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- EMDB-7076: Mechanisms of Opening and Closing of the Bacterial Replicative He... -
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Open data
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Basic information
Entry | Database: EMDB / ID: EMD-7076 | |||||||||||||||||||||||||||||||||||||||
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Title | Mechanisms of Opening and Closing of the Bacterial Replicative Helicase: The DnaB Helicase and Lambda P Helicase Loader Complex | |||||||||||||||||||||||||||||||||||||||
![]() | DnaB-LambdaP helicase-helicase loader complex from single particle cryoEM at 4.1A. The suggested viewing thresholds are 0.0276 (Chimera) or an isomesh level of 6 (PyMol). | |||||||||||||||||||||||||||||||||||||||
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![]() | Helicase Loader / Helicase / DNA replication / ATPase / DNA Replication Initiation / Bacteriophage Lambda / REPLICATION | |||||||||||||||||||||||||||||||||||||||
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 / DNA 5'-3' helicase / bidirectional double-stranded viral DNA replication / primosome complex / 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 / DNA 5'-3' helicase / bidirectional double-stranded viral DNA replication / primosome complex / DNA replication, synthesis of primer / replisome / DNA duplex unwinding / response to ionizing radiation / DNA unwinding involved in DNA replication / replication fork processing / DNA replication initiation / DNA helicase activity / helicase activity / 5'-3' DNA helicase activity / DNA replication / DNA helicase / hydrolase activity / ATP hydrolysis activity / DNA binding / ATP binding / identical protein binding / cytosol Similarity search - Function | |||||||||||||||||||||||||||||||||||||||
Biological species | ![]() ![]() ![]() ![]() ![]() ![]() | |||||||||||||||||||||||||||||||||||||||
Method | single particle reconstruction / cryo EM / Resolution: 4.1 Å | |||||||||||||||||||||||||||||||||||||||
![]() | Chase J / Catalano A | |||||||||||||||||||||||||||||||||||||||
Funding support | ![]()
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![]() | ![]() Title: Mechanisms of opening and closing of the bacterial replicative helicase. Authors: Jillian Chase / Andrew Catalano / Alex J Noble / Edward T Eng / Paul Db Olinares / Kelly Molloy / Danaya Pakotiprapha / Martin Samuels / Brian Chait / Amedee des Georges / David Jeruzalmi / ![]() ![]() Abstract: Assembly of bacterial ring-shaped hexameric replicative helicases on single-stranded (ss) DNA requires specialized loading factors. However, mechanisms implemented by these factors during opening and ...Assembly of bacterial ring-shaped hexameric replicative helicases on single-stranded (ss) DNA requires specialized loading factors. However, mechanisms implemented by these factors during opening and closing of the helicase, which enable and restrict access to an internal chamber, are not known. Here, we investigate these mechanisms in the DnaB helicase•bacteriophage λ helicase loader (λP) complex. We show that five copies of λP bind at DnaB subunit interfaces and reconfigure the helicase into an open spiral conformation that is intermediate to previously observed closed ring and closed spiral forms; reconfiguration also produces openings large enough to admit ssDNA into the inner chamber. The helicase is also observed in a restrained inactive configuration that poises it to close on activating signal, and transition to the translocation state. Our findings provide insights into helicase opening, delivery to the origin and ssDNA entry, and closing in preparation for translocation. | |||||||||||||||||||||||||||||||||||||||
History |
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Structure visualization
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Structure viewer | EM map: ![]() ![]() ![]() |
Supplemental images |
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Downloads & links
-EMDB archive
Map data | ![]() | 59.9 MB | ![]() | |
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Header (meta data) | ![]() ![]() | 26.1 KB 26.1 KB | Display Display | ![]() |
FSC (resolution estimation) | ![]() | 9.2 KB | Display | ![]() |
Images | ![]() | 157.3 KB | ||
Filedesc metadata | ![]() | 9.8 KB | ||
Archive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Related structure data | ![]() 6bbmMC M: atomic model generated by this map C: citing same article ( |
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Similar structure data |
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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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Annotation | DnaB-LambdaP helicase-helicase loader complex from single particle cryoEM at 4.1A. The suggested viewing thresholds are 0.0276 (Chimera) or an isomesh level of 6 (PyMol). | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 1.07 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
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Sample components
-Entire : DnaB helicase - Lambda P helicase loader DNA replication complex
Entire | Name: DnaB helicase - Lambda P helicase loader DNA replication complex |
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Components |
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-Supramolecule #1: DnaB helicase - Lambda P helicase loader DNA replication complex
Supramolecule | Name: DnaB helicase - Lambda P helicase loader DNA replication complex type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2 Details: pET24a containing full-length DnaB was co-expressed with pCDFDuet containing full-length LambdaP in BL21(DE3) cells. The resolution of the LambdaP portion of our EM map did not permit the ...Details: pET24a containing full-length DnaB was co-expressed with pCDFDuet containing full-length LambdaP in BL21(DE3) cells. The resolution of the LambdaP portion of our EM map did not permit the unambiguous assignment of the amino acid sequence to the structure. As such, the model for LambdaP was built as a poly alanine model. Additionally, only half of LambdaP was observed in our maps due to the intrinsic flexibility of the amino and carboxy terminal domains of LambdaP. Subsequent experiments determined that the observed portion of LambdaP in our maps corresponds to the C-terminal domain. |
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Source (natural) | Organism: ![]() ![]() |
-Supramolecule #2: E coli DnaB helicase
