EMDB-3316: Cryo-EM structure of CSN-N8-CRL4ADDB2 at 8.3 A resolution

Basic information

• Entry: Database: EMDB / ID: EMD-3316
• Title: Cryo-EM structure of CSN-N8-CRL4ADDB2 at 8.3 A resolution
• Map data: Cryo-EM of the human CSN-N8-CRL4ADDB2 complex at 8.3 A.

Sample

• Sample: Recombinant human CSN-N8-CRL4ADDB2 complex.
• Protein or peptide: x 13 types

• Keywords: COP9 Signalosome / Cullin-RING ligases / Cryo-EM

Function / homology

Function:

Dual Incision in GG-NER / DNA Damage Recognition in GG-NER / Formation of Incision Complex in GG-NER / Neddylation / Ub-specific processing proteases / Prolactin receptor signaling / regulation of mitotic cytokinesis / Recognition of DNA damage by PCNA-containing replication complex / Formation of TC-NER Pre-Incision Complex / DNA Damage Recognition in GG-NER / Dual Incision in GG-NER / Dual incision in TC-NER / Gap-filling DNA repair synthesis and ligation in TC-NER / Formation of Incision Complex in GG-NER / Regulation of BACH1 activity / COP9 signalosome assembly / regulation of miRNA-mediated gene silencing / Regulation of RAS by GAPs / SPOP-mediated proteasomal degradation of PD-L1(CD274) / Degradation of DVL / macrophage migration inhibitory factor binding / Ribosome Quality Control (RQC) complex extracts and degrades nascent peptide / Regulation of RUNX2 expression and activity / Degradation of GLI1 by the proteasome / Ubiquitin-Mediated Degradation of Phosphorylated Cdc25A / GSK3B-mediated proteasomal degradation of PD-L1(CD274) / Hedgehog 'on' state / FBXL7 down-regulates AURKA during mitotic entry and in early mitosis / Orc1 removal from chromatin / GSK3B and BTRC:CUL1-mediated-degradation of NFE2L2 / nucleotide-excision repair complex / eukaryotic initiation factor 4E binding / Oxygen-dependent proline hydroxylation of Hypoxia-inducible Factor Alpha / regulation of IRE1-mediated unfolded protein response / regulation of cell cycle phase transition / Degradation of beta-catenin by the destruction complex / base-excision repair, AP site formation via deaminated base removal / miRNA-mediated gene silencing by mRNA destabilization / regulation of stem cell population maintenance / exosomal secretion / regulation of natural killer cell activation / GTPase inhibitor activity / anaphase-promoting complex / deNEDDylase activity / Interleukin-1 signaling / activation of NF-kappaB-inducing kinase activity / protein deneddylation / regulation of protein neddylation / GLI3 is processed to GLI3R by the proteasome / eukaryotic translation initiation factor 3 complex / negative regulation of adipose tissue development / cellular response to camptothecin / Neddylation / regulation of cellular response to stress / COP9 signalosome / cullin-RING-type E3 NEDD8 transferase / deubiquitinase activity / KEAP1-NFE2L2 pathway / cullin-RING ubiquitin ligase complex / positive regulation of epithelial cell apoptotic process / Cul7-RING ubiquitin ligase complex / Antigen processing: Ubiquitination & Proteasome degradation / positive regulation of protein autoubiquitination / positive regulation by virus of viral protein levels in host cell / RNA polymerase II transcription initiation surveillance / protein neddylation / spindle assembly involved in female meiosis / epigenetic programming in the zygotic pronuclei / Hydrolases; Acting on peptide bonds (peptidases) / NEDD8 ligase activity / lysosome organization / regulation of JNK cascade / regulation of DNA damage response, signal transduction by p53 class mediator / negative regulation of response to oxidative stress / RHOBTB1 GTPase cycle / UV-damage excision repair / protein K27-linked ubiquitination / VCB complex / regulation of DNA-templated DNA replication initiation / Cul5-RING ubiquitin ligase complex / ubiquitin-ubiquitin ligase activity / metal-dependent deubiquitinase activity / type I interferon-mediated signaling pathway / ubiquitin-dependent protein catabolic process via the C-end degron rule pathway / SCF ubiquitin ligase complex / Cul2-RING ubiquitin ligase complex / biological process involved in interaction with symbiont / Cul3-RING ubiquitin ligase complex / regulation of mitotic cell cycle phase transition / intercellular bridge / negative regulation of type I interferon production / WD40-repeat domain binding / p38MAPK cascade / SCF-dependent proteasomal ubiquitin-dependent protein catabolic process / Cul4A-RING E3 ubiquitin ligase complex / Cul4-RING E3 ubiquitin ligase complex / TORC1 signaling / negative regulation of reproductive process / negative regulation of developmental process / Cul4B-RING E3 ubiquitin ligase complex

