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- EMDB-21958: The negative stain EM structure of the human DNA LigI-PCNA-DNA complex -

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Basic information

Entry
Database: EMDB / ID: EMD-21958
TitleThe negative stain EM structure of the human DNA LigI-PCNA-DNA complex
Map dataRefined 3D map of full-length human DNA LigI with human PCNA and 32bp non-ligatable nicked DNA.
Sample
  • Complex: Full-length DNA LigI in complex with nicked duplex DNA and PCNA
    • Complex: DNA ligase I
      • Protein or peptide: DNA ligase I
    • Complex: Proliferating Cell Nuclear Antigen
      • Protein or peptide: Proliferating Cell Nuclear Antigen
    • Complex: Nicked DNA duplex
      • DNA: Nicked DNA duplex
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / negative staining / Resolution: 19.0 Å
AuthorsSverzhinsky A / Pascal JM
Funding support United States, Canada, 2 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01 GM57479 United States
Natural Sciences and Engineering Research Council (NSERC, Canada)RGPIN-2015-05776 Canada
CitationJournal: J Mol Biol / Year: 2020
Title: Dynamic DNA-bound PCNA complexes co-ordinate Okazaki fragment synthesis, processing and ligation.
Authors: Yoshihiro Matsumoto / Rhys C Brooks / Aleksandr Sverzhinsky / John M Pascal / Alan E Tomkinson /
Abstract: More than a million Okazaki fragments are synthesized, processed and joined during replication of the human genome. After synthesis of an RNA-DNA oligonucleotide by DNA polymerase α holoenzyme, ...More than a million Okazaki fragments are synthesized, processed and joined during replication of the human genome. After synthesis of an RNA-DNA oligonucleotide by DNA polymerase α holoenzyme, proliferating cell nuclear antigen (PCNA), a homotrimeric DNA sliding clamp and polymerase processivity factor, is loaded onto the primer-template junction by replication factor C (RFC). Although PCNA interacts with the enzymes DNA polymerase δ (Pol δ), flap endonuclease 1 (FEN1) and DNA ligase I (LigI) that complete Okazaki fragment processing and joining, it is not known how the activities of these enzymes are coordinated. Here we describe a novel interaction between Pol δ and LigI that is critical for Okazaki fragment joining in vitro. Both LigI and FEN1 associate with PCNA-Pol δ during gap-filling synthesis, suggesting that gap-filling synthesis is carried out by a complex of PCNA, Pol δ, FEN1 and LigI. Following ligation, PCNA and LigI remain on the DNA, indicating that Pol δ and FEN1 dissociate during 5' end processing and that LigI engages PCNA at the DNA nick generated by FEN1 and Pol δ. Thus, dynamic PCNA complexes coordinate Okazaki fragment synthesis and processing with PCNA and LigI forming a terminal structure of two linked protein rings encircling the ligated DNA.
History
DepositionMay 10, 2020-
Header (metadata) releaseJan 27, 2021-
Map releaseJan 27, 2021-
UpdateJan 27, 2021-
Current statusJan 27, 2021Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.023
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.023
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_21958.png

Downloads & links

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Map

FileDownload / File: emd_21958.map.gz / Format: CCP4 / Size: 1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationRefined 3D map of full-length human DNA LigI with human PCNA and 32bp non-ligatable nicked DNA.
Voxel sizeX=Y=Z: 3.28 Å
Density
Contour LevelBy AUTHOR: 0.023 / Movie #1: 0.023
Minimum - Maximum-0.06672185 - 0.12201776
Average (Standard dev.)0.0012391106 (±0.012408545)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions646464
Spacing646464
CellA=B=C: 209.92 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z3.283.283.28
M x/y/z646464
origin x/y/z0.0000.0000.000
length x/y/z209.920209.920209.920
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ400400400
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS646464
D min/max/mean-0.0670.1220.001

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Supplemental data

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Sample components

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Entire : Full-length DNA LigI in complex with nicked duplex DNA and PCNA

EntireName: Full-length DNA LigI in complex with nicked duplex DNA and PCNA
Components
  • Complex: Full-length DNA LigI in complex with nicked duplex DNA and PCNA
    • Complex: DNA ligase I
      • Protein or peptide: DNA ligase I
    • Complex: Proliferating Cell Nuclear Antigen
      • Protein or peptide: Proliferating Cell Nuclear Antigen
    • Complex: Nicked DNA duplex
      • DNA: Nicked DNA duplex

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Supramolecule #1: Full-length DNA LigI in complex with nicked duplex DNA and PCNA

SupramoleculeName: Full-length DNA LigI in complex with nicked duplex DNA and PCNA
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Molecular weightExperimental: 220 KDa

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Supramolecule #2: DNA ligase I

SupramoleculeName: DNA ligase I / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria) / Recombinant strain: Rosetta2

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Supramolecule #3: Proliferating Cell Nuclear Antigen

