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- EMDB-30309: Human DMC1 post-synaptic complexes with mismatched dsDNA -

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Basic information

Entry
Database: EMDB / ID: EMD-30309
TitleHuman DMC1 post-synaptic complexes with mismatched dsDNA
Map data
Sample
  • Complex: DMC1-dsDNA filament with mismatched dsDNA
    • Complex: DMC1
      • Protein or peptide: Meiotic recombination protein DMC1/LIM15 homolog
    • Complex: DNA
      • DNA: DNA (5'-D(P*TP*TP*TP*TP*TP*TP*TP*TP*T)-3')
      • DNA: DNA (5'-D(P*AP*AP*AP*AP*AP*AP*AP*AP*A)-3')
  • Ligand: CALCIUM ION
  • Ligand: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER
Keywordsmeiotic homologous recombination / DNA repair / ATPase / RECOMBINATION / RECOMBINATION-DNA complex
Function / homology
Function and homology information


female gamete generation / chromosome organization involved in meiotic cell cycle / DNA recombinase assembly / double-strand break repair involved in meiotic recombination / homologous chromosome pairing at meiosis / DNA strand invasion / mitotic recombination / lateral element / DNA strand exchange activity / reciprocal meiotic recombination ...female gamete generation / chromosome organization involved in meiotic cell cycle / DNA recombinase assembly / double-strand break repair involved in meiotic recombination / homologous chromosome pairing at meiosis / DNA strand invasion / mitotic recombination / lateral element / DNA strand exchange activity / reciprocal meiotic recombination / oocyte maturation / ATP-dependent DNA damage sensor activity / male meiosis I / spermatid development / ATP-dependent activity, acting on DNA / ovarian follicle development / condensed nuclear chromosome / meiotic cell cycle / Meiotic recombination / single-stranded DNA binding / chromosome / site of double-strand break / double-stranded DNA binding / spermatogenesis / chromosome, telomeric region / ATP hydrolysis activity / DNA binding / nucleoplasm / ATP binding / identical protein binding / nucleus
Similarity search - Function
Meiotic recombination protein Dmc1 / 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 ...Meiotic recombination protein Dmc1 / 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
Meiotic recombination protein DMC1/LIM15 homolog
Similarity search - Component
Biological speciesHomo sapiens (human) / synthetic construct (others)
Methodhelical reconstruction / cryo EM / Resolution: 3.36 Å
AuthorsLuo SC / Yeh HY / Chi P / Ho MC / Tsai MD
Funding support Taiwan, 3 items
OrganizationGrant numberCountry
Academia Sinica (Taiwan)AS-KPQ-109-TPP2 Taiwan
Academia Sinica (Taiwan)AS-CFII-108-110 Taiwan
Academia Sinica (Taiwan)AS-KPQ-105-TPP Taiwan
CitationJournal: Nat Commun / Year: 2021
Title: Identification of fidelity-governing factors in human recombinases DMC1 and RAD51 from cryo-EM structures.
Authors: Shih-Chi Luo / Hsin-Yi Yeh / Wei-Hsuan Lan / Yi-Min Wu / Cheng-Han Yang / Hao-Yen Chang / Guan-Chin Su / Chia-Yi Lee / Wen-Jin Wu / Hung-Wen Li / Meng-Chiao Ho / Peter Chi / Ming-Daw Tsai /
Abstract: Both high-fidelity and mismatch-tolerant recombination, catalyzed by RAD51 and DMC1 recombinases, respectively, are indispensable for genomic integrity. Here, we use cryo-EM, MD simulation and ...Both high-fidelity and mismatch-tolerant recombination, catalyzed by RAD51 and DMC1 recombinases, respectively, are indispensable for genomic integrity. Here, we use cryo-EM, MD simulation and functional analysis to elucidate the structural basis for the mismatch tolerance of DMC1. Structural analysis of DMC1 presynaptic and postsynaptic complexes suggested that the lineage-specific Loop 1 Gln244 (Met243 in RAD51) may help stabilize DNA backbone, whereas Loop 2 Pro274 and Gly275 (Val273/Asp274 in RAD51) may provide an open "triplet gate" for mismatch tolerance. In support, DMC1-Q244M displayed marked increase in DNA dynamics, leading to unobservable DNA map. MD simulation showed highly dispersive mismatched DNA ensemble in RAD51 but well-converged DNA in DMC1 and RAD51-V273P/D274G. Replacing Loop 1 or Loop 2 residues in DMC1 with RAD51 counterparts enhanced DMC1 fidelity, while reciprocal mutations in RAD51 attenuated its fidelity. Our results show that three Loop 1/Loop 2 residues jointly enact contrasting fidelities of DNA recombinases.
History
DepositionJun 5, 2020-
Header (metadata) releaseNov 18, 2020-
Map releaseNov 18, 2020-
UpdateMar 27, 2024-
Current statusMar 27, 2024Processing site: PDBj / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.012
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.012
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7c99
  • Surface level: 0.012
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-7c99
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_30309.png

Downloads & links

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Map

FileDownload / File: emd_30309.map.gz / Format: CCP4 / Size: 216 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.84 Å/pix.
x 384 pix.
= 322.56 Å		0.84 Å/pix.
x 384 pix.
= 322.56 Å		0.84 Å/pix.
x 384 pix.
= 322.56 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.84 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.012 / Movie #1: 0.012
Minimum - Maximum-0.040434495 - 0.06662193
Average (Standard dev.)-0.000047774865 (±0.0020698674)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions384384384
Spacing384384384
CellA=B=C: 322.56 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.840.840.84
M x/y/z384384384
origin x/y/z0.0000.0000.000
length x/y/z322.560322.560322.560
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ256256256
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS384384384
D min/max/mean-0.0400.067-0.000