Domain/homology:

DNA damage-binding protein 2 / COP9 signalosome complex subunit 7, helix I / : / COP9 signalosome complex subunit 7a helix I domain / COP9 signalosome complex subunit 3-like, C-terminal helix / CSN7 helical bundle subdomain / COP9 signalosome, subunit CSN8 / COP9 signalosome complex subunit 4, helix turn helix domain / : / CSN4/RPN5/eIF3a helix turn helix domain / COP9 signalosome complex subunit 3, N-terminal helical repeats / COP9 signalosome subunit 6 / : / : / COP9 signalosome complex subunit 1, C-terminal helix / COP9 signalosome complex subunit 2, C-terminal helix / Cop9 signalosome subunit 5 C-terminal domain / Cop9 signalosome subunit 5 C-terminal domain / Nedd8-like ubiquitin / Eukaryotic translation initiation factor 3 subunit M eIF3m/COP9 signalosome complex subunit 7 COPS7 / Zinc finger, RING-H2-type / RING-H2 zinc finger domain / : / Cullin protein neddylation domain / Cullin, conserved site / Cullin family signature. / Cullin, N-terminal / Cullin repeat-like-containing domain superfamily / Cullin protein, neddylation domain / Cullin / Cullin protein neddylation domain / Cullin alpha solenoid domain / Cullin / : / : / : / PSMD12/CSN4, N-terminal / 26S proteasome regulatory subunit Rpn7/COP9 signalosome complex subunit 1 / 26S proteasome regulatory subunit Rpn7, N-terminal / 26S proteasome subunit RPN7 / 26S Proteasome non-ATPase regulatory subunit 12/COP9 signalosome complex subunit 4 / : / Cullin alpha+beta domain / Cullin homology domain / Cullin homology domain superfamily / Cullin family profile. / PCI/PINT associated module / CSN8/PSMD8/EIF3K / CSN8/PSMD8/EIF3K family / Rpn11/EIF3F, C-terminal / Maintenance of mitochondrial structure and function / motif in proteasome subunits, Int-6, Nip-1 and TRIP-15 / : / PCI domain / : / RSE1/DDB1/CPSF1 second beta-propeller / Cleavage/polyadenylation specificity factor, A subunit, C-terminal / Cleavage/polyadenylation specificity factor, A subunit, N-terminal / : / CPSF A subunit region / RSE1/DDB1/CPSF1 first beta-propeller / Proteasome component (PCI) domain / PCI domain profile. / JAB1/Mov34/MPN/PAD-1 ubiquitin protease / JAB/MPN domain / JAB1/MPN/MOV34 metalloenzyme domain / MPN domain / MPN domain profile. / Zinc finger RING-type profile. / Zinc finger, RING-type / : / Ubiquitin domain signature. / Ubiquitin conserved site / Ubiquitin domain / Ubiquitin family / Tetratricopeptide-like helical domain superfamily / Ubiquitin homologues / Ubiquitin domain profile. / Ubiquitin-like domain / WD domain, G-beta repeat / Zinc finger, RING/FYVE/PHD-type / Ubiquitin-like domain superfamily / Winged helix DNA-binding domain superfamily / WD40 repeat, conserved site / Trp-Asp (WD) repeats signature. / Trp-Asp (WD) repeats profile. / Trp-Asp (WD) repeats circular profile. / WD40 repeats / WD40 repeat / Winged helix-like DNA-binding domain superfamily / WD40-repeat-containing domain superfamily / WD40/YVTN repeat-like-containing domain superfamily