SupramoleculeName: Proliferating Cell Nuclear Antigen / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria) / Recombinant strain: BL21(DE3) / Recombinant cell: Rosetta2

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Supramolecule #4: Nicked DNA duplex

SupramoleculeName: Nicked DNA duplex / type: complex / ID: 4 / Parent: 1 / Macromolecule list: #3
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: synthetic (others)

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Macromolecule #1: DNA ligase I

MacromoleculeName: DNA ligase I / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO / EC number: DNA ligase (ATP)
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MGSSHHHHHH SSGLVPRGSH MtRSIMSFFH PKKEGKAKKP EKEASNSSRE TEPPPKAALK EWNGVVSES DSPVKRPGRK AARVLGSEGE EEDEALSPAK GQKPALDCSQ VSPPRPATSP E NNASLSDT SPMDSSPSGI PKRRTARKQL PKRTIQEVLE EQSEDEDREA ...String:
MGSSHHHHHH SSGLVPRGSH MtRSIMSFFH PKKEGKAKKP EKEASNSSRE TEPPPKAALK EWNGVVSES DSPVKRPGRK AARVLGSEGE EEDEALSPAK GQKPALDCSQ VSPPRPATSP E NNASLSDT SPMDSSPSGI PKRRTARKQL PKRTIQEVLE EQSEDEDREA KRKKEEEEEE TP KESLTEA EVATEKEGED GDQPTTPPKP LKTSKAETPT ESVSEPEVAT KQELQEEEEQ TKP PRRAPK TLSSFFTPRK PAVKKEVKEE EPGAPGKEGA AEGPLDPSGY NPAKNNYHPV EDAC WKPGQ KVPYLAVART FEKIEEVSAR LRMVETLSNL LRSVVALSPP DLLPVLYLSL NHLGP PQQG LELGVGDGVL LKAVAQATGR QLESVRAEAA EKGDVGLVAE NSRSTQRLML PPPPLT ASG VFSKFRDIAR LTGSASTAKK IDIIKGLFVA CRHSEARFIA RSLSGRLRLG LAEQSVL AA LSQAVSLTPP GQEFPPAMVD AGKGKTAEAR KTWLEEQGMI LKQTFCEVPD LDRIIPVL L EHGLERLPEH CKLSPGIPLK PMLAHPTRGI SEVLKRFEEA AFTCEYKYDG QRAQIHALE GGEVKIFSRN QEDNTGKYPD IISRIPKIKL PSVTSFILDT EAVAWDREKK QIQPFQVLTT RKRKEVDAS EIQVQVCLYA FDLIYLNGES LVREPLSRRR QLLRENFVET EGEFVFATSL D TKDIEQIA EFLEQSVKDS CEGLMVKTLD VDATYEIAKR SHNWLKLKKD YLDGVGDTLD LV VIGAYLG RGKRAGRYGG FLLASYDEDS EELQAICKLG TGFSDEELEE HHQSLKALVL PSP RPYVRI DGAVIPDHWL DPSAVWEVKC ADLSLSPIYP AARGLVDSDK GISLRFPRFI RVRE DKQPE QATTSAQVAC LYRKQSQIQN QQGEDSGSDP EDTY

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Macromolecule #2: Proliferating Cell Nuclear Antigen

MacromoleculeName: Proliferating Cell Nuclear Antigen / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MGHHHHHHHH HHSSGHIDDD DKHTSLEVVF QGPHMFEARL VQGSILKKVL EALKDLINEA CWDISSSGVN LQSMDSSHVS LVQLTLRSEG FDTYRCDRNL AMGVNLTSMS KILKCAGNED IITLRAEDNA DTLALVFEAP NQEKVSDYEM KLMD LDVEQ LGIPEQEYSC ...String:
MGHHHHHHHH HHSSGHIDDD DKHTSLEVVF QGPHMFEARL VQGSILKKVL EALKDLINEA CWDISSSGVN LQSMDSSHVS LVQLTLRSEG FDTYRCDRNL AMGVNLTSMS KILKCAGNED IITLRAEDNA DTLALVFEAP NQEKVSDYEM KLMD LDVEQ LGIPEQEYSC VVKMPSGEFA RICRDLSHIG DAVVISCAKD GVKFSASGEL GNGNI KLSQ TSNVDKEEEA VTIEMNEPVQ LTFALRYLNF FTKATPLSST VTLSMSADVP LVVEYK IAD MGHLKYYLAP KIEDEEGS

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Macromolecule #3: Nicked DNA duplex

MacromoleculeName: Nicked DNA duplex / type: dna / ID: 3 / Classification: DNA
Source (natural)Organism: Homo sapiens (human)
SequenceString:
GGTTCAGTCC GACGACGCAT CAGCACAGAA GC

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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: 8
StainingType: NEGATIVE / Material: Uranyl Formate
GridMaterial: COPPER / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR

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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: 228451
Startup modelType of model: OTHER / Details: SGD
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 19.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: RELION (ver. 2.1) / Number images used: 49504
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION

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