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- EMDB-9566: Human RAD51 presynaptic complex -

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Basic information

Entry
Database: EMDB / ID: EMD-9566
TitleHuman RAD51 presynaptic complex
Map dataHuman RAD51-ssDNA formed presynaptic complex
Sample
  • Complex: Human RAD51 and ssDNA formed presynaptic complex
    • Complex: Human RAD51
      • Protein or peptide: DNA repair protein RAD51 homolog 1
    • Complex: ssDNADNA
      • DNA: DNA (5'-D(P*TP*TP*TP*TP*TP*TP*TP*TP*T)-3')
  • Ligand: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER
  • Ligand: MAGNESIUM ION
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 / mitotic recombination / double-strand break repair involved in meiotic recombination / nuclear ubiquitin ligase complex ...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 / mitotic recombination / double-strand break repair involved in meiotic recombination / nuclear ubiquitin ligase complex / 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 / nuclear chromosome / replication fork processing / 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 specieshuman (human) / Homo sapiens (human)
Methodhelical reconstruction / cryo EM / Resolution: 4.4 Å
AuthorsXu J / Zhao L / Xu Y / Zhao W / Sung P / Wang HW
Funding support China, United States, 6 items
OrganizationGrant numberCountry
National Science Foundation of China31270765 China
The Key Research and Development Program of MOST2016YFA0501101 China
The Beijing Municipal Science & Technology CommissionZ161100000116034 China
National Institutes of Health/National Cancer Institute (NIH/NCI)CA168635 United States
National Institutes of Health/National Institute of Environmental Health Sciences (NIH/NIEHS)ES007061 United States
National Institutes of Health/National Institute of Environmental Health Sciences (NIH/NIEHS)ES015252 United States
CitationJournal: Nat Struct Mol Biol / Year: 2017
Title: Cryo-EM structures of human RAD51 recombinase filaments during catalysis of DNA-strand exchange.
Authors: Jingfei Xu / Lingyun Zhao / Yuanyuan Xu / Weixing Zhao / Patrick Sung / Hong-Wei Wang /
Abstract: The central step in eukaryotic homologous recombination (HR) is ATP-dependent DNA-strand exchange mediated by the Rad51 recombinase. In this process, Rad51 assembles on single-stranded DNA (ssDNA) ...The central step in eukaryotic homologous recombination (HR) is ATP-dependent DNA-strand exchange mediated by the Rad51 recombinase. In this process, Rad51 assembles on single-stranded DNA (ssDNA) and generates a helical filament that is able to search for and invade homologous double-stranded DNA (dsDNA), thus leading to strand separation and formation of new base pairs between the initiating ssDNA and the complementary strand within the duplex. Here, we used cryo-EM to solve the structures of human RAD51 in complex with DNA molecules, in presynaptic and postsynaptic states, at near-atomic resolution. Our structures reveal both conserved and distinct structural features of the human RAD51-DNA complexes compared with their prokaryotic counterpart. Notably, we also captured the structure of an arrested synaptic complex. Our results provide new insight into the molecular mechanisms of the DNA homology search and strand-exchange processes.
History
DepositionOct 8, 2016-
Header (metadata) releaseNov 2, 2016-
Map releaseDec 21, 2016-
UpdateMar 23, 2022-
Current statusMar 23, 2022Processing site: PDBj / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.013
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.013
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  • Surface view with fitted model
  • Atomic models: PDB-5h1b
  • Surface level: 0.013
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-5h1b
  • Surface level: 0.013
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-5h1b
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_9566.png

Downloads & links

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Map

FileDownload / File: emd_9566.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationHuman RAD51-ssDNA formed presynaptic complex
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.31 Å/pix.
x 256 pix.
= 334.336 Å		1.31 Å/pix.
x 256 pix.
= 334.336 Å		1.31 Å/pix.
x 256 pix.
= 334.336 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.306 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.013 / Movie #1: 0.013
Minimum - Maximum-0.014779515 - 0.04188662
Average (Standard dev.)0.00021341213 (±0.002159576)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 334.336 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.3061.3061.306
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z334.336334.336334.336
α/β/γ90.00090.00090.000
start NX/NY/NZ-2600
NX/NY/NZ264044
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-0.0150.0420.000