Component:

COP9 signalosome complex subunit 2 / E3 ubiquitin-protein ligase RBX1 / COP9 signalosome complex subunit 1 / Cullin-4A / Ubiquitin-like protein NEDD8 / DNA damage-binding protein 1 / DNA damage-binding protein 2 / COP9 signalosome complex subunit 6 / COP9 signalosome complex subunit 5 / COP9 signalosome complex subunit 8 / COP9 signalosome complex subunit 4 / COP9 signalosome complex subunit 7a / COP9 signalosome complex subunit 3

• Biological species: Homo sapiens (human) / Danio rerio (zebrafish)
• Method: single particle reconstruction / cryo EM / Resolution: 8.3 Å
• Authors: Cavadini S / Fischer ES / Bunker RD / Potenza A / Lingaraju GM / Goldie KN / Mohamed WI / Faty M / Petzold G / Beckwith REJ / Tichkule R / Hassiepen U / Abdulrahman W / Pantelic RS / Matsumoto S / Sugasawa K / Stahlberg H / Thoma NH
• Citation: Journal: Nature / Year: 2016; Title: Cullin-RING ubiquitin E3 ligase regulation by the COP9 signalosome.; Authors: Simone Cavadini / Eric S Fischer / Richard D Bunker / Alessandro Potenza / Gondichatnahalli M Lingaraju / Kenneth N Goldie / Weaam I Mohamed / Mahamadou Faty / Georg Petzold / Rohan E J Beckwith / Ritesh B Tichkule / Ulrich Hassiepen / Wassim Abdulrahman / Radosav S Pantelic / Syota Matsumoto / Kaoru Sugasawa / Henning Stahlberg / Nicolas H Thomä / ; Abstract: The cullin-RING ubiquitin E3 ligase (CRL) family comprises over 200 members in humans. The COP9 signalosome complex (CSN) regulates CRLs by removing their ubiquitin-like activator NEDD8. The CUL4A-RBX1-DDB1-DDB2 complex (CRL4A(DDB2)) monitors the genome for ultraviolet-light-induced DNA damage. CRL4A(DBB2) is inactive in the absence of damaged DNA and requires CSN to regulate the repair process. The structural basis of CSN binding to CRL4A(DDB2) and the principles of CSN activation are poorly understood. Here we present cryo-electron microscopy structures for CSN in complex with neddylated CRL4A ligases to 6.4 Å resolution. The CSN conformers defined by cryo-electron microscopy and a novel apo-CSN crystal structure indicate an induced-fit mechanism that drives CSN activation by neddylated CRLs. We find that CSN and a substrate cannot bind simultaneously to CRL4A, favouring a deneddylated, inactive state for substrate-free CRL4 complexes. These architectural and regulatory principles appear conserved across CRL families, allowing global regulation by CSN.

