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- EMDB-16197: CryoEM structure of the post-synaptic RAD51 nucleoprotein filamen... -

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Basic information

Entry
Database: EMDB / ID: EMD-16197
TitleCryoEM structure of the post-synaptic RAD51 nucleoprotein filament in the presence of ATP and Ca2+
Map dataPostprocessed map
Sample
  • Complex: Post-synaptic RAD51 nucleoprotein filament in the presence of ATP and Ca2+
    • Protein or peptide: DNA repair protein RAD51 homolog 1
    • DNA: DNA (5'-D(P*TP*GP*GP*AP*GP*GP*TP*GP*CP*AP*TP*CP*GP*AP*GP*CP*TP*CP*GP*C)-3')
    • DNA: DNA (5'-D(P*GP*CP*GP*AP*GP*CP*TP*CP*GP*AP*TP*GP*CP*AP*CP*CP*TP*CP*CP*A)-3')
  • Ligand: CALCIUM IONCalcium
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE
  • Ligand: water
KeywordsDNA repair / Homologous Recombination / DNA-strand exchange / ATPase / DNA BINDING PROTEIN
Function / homology
Function and homology information


presynaptic intermediate filament cytoskeleton / mitotic recombination-dependent replication fork processing / chromosome organization involved in meiotic cell cycle / cellular response to camptothecin / DNA recombinase assembly / telomere maintenance via telomere lengthening / positive regulation of DNA ligation / double-strand break repair involved in meiotic recombination / nuclear ubiquitin ligase complex / mitotic recombination ...presynaptic intermediate filament cytoskeleton / mitotic recombination-dependent replication fork processing / chromosome organization involved in meiotic cell cycle / cellular response to camptothecin / DNA recombinase assembly / telomere maintenance via telomere lengthening / positive regulation of DNA ligation / double-strand break repair involved in meiotic recombination / nuclear ubiquitin ligase complex / mitotic recombination / DNA strand invasion / replication-born double-strand break repair via sister chromatid exchange / cellular response to hydroxyurea / DNA strand exchange activity / lateral element / telomere maintenance via recombination / regulation of DNA damage checkpoint / Impaired BRCA2 binding to PALB2 / single-stranded DNA helicase activity / reciprocal meiotic recombination / Defective homologous recombination repair (HRR) due to BRCA1 loss of function / Defective HDR through Homologous Recombination Repair (HRR) due to PALB2 loss of BRCA1 binding function / Defective HDR through Homologous Recombination Repair (HRR) due to PALB2 loss of BRCA2/RAD51/RAD51C binding function / Homologous DNA Pairing and Strand Exchange / Resolution of D-loop Structures through Synthesis-Dependent Strand Annealing (SDSA) / Resolution of D-loop Structures through Holliday Junction Intermediates / HDR through Single Strand Annealing (SSA) / Impaired BRCA2 binding to RAD51 / ATP-dependent DNA damage sensor activity / regulation of double-strand break repair via homologous recombination / replication fork processing / nuclear chromosome / DNA unwinding involved in DNA replication / Transcriptional Regulation by E2F6 / Presynaptic phase of homologous DNA pairing and strand exchange / ATP-dependent activity, acting on DNA / interstrand cross-link repair / DNA polymerase binding / condensed chromosome / meiotic cell cycle / condensed nuclear chromosome / male germ cell nucleus / cellular response to ionizing radiation / double-strand break repair via homologous recombination / regulation of protein phosphorylation / HDR through Homologous Recombination (HRR) / PML body / Meiotic recombination / single-stranded DNA binding / site of double-strand break / double-stranded DNA binding / DNA recombination / chromosome, telomeric region / mitochondrial matrix / DNA repair / centrosome / DNA damage response / chromatin binding / chromatin / nucleolus / perinuclear region of cytoplasm / enzyme binding / ATP hydrolysis activity / protein-containing complex / mitochondrion / nucleoplasm / ATP binding / identical protein binding / nucleus / cytosol / cytoplasm
Similarity search - Function
DNA recombination/repair protein Rad51 / DNA recombination and repair protein, RecA-like / DNA recombination and repair protein Rad51-like, C-terminal / Rad51 / 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. / DNA repair Rad51/transcription factor NusA, alpha-helical / Helix-hairpin-helix domain ...DNA recombination/repair protein Rad51 / DNA recombination and repair protein, RecA-like / DNA recombination and repair protein Rad51-like, C-terminal / Rad51 / 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. / DNA repair Rad51/transcription factor NusA, alpha-helical / Helix-hairpin-helix domain / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
DNA repair protein RAD51 homolog 1
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.9 Å
AuthorsAppleby R / Bollschweiler D / Pellegrini L
Funding support United Kingdom, 1 items
OrganizationGrant numberCountry
Wellcome Trust221892/Z/20/Z United Kingdom
CitationJournal: iScience / Year: 2023
Title: A metal ion-dependent mechanism of RAD51 nucleoprotein filament disassembly.
Authors: Robert Appleby / Daniel Bollschweiler / Dimitri Y Chirgadze / Luay Joudeh / Luca Pellegrini /
Abstract: The RAD51 ATPase polymerizes on single-stranded DNA to form nucleoprotein filaments (NPFs) that are critical intermediates in the reaction of homologous recombination. ATP binding maintains the NPF ...The RAD51 ATPase polymerizes on single-stranded DNA to form nucleoprotein filaments (NPFs) that are critical intermediates in the reaction of homologous recombination. ATP binding maintains the NPF in a competent conformation for strand pairing and exchange. Once strand exchange is completed, ATP hydrolysis licenses the filament for disassembly. Here we show that the ATP-binding site of the RAD51 NPF contains a second metal ion. In the presence of ATP, the metal ion promotes the local folding of RAD51 into the conformation required for DNA binding. The metal ion is absent in the ADP-bound RAD51 filament, that rearranges in a conformation incompatible with DNA binding. The presence of the second metal ion explains how RAD51 couples the nucleotide state of the filament to DNA binding. We propose that loss of the second metal ion upon ATP hydrolysis drives RAD51 dissociation from the DNA and weakens filament stability, contributing to NPF disassembly.
History
DepositionNov 22, 2022-
Header (metadata) releaseMay 3, 2023-
Map releaseMay 3, 2023-
UpdateMay 31, 2023-
Current statusMay 31, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_16197.png

