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- EMDB-28736: XRCC4-XLF-XRCC4 subcomplex of PAXX-LR complex -

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Basic information

Entry
Database: EMDB / ID: EMD-28736
TitleXRCC4-XLF-XRCC4 subcomplex of PAXX-LR complex
Map dataXXL in PAXX-LR
Sample
  • Complex: NHEJ Long-range complex with PAXX
    • Protein or peptide: XRCC4_HUMAN DNA repair protein XRCC4
    • Protein or peptide: NHEJ1_HUMAN Non-homologous end-joining factor 1
KeywordsComplex / Kinase / NHEJ / DNA BINDING PROTEIN
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 14.0 Å
AuthorsChen S / He Y
Funding support United States, 2 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01 GM135651 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)U24GM129547 United States
CitationJournal: Sci Adv / Year: 2023
Title: Cryo-EM visualization of DNA-PKcs structural intermediates in NHEJ.
Authors: Siyu Chen / Alex Vogt / Linda Lee / Tasmin Naila / Ryan McKeown / Alan E Tomkinson / Susan P Lees-Miller / Yuan He /
Abstract: DNA double-strand breaks (DSBs), one of the most cytotoxic forms of DNA damage, can be repaired by the tightly regulated nonhomologous end joining (NHEJ) machinery (Stinson and Loparo and Zhao ). ...DNA double-strand breaks (DSBs), one of the most cytotoxic forms of DNA damage, can be repaired by the tightly regulated nonhomologous end joining (NHEJ) machinery (Stinson and Loparo and Zhao ). Core NHEJ factors form an initial long-range (LR) synaptic complex that transitions into a DNA-PKcs (DNA-dependent protein kinase, catalytic subunit)-free, short-range state to align the DSB ends (Chen ). Using single-particle cryo-electron microscopy, we have visualized three additional key NHEJ complexes representing different transition states, with DNA-PKcs adopting distinct dimeric conformations within each of them. Upon DNA-PKcs autophosphorylation, the LR complex undergoes a substantial conformational change, with both Ku and DNA-PKcs rotating outward to promote DNA break exposure and DNA-PKcs dissociation. We also captured a dimeric state of catalytically inactive DNA-PKcs, which resembles structures of other PIKK (Phosphatidylinositol 3-kinase-related kinase) family kinases, revealing a model of the full regulatory cycle of DNA-PKcs during NHEJ.
History
DepositionOct 31, 2022-
Header (metadata) releaseJun 14, 2023-
Map releaseJun 14, 2023-
UpdateJun 14, 2023-
Current statusJun 14, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_28736.png

Downloads & links

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Map

FileDownload / File: emd_28736.map.gz / Format: CCP4 / Size: 43.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationXXL in PAXX-LR
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.16 Å/pix.
x 225 pix.
= 485.55 Å		2.16 Å/pix.
x 225 pix.
= 485.55 Å		2.16 Å/pix.
x 225 pix.
= 485.55 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2.158 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.02
Minimum - Maximum-0.13531063 - 0.27572688
Average (Standard dev.)-0.00011912254 (±0.003984754)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions225225225
Spacing225225225
CellA=B=C: 485.55 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: half1

Fileemd_28736_half_map_1.map
Annotationhalf1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half2

Fileemd_28736_half_map_2.map
Annotationhalf2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : NHEJ Long-range complex with PAXX

EntireName: NHEJ Long-range complex with PAXX
Components
  • Complex: NHEJ Long-range complex with PAXX
    • Protein or peptide: XRCC4_HUMAN DNA repair protein XRCC4
    • Protein or peptide: NHEJ1_HUMAN Non-homologous end-joining factor 1

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Supramolecule #1: NHEJ Long-range complex with PAXX

SupramoleculeName: NHEJ Long-range complex with PAXX / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 1.75 MDa

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Macromolecule #1: XRCC4_HUMAN DNA repair protein XRCC4

MacromoleculeName: XRCC4_HUMAN DNA repair protein XRCC4 / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MERKISRIHL VSEPSITHFL QVSWEKTLES GFVITLTDGH SAWTGTVSES EISQEADDMA MEKGKYVGEL RKALLSGAGP ADVYTFNFSK ESCYFFFEKN LKDVSFRLGS FNLEKVENPA EVIRELICYC LDTIAENQAK NEHLQKENER LLRDWNDVQG RFEKCVSAKE ...String:
MERKISRIHL VSEPSITHFL QVSWEKTLES GFVITLTDGH SAWTGTVSES EISQEADDMA MEKGKYVGEL RKALLSGAGP ADVYTFNFSK ESCYFFFEKN LKDVSFRLGS FNLEKVENPA EVIRELICYC LDTIAENQAK NEHLQKENER LLRDWNDVQG RFEKCVSAKE ALETDLYKRF ILVLNEKKTK IRSLHNKLLN AAQEREKDIK QEGETAICSE MTADRDPVYD ESTDEESENQ TDLSGLASAA VSKDDSIISS LDVTDIAPSR KRRQRMQRNL GTEPKMAPQE NQLQEKENSR PDSSLPETSK KEHISAENMS LETLRNSSPE DLFDEI

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Macromolecule #2: NHEJ1_HUMAN Non-homologous end-joining factor 1

MacromoleculeName: NHEJ1_HUMAN Non-homologous end-joining factor 1 / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MEELEQGLLM QPWAWLQLAE NSLLAKVFIT KQGYALLVSD LQQVWHEQVD TSVVSQRAKE LNKRLTAPPA AFLCHLDNLL RPLLKDAAHP SEATFSCDCV ADALILRVRS ELSGLPFYWN FHCMLASPSL VSQHLIRPLM GMSLALQCQV RELATLLHMK DLEIQDYQES ...String:
MEELEQGLLM QPWAWLQLAE NSLLAKVFIT KQGYALLVSD LQQVWHEQVD TSVVSQRAKE LNKRLTAPPA AFLCHLDNLL RPLLKDAAHP SEATFSCDCV ADALILRVRS ELSGLPFYWN FHCMLASPSL VSQHLIRPLM GMSLALQCQV RELATLLHMK DLEIQDYQES GATLIRDRLK TEPFEENSFL EQFMIEKLPE ACSIGDGKPF VMNLQDLYMA VTTQEVQVGQ KHQGAGDPHT SNSASLQGID SQCVNQPEQL VSSAPTLSAP EKESTGTSGP LQRPQLSKVK RKKPRGLFS

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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

Concentration2 mg/mL
BufferpH: 7.9
GridModel: Quantifoil R2/1 / Material: COPPER / Mesh: 200 / Support film - Material: GRAPHENE OXIDE / Support film - topology: CONTINUOUS / Support film - Film thickness: 200 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 10 sec. / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 302 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
TemperatureMin: 70.0 K / Max: 70.0 K
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: 11006 / Average exposure time: 4.0 sec. / Average electron dose: 65.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Calibrated defocus max: 4.0 µm / Calibrated defocus min: 2.0 µm / Calibrated magnification: 60000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.0 mm / Nominal defocus max: 4.0 µm / Nominal defocus min: 2.0 µm / Nominal magnification: 60000
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

Particle selectionNumber selected: 1101255
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

7lt3

Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C2 (2 fold cyclic) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 14.0 Å / Resolution method: OTHER / Software - Name: RELION (ver. 3.1.3) / Number images used: 44866
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1.3)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1.3)
Final 3D classificationNumber classes: 10 / Software - Name: RELION (ver. 3.1.3)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Target criteria: Cross-correlation coefficient

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