History

Deposition	Jan 30, 2016	-
Header (metadata) release	Feb 24, 2016	-
Map release	Apr 6, 2016	-
Update	Apr 13, 2016	-
Current status	Apr 13, 2016	Processing site: PDBe / Status: Released

Map

• File: Download / File: emd_3316.map.gz / Format: CCP4 / Size: 7.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
• Annotation: Cryo-EM of the human CSN-N8-CRL4ADDB2 complex at 8.3 A.
• Voxel size: X=Y=Z: 2.6 Å

Density

Contour Level	By AUTHOR: 0.045 / Movie #1: 0.045
Minimum - Maximum	-0.05252366 - 0.12985988
Average (Standard dev.)	0.00000905 (±0.00986453)

• Symmetry: Space group: 1

Details

EMDB XML:

Map geometry	Axis order X Y Z Origin -128 -128 -128 Dimensions 128 128 128 Spacing 128 128 128
Cell	A=B=C: 332.8 Å α=β=γ: 90.0 °

CCP4 map header:

mode	Image stored as Reals
Å/pix. X/Y/Z	2.6	2.6	2.6
M x/y/z	128	128	128
origin x/y/z	0.000	0.000	0.000
length x/y/z	332.800	332.800	332.800
α/β/γ	90.000	90.000	90.000
start NX/NY/NZ	-30	0	-64
NX/NY/NZ	61	61	129
MAP C/R/S	1	2	3
start NC/NR/NS	-128	-128	-128
NC/NR/NS	128	128	128
D min/max/mean	-0.053	0.130	0.000

Supplemental data

Sample components

Entire : Recombinant human CSN-N8-CRL4ADDB2 complex.

• Entire: Name: Recombinant human CSN-N8-CRL4ADDB2 complex.

Components

• Sample: Recombinant human CSN-N8-CRL4ADDB2 complex.
• Protein or peptide: COP9 signalosome subunit 1
• Protein or peptide: COP9 signalosome subunit 2
• Protein or peptide: COP9 signalosome subunit 3
• Protein or peptide: COP9 signalosome subunit 4
• Protein or peptide: COP9 signalosome subunit 5
• Protein or peptide: COP9 signalosome subunit 6
• Protein or peptide: COP9 signalosome subunit 7A
• Protein or peptide: COP9 signalosome subunit 8
• Protein or peptide: CUL4A
• Protein or peptide: DNA DAMAGE-BINDING PROTEIN 1
• Protein or peptide: E3 UBIQUITIN-PROTEIN LIGASE RBX1
• Protein or peptide: NEDD8
• Protein or peptide: DNA DAMAGE-BINDING PROTEIN 2

Supramolecule #1000: Recombinant human CSN-N8-CRL4ADDB2 complex.

• Supramolecule: Name: Recombinant human CSN-N8-CRL4ADDB2 complex. / type: sample / ID: 1000 / Details: Sample was subjected to GraFix. / Number unique components: 13
• Molecular weight: Theoretical: 533 KDa

Macromolecule #1: COP9 signalosome subunit 1

• Macromolecule: Name: COP9 signalosome subunit 1 / type: protein_or_peptide / ID: 1 / Name.synonym: CSN1 / Number of copies: 1 / Recombinant expression: Yes
• Source (natural): Organism: Homo sapiens (human)
• Molecular weight: Theoretical: 52 KDa
• Recombinant expression: Organism: Trichoplusia ni (cabbage looper)
• Sequence: UniProtKB: COP9 signalosome complex subunit 1

Macromolecule #2: COP9 signalosome subunit 2

• Macromolecule: Name: COP9 signalosome subunit 2 / type: protein_or_peptide / ID: 2 / Name.synonym: CSN2 / Number of copies: 1 / Recombinant expression: Yes
• Source (natural): Organism: Homo sapiens (human)
• Molecular weight: Theoretical: 49 KDa
• Recombinant expression: Organism: Trichoplusia ni (cabbage looper)
• Sequence: UniProtKB: COP9 signalosome complex subunit 2

Macromolecule #3: COP9 signalosome subunit 3

• Macromolecule: Name: COP9 signalosome subunit 3 / type: protein_or_peptide / ID: 3 / Name.synonym: CSN3 / Number of copies: 1 / Recombinant expression: Yes
• Source (natural): Organism: Homo sapiens (human)
• Molecular weight: Theoretical: 46 KDa
• Recombinant expression: Organism: Trichoplusia ni (cabbage looper)
• Sequence: UniProtKB: COP9 signalosome complex subunit 3

