EMDB-4765: Cryo-EM structure of NCP_THF2(-3)-UV-DDB

Basic information

• Entry: Database: EMDB / ID: EMD-4765
• Title: Cryo-EM structure of NCP_THF2(-3)-UV-DDB
• Sample: UV-DDB bound to a THF2 (-3) containing nucleosome:; Histone H3.1, Histone H2A, Histone H2B / (Histone H4) x 2 / DNA / (DNA damage-binding protein ...) x 3 / Histone H3.1 / Histone H2A type 1-B/E / Histone H2B type 1-J / (nucleic-acidNucleic acid) x 2

Function / homology

Function:

Cleavage of the damaged purine / Recognition of DNA damage by PCNA-containing replication complex / Recognition and association of DNA glycosylase with site containing an affected pyrimidine / Cleavage of the damaged pyrimidine / Recognition and association of DNA glycosylase with site containing an affected purine / Meiotic synapsis / Interleukin-7 signaling / Packaging Of Telomere Ends / Pre-NOTCH Transcription and Translation / Formation of the beta-catenin:TCF transactivating complex / PRC2 methylates histones and DNA / Condensation of Prophase Chromosomes / Oxidative Stress Induced Senescence / DNA Damage/Telomere Stress Induced Senescence / HDACs deacetylate histones / HDMs demethylate histones / PKMTs methylate histone lysines / Senescence-Associated Secretory Phenotype (SASP) / HATs acetylate histones / Recruitment and ATM-mediated phosphorylation of repair and signaling proteins at DNA double strand breaks / G2/M DNA damage checkpoint / Activated PKN1 stimulates transcription of AR (androgen receptor) regulated genes KLK2 and KLK3 / UCH proteinases / Ub-specific processing proteases / TP53 Regulates Transcription of DNA Repair Genes / Gap-filling DNA repair synthesis and ligation in TC-NER / Metalloprotease DUBs / Dual incision in TC-NER / Formation of Incision Complex in GG-NER / RMTs methylate histone arginines / Nonhomologous End-Joining (NHEJ) / Processing of DNA double-strand break ends / Transcription-Coupled Nucleotide Excision Repair (TC-NER) / DNA Damage Recognition in GG-NER / Formation of TC-NER Pre-Incision Complex / Deposition of new CENPA-containing nucleosomes at the centromere / RNA Polymerase I Promoter Opening / Activation of anterior HOX genes in hindbrain development during early embryogenesis / Transcriptional regulation by small RNAs / Neddylation / Chromatin modifying enzymes / SIRT1 negatively regulates rRNA expression / Factors involved in megakaryocyte development and platelet production / ERCC6 (CSB) and EHMT2 (G9a) positively regulate rRNA expression / Amyloid fiber formation / Meiotic recombination / RNA Polymerase I Promoter Escape / Estrogen-dependent gene expression / NoRC negatively regulates rRNA expression / SUMOylation of chromatin organization proteins / B-WICH complex positively regulates rRNA expression / RUNX1 regulates transcription of genes involved in differentiation of HSCs / DNA methylation / RUNX1 regulates genes involved in megakaryocyte differentiation and platelet function / E3 ubiquitin ligases ubiquitinate target proteins / Dual Incision in GG-NER / positive regulation by virus of viral protein levels in host cell / Cul4B-RING E3 ubiquitin ligase complex / Cul4A-RING E3 ubiquitin ligase complex / UV-damage excision repair / positive regulation of viral release from host cell / histone H2A monoubiquitination / WD40-repeat domain binding / positive regulation of gluconeogenesis / interaction with symbiont / response to UV-B / Cul4-RING E3 ubiquitin ligase complex / cullin family protein binding / nucleotide-excision repair, preincision complex stabilization / nucleotide-excision repair, DNA incision, 3'-to lesion / negative regulation of megakaryocyte differentiation / CENP-A containing nucleosome assembly / DNA replication-independent nucleosome assembly / telomere capping / negative regulation of tumor necrosis factor-mediated signaling pathway / interleukin-7-mediated signaling pathway / positive regulation of viral genome replication / regulation of mitotic cell cycle phase transition / protein autoubiquitination / DNA replication-dependent nucleosome assembly / telomere organization / innate immune response in mucosa / response to UV / nuclear nucleosome / pyrimidine dimer repair / chromatin silencing at rDNA / negative regulation of gene expression, epigenetic / nucleosomal DNA binding / DNA-templated transcription, initiation / regulation of gene silencing by miRNA / nuclear chromosome / nucleotide-excision repair, DNA damage recognition / nucleotide-excision repair, DNA duplex unwinding / regulation of gene silencing / global genome nucleotide-excision repair / nucleotide-excision repair, preincision complex assembly / proteasomal protein catabolic process / regulation of megakaryocyte differentiation / nucleotide-excision repair / nucleotide-excision repair, DNA incision, 5'-to lesion

