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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 P
  • Replicative DNA helicase
KeywordsREPLICATION / Helicase Loader / Helicase / DNA replication / ATPase / DNA Replication Initiation / Bacteriophage Lambda
Function / homology
Function and homology information


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 / bidirectional double-stranded viral DNA replication / DNA 5'-3' helicase / 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 / bidirectional double-stranded viral DNA replication / DNA 5'-3' helicase / 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 / isomerase activity / helicase activity / 5'-3' DNA helicase activity / DNA helicase / DNA replication / hydrolase activity / ATP hydrolysis activity / DNA binding / ATP binding / identical protein binding / cytosol
Similarity search - Function
Replication P / Replication protein P / DNA helicase, DnaB type / DNA helicase, DnaB-like, N-terminal / DnaB-like helicase N terminal domain / DNA helicase, DnaB-like, N-terminal domain superfamily / DNA helicase DnaB, N-terminal/DNA primase DnaG, C-terminal / DnaB-like helicase C terminal domain / DNA helicase, DnaB-like, C-terminal / Superfamily 4 helicase domain profile. ...Replication P / Replication protein P / DNA helicase, DnaB type / DNA helicase, DnaB-like, N-terminal / DnaB-like helicase N terminal domain / DNA helicase, DnaB-like, N-terminal domain superfamily / DNA helicase DnaB, N-terminal/DNA primase DnaG, C-terminal / DnaB-like helicase C terminal domain / DNA helicase, DnaB-like, C-terminal / Superfamily 4 helicase domain profile. / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
ADENOSINE-5'-DIPHOSPHATE / Replicative DNA helicase / Helicase loader / Replicative DNA helicase DnaB
Similarity search - Component
Biological speciesEscherichia coli O111:NM (bacteria)
Escherichia phage lambda (virus)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.1 Å
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.
Funding support United States, 12items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01 GM084162 United States
National Science Foundation (NSF, United States)MCB 1818255 United States
National Institutes of Health/National Institute on Minority Health and Health Disparities (NIH/NIMHD)5G12MD007603-30 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)P41 GM109824 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)P41 GM103314 United States
Other governmentPA200A150068 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)F32GM128303 United States
Other privateSF349247 (Simons Foundation) United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM103310 United States
Other privateF00316 (Agouron Institute) United States
National Institutes of Health/Office of the DirectorOD019994 United States
Other privateSM-2015-289297 (Silicon Mechanics/Research Cluster Grant program) United States
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.
History
DepositionOct 18, 2017Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 6, 2019Provider: repository / Type: Initial release
Revision 1.1Mar 13, 2019Group: Data collection
Category: em_admin / pdbx_database_proc / pdbx_seq_map_depositor_info
Item: _em_admin.last_update / _pdbx_seq_map_depositor_info.one_letter_code_mod
Revision 1.2Aug 14, 2019Group: Data collection / Data processing / Experimental preparation
Category: em_3d_reconstruction / em_ctf_correction ...em_3d_reconstruction / em_ctf_correction / em_experiment / em_image_processing / em_software / em_specimen
Item: _em_experiment.reconstruction_method / _em_software.image_processing_id / _em_specimen.details
Revision 1.3Nov 27, 2019Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.4Dec 18, 2019Group: Other / Category: atom_sites
Item: _atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] / _atom_sites.fract_transf_matrix[3][3]
Revision 1.5Mar 13, 2024Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_3d_fitting_list / pdbx_initial_refinement_model
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type

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Structure visualization

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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
Polymers430,41211
Non-polymers2,1365
Water00
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 ...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.
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein
Replicative DNA helicase


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
#2: Protein
Replication protein P


Mass: 23141.221 Da / Num. of mol.: 5
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia phage lambda (virus) / Plasmid: 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 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / 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 / 3D reconstruction method: single particle reconstruction

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Sample preparation

Component
IDNameTypeDetailsEntity IDParent-IDSource
1DnaB helicase - Lambda P helicase loader DNA replication complexCOMPLEXpET24a 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-#20RECOMBINANT
2E coli DnaB helicaseCOMPLEXE 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.#11RECOMBINANT
3Lambda P helicase loaderCOMPLEXFive 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.#21RECOMBINANT
Molecular weight
IDEntity assembly-IDValue (°)Experimental value
110.4465 MDaYES
21NO
31NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
21Escherichia coli (E. coli)562
32Escherichia coli (E. coli)562
43Enterobacteria phage lambda (virus)10710
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-IDCellPlasmid
21Escherichia coli (E. coli)562BL21(DE3)pET24a and pCDFDuet
32Escherichia coli (E. coli)562BL21(DE3)pET24a
43Escherichia coli (E. coli)562BL21(DE3)pCDFDuet
Buffer solutionpH: 7.5
Details: Concentrated BP sample (18mg/mL) was diluted with freshly prepared buffer to desired concentration (~1.5 micromolar) for grid preparation.
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 / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
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.
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 divisions/in. / Grid type: Quantifoil R0.6/1
VitrificationInstrument: 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.

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: 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 FIELD / 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 holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 70 K / Temperature (min): 70 K
Image recordingAverage 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 opticsSpherical 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
IDNameVersionCategoryDetails
1Leginon0image acquisitionLeginon was used for automatic hole targeting during single particle movie collection.
2Appion0image acquisitionSingle particle micrograph movies were aligned on-the-fly using MotionCorr in Appion.
3Appion0image acquisitionTilt angle
5GctfCTF correctionGctf was used to estimate CTF for summed micrographs.
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.
12Gautomatchseries alignmentTemplate-based particle picking was performed with Gautomatch.
13EMAN2series alignmentEMAN2 was used to project tomography-derived initial model for templates used in auto picking procedures.
14RELION3D reconstructionAuto refine and post processing was performed in Relion.
16TOMO3Dseries alignmentTomo3D was used to reconstruct tomograms from aligned images.
17Dynamoseries alignmentDynamo was used to pick and align particles from tomograms.
18EMAN2series alignmentEMAN2 was used to generate 2D projections of sub-tomogram average.
19Dynamo3D reconstructionDynamo was used to generate sub-tomogram average, later used as initial model.
20PHENIXmodel refinementPhenix real_space_refine was used to assess model validity.
Image processingDetails: 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.
CTF correctionDetails: CTF correction was performed before data processing on summed micrographs using Gctf.
Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 4.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 90883
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.
Atomic model buildingProtocol: FLEXIBLE FIT / 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 ...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.
Atomic model building

3D fitting-ID: 1 / Source name: PDB / Type: experimental model

IDPDB-IDAccession codeInitial refinement model-IDPdb chain residue range
12R5U

2r5u

2R5U11-173
23BH0

3bh0

3BH02203-441

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