Supramolecule | Name: E coli DnaB helicase / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1 Details: E coli DnaB helicase is observed as an open-spiral hexamer, in which one of the interfaces is breached. Five ADP molecules are observed at the five intact ATP binding sites. Additionally, ...Details: E coli DnaB helicase is observed as an open-spiral hexamer, in which one of the interfaces is breached. Five ADP molecules are observed at the five intact ATP binding sites. Additionally, clear density is observed for five of six linkers permitting unambiguous assignment of NTD to parent CTD domain. |
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Source (natural) | Organism: ![]() ![]() |
-Supramolecule #3: Lambda P helicase loader
Supramolecule | Name: Lambda P helicase loader / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2 Details: Five lambda P molecules were observed bound to the five intact DnaB subunit interfaces. Unambiguous assignment of side chain density for lambda P was not possible due to the resolution of ...Details: Five lambda P molecules were observed bound to the five intact DnaB subunit interfaces. Unambiguous assignment of side chain density for lambda P was not possible due to the resolution of this region of the EM map. Instead, a polyalanine model was built for each lambda P molecule. Additionally, density for approximately half of the expected 233 residues of lambda P was observed owing to flexibility between domains. Subsequent experiments confirmed that the observed region of Lambda P is the C-terminal domain, which interacts with DnaB. |
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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 |
-Macromolecule #2: Replication protein P
Macromolecule | Name: Replication protein P / type: protein_or_peptide / ID: 2 / Number of copies: 5 / Enantiomer: LEVO |
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Source (natural) | Organism: ![]() |
Molecular weight | Theoretical: 23.141221 KDa |
Recombinant expression | Organism: ![]() ![]() |
Sequence | String: MKNIAAQMVN FDREQMRRIA NNMPEQYDEK PQVQQVAQII NGVFSQLLAT FPASLANRDQ NEVNEIRRQW VLAFRENGIT TMEQVNAGM RVARRQNRPF LPSPGQFV(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK) (UNK)(UNK)(UNK) ...String: MKNIAAQMVN FDREQMRRIA NNMPEQYDEK PQVQQVAQII NGVFSQLLAT FPASLANRDQ NEVNEIRRQW VLAFRENGIT TMEQVNAGM RVARRQNRPF LPSPGQFV(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK) (UNK)(UNK) UniProtKB: Replication protein P |
-Macromolecule #3: ADENOSINE-5'-DIPHOSPHATE
Macromolecule | Name: ADENOSINE-5'-DIPHOSPHATE / type: ligand / ID: 3 / Number of copies: 5 / Formula: ADP |
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Molecular weight | Theoretical: 427.201 Da |
Chemical component information | ![]() ChemComp-ADP: |
-Experimental details
-Structure determination
Method | cryo EM |
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![]() | single particle reconstruction |
Aggregation state | particle |
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Sample preparation
Concentration | 0.48 mg/mL | ||||||||||||||||||
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Buffer | pH: 7.5 Component:
Details: Concentrated BP sample (18mg/mL) was diluted with freshly prepared buffer to desired concentration (~1.5 micromolar) for grid preparation. | ||||||||||||||||||
Grid | Model: Quantifoil R0.6/1 / Material: GOLD / Mesh: 400 / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Time: 60 sec. / Pretreatment - Atmosphere: OTHER Details: The grid was coated with 50 nm of evaporated gold prior to use. All remaining carbon was removed by plasma cleaning for 5 minutes in a Gatan Solarus plasma cleaner. | ||||||||||||||||||
Vitrification | Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV Details: 3uL of sample was adhered to a fresh plasma cleaned grid and allowed to adsorb for 30 seconds, blotted for 3 seconds with a blot force of 4 and plunge frozen into liquid nitrogen-cooled ethane.. | ||||||||||||||||||
Details | The sample was monodisperse. Side views were more electron weak than top or bottom views creating challenges for particle picking. This issue was overcome with cryo-electron tomography techniques used for 1) initial model generation and 2) template generation for particle picking. |
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Electron microscopy
Microscope | FEI TITAN KRIOS |
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Temperature | Min: 70.0 K / Max: 70.0 K |
Specialist optics | Spherical aberration corrector: The Krios this data was collected on has a Cs of 2.7. |
Details | Preliminary grid screening was performed prior to Krios data collections. All microscope alignments were completed by the New York Structural Biology SEMC team. |
Image recording | Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 3838 pixel / Digitization - Dimensions - Height: 3710 pixel / Digitization - Frames/image: 1-50 / Number grids imaged: 3 / Number real images: 2426 / Average exposure time: 10.0 sec. / Average electron dose: 8.0 e/Å2 Details: Single particle movies were recorded at a pixel size of 1.07 angstroms/pixel. Three 24-hour sessions produced 2,426 micrograph movies. In addition, five tilt series were collected from the ...Details: Single particle movies were recorded at a pixel size of 1.07 angstroms/pixel. Three 24-hour sessions produced 2,426 micrograph movies. In addition, five tilt series were collected from the same grids bi-directionally over a tilt range of -45 degrees to +45 degrees in 3 degree increments at a dose of 2.57 to 3.3 electrons per angstrom squared (total accumulated dose of 90 electrons per angstrom squared). Tilt series were collected at a pixel size of 1.76 angstroms and at defocus values of -2.8um, -6.1um and -9.3um. |
Electron beam | Acceleration voltage: 300 kV / Electron source: ![]() |
Electron optics | C2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: -0.003 µm / Nominal defocus min: -0.001 µm / Nominal magnification: 22500 |
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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Details | The initial fitting was done with the 2R5U and 3BH0 models onto which the E. coli amino sequence had been built. The linker segments that connected these segments were built by hand. PHENIX real_space_refine was used to refine the complete model for the B6P5 entity. | ||||||
Refinement | Space: REAL / Protocol: FLEXIBLE FIT | ||||||
Output model | ![]() PDB-6bbm: |