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- EMDB-22524: Analysis of a strand exchange reaction with a mini filament of 9-... -

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Basic information

Entry
Database: EMDB / ID: EMD-22524
TitleAnalysis of a strand exchange reaction with a mini filament of 9-RecA, 27-mer ssDNA, partially-homologous 67 bp dsDNA and ATPgammaS.
Map dataconsensus reconstruction postprocessed with relion
Sample
  • Complex: Ensemble of products of a strand exchange reaction with partially-homologous 67 bp dsDNA and ATPgammaS
    • Protein or peptide: Protein RecA
    • DNA: DNA (27-MER)
    • DNA: DNA (5'-D(P*AP*AP*AP*AP*AP*AP*AP*AP*AP*A)-3')
  • Ligand: MAGNESIUM ION
  • Ligand: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER
KeywordsRecombination / DNA repair / DNA BINDING PROTEIN / DNA BINDING PROTEIN-DNA complex
Function / homology
Function and homology information


DNA polymerase V complex / homologous recombination / recombinational repair / SOS response / ATP-dependent DNA damage sensor activity / response to ionizing radiation / translesion synthesis / ATP-dependent activity, acting on DNA / cell motility / single-stranded DNA binding ...DNA polymerase V complex / homologous recombination / recombinational repair / SOS response / ATP-dependent DNA damage sensor activity / response to ionizing radiation / translesion synthesis / ATP-dependent activity, acting on DNA / cell motility / single-stranded DNA binding / DNA-binding transcription factor binding / DNA recombination / damaged DNA binding / DNA repair / DNA damage response / ATP hydrolysis activity / ATP binding / cytoplasm
Similarity search - Function
: / : / RecA C-terminal domain / DNA recombination/repair protein RecA, conserved site / DNA recombination and repair protein RecA, C-terminal / recA signature. / DNA recombination and repair protein RecA / recA bacterial DNA recombination protein / DNA recombination and repair protein RecA, monomer-monomer interface / RecA family profile 2. ...: / : / RecA C-terminal domain / DNA recombination/repair protein RecA, conserved site / DNA recombination and repair protein RecA, C-terminal / recA signature. / DNA recombination and repair protein RecA / recA bacterial DNA recombination protein / DNA recombination and repair protein RecA, monomer-monomer interface / RecA family profile 2. / DNA recombination and repair protein RecA-like, ATP-binding domain / RecA family profile 1. / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Protein RecA / Protein RecA
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.4 Å
AuthorsPavletich NP
Funding support United States, 1 items
OrganizationGrant numberCountry
Howard Hughes Medical Institute (HHMI) United States
CitationJournal: Nature / Year: 2020
Title: Mechanism of strand exchange from RecA-DNA synaptic and D-loop structures.
Authors: Haijuan Yang / Chun Zhou / Ankita Dhar / Nikola P Pavletich /
Abstract: The strand-exchange reaction is central to homologous recombination. It is catalysed by the RecA family of ATPases, which form a helical filament with single-stranded DNA (ssDNA) and ATP. This ...The strand-exchange reaction is central to homologous recombination. It is catalysed by the RecA family of ATPases, which form a helical filament with single-stranded DNA (ssDNA) and ATP. This filament binds to a donor double-stranded DNA (dsDNA) to form synaptic filaments, which search for homology and then catalyse the exchange of the complementary strand, forming either a new heteroduplex or-if homology is limited-a D-loop. How synaptic filaments form, search for homology and catalyse strand exchange is poorly understood. Here we report the cryo-electron microscopy analysis of synaptic mini-filaments with both non-complementary and partially complementary dsDNA, and structures of RecA-D-loop complexes containing a 10- or a 12-base-pair heteroduplex. The C-terminal domain of RecA binds to dsDNA and directs it to the RecA L2 loop, which inserts into and opens up the duplex. The opening propagates through RecA sequestering the homologous strand at a secondary DNA-binding site, which frees the complementary strand to sample pairing with the ssDNA. At each RecA step, there is a roughly 20% probability that duplex opening will terminate and the as-yet-unopened dsDNA portion will bind to another C-terminal domain. Homology suppresses this process, through the cooperation of heteroduplex pairing with the binding of ssDNA to the secondary site, to extend dsDNA opening. This mechanism locally limits the length of ssDNA sampled for pairing if homology is not encountered, and could allow for the formation of multiple, widely separated synapses on the donor dsDNA, which would increase the likelihood of encountering homology. These findings provide key mechanistic insights into homologous recombination.
History
DepositionAug 29, 2020-
Header (metadata) releaseNov 4, 2020-
Map releaseNov 4, 2020-
UpdateMar 6, 2024-
Current statusMar 6, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.028
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.028
  • Imaged by UCSF Chimera
  • Download
  • Surface view with fitted model
  • Atomic models: PDB-7jy8
  • Surface level: 0.028
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_22524.png

Downloads & links

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Map

FileDownload / File: emd_22524.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationconsensus reconstruction postprocessed with relion
Voxel sizeX=Y=Z: 1.078 Å
Density
Contour LevelBy AUTHOR: 0.028 / Movie #1: 0.028
Minimum - Maximum-0.21980011 - 0.5270352
Average (Standard dev.)-0.000008914903 (±0.007933587)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 275.968 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0781.0781.078
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z275.968275.968275.968
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-0.2200.527-0.000

