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- EMDB-70269: Ecoli DnaB helicase and Phage Lambda loader P with ADP-Mg in a 6:... -
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Open data
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Basic information
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Title | Ecoli DnaB helicase and Phage Lambda loader P with ADP-Mg in a 6:5 stoichiometry ratio. | |||||||||
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![]() | Hexameric DnaB helicase / Phage Lambda P helicase loader / bacterial DNA replication initiation / auto inhibition / DNA BINDING PROTEIN | |||||||||
Function / homology | ![]() DnaB-DnaC complex / DnaB-DnaC-Rep-PriC complex / DnaB-DnaG complex / DnaB-DnaC-DnaT-PriA-PriC complex / DnaB-DnaC-DnaT-PriA-PriB complex / DNA helicase complex / primosome complex / DNA 5'-3' helicase / bidirectional double-stranded viral DNA replication / DNA replication, synthesis of primer ...DnaB-DnaC complex / DnaB-DnaC-Rep-PriC complex / DnaB-DnaG complex / DnaB-DnaC-DnaT-PriA-PriC complex / DnaB-DnaC-DnaT-PriA-PriB complex / DNA helicase complex / primosome complex / DNA 5'-3' helicase / bidirectional double-stranded viral DNA replication / DNA replication, synthesis of primer / replisome / response to ionizing radiation / replication fork processing / DNA replication initiation / DNA helicase activity / helicase activity / 5'-3' DNA helicase activity / DNA replication / ATP hydrolysis activity / DNA binding / ATP binding / identical protein binding / cytosol Similarity search - Function | |||||||||
Biological species | ![]() ![]() ![]() | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 2.66 Å | |||||||||
![]() | Shatarupa A / Brown D / Olinares PDB / Chase J / Isiorho E / Chait BT / Jeruzalmi D | |||||||||
Funding support | ![]()
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![]() | ![]() Title: Distinct Quaternary States, Intermediates, and Autoinhibition During Loading of the DnaB-Replicative Helicase by the Phage λP Helicase Loader. Authors: Abhipsa Shatarupa / Dhanjai Brown / Paul Dominic B Olinares / Jillian Chase / Eta Isiorho / Brian T Chait / David Jeruzalmi Abstract: Replicative helicases require loader proteins for assembly at the origins of DNA replication. Multiple copies of the bacteriophage λP (P) loader bind to and load the DnaB (B) replicative helicase ...Replicative helicases require loader proteins for assembly at the origins of DNA replication. Multiple copies of the bacteriophage λP (P) loader bind to and load the DnaB (B) replicative helicase on replication-origin-derived single-stranded DNA. We find that the DnaB•λP complex exists in two forms: B P and B P . In the 2.66 Å cryo-EM model of B P , five copies of the λP loader assemble into a crown-like shape that tightly grips DnaB. In this complex, closed planar DnaB is reconfigured into an open spiral with a sufficiently sized breach to permit ssDNA to enter an internal chamber. The transition to the open spiral involves λP-mediated changes to the Docking Helix (DH)-Linker Helix (LH) interface. The loader directly stabilizes the open spiral. Unexpectedly, one λP chain in B P is bound across the breach, precluding entry of replication-origin-derived ssDNA into DnaB's central chamber. We suggest that the B P complex is an early intermediate in the helicase activation pathway wherein neither the DnaB helicase nor the λP loader has attained its final form. DnaB in this complex adopts a partially open planar configuration, termed ajar planar. The partially ordered λP loader assembly features a much looser interaction with DnaB. The ssDNA and ATP sites in both complexes are in a configuration ill-suited for binding or hydrolysis. Our work specifies the conformational changes required for the intermediate B P to transition to B P on the pathway to recruitment by the initiator protein complex to the replication origin. #1: ![]() Title: An Autoinhibited Conformation of the DnaB-Replicative Helicase- phage LP Helicase Loader Complex Authors: Brown D / Shatarupa A / Olinares PDB / Chase J / Isiorho E / Chait BT / Jeruzalmi D | |||||||||
