ID or keywords:
|Entry||Database: PDB / ID: 6bbm|
|Title||Mechanisms of Opening and Closing of the Bacterial Replicative Helicase: The DnaB Helicase and Lambda P Helicase Loader Complex|
|Keywords||REPLICATION / Helicase Loader / Helicase / DNA replication / ATPase / DNA Replication Initiation / Bacteriophage Lambda|
|Function / homology|
Function and homology information
primosome complex / DNA replication, synthesis of RNA primer / bidirectional double-stranded viral DNA replication / DNA unwinding involved in DNA replication / response to ionizing radiation / DNA replication initiation / DNA helicase / helicase activity / DNA helicase activity / DNA replication ...primosome complex / DNA replication, synthesis of RNA primer / bidirectional double-stranded viral DNA replication / DNA unwinding involved in DNA replication / response to ionizing radiation / DNA replication initiation / DNA helicase / helicase activity / DNA helicase activity / DNA replication / DNA binding / ATP binding / identical protein binding / cytosol
DnaB-like helicase C terminal domain / AAA+ ATPase domain / DnaB-like helicase N terminal domain / DNA helicase, DnaB-like, N-terminal domain superfamily / P-loop containing nucleoside triphosphate hydrolase / DNA helicase DnaB, N-terminal/DNA primase DnaG, C-terminal / Replication P / DNA helicase, DnaB-like, C-terminal / DNA helicase, DnaB-like, N-terminal / DNA helicase, DnaB type
Replicative DNA helicase / Replication protein P / Replicative DNA helicase
|Biological species||Escherichia coli O111:NM (bacteria)|
Escherichia phage lambda (bacteriophage)
|Method||ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.1 Å|
|Authors||Chase, J. / Catalano, A. / Noble, A.J. / Eng, E.T. / Olinares, P.D.B. / Molloy, K. / Pakotiprapha, D. / Samuels, M. / Chain, B. / des Georges, A. / Jeruzalmi, D.|
|Funding support|| United States, 12items |
|Citation||Journal: Elife / Year: 2018|
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.
SummaryFull reportAbout validation report
|Structure viewer||Molecule: |
Downloads & links
A: Replicative DNA helicase
B: Replicative DNA helicase
C: Replicative DNA helicase
D: Replicative DNA helicase
E: Replicative DNA helicase
F: Replicative DNA helicase
V: Replication protein P
W: Replication protein P
X: Replication protein P
Y: Replication protein P
Z: Replication protein P
Mass: 52450.945 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli O111:NM (bacteria) / Gene: DQW49_15020 / Plasmid: pET24a / Cell line (production host): BL21(DE3) / Production host: Escherichia coli BL21(DE3) (bacteria)
References: UniProt: A0A365Q7M1, UniProt: P0ACB0*PLUS, DNA helicase
Mass: 23141.221 Da / Num. of mol.: 5
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia phage lambda (bacteriophage)
Plasmid: pET24a / Cell line (production host): BL21(DE3) / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P03689
Mass: 427.201 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Comment: ADP, energy-carrying molecule*YM
|Sequence details||The complete sequence of Bacteriophage Lambda P is ...The complete sequence of Bacteriophage Lambda P is MKNIAAQMVN|
|Experiment||Method: ELECTRON MICROSCOPY|
|EM experiment||Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction|
|Buffer solution||pH: 7.5 |
Details: Concentrated BP sample (18mg/mL) was diluted with freshly prepared buffer to desired concentration (~1.5 micromolar) for grid preparation.
|Specimen||Conc.: 0.48 mg/ml|
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 ...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.
Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
|Specimen support||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.|
Grid material: GOLD / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil R0.6/1
|Vitrification||Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 K|
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.
-Electron microscopy imaging
Model: Titan Krios / Image courtesy: FEI Company
|Microscopy||Model: FEI TITAN KRIOS|
Details: Preliminary grid screening was performed prior to Krios data collections. All microscope alignments were completed by the New York Structural Biology SEMC team.
|Electron gun||Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM|
|Electron lens||Mode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 22500 X / Nominal defocus max: -3 nm / Nominal defocus min: -1 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm / Alignment procedure: COMA FREE|
|Specimen holder||Cryogen: NITROGEN / Model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 70 K / Temperature (min): 70 K|
|Image recording||Average exposure time: 10 sec. / Electron dose: 8 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 3 / Num. of real images: 2426 |
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.
|EM imaging optics||Phase plate: No phase plates were used in data collection.|
Spherical aberration corrector: The Krios this data was collected on has a Cs of 2.7.
|Image scans||Width: 3838 / Height: 3710 / Movie frames/image: 50 / Used frames/image: 1-50|
|Software||Name: PHENIX / Version: 1.14_3260: / Classification: refinement|