Downloads & links

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Map

FileDownload / File: emd_16197.map.gz / Format: CCP4 / Size: 32.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationPostprocessed map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.3 Å/pix.
x 204 pix.
= 266.016 Å		1.3 Å/pix.
x 204 pix.
= 266.016 Å		1.3 Å/pix.
x 204 pix.
= 266.016 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.304 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0466
Minimum - Maximum-0.19773227 - 0.43551227
Average (Standard dev.)0.000025621539 (±0.011145135)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions204204204
Spacing204204204
CellA=B=C: 266.016 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_16197_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_16197_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Post-synaptic RAD51 nucleoprotein filament in the presence of ATP...

EntireName: Post-synaptic RAD51 nucleoprotein filament in the presence of ATP and Ca2+
Components
  • Complex: Post-synaptic RAD51 nucleoprotein filament in the presence of ATP and Ca2+
    • Protein or peptide: DNA repair protein RAD51 homolog 1
    • DNA: DNA (5'-D(P*TP*GP*GP*AP*GP*GP*TP*GP*CP*AP*TP*CP*GP*AP*GP*CP*TP*CP*GP*C)-3')
    • DNA: DNA (5'-D(P*GP*CP*GP*AP*GP*CP*TP*CP*GP*AP*TP*GP*CP*AP*CP*CP*TP*CP*CP*A)-3')
  • Ligand: CALCIUM IONCalcium
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE
  • Ligand: water

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Supramolecule #1: Post-synaptic RAD51 nucleoprotein filament in the presence of ATP...