Domain/homology:

CPSF A subunit region / Histone H2B signature. / Trp-Asp (WD) repeats signature. / Histone H3 signature 2. / Trp-Asp (WD) repeats profile. / Trp-Asp (WD) repeats circular profile. / Histone H4 signature. / Centromere kinetochore component CENP-T histone fold / Histone H3 signature 1. / Histone H2A signature. / WD40 repeat, conserved site / WD domain, G-beta repeat / Core histone H2A/H2B/H3/H4 / WD40-repeat-containing domain superfamily / CENP-T/Histone H4, histone fold / DNA damage-binding protein 2 / Histone H2A conserved site / Histone H2A, C-terminal domain / DNA damage-binding protein 1 / Histone H4, conserved site / WD40-repeat-containing domain / C-terminus of histone H2A / WD40/YVTN repeat-like-containing domain superfamily / Histone-fold / Histone H2A/H2B/H3 / Cleavage/polyadenylation specificity factor, A subunit, C-terminal / TATA box binding protein associated factor (TAF) / Histone H2A / Histone H4 / WD40 repeat / Histone H2B / Histone H3/CENP-A

Component:

Histone H2A type 1-B/E / Histone H2B type 1-J / Histone H4 / Histone H3.1 / DNA damage-binding protein 1 / DNA damage-binding protein 2

• Biological species: Homo sapiens (human)
• Method: single particle reconstruction / cryo EM / Resolution: 4.1 Å
• Authors: Matsumoto S / Cavadini S / Bunker RD / Thoma NH
• Citation: Journal: Nature / Year: 2019; Title: DNA damage detection in nucleosomes involves DNA register shifting.; Authors: Syota Matsumoto / Simone Cavadini / Richard D Bunker / Ralph S Grand / Alessandro Potenza / Julius Rabl / Junpei Yamamoto / Andreas D Schenk / Dirk Schübeler / Shigenori Iwai / Kaoru Sugasawa / Hitoshi Kurumizaka / Nicolas H Thomä / ; Abstract: Access to DNA packaged in nucleosomes is critical for gene regulation, DNA replication and DNA repair. In humans, the UV-damaged DNA-binding protein (UV-DDB) complex detects UV-light-induced pyrimidine dimers throughout the genome; however, it remains unknown how these lesions are recognized in chromatin, in which nucleosomes restrict access to DNA. Here we report cryo-electron microscopy structures of UV-DDB bound to nucleosomes bearing a 6-4 pyrimidine-pyrimidone dimer or a DNA-damage mimic in various positions. We find that UV-DDB binds UV-damaged nucleosomes at lesions located in the solvent-facing minor groove without affecting the overall nucleosome architecture. In the case of buried lesions that face the histone core, UV-DDB changes the predominant translational register of the nucleosome and selectively binds the lesion in an accessible, exposed position. Our findings explain how UV-DDB detects occluded lesions in strongly positioned nucleosomes, and identify slide-assisted site exposure as a mechanism by which high-affinity DNA-binding proteins can access otherwise occluded sites in nucleosomal DNA.
• Validation Report: PDB-ID: 6r91; ; SummaryFull reportAbout validation report
• Date: Deposition: Apr 2, 2019 / Header (metadata) release: May 22, 2019 / Map release: Jun 12, 2019 / Update: Jun 12, 2019

Map

• File: Download / File: emd_4765.map.gz / Format: CCP4 / Size: 27 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
• Voxel size: X=Y=Z: 1.72 Å

Density

Contour Level	By AUTHOR: 5.12 / Movie #1: 5.12
Minimum - Maximum	-19.132261 - 45.49785
Average (Standard dev.)	-0.000000000004188 (±1.0)

• Symmetry: Space group: 1

Details

EMDB XML:

Map geometry	Axis order Z Y X Origin 0 0 0 Dimensions 192 192 192 Spacing 192 192 192
Cell	A=B=C: 330.24 Å α=β=γ: 90.0 °