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Supplemental data

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Sample components

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Entire : DMC1-dsDNA filament with mismatched dsDNA

EntireName: DMC1-dsDNA filament with mismatched dsDNA
Components
  • Complex: DMC1-dsDNA filament with mismatched dsDNA
    • Complex: DMC1
      • Protein or peptide: Meiotic recombination protein DMC1/LIM15 homolog
    • Complex: DNA
      • DNA: DNA (5'-D(P*TP*TP*TP*TP*TP*TP*TP*TP*T)-3')
      • DNA: DNA (5'-D(P*AP*AP*AP*AP*AP*AP*AP*AP*A)-3')
  • Ligand: CALCIUM ION
  • Ligand: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER

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Supramolecule #1: DMC1-dsDNA filament with mismatched dsDNA

SupramoleculeName: DMC1-dsDNA filament with mismatched dsDNA / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Source (natural)Organism: Homo sapiens (human)

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Supramolecule #2: DMC1

SupramoleculeName: DMC1 / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1

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Supramolecule #3: DNA

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

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Macromolecule #1: Meiotic recombination protein DMC1/LIM15 homolog

MacromoleculeName: Meiotic recombination protein DMC1/LIM15 homolog / type: protein_or_peptide / ID: 1 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 37.731031 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MKEDQVVAEE PGFQDEEESL FQDIDLLQKH GINVADIKKL KSVGICTIKG IQMTTRRALC NVKGLSEAKV DKIKEAANKL IEPGFLTAF EYSEKRKMVF HITTGSQEFD KLLGGGIESM AITEAFGEFR TGKTQLSHTL CVTAQLPGAG GYPGGKIIFI D TENTFRPD ...String:
MKEDQVVAEE PGFQDEEESL FQDIDLLQKH GINVADIKKL KSVGICTIKG IQMTTRRALC NVKGLSEAKV DKIKEAANKL IEPGFLTAF EYSEKRKMVF HITTGSQEFD KLLGGGIESM AITEAFGEFR TGKTQLSHTL CVTAQLPGAG GYPGGKIIFI D TENTFRPD RLRDIADRFN VDHDAVLDNV LYARAYTSEH QMELLDYVAA KFHEEAGIFK LLIIDSIMAL FRVDFSGRGE LA ERQQKLA QMLSRLQKIS EEYNVAVFVT NQMTADPGAT MTFQADPKKP IGGHILAHAS TTRISLRKGR GELRIAKIYD SPE MPENEA TFAITAGGIG DAKE

UniProtKB: Meiotic recombination protein DMC1/LIM15 homolog

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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: synthetic construct (others)
Molecular weightTheoretical: 2.692778 KDa
SequenceString:
(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*A)-3')

MacromoleculeName: DNA (5'-D(P*AP*AP*AP*AP*AP*AP*AP*AP*A)-3') / type: dna / ID: 3 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 2.773904 KDa
SequenceString:
(DA)(DA)(DA)(DA)(DA)(DA)(DA)(DA)(DA)

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Macromolecule #4: CALCIUM ION

MacromoleculeName: CALCIUM ION / type: ligand / ID: 4 / Number of copies: 3 / Formula: CA
Molecular weightTheoretical: 40.078 Da

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Macromolecule #5: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER

MacromoleculeName: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER / type: ligand / ID: 5 / 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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Experimental details

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Structure determination

Methodcryo EM
Processinghelical reconstruction
Aggregation statefilament

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Sample preparation

BufferpH: 7.5
Details: 25 mM Tris-HCl, pH 7.5, 50 mM KCl and 1 mM dithiothreitol) containing 2 mM AMP-PNP and 5 mM CaCl2
GridModel: Quantifoil R1.2/1.3 / Material: GRAPHENE OXIDE / Mesh: 200 / Support film - Material: GRAPHENE OXIDE / Support film - topology: CONTINUOUS
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 295 K / Instrument: FEI VITROBOT MARK IV
Details: The grids were blotted for 1 sec at 22 degree C with 100% relative humidity and plunge-frozen in liquid ethane cooled by liquid nitrogen using a Vitrobot Mark IV (Thermo Fisher)..
Detailsprotein sample were applied onto a pre-glow-discharged graphene-oxide coated Quantifoil holey carbon grid (1.2/1.3, 200 mesh) using published protocol

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Alignment procedureComa free - Residual tilt: 10.0 mrad
Specialist opticsEnergy filter - Name: GIF Quantum LS / Energy filter - Slit width: 30 eV
Image recordingFilm or detector model: GATAN K2 QUANTUM (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 3838 pixel / Digitization - Dimensions - Height: 3710 pixel / Digitization - Frames/image: 1-50 / Number grids imaged: 1 / Average exposure time: 5.0 sec. / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal magnification: 165000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionNumber classes used: 1
Applied symmetry - Helical parameters - Δz: 15.72 Å
Applied symmetry - Helical parameters - Δ&Phi: 55.49 °
Applied symmetry - Helical parameters - Axial symmetry: C1 (asymmetric)
Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.36 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0) / Number images used: 99500
Segment selectionNumber selected: 140823
Details: Filaments were manually picked, and segments were extracted using a box size of 384 pixel and an inter-box distance of ~10% of the box length.
Startup modelType of model: INSILICO MODEL
In silico model: A simple cylinder was used as initial model to prevent model bias.
Final angle assignmentType: NOT APPLICABLE
FSC plot (resolution estimation)

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