCountry
Howard Hughes Medical Institute (HHMI) United States
CitationJournal: Nat Struct Mol Biol / Year: 2024
Title: Autoinhibition of a clamp-loader ATPase revealed by deep mutagenesis and cryo-EM.
Authors: Kendra Marcus / Yongjian Huang / Subu Subramanian / Christine L Gee / Kent Gorday / Sam Ghaffari-Kashani / Xiao Ran Luo / Lisa Zheng / Michael O'Donnell / Sriram Subramaniam / John Kuriyan /
Abstract: Clamp loaders are AAA+ ATPases that facilitate high-speed DNA replication. In eukaryotic and bacteriophage clamp loaders, ATP hydrolysis requires interactions between aspartate residues in one ...Clamp loaders are AAA+ ATPases that facilitate high-speed DNA replication. In eukaryotic and bacteriophage clamp loaders, ATP hydrolysis requires interactions between aspartate residues in one protomer, present in conserved 'DEAD-box' motifs, and arginine residues in adjacent protomers. We show that functional defects resulting from a DEAD-box mutation in the T4 bacteriophage clamp loader can be compensated by widely distributed single mutations in the ATPase domain. Using cryo-EM, we discovered an unsuspected inactive conformation of the clamp loader, in which DNA binding is blocked and the catalytic sites are disassembled. Mutations that restore function map to regions of conformational change upon activation, suggesting that these mutations may increase DNA affinity by altering the energetic balance between inactive and active states. Our results show that there are extensive opportunities for evolution to improve catalytic efficiency when an inactive intermediate is involved.
History
DepositionOct 19, 2023-
Header (metadata) releaseDec 13, 2023-
Map releaseDec 13, 2023-
UpdateApr 3, 2024-
Current statusApr 3, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_42402.png

Downloads & links

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Map

FileDownload / File: emd_42402.map.gz / Format: CCP4 / Size: 30.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.05 Å/pix.
x 200 pix.
= 209.6 Å		1.05 Å/pix.
x 200 pix.
= 209.6 Å		1.05 Å/pix.
x 200 pix.
= 209.6 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.048 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.1
Minimum - Maximum-0.2116968 - 0.5224986
Average (Standard dev.)0.0029300912 (±0.024919763)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions200200200
Spacing200200200
CellA=B=C: 209.59999 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_42402_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_42402_half_map_2.map
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 Clamp Loader with Sliding Clamp

EntireName: T4 Bacteriophage Clamp Loader with Sliding Clamp
Components
  • Complex: T4 Bacteriophage Clamp Loader with Sliding Clamp
    • Complex: T4 Bacteriophage Sliding Clamp (gp45)
      • Protein or peptide: Sliding clamp
    • Complex: T4 Bacteriophage Clamp Loader (gp44 + gp62)
      • Protein or peptide: Sliding-clamp-loader large subunit
      • Protein or peptide: Sliding-clamp-loader small subunit
  • Ligand: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER
  • Ligand: MAGNESIUM ION

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Supramolecule #1: T4 Bacteriophage Clamp Loader with Sliding Clamp

SupramoleculeName: T4 Bacteriophage Clamp Loader with Sliding Clamp / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Source (natural)Organism: Tequatrovirus T4

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Supramolecule #2: T4 Bacteriophage Sliding Clamp (gp45)

SupramoleculeName: T4 Bacteriophage Sliding Clamp (gp45) / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #3
Source (natural)Organism: Tequatrovirus T4

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Supramolecule #3: T4 Bacteriophage Clamp Loader (gp44 + gp62)

SupramoleculeName: T4 Bacteriophage Clamp Loader (gp44 + gp62) / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #1-#2
Source (natural)Organism: Tequatrovirus T4

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Macromolecule #1: Sliding-clamp-loader large subunit

MacromoleculeName: Sliding-clamp-loader large subunit / type: protein_or_peptide / ID: 1 / Number of copies: 4 / Enantiomer: LEVO
Source (natural)Organism: Tequatrovirus T4
Molecular weightTheoretical: 35.834254 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MITVNEKEHI LEQKYRPSTI DECILPAFDK ETFKSITSKG KIPHIILHSP SPGTGKTTVA KALCHDVNAD MMFVNGSDCK IDFVRGPLT NFASAASFDG RQKVIVIDEF DRSGLAESQR HLRSFMEAYS SNCSIIITAN NIDGIIKPLQ SRCRVITFGQ P TDEDKIEM ...String:
MITVNEKEHI LEQKYRPSTI DECILPAFDK ETFKSITSKG KIPHIILHSP SPGTGKTTVA KALCHDVNAD MMFVNGSDCK IDFVRGPLT NFASAASFDG RQKVIVIDEF DRSGLAESQR HLRSFMEAYS SNCSIIITAN NIDGIIKPLQ SRCRVITFGQ P TDEDKIEM MKQMIRRLTE ICKHEGIAIA DMKVVAALVK KNFPDFRKTI GELDSYSSKG VLDAGILSLV TNDRGAIDDV LE SLKNKDV KQLRALAPKY AADYSWFVGK LAEEIYSRVT PQSIIRMYEI VGENNQYHGI AANTELHLAY LFIQLACEMQ WK

UniProtKB: Sliding-clamp-loader large subunit

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Macromolecule #2: Sliding-clamp-loader small subunit

MacromoleculeName: Sliding-clamp-loader small subunit / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Tequatrovirus T4
Molecular weightTheoretical: 21.391703 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MSLFKDDIQL NEHQVAWYSK DWTAVQSAAD SFKEKAENEF FEIIGAINNK TKCSIAQKDY SKFMVENALS QFPECMPAVY AMNLIGSGL SDEAHFNYLM AAVPRGKRYG KWAKLVEDST EVLIIKLLAK RYQVNTNDAI NYKSILTKNG KLPLVLKELK G LVTDDFLK EVTKNVKEQK QLKKLALEW

UniProtKB: Sliding-clamp-loader small subunit

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Macromolecule #3: Sliding clamp

MacromoleculeName: Sliding clamp / type: protein_or_peptide / ID: 3 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Tequatrovirus T4
Molecular weightTheoretical: 24.881223 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
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 #4: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER

MacromoleculeName: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / type: ligand / ID: 4 / Number of copies: 2 / Formula: AGS
Molecular weightTheoretical: 523.247 Da
Chemical component information

ChemComp-AGS:
PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / ATP-gamma-S, energy-carrying molecule analogue*YM

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Macromolecule #5: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 5 / Number of copies: 2 / 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
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 300
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 283 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (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: 0.8 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 17937844
Startup modelType of model: INSILICO MODEL
In silico model: 3D Ab-initio reconstruction from selected particles after 2D classification in cryoSPARC
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.21 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3) / Software - details: Non-uniform refinement / Number images used: 455041
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3) / Software - details: ab-initio reconstruction
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3) / Software - details: heterogenous refinement
Final 3D classificationNumber classes: 10 / Software - Name: cryoSPARC (ver. 3) / Software - details: heterogenous refinement

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: OTHER
Output model

PDB-8unh:
Cryo-EM structure of T4 Bacteriophage Clamp Loader with Sliding Clamp

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