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- EMDB-22130: The negative stain EM structure of the human DNA LigIIIalpha-XRCC... -

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Basic information

Entry
Database: EMDB / ID: EMD-22130
TitleThe negative stain EM structure of the human DNA LigIIIalpha-XRCC1 complex; conformer 1
Map dataRefined 3D map of full-length DNA LigaseIII alpha in complex with full-length XRCC1; conformer 1
Sample
  • Complex: Full-length DNA LigIII alpha in complex with full-length XRCC1; conformer 1
    • Protein or peptide: DNA LigIII-alpha-nuclear
    • Protein or peptide: XRCC1
KeywordsDNA repair / protein-protein interactions / DNA BINDING PROTEIN
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / negative staining / Resolution: 31.0 Å
AuthorsSverzhinsky A / Pascal JM
Funding support United States, 4 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01 ES012512 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35 CA22043 United States
Other governmentP01 CA92584 United States
Other governmentV2018-25 United States
CitationJournal: Nucleic Acids Res / Year: 2021
Title: An atypical BRCT-BRCT interaction with the XRCC1 scaffold protein compacts human DNA Ligase IIIα within a flexible DNA repair complex.
Authors: Michal Hammel / Ishtiaque Rashid / Aleksandr Sverzhinsky / Yasin Pourfarjam / Miaw-Sheue Tsai / Tom Ellenberger / John M Pascal / In-Kwon Kim / John A Tainer / Alan E Tomkinson /
Abstract: The XRCC1-DNA ligase IIIα complex (XL) is critical for DNA single-strand break repair, a key target for PARP inhibitors in cancer cells deficient in homologous recombination. Here, we combined ...The XRCC1-DNA ligase IIIα complex (XL) is critical for DNA single-strand break repair, a key target for PARP inhibitors in cancer cells deficient in homologous recombination. Here, we combined biophysical approaches to gain insights into the shape and conformational flexibility of the XL as well as XRCC1 and DNA ligase IIIα (LigIIIα) alone. Structurally-guided mutational analyses based on the crystal structure of the human BRCT-BRCT heterodimer identified the network of salt bridges that together with the N-terminal extension of the XRCC1 C-terminal BRCT domain constitute the XL molecular interface. Coupling size exclusion chromatography with small angle X-ray scattering and multiangle light scattering (SEC-SAXS-MALS), we determined that the XL is more compact than either XRCC1 or LigIIIα, both of which form transient homodimers and are highly disordered. The reduced disorder and flexibility allowed us to build models of XL particles visualized by negative stain electron microscopy that predict close spatial organization between the LigIIIα catalytic core and both BRCT domains of XRCC1. Together our results identify an atypical BRCT-BRCT interaction as the stable nucleating core of the XL that links the flexible nick sensing and catalytic domains of LigIIIα to other protein partners of the flexible XRCC1 scaffold.
History
DepositionJun 9, 2020-
Header (metadata) releaseDec 2, 2020-
Map releaseDec 2, 2020-
UpdateNov 29, 2023-
Current statusNov 29, 2023Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.04
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.04
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_22130.png

Downloads & links

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Map

FileDownload / File: emd_22130.map.gz / Format: CCP4 / Size: 2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationRefined 3D map of full-length DNA LigaseIII alpha in complex with full-length XRCC1; conformer 1
Voxel sizeX=Y=Z: 3.3 Å
Density
Contour LevelBy AUTHOR: 0.04 / Movie #1: 0.04
Minimum - Maximum-0.037102547 - 0.12899613
Average (Standard dev.)0.0010139918 (±0.009402151)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions808080
Spacing808080
CellA=B=C: 264.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z3.33.33.3
M x/y/z808080
origin x/y/z0.0000.0000.000
length x/y/z264.000264.000264.000
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ192192192
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS808080
D min/max/mean-0.0370.1290.001

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Supplemental data

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Sample components

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Entire : Full-length DNA LigIII alpha in complex with full-length XRCC1; c...

EntireName: Full-length DNA LigIII alpha in complex with full-length XRCC1; conformer 1
Components
  • Complex: Full-length DNA LigIII alpha in complex with full-length XRCC1; conformer 1
    • Protein or peptide: DNA LigIII-alpha-nuclear
    • Protein or peptide: XRCC1

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Supramolecule #1: Full-length DNA LigIII alpha in complex with full-length XRCC1; c...

SupramoleculeName: Full-length DNA LigIII alpha in complex with full-length XRCC1; conformer 1
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 180 KDa

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Macromolecule #1: DNA LigIII-alpha-nuclear