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Supplemental data

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Sample components

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Entire : Human RAD51 and ssDNA formed presynaptic complex

EntireName: Human RAD51 and ssDNA formed presynaptic complex
Components
  • Complex: Human RAD51 and ssDNA formed presynaptic complex
    • Complex: Human RAD51
      • Protein or peptide: DNA repair protein RAD51 homolog 1
    • Complex: ssDNADNA
      • DNA: DNA (5'-D(P*TP*TP*TP*TP*TP*TP*TP*TP*T)-3')
  • Ligand: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER
  • Ligand: MAGNESIUM ION

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Supramolecule #1: Human RAD51 and ssDNA formed presynaptic complex

SupramoleculeName: Human RAD51 and ssDNA formed presynaptic complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: human (human)
Molecular weightTheoretical: 23.56 kDa/nm

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Supramolecule #2: Human RAD51

SupramoleculeName: Human RAD51 / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1

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Supramolecule #3: ssDNA

SupramoleculeName: ssDNA / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2

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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: 3 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 37.008074 KDa
Recombinant expressionOrganism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
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 ETRICQIYDS PCL PEAEAM FAINADGVGD AKD

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Macromolecule #2: DNA (5'-D(P*TP*TP*TP*TP*TP*TP*TP*TP*T)-3')

MacromoleculeName: DNA (5'-D(P*TP*TP*TP*TP*TP*TP*TP*TP*T)-3') / type: dna / ID: 2 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 2.692778 KDa
SequenceString:
(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)

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Macromolecule #3: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER

MacromoleculeName: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER / type: ligand / ID: 3 / Number of copies: 3 / Formula: ANP
Molecular weightTheoretical: 506.196 Da
Chemical component information

ChemComp-ANP:
PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER / AMP-PNP, energy-carrying molecule analogue*YM

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Macromolecule #4: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 4 / Number of copies: 3 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Experimental details

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Structure determination

Methodcryo EM
Processinghelical reconstruction
Aggregation statefilament

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Sample preparation

Concentration0.075 mg/mL
BufferpH: 7.5
Details: 25mM Tris-HCl, pH 7.5, 50mM KCl, 1mM dithiothreitol, 1mM AMP-PNP and 2mM MgCl2
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Support film - Film thickness: 20.0 nm / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 101.325 kPa
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 289 K / 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 opticsC2 aperture diameter: 70.0 µm / Calibrated defocus max: 2.5 µm / Calibrated defocus min: 1.5 µm / Calibrated magnification: 22500 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 22500
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
TemperatureMin: 80.0 K / Max: 80.0 K
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Digitization - Dimensions - Width: 7676 pixel / Digitization - Dimensions - Height: 7420 pixel / Digitization - Sampling interval: 5.0 µm / Digitization - Frames/image: 3-14 / Number grids imaged: 2 / Number real images: 40404 / Average exposure time: 8.0 sec. / Average electron dose: 50.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Segment selectionNumber selected: 540
CTF correctionSoftware - Name: CTFFIND3
Startup modelType of model: OTHER
Details: Randomly assigned euler angle for each segment and using back projection to generate initial model.
Final angle assignmentType: NOT APPLICABLE
Software:
Namedetails
SPIDER (ver. 20.02)
RELION (ver. 1.2)with helix option
Final reconstructionNumber classes used: 82
Applied symmetry - Helical parameters - Δz: 15.88 Å
Applied symmetry - Helical parameters - Δ&Phi: 56.77 °
Applied symmetry - Helical parameters - Axial symmetry: C1 (asymmetric)
Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 4.4 Å / Resolution method: FSC 0.143 CUT-OFF
Software:
Namedetails
SPIDER (ver. 20.02)
RELION (ver. 1.2)with helix option

Number images used: 33838

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Atomic model buiding 1

Initial modelPDB ID:

1szp


Chain - Chain ID: E / Chain - Residue range: 80-395
RefinementSpace: REAL
Output model

PDB-5h1b:
Human RAD51 presynaptic complex

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