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- PDB-6bbm: Mechanisms of Opening and Closing of the Bacterial Replicative He... -

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Entry
Database: PDB / ID: 6bbm
TitleMechanisms of Opening and Closing of the Bacterial Replicative Helicase: The DnaB Helicase and Lambda P Helicase Loader Complex
Components
  • Replication protein PDNA replication
  • Replicative DNA helicase
KeywordsREPLICATION / Helicase Loader / Helicase / DNA replication / ATPase / DNA Replication Initiation / Bacteriophage Lambda
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
Specimen sourceEscherichia coli O111:NM (bacteria)
Escherichia phage lambda (bacteriophage)
MethodELECTRON MICROSCOPY / electron tomography / cryo EM / 4.1 Å resolution
AuthorsChase, 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.
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 Report
SummaryFull reportAbout validation report
DateDeposition: Oct 18, 2017 / Release: Mar 6, 2019
RevisionDateData content typeGroupCategoryItemProviderType
1.0Mar 6, 2019Structure modelrepositoryInitial release
1.1Mar 13, 2019Structure modelData collectionem_admin / pdbx_database_proc / pdbx_seq_map_depositor_info_em_admin.last_update / _pdbx_seq_map_depositor_info.one_letter_code_mod

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Structure visualization

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  • Deposited structure unit
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Structure viewerMolecule:
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Assembly

Deposited unit
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
hetero molecules


Theoretical massNumber of molelcules
Total (without water)432,54816
Polyers430,41211
Non-polymers2,1365
Water0
1


  • idetical with deposited unit
  • defined by author
  • Evidence: mass spectrometry, Native mass spectrometry of DnaB-LambdaP complexes point to a physiological stoichiometry of DnaB6-LambdaP5, which agrees with the data derived from our cryoEM map., mass spectrometry, Native mass spectrometry of DnaB-LambdaP in the presence of origin-derived single stranded DNA reveals a single DnaB6-LambdaP5-ssDNA complex, supporting this is the physiologically relevant stoichiometry., cross-linking, Cross-linking mass spectrometry experiments validated that the C-terminus of Lambda P interacts with DnaB. This finding was also supplemented with binding studies.
  • Download structure data
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein/peptide
Replicative DNA helicase


Mass: 52450.945 Da / Num. of mol.: 6 / Source: (gene. exp.) Escherichia coli O111:NM (bacteria) / Gene: DQW49_15020 / Plasmid name: pET24a / Cell line (production host): BL21(DE3) / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: A0A365Q7M1, DNA helicase
#2: Protein/peptide
Replication protein P / DNA replication


Mass: 23141.221 Da / Num. of mol.: 5
Source: (gene. exp.) Escherichia phage lambda (bacteriophage)
Plasmid name: pET24a / Cell line (production host): BL21(DE3) / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P03689
#3: Chemical
ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE


Mass: 427.201 Da / Num. of mol.: 5 / Formula: C10H15N5O10P2 / Adenosine diphosphate / Comment: ADP (energy-carrying molecule) *YM
Sequence detailsThe complete sequence of Bacteriophage Lambda P is ...The complete sequence of Bacteriophage Lambda P is MKNIAAQMVNFDREQMRRIANNMPEQYDEKPQVQQVAQIINGVFSQLLATFPASLANRDQ NEVNEIRRQWVLAFRENGITTMEQVNAGMRVARRQNRPFLPSPGQFVAWCREEASVTAGL PNVSELVDMVYEYCRKRGLYPDAESYPWKSNAHYWLVTNLYQNMRANALTDAELRRKAAD ELVHMTARINRGEAIPEPVKQLPVMGGRPLNRAQALAKIAEIKAKFGLKGASV

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / Reconstruction method: electron tomography