CCP4 map header:

mode	Image stored as Reals
Å/pix. X/Y/Z	1.72	1.72	1.72
M x/y/z	192	192	192
origin x/y/z	0.000	0.000	0.000
length x/y/z	330.240	330.240	330.240
α/β/γ	90.000	90.000	90.000
start NX/NY/NZ	0	0	0
NX/NY/NZ	192	192	192
MAP C/R/S	3	2	1
start NC/NR/NS	0	0	0
NC/NR/NS	192	192	192
D min/max/mean	-19.132	45.498	-0.000

Supplemental data

Sample components

Entire UV-DDB bound to a THF2 (-3) containing nucleosome

• Entire: Name: UV-DDB bound to a THF2 (-3) containing nucleosome / Number of components: 13

Component #1: protein, UV-DDB bound to a THF2 (-3) containing nucleosome

• Protein: Name: UV-DDB bound to a THF2 (-3) containing nucleosome / Recombinant expression: No

Component #2: protein, Histone H3.1, Histone H2A, Histone H2B

• Protein: Name: Histone H3.1, Histone H2A, Histone H2B / Recombinant expression: No
• Source: Species: Homo sapiens (human)
• Source (engineered): Expression System: Escherichia coli BL21(DE3) (bacteria)

Component #3: protein, Histone H4

• Protein: Name: Histone H4 / Recombinant expression: No
• Source: Species: Homo sapiens (human)
• Source (engineered): Expression System: Escherichia coli (E. coli)

Component #4: protein, DNA

• Protein: Name: DNA / Recombinant expression: No
• Source: Species: Homo sapiens (human)
• Source (engineered): Expression System: synthetic construct (others)

Component #5: protein, DNA damage-binding protein 1(2), DNA damage-binding protein 2

• Protein: Name: DNA damage-binding protein 1(2), DNA damage-binding protein 2; Recombinant expression: No
• Source: Species: Homo sapiens (human)
• Source (engineered): Expression System: Trichoplusia ni (cabbage looper)

Component #6: protein, Histone H3.1

• Protein: Name: Histone H3.1 / Number of Copies: 2 / Recombinant expression: No
• Mass: Theoretical: 15.719445 kDa
• Source: Species: Homo sapiens (human)
• Source (engineered): Expression System: Escherichia coli BL21(DE3) (bacteria)

Component #7: protein, Histone H4

• Protein: Name: Histone H4 / Number of Copies: 2 / Recombinant expression: No
• Mass: Theoretical: 11.676703 kDa
• Source: Species: Homo sapiens (human)
• Source (engineered): Expression System: Escherichia coli (E. coli)

Component #8: protein, Histone H2A type 1-B/E

• Protein: Name: Histone H2A type 1-B/E / Number of Copies: 2 / Recombinant expression: No
• Mass: Theoretical: 14.447825 kDa
• Source: Species: Homo sapiens (human)
• Source (engineered): Expression System: Escherichia coli BL21(DE3) (bacteria)

Component #9: protein, Histone H2B type 1-J

• Protein: Name: Histone H2B type 1-J / Number of Copies: 2 / Recombinant expression: No
• Mass: Theoretical: 14.217516 kDa
• Source: Species: Homo sapiens (human)
• Source (engineered): Expression System: Escherichia coli BL21(DE3) (bacteria)

Component #10: nucleic-acid, Human alpha-satellite DNA (145-MER)

nucleic acid

Name: Human alpha-satellite DNA (145-MER) / Class: DNA / Structure: OTHER / Synthetic: No
Sequence:

(DA)(DT)(DC)(DA)(DA)(DT)(DA)(DT)(DC)(DC) (DA)(DC)(DC)(DT)(DG)(DC)(DA)(DG)(DA)(DT) (DT)(DC)(DT)(DA)(DC)(DC)(DA)(DA)(DA)(DA) (DG)(DT)(DG)(DT)(DA)(DT)(DT)(DT)(DG)(DG) (DA)(DA)(DA)(DC)(DT)(DG)(DC)(DT)(DC)(DC) (DA)(DT)(DC)(DA)(DA)(DA)(DA)(DG)(DG)(DC) (DA)(DT)(DG)(DT)(DT)(DC)(DA)(DG)(DC)(DT) (DG)(DG)(DT)(DT)(DC)(DA)(DG)(DC)(DT)(DG) (DA)(DA)(DC)(DA)(DT)(DG)(DC)(DC)(DT)(DT) (DT)(DT)(DG)(DA)(DT)(DG)(DG)(DA)(DG)(DC) (DA)(DG)(DT)(DT)(DT)(DC)(DC)(DA)(DA)(DA) (DT)(DA)(DC)(DA)(DC)(DT)(DT)(DT)(DT)(DG) (DG)(DT)(DA)(DG)(DA)(DA)(DT)(DC)(DT)(DG) (DC)(DA)(DG)(DG)(DT)(DG)(DG)(DA)(DT)(DA) (DT)(DT)(DG)(DA)(DT)