History |
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Structure visualization
Supplemental images |
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Downloads & links
-EMDB archive
Map data | ![]() | 123.3 MB | ![]() | |
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Header (meta data) | ![]() ![]() | 24.8 KB 24.8 KB | Display Display | ![]() |
FSC (resolution estimation) | ![]() | 13.2 KB | Display | ![]() |
Images | ![]() | 74.3 KB | ||
Filedesc metadata | ![]() | 7.6 KB | ||
Others | ![]() ![]() | 226.4 MB 226.4 MB | ||
Archive directory | ![]() ![]() | HTTPS FTP |
-Validation report
Summary document | ![]() | 728.8 KB | Display | ![]() |
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Full document | ![]() | 728.3 KB | Display | |
Data in XML | ![]() | 21.6 KB | Display | |
Data in CIF | ![]() | 28.1 KB | Display | |
Arichive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Related structure data | ![]() 9oa1MC ![]() 8v9sC ![]() 9oa2C M: atomic model generated by this map C: citing same article ( |
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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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Projections & slices | Image control
Images are generated by Spider. | ||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 1.083 Å | ||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
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-Supplemental data
-Half map: #2
File | emd_70269_half_map_1.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Half map: #1
File | emd_70269_half_map_2.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
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Sample components
-Entire : Ecoli DnaB helicase and Phage Lambda loader P with ADP-Mg in a 6:...
Entire | Name: Ecoli DnaB helicase and Phage Lambda loader P with ADP-Mg in a 6:5 stoichiometry ratio. |
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Components |
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-Supramolecule #1: Ecoli DnaB helicase and Phage Lambda loader P with ADP-Mg in a 6:...
Supramolecule | Name: Ecoli DnaB helicase and Phage Lambda loader P with ADP-Mg in a 6:5 stoichiometry ratio. type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2 |
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Molecular weight | Theoretical: 446.94 KDa |
-Supramolecule #2: Ecoli DnaB helicase
Supramolecule | Name: Ecoli DnaB helicase / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1 |
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Source (natural) | Organism: ![]() ![]() |
-Supramolecule #3: Phage Lambda loader P
Supramolecule | Name: Phage Lambda loader P / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2 |
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Source (natural) | Organism: ![]() |
-Macromolecule #1: Replicative DNA helicase
Macromolecule | Name: Replicative DNA helicase / type: protein_or_peptide / ID: 1 / Number of copies: 6 / Enantiomer: LEVO / EC number: DNA helicase |
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Source (natural) | Organism: ![]() ![]() |
Molecular weight | Theoretical: 52.450945 KDa |
Recombinant expression | Organism: ![]() ![]() |
Sequence | String: MAGNKPFNKQ QAEPRERDPQ VAGLKVPPHS IEAEQSVLGG LMLDNERWDD VAERVVADDF YTRPHRHIFT EMARLQESGS PIDLITLAE SLERQGQLDS VGGFAYLAEL SKNTPSAANI SAYADIVRER AVVREMISVA NEIAEAGFDP QGRTSEDLLD L AESRVFKI ...String: MAGNKPFNKQ QAEPRERDPQ VAGLKVPPHS IEAEQSVLGG LMLDNERWDD VAERVVADDF YTRPHRHIFT EMARLQESGS PIDLITLAE SLERQGQLDS VGGFAYLAEL SKNTPSAANI SAYADIVRER AVVREMISVA NEIAEAGFDP QGRTSEDLLD L AESRVFKI AESRANKDEG PKNIADVLDA TVARIEQLFQ QPHDGVTGVN TGYDDLNKKT AGLQPSDLII VAARPSMGKT TF AMNLVEN AAMLQDKPVL IFSLEMPSEQ IMMRSLASLS RVDQTKIRTG QLDDEDWARI SGTMGILLEK RNIYIDDSSG LTP TEVRSR ARRIAREHGG IGLIMIDYLQ LMRVPALSDN RTLEIAEISR SLKALAKELN VPVVALSQLN RSLEQRADKR PVNS DLRES GSIEQDADLI MFIYRDEVYH ENSDLKGIAE IIIGKQRNGP IGTVRLTFNG QWSRFDNYAG PQYDDE UniProtKB: Replicative DNA helicase DnaB |