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Sample preparation

Component

Source: RECOMBINANT / Type: COMPLEX

IDNameDetailsEntity IDParent ID
1DnaB helicase - Lambda P helicase loader DNA replication complexpET24a 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.1, 20
2E coli DnaB helicaseE 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.11
3Lambda P helicase loaderFive 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.21
Molecular weight
IDValueEntity assembly IDExperimental value
10.4465 MDa1YES
21NO
31NO
Source (natural)
IDEntity assembly IDNcbi tax IDOrganism
21562Escherichia coli (E. coli)
32562Escherichia coli (E. coli)
4310710Enterobacteria phage lambda (bacteriophage)
Source (recombinant)
IDEntity assembly IDCellNcbi tax IDOrganismPlasmid
21BL21(DE3)562Escherichia coli (E. coli)pET24a and pCDFDuet
32BL21(DE3)562Escherichia coli (E. coli)pET24a
43BL21(DE3)562Escherichia coli (E. coli)pCDFDuet
Buffer solutionDetails: Concentrated BP sample (18mg/mL) was diluted with freshly prepared buffer to desired concentration (~1.5 micromolar) for grid preparation.
pH: 7.5
Buffer component
IDConc.NameFormulaBuffer ID
120 mMSodium HEPESNa-HEPES1
2450 mMSodium ChlorideNaCl1
32 mMDithiothreitolDTT1
40.5 mMMagnesium ChlorideMgCl21
50.5 mMAdenosine triphosphateATP1
SpecimenConc.: 0.48 mg/ml
Details: The sample was monodispersed. 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 supportDetails: 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 / Grid type: Quantifoil R0.6/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 kelvins
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
MicroscopyMicroscope 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 gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 22500 / Nominal defocus max: -3 nm / Nominal defocus min: -1 nm / Cs: 2.7 mm / C2 aperture diameter: 70 microns / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 70 kelvins / Temperature (min): 70 kelvins
Image recordingAverage exposure time: 10 sec. / Electron dose: 8 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 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.
Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Number of grids imaged: 3 / Number of real images: 2426
EM imaging opticsPhase plate: No phase plates were used in data collection.
Sph aberration corrector: The Krios this data was collected on has a Cs of 2.7.
Image scansWidth: 3838 / Height: 3710 / Movie frames/image: 50 / Used frames/image: 1-50

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Processing

SoftwareName: PHENIX / Version: 1.14_3260: / Classification: refinement
EM software
IDNameVersionCategoryDetailsImage processing ID
1Leginon0.0image acquisitionLeginon was used for automatic hole targeting during single particle movie collection.
2Appion0.0image acquisitionSingle particle micrograph movies were aligned on-the-fly using MotionCorr in Appion.
3Appion0.0image acquisitionTilt angle
5GctfCTF correctionGctf was used to estimate CTF for summed micrographs.1
8MOLREPmodel fittingMolrep was used to unambiguously assign six DnaB CTD and six DnaB NTD homology models into our BP map.
9Cootmodel fittingCoot was used to manually build 5 lambda P protomers and five DnaB NTD-CTD linkers
11RELIONseries alignment2D and 3D classifications and auto-refinement were performed in Relion.1
12Gautomatchseries alignmentTemplate-based particle picking was performed with Gautomatch.1
13EMAN2series alignmentEMAN2 was used to project tomography-derived initial model for templates used in auto picking procedures.1
14RELION3D reconstructionAuto refine and post processing was performed in Relion.1
16TOMO3Dseries alignmentTomo3D was used to reconstruct tomograms from aligned images.2
17Dynamoseries alignmentDynamo was used to pick and align particles from tomograms.2
18EMAN2series alignmentEMAN2 was used to generate 2D projections of sub-tomogram average.2
19Dynamo3D reconstructionDynamo was used to generate sub-tomogram average, later used as initial model.2
20PHENIXmodel refinementPhenix real_space_refine was used to assess model validity.
Image processing
IDImage recording IDDetails
11Images 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.
21Three tilt series were collected from same single-particle grids at a pixel size of 1.76 angstroms per pixel and defocus values of -2.8um, -6.1um and -9.3um. Tilt series were collected bi-directionally over a tilt range of -45 degrees to +45 degrees in 3 degree increments, with a dose of 2.57 to 3.3 electrons per angstrom squared per tilt increment (subdivided over seven to nine frames.)
CTF correction
IDDetailsEM image processing IDType
1CTF correction was performed before data processing on summed micrographs using Gctf.1PHASE FLIPPING AND AMPLITUDE CORRECTION
22NONE
3D reconstruction

Entry ID: 6BBM

IDResolutionResolution methodNumber of particlesImage processing IDDetailsAlgorithm
14.1FSC 0.143 CUT-OFF908831Relion 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.
240OTHER10002A 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.BACK PROJECTION
Atomic model buildingDetails: 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.
Ref protocol: FLEXIBLE FIT / Ref space: REAL
Atomic model building
IDPDB-ID 3D fitting IDPdb chain residue range
12R5U11-173
23BH01203-441

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