MacromoleculeName: DNA LigIII-alpha-nuclear / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO / EC number: DNA ligase (ATP)
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MAEQRFCVDY AKRGTAGCKK CKEKIVKGVC RIGKVVPNPF SESGGDMKEW YHIKCMFEKL ERARATTKK IEDLTELEGW EELEDNEKEQ ITQHIADLSS KAAGTPKKKA VVQAKLTTTG Q VTSPVKGA SFVTSTNPRK FSGFSAKPNN SGEAPSSPTP KRSLSSSKCD ...String:
MAEQRFCVDY AKRGTAGCKK CKEKIVKGVC RIGKVVPNPF SESGGDMKEW YHIKCMFEKL ERARATTKK IEDLTELEGW EELEDNEKEQ ITQHIADLSS KAAGTPKKKA VVQAKLTTTG Q VTSPVKGA SFVTSTNPRK FSGFSAKPNN SGEAPSSPTP KRSLSSSKCD PRHKDCLLRE FR KLCAMVA DNPSYNTKTQ IIQDFLRKGS AGDGFHGDVY LTVKLLLPGV IKTVYNLNDK QIV KLFSRI FNCNPDDMAR DLEQGDVSET IRVFFEQSKS FPPAAKSLLT IQEVDEFLLR LSKL TKEDE QQQALQDIAS RCTANDLKCI IRLIKHDLKM NSGAKHVLDA LDPNAYEAFK ASRNL QDVV ERVLHNAQEV EKEPGQRRAL SVQASLMTPV QPMLAEACKS VEYAMKKCPN GMFSEI KYD GERVQVHKNG DHFSYFSRSL KPVLPHKVAH FKDYIPQAFP GGHSMILDSE VLLIDNK TG KPLPFGTLGV HKKAAFQDAN VCLFVFDCIY FNDVSLMDRP LCERRKFLHD NMVEIPNR I MFSEMKRVTK ALDLADMITR VIQEGLEGLV LKDVKGTYEP GKRHWLKVKK DYLNEGAMA DTADLVVLGA FYGQGSKGGM MSIFLMGCYD PGSQKWCTVT KCAGGHDDAT LARLQNELDM VKISKDPSK IPSWLKVNKI YYPDFIVPDP KKAAVWEITG AEFSKSEAHT ADGISIRFPR C TRIRDDKD WKSATNLPQL KELYQLSKEK ADFTVVAGDE GSSTTGGSSE ENKGPSGSAV SR KAPSKPS ASTKKAEGKL SNSNSKDGNM QTAKPSAMKV GEKLATKSSP VKVGEKRKAA DET LCQTKV LLDIFTGVRL YLPPSTPDFS RLRRYFVAFD GDLVQEFDMT SATHVLGSRD KNPA AQQVS PEWIWACIRK RRLVAPCWSH PQFEK

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Macromolecule #2: XRCC1

MacromoleculeName: XRCC1 / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: HHHHHHMPEI RLRHVVSCSS QDSTHCAENL LKADTYRKWR AAKAGEKTIS VVLQLEKEEQ IHSVDI GND GSAFVEVLVG SSAGGAGEQD YEVLLVTSSF MSPSESRSGS NPNRVRMFGP DKLVRAA AE KRWDRVKIVC SQPYSKDSPF GLSFVRFHSP PDKDEAEAPS ...String:
HHHHHHMPEI RLRHVVSCSS QDSTHCAENL LKADTYRKWR AAKAGEKTIS VVLQLEKEEQ IHSVDI GND GSAFVEVLVG SSAGGAGEQD YEVLLVTSSF MSPSESRSGS NPNRVRMFGP DKLVRAA AE KRWDRVKIVC SQPYSKDSPF GLSFVRFHSP PDKDEAEAPS QKVTVTKLGQ FRVKEEDE S ANSLRPGALF FSRINKTSPV TASDPAGPSY AAATLQASSA ASSASPVSRA IGSTSKPQE SPKGKRKLDL NQEEKKTPSK PPAQLSPSVP KRPKLPAPTR TPATAPVPAR AQGAVTGKPR GEGTEPRRP RAGPEELGKI LQGVVVVLSG FQNPFRSELR DKALELGAKY RPDWTRDSTH L ICAFANTP KYSQVLGLGG RIVRKEWVLD CHRMRRRLPS RRYLMAGPGS SSEEDEASHS GG SGDEAPK LPQKQPQTKT KPTQAAGPSS PQKPPTPEET KAASPVLQED IDIEGVQSEG QDN GAEDSG DTEDELRRVA EQKEHRLPPG QEENGEDPYA GSTDENTDSE EHQEPPDLPV PELP DFFQG KHFFLYGEFP GDERRKLIRY VTAFNGELED NMSDRVQFVI TAQEWDPSFE EALMD NPSL AFVRPRWIYS CNEKQKLLPH QLYGVVPQA

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Experimental details

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Structure determination

Methodnegative staining
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.01 mg/mL
BufferpH: 7.5
StainingType: NEGATIVE / Material: Uranyl Formate
GridMaterial: COPPER / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec. / Pretreatment - Atmosphere: AIR
DetailsCo-purified proteins were crosslinked with glutaraldehyde, then buffer exchanged to remove the crosslinker before negative staining.

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Electron microscopy

MicroscopeFEI TECNAI 12
Image recordingFilm or detector model: FEI EAGLE (4k x 4k) / Average exposure time: 1.0 sec. / Average electron dose: 70.0 e/Å2
Electron beamAcceleration voltage: 120 kV / Electron source: LAB6
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal magnification: 67000

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Image processing

Particle selectionNumber selected: 78400
Startup modelType of model: OTHER / Details: SGD
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 31.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: RELION (ver. 2.1) / Number images used: 4516
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION

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