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Supplemental data

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Additional map: composite REFMAC5 map (masked) used in coordinate refinement

Fileemd_22524_additional_1.map
Annotationcomposite REFMAC5 map (masked) used in coordinate refinement
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: reconstruction (masked) focused at 5' end postprocessed with relion

Fileemd_22524_additional_2.map
Annotationreconstruction (masked) focused at 5' end postprocessed with relion
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: reconstruction (masked) focused at 3' end postprocessed with relion

Fileemd_22524_additional_3.map
Annotationreconstruction (masked) focused at 3' end postprocessed with relion
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: unsharpened reconstruction of particles containing DloopDG (Fig. 2k)

Fileemd_22524_additional_4.map
Annotationunsharpened reconstruction of particles containing DloopDG (Fig. 2k)
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Ensemble of products of a strand exchange reaction with partially...

EntireName: Ensemble of products of a strand exchange reaction with partially-homologous 67 bp dsDNA and ATPgammaS
Components
  • Complex: Ensemble of products of a strand exchange reaction with partially-homologous 67 bp dsDNA and ATPgammaS
    • Protein or peptide: Protein RecA
    • DNA: DNA (27-MER)
    • DNA: DNA (5'-D(P*AP*AP*AP*AP*AP*AP*AP*AP*AP*A)-3')
  • Ligand: MAGNESIUM ION
  • Ligand: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER

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Supramolecule #1: Ensemble of products of a strand exchange reaction with partially...

SupramoleculeName: Ensemble of products of a strand exchange reaction with partially-homologous 67 bp dsDNA and ATPgammaS
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Source (natural)Organism: Escherichia coli (E. coli)

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Macromolecule #1: Protein RecA

MacromoleculeName: Protein RecA / type: protein_or_peptide / ID: 1 / Number of copies: 9 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 35.960281 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MAIDENKQKA LAAALGQIEK QFGKGSIMRL GEDRSMDVET ISTGSLSLDI ALGAGGLPMG RIVEIYGPES SGKTTLTLQV IAAAQREGK TCAFIDAEHA LDPIYARKLG VDIDNLLCSQ PDTGEQALEI CDALARSGAV DVIVVDSVAA LTPKAEIEGE I GDSHMGLA ...String:
MAIDENKQKA LAAALGQIEK QFGKGSIMRL GEDRSMDVET ISTGSLSLDI ALGAGGLPMG RIVEIYGPES SGKTTLTLQV IAAAQREGK TCAFIDAEHA LDPIYARKLG VDIDNLLCSQ PDTGEQALEI CDALARSGAV DVIVVDSVAA LTPKAEIEGE I GDSHMGLA ARMMSQAMRK LAGNLKQSNT LLIFINQIRM KIGVMFGNPE TTTGGNALKF YASVRLDIRR IGAVKEGENV VG SETRVKV VKNKIAAPFK QAEFQILYGE GINFYGELVD LGVKEKLIEK AGAWYSYKGE KIGQGKANAT AWLKDNPETA KEI EKKVRE LLLSNPNS

UniProtKB: Protein RecA

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Macromolecule #2: DNA (27-MER)

MacromoleculeName: DNA (27-MER) / type: dna / ID: 2 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 8.168248 KDa
SequenceString:
(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT) (DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT) (DT)(DT)(DT)(DT)(DT)(DT)(DT)

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Macromolecule #3: DNA (5'-D(P*AP*AP*AP*AP*AP*AP*AP*AP*AP*A)-3')

MacromoleculeName: DNA (5'-D(P*AP*AP*AP*AP*AP*AP*AP*AP*AP*A)-3') / type: dna / ID: 3 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 14.049344 KDa
SequenceString:
(DA)(DA)(DA)(DA)(DA)(DA)(DA)(DA)(DA)(DA) (DA)(DA)(DA)(DA)(DA)(DA)(DA)(DA)(DA)(DA) (DA)(DA)(DA)(DA)(DA)(DA)(DA)(DA)(DA) (DA)(DA)(DA)(DA)(DA)(DA)(DA)(DA)(DA)(DA) (DA) (DA)(DA)(DA)(DA)(DA)

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Macromolecule #4: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 4 / Number of copies: 9 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #5: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER

MacromoleculeName: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / type: ligand / ID: 5 / Number of copies: 9 / Formula: AGS
Molecular weightTheoretical: 523.247 Da
Chemical component information

ChemComp-AGS:
PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / ATP-gamma-S, energy-carrying molecule analogue*YM

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 8
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeTFS KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 52.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.4 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 1697726

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Atomic model buiding 1

Initial modelPDB ID:

3cmw


Chain - Source name: PDB / Chain - Initial model type: experimental model
Output model

PDB-7jy8:
Analysis of a strand exchange reaction with a mini filament of 9-RecA, 27-mer ssDNA, partially-homologous 67 bp dsDNA and ATPgammaS

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