SupramoleculeName: Post-synaptic RAD51 nucleoprotein filament in the presence of ATP and Ca2+
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: DNA repair protein RAD51 homolog 1

MacromoleculeName: DNA repair protein RAD51 homolog 1 / type: protein_or_peptide / ID: 1 / Number of copies: 6 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 37.009125 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MAMQMQLEAN ADTSVEEESF GPQPISRLEQ CGINANDVKK LEEAGFHTVE AVAYAPKKEL INIKGISEAK ADKILAEAAK LVPMGFTTA TEFHQRRSEI IQITTGSKEL DKLLQGGIET GSITEMFGEF RTGKTQICHT LAVTCQLPID RGGGEGKAMY I DTEGTFRP ...String:
MAMQMQLEAN ADTSVEEESF GPQPISRLEQ CGINANDVKK LEEAGFHTVE AVAYAPKKEL INIKGISEAK ADKILAEAAK LVPMGFTTA TEFHQRRSEI IQITTGSKEL DKLLQGGIET GSITEMFGEF RTGKTQICHT LAVTCQLPID RGGGEGKAMY I DTEGTFRP ERLLAVAERY GLSGSDVLDN VAYARAFNTD HQTQLLYQAS AMMVESRYAL LIVDSATALY RTDYSGRGEL SA RQMHLAR FLRMLLRLAD EFGVAVVITN QVVAQVDGAA MFAADPKKPI GGNIIAHAST TRLYLRKGRG ETRICKIYDS PCL PEAEAM FAINADGVGD AKD

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Macromolecule #2: DNA (5'-D(P*TP*GP*GP*AP*GP*GP*TP*GP*CP*AP*TP*CP*GP*AP*GP*CP*TP*CP...

MacromoleculeName: DNA (5'-D(P*TP*GP*GP*AP*GP*GP*TP*GP*CP*AP*TP*CP*GP*AP*GP*CP*TP*CP*GP*C)-3')
type: dna / ID: 2 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 6.19099 KDa
SequenceString:
(DT)(DG)(DG)(DA)(DG)(DG)(DT)(DG)(DC)(DA) (DT)(DC)(DG)(DA)(DG)(DC)(DT)(DC)(DG)(DC)

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Macromolecule #3: DNA (5'-D(P*GP*CP*GP*AP*GP*CP*TP*CP*GP*AP*TP*GP*CP*AP*CP*CP*TP*CP...

MacromoleculeName: DNA (5'-D(P*GP*CP*GP*AP*GP*CP*TP*CP*GP*AP*TP*GP*CP*AP*CP*CP*TP*CP*CP*A)-3')
type: dna / ID: 3 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 6.079931 KDa
SequenceString:
(DG)(DC)(DG)(DA)(DG)(DC)(DT)(DC)(DG)(DA) (DT)(DG)(DC)(DA)(DC)(DC)(DT)(DC)(DC)(DA)

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Macromolecule #4: CALCIUM ION

MacromoleculeName: CALCIUM ION / type: ligand / ID: 4 / Number of copies: 12 / Formula: CA
Molecular weightTheoretical: 40.078 Da

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Macromolecule #5: ADENOSINE-5'-TRIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 5 / Number of copies: 6 / Formula: ATP
Molecular weightTheoretical: 507.181 Da
Chemical component information

ChemComp-ATP:
ADENOSINE-5'-TRIPHOSPHATE / ATP, energy-carrying molecule*YM / Adenosine triphosphate

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Macromolecule #6: water

MacromoleculeName: water / type: ligand / ID: 6 / Number of copies: 120 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER / Water

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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: 7.5
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2.5 µm / Nominal defocus min: 0.9 µm / Nominal magnification: 130000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Digitization - Dimensions - Width: 5760 pixel / Digitization - Dimensions - Height: 4092 pixel / Number grids imaged: 1 / Number real images: 3851 / Average exposure time: 1.61 sec. / Average electron dose: 47.22 e/Å2
Details: Images were collected in movie-mode at 40 frames per movie.
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 1051037
Startup modelType of model: OTHER / Details: An initial 3D model was generated in Relion 3.1
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Details: 3D refine and postprocess in Relion 3.1 / Number images used: 543102

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: OTHER
Output model

PDB-8br2:
CryoEM structure of the post-synaptic RAD51 nucleoprotein filament in the presence of ATP and Ca2+

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