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- EMDB-7076: Mechanisms of Opening and Closing of the Bacterial Replicative He... -

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Entry
Database: EMDB / ID: 7076
TitleMechanisms of Opening and Closing of the Bacterial Replicative Helicase: The DnaB Helicase and Lambda P Helicase Loader Complex
Map dataDnaB-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).
SampleDnaB helicase - Lambda P helicase loader DNA replication complex:
E coli DnaB helicase / Lambda P helicase loader / Replicative DNA helicase / Replication protein PDNA replication / ligand
Function / homologyDNA helicase, DnaB-like, C-terminal / DNA helicase, DnaB type / 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 / Replication protein P / DNA helicase, DnaB-like, N-terminal / AAA+ ATPase domain / DnaB-like helicase C terminal domain ...DNA helicase, DnaB-like, C-terminal / DNA helicase, DnaB type / 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 / Replication protein P / DNA helicase, DnaB-like, N-terminal / AAA+ ATPase domain / DnaB-like helicase C terminal domain / Superfamily 4 helicase domain profile. / DnaB-like helicase N terminal domain / primosome complex / DNA replication, synthesis of RNA primer / bidirectional double-stranded viral DNA replication / DNA helicase activity / DNA replication initiation / DNA helicase / DNA binding / ATP binding / Replicative DNA helicase / Replication protein P
Function and homology information
SourceEscherichia coli (E. coli) / Enterobacteria phage lambda (bacteriophage) / Escherichia coli O111:NM (bacteria) / Escherichia phage lambda (bacteriophage)
Methodelectron tomography / cryo EM / 4.1 Å resolution
AuthorsChase J / Catalano A / Noble AJ / Eng ET / Olinares PDB / Molloy K / Pakotiprapha D / Samuels M / Chain B / des Georges A / Jeruzalmi D
CitationJournal: 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.
Validation ReportPDB-ID: 6bbm

SummaryFull reportAbout validation report
DateDeposition: Oct 18, 2017 / Header (metadata) release: Mar 6, 2019 / Map release: Mar 6, 2019 / Last update: Mar 13, 2019

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.03
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.03
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: : PDB-6bbm
  • Surface level: 0.03
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

Fileemd_7076.map.gz (map file in CCP4 format, 67109 KB)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
256 pix
1.07 Å/pix.
= 273.92 Å
256 pix
1.07 Å/pix.
= 273.92 Å
256 pix
1.07 Å/pix.
= 273.92 Å

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.07 Å
Density
Contour Level:0.03 (movie #1):
Minimum - Maximum-0.09008105 - 0.161977
Average (Standard dev.)0.000051110845 (0.0061288467)
Details

EMDB XML:

Space Group Number1
Map Geometry
Axis orderXYZ
Dimensions256256256
Origin0.00.00.0
Limit255.0255.0255.0
Spacing256256256
CellA=B=C: 273.92 Å
α=β=γ: 90.0 deg.

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.071.071.07
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z273.920273.920273.920
α/β/γ90.00090.00090.000
start NX/NY/NZ
NX/NY/NZ
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-0.0900.1620.000

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Supplemental data

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Sample components

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Entire DnaB helicase - Lambda P helicase loader DNA replication complex

EntireName: DnaB helicase - Lambda P helicase loader DNA replication complex
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.
Number of components: 6

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Component #1: protein, DnaB helicase - Lambda P helicase loader DNA replication...

ProteinName: DnaB helicase - Lambda P helicase loader DNA replication complex
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.
Recombinant expression: No
SourceSpecies: Escherichia coli (E. coli)
Source (engineered)Expression System: Escherichia coli (E. coli) / Vector: pET24a and pCDFDuet / Cell of expression system: BL21(DE3)

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Component #2: protein, E coli DnaB helicase

ProteinName: E coli DnaB helicase
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.
Recombinant expression: No
SourceSpecies: Escherichia coli (E. coli)
Source (engineered)Expression System: Escherichia coli (E. coli) / Vector: pET24a / Cell of expression system: BL21(DE3)

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Component #3: protein, Lambda P helicase loader

ProteinName: Lambda P helicase loader
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.
Recombinant expression: No
SourceSpecies: Enterobacteria phage lambda (bacteriophage)
Source (engineered)Expression System: Escherichia coli (E. coli) / Vector: pCDFDuet / Cell of expression system: BL21(DE3)

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Component #4: protein, Replicative DNA helicase

ProteinName: Replicative DNA helicase / Number of Copies: 6 / Recombinant expression: No
MassTheoretical: 52.450945 kDa
SourceSpecies: Escherichia coli O111:NM (bacteria)
Source (engineered)Expression System: Escherichia coli BL21(DE3) (bacteria)

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Component #5: protein, Replication protein P

ProteinName: Replication protein PDNA replication / Number of Copies: 5 / Recombinant expression: No
MassTheoretical: 23.141221 kDa
SourceSpecies: Escherichia phage lambda (bacteriophage)
Source (engineered)Expression System: Escherichia coli BL21(DE3) (bacteria)

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Component #6: ligand, ADENOSINE-5'-DIPHOSPHATE

LigandName: ADENOSINE-5'-DIPHOSPHATE / Number of Copies: 5 / Recombinant expression: No
MassTheoretical: 0.427201 kDa

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Experimental details

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Sample preparation

SpecimenSpecimen state: particle / Method: cryo EM
Sample solutionSpecimen conc.: 0.48 mg/ml
Buffer solution: Concentrated BP sample (18mg/mL) was diluted with freshly prepared buffer to desired concentration (~1.5 micromolar) for grid preparation.
pH: 7.5
Support filmThe 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.
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Temperature: 277.15 K / Humidity: 100 %
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..

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
ImagingMicroscope: 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 gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 8 e/Å2 / Illumination mode: FLOOD BEAM
LensMagnification: 22500.0 X (nominal) / Cs: 2.7 mm / Imaging mode: BRIGHT FIELD / Defocus: -1.0 - -3.0 nm
Specimen HolderModel: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature: K ( 70.0 - 70.0 K)
CameraDetector: GATAN K2 SUMMIT (4k x 4k)

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Image acquisition

Image acquisitionNumber of digital 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 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.

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Image processing

ProcessingMethod: electron tomography / Number of sections: 90883
Details: Images collected were CTF corrected and selected based on CTF estimates of less than 10 angstrom at a confidence cutoff of 0.8 for subsequent processing.
3D reconstruction #1Software: RELION
CTF correction: CTF correction was performed before data processing on summed micrographs using Gctf.
Resolution: 4.1 Å / Resolution method: FSC 0.143 CUT-OFF
Details: Relion was used to independently refine half sets using 0.143 gold-standard to a resolution of 4.1A. A total of 90,883 particles went into this reconstruction.
3D reconstruction #2Algorithm: BACK PROJECTION / Software: Dynamo / Resolution: 4 Å / Resolution method: OTHER
Details: A low resolution initial model was generated from ~1,000 particles picked from three tilt series. These tilt series were first aligned using a fiducial-less algorithm implemented in Appion-Portomo, then reconstructed using Tomo3D. 1,000 particles were picked from resulting tomograms (binned 4 x 4) to generate a ~40 angstrom initial model. Resolution was estimated by filtering procedures. The resulting volume served as an initial model and was projected to generate templates for template-based particle picking of single particle micrographs.
FSC plot
(resolution estimation)

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Atomic model buiding

Modeling #1Refinement protocol: flexible / Refinement space: REAL
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.
Input PDB model: 2R5U, 3BH0
Output model

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