-Macromolecule #2: Helicase loader
Macromolecule | Name: Helicase loader / type: protein_or_peptide / ID: 2 / Number of copies: 5 / Enantiomer: LEVO |
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Source (natural) | Organism: ![]() |
Molecular weight | Theoretical: 26.551326 KDa |
Recombinant expression | Organism: ![]() ![]() |
Sequence | String: MENIAAQMVN FDREQMRRIA NNMPEQYDEK PQVQQVAQII NGVFSQLLAT FPASLANRDQ NEVNEIRRQW VLAFRENGIT TMEQVNAGM RVARRQNRPF LPSPGQFVAW CREEASVTAG LPNVSELVDM VYEYCRKRGL YPDAESYPWK SNAHYWLVTN L YQNMRANA ...String: MENIAAQMVN FDREQMRRIA NNMPEQYDEK PQVQQVAQII NGVFSQLLAT FPASLANRDQ NEVNEIRRQW VLAFRENGIT TMEQVNAGM RVARRQNRPF LPSPGQFVAW CREEASVTAG LPNVSELVDM VYEYCRKRGL YPDAESYPWK SNAHYWLVTN L YQNMRANA LTDAELRRKA ADELVHMTAR INRGEAIPEP VKQLPVMGGR PLNRAQALAK IAEIKAKFGL KGASV UniProtKB: Helicase loader |
-Macromolecule #3: ADENOSINE-5'-DIPHOSPHATE
Macromolecule | Name: ADENOSINE-5'-DIPHOSPHATE / type: ligand / ID: 3 / Number of copies: 6 / Formula: ADP |
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Molecular weight | Theoretical: 427.201 Da |
Chemical component information | ![]() ChemComp-ADP: |
-Macromolecule #4: MAGNESIUM ION
Macromolecule | Name: MAGNESIUM ION / type: ligand / ID: 4 / Number of copies: 6 / Formula: MG |
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Molecular weight | Theoretical: 24.305 Da |
-Experimental details
-Structure determination
Method | cryo EM |
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![]() | single particle reconstruction |
Aggregation state | particle |
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Sample preparation
Concentration | 4.98 mg/mL | |||||||||||||||||||||
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Buffer | pH: 7.5 Component:
Details: 20 mM Na-HEPES pH 7.5, 450mM NaCl, 2mM DTT, 0.5mM MgCl2, 0.2mM ATP, 0.25% Glycerol | |||||||||||||||||||||
Grid | Model: Quantifoil R0.6/1 / Material: GOLD / Mesh: 300 / Support film - Material: GOLD / Support film - topology: HOLEY / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Time: 120 sec. / Pretreatment - Atmosphere: OTHER | |||||||||||||||||||||
Vitrification | Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV Details: Protein BP (1.5 uM) and DNA (1.875 uM) was mixed in a 1.25 molar excess. 3uL of the sample was added to a plasma-cleaned grid at 4 degrees celsius, 100 percent humidity, blot force 4, blot ...Details: Protein BP (1.5 uM) and DNA (1.875 uM) was mixed in a 1.25 molar excess. 3uL of the sample was added to a plasma-cleaned grid at 4 degrees celsius, 100 percent humidity, blot force 4, blot time 4s, wait time 30s, total blots 1, and plunge-frozen into liquid nitrogen-cooled ethane.. |
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Electron microscopy
Microscope | TFS KRIOS |
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Specialist optics | Energy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV |
Image recording | Film or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 1 / Number real images: 7863 / Average exposure time: 2.0 sec. / Average electron dose: 51.01 e/Å2 |
Electron beam | Acceleration voltage: 300 kV / Electron source: ![]() |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 81000 |
Sample stage | Specimen 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
-Atomic model buiding 1
Initial model |
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Refinement | Space: REAL / Protocol: OTHER / Overall B value: 160.67 | ||||||||||||
Output model | ![]() PDB-9oa1: |