• Mass: Theoretical: 44.756648 kDa
• Source: Species: Homo sapiens (human)

Component #11: nucleic-acid, Human alpha-satellite DNA (145-MER) with abasic sit...

nucleic acid

Name: Human alpha-satellite DNA (145-MER) with abasic sites at positions 97-98
Class: DNA / Structure: OTHER / Synthetic: No
Sequence:

(DA)(DT)(DC)(DA)(DA)(DT)(DA)(DT)(DC)(DC) (DA)(DC)(DC)(DT)(DG)(DC)(DA)(DG)(DA)(DT) (DT)(DC)(DT)(DA)(DC)(DC)(DA)(DA)(DA)(DA) (DG)(DT)(DG)(DT)(DA)(DT)(DT)(DT)(DG)(DG) (DA)(DA)(DA)(DC)(DT)(DG)(DC)(DT)(DC)(DC) (DA)(DT)(DC)(DA)(DA)(DA)(DA)(DG)(DG)(DC) (DA)(DT)(DG)(DT)(DT)(DC)(DA)(DG)(DC)(DT) (DG)(DA)(DA)(DC)(DC)(DA)(DG)(DC)(DT)(DG) (DA)(DA)(DC)(DA)(DT)(DG)(DC)(DC)(DT)(DT) (DT)(DT)(DG)(DA)(DT)(DG)(3DR)(3DR)(DG)(DC) (DA)(DG)(DT)(DT)(DT)(DC)(DC)(DA)(DA)(DA) (DT)(DA)(DC)(DA)(DC)(DT)(DT)(DT)(DT)(DG) (DG)(DT)(DA)(DG)(DA)(DA)(DT)(DC)(DT)(DG) (DC)(DA)(DG)(DG)(DT)(DG)(DG)(DA)(DT)(DA) (DT)(DT)(DG)(DA)(DT)

• Mass: Theoretical: 44.452434 kDa
• Source: Species: Homo sapiens (human)

Component #12: protein, DNA damage-binding protein 1

• Protein: Name: DNA damage-binding protein 1 / Number of Copies: 1 / Recombinant expression: No
• Mass: Theoretical: 129.766305 kDa
• Source: Species: Homo sapiens (human)
• Source (engineered): Expression System: Trichoplusia ni (cabbage looper)

Component #13: protein, DNA damage-binding protein 2

• Protein: Name: DNA damage-binding protein 2 / Number of Copies: 1 / Recombinant expression: No
• Mass: Theoretical: 50.601844 kDa
• Source: Species: Homo sapiens (human)
• Source (engineered): Expression System: Trichoplusia ni (cabbage looper)

Experimental details

Sample preparation

• Specimen: Specimen state: Particle / Method: cryo EM
• Sample solution: pH: 7.4
• Vitrification: Cryogen name: ETHANE / Temperature: 277 K / Humidity: 85 %

Electron microscopy imaging

• Experimental equipment: Model: Titan Krios / Image courtesy: FEI Company
• Imaging: Microscope: FEI TITAN KRIOS
• Electron gun: Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 40 e/Ų / Illumination mode: SPOT SCAN
• Lens: Imaging mode: BRIGHT FIELD
• Specimen Holder: Model: OTHER
• Camera: Detector: GATAN K2 SUMMIT (4k x 4k)

Image processing

• Processing: Method: single particle reconstruction / Number of projections: 119309
• 3D reconstruction: Resolution: 4.1 Å / Resolution method: FSC 0.143 CUT-OFF

Atomic model buiding

Modeling #1

Refinement protocol: flexible / Target criteria: Correlation coefficient / Refinement space: REAL
Input PDB model: 4ZUX, 4ZUX, 5Y0C, 5Y0C, 5Y0C, 5Y0C, 5Y0C, 5Y0C, 5Y0C, 5Y0C, 4E54, 3EI4

4zux

4zux

5y0c

5y0c

5y0c

5y0c

5y0c

5y0c

5y0c

5y0c

4e54

3ei4

Chain ID: I, J, A, B, C, D, E, F, G, H, B, A

Output model

6r91