Macromolecule #4: COP9 signalosome subunit 4

• Macromolecule: Name: COP9 signalosome subunit 4 / type: protein_or_peptide / ID: 4 / Name.synonym: CSN4 / Number of copies: 1 / Recombinant expression: Yes
• Source (natural): Organism: Homo sapiens (human)
• Molecular weight: Theoretical: 44.6 KDa
• Recombinant expression: Organism: Trichoplusia ni (cabbage looper)
• Sequence: UniProtKB: COP9 signalosome complex subunit 4

Macromolecule #5: COP9 signalosome subunit 5

• Macromolecule: Name: COP9 signalosome subunit 5 / type: protein_or_peptide / ID: 5 / Name.synonym: CSN5 / Number of copies: 1 / Recombinant expression: Yes
• Source (natural): Organism: Homo sapiens (human)
• Molecular weight: Theoretical: 35.4 KDa
• Recombinant expression: Organism: Trichoplusia ni (cabbage looper)
• Sequence: UniProtKB: COP9 signalosome complex subunit 5

Macromolecule #6: COP9 signalosome subunit 6

• Macromolecule: Name: COP9 signalosome subunit 6 / type: protein_or_peptide / ID: 6 / Name.synonym: CSN6 / Number of copies: 1 / Recombinant expression: Yes
• Source (natural): Organism: Homo sapiens (human)
• Molecular weight: Theoretical: 35.6 KDa
• Recombinant expression: Organism: Trichoplusia ni (cabbage looper)
• Sequence: UniProtKB: COP9 signalosome complex subunit 6

Macromolecule #7: COP9 signalosome subunit 7A

• Macromolecule: Name: COP9 signalosome subunit 7A / type: protein_or_peptide / ID: 7 / Name.synonym: CSN7A / Number of copies: 1 / Recombinant expression: Yes
• Source (natural): Organism: Homo sapiens (human)
• Molecular weight: Theoretical: 30 KDa
• Recombinant expression: Organism: Trichoplusia ni (cabbage looper)
• Sequence: UniProtKB: COP9 signalosome complex subunit 7a

Macromolecule #8: COP9 signalosome subunit 8

• Macromolecule: Name: COP9 signalosome subunit 8 / type: protein_or_peptide / ID: 8 / Name.synonym: CSN8 / Number of copies: 1 / Recombinant expression: Yes
• Source (natural): Organism: Homo sapiens (human)
• Molecular weight: Theoretical: 23 KDa
• Recombinant expression: Organism: Trichoplusia ni (cabbage looper)
• Sequence: UniProtKB: COP9 signalosome complex subunit 8

Macromolecule #9: CUL4A

• Macromolecule: Name: CUL4A / type: protein_or_peptide / ID: 9 / Number of copies: 1 / Recombinant expression: Yes
• Source (natural): Organism: Homo sapiens (human)
• Molecular weight: Theoretical: 79.3 KDa
• Recombinant expression: Organism: Trichoplusia ni (cabbage looper)
• Sequence: UniProtKB: Cullin-4A

Macromolecule #10: DNA DAMAGE-BINDING PROTEIN 1

• Macromolecule: Name: DNA DAMAGE-BINDING PROTEIN 1 / type: protein_or_peptide / ID: 10 / Name.synonym: DDB1 / Number of copies: 1 / Recombinant expression: Yes
• Source (natural): Organism: Homo sapiens (human)
• Molecular weight: Theoretical: 125.4 KDa
• Recombinant expression: Organism: Trichoplusia ni (cabbage looper)
• Sequence: UniProtKB: DNA damage-binding protein 1

Macromolecule #11: E3 UBIQUITIN-PROTEIN LIGASE RBX1

• Macromolecule: Name: E3 UBIQUITIN-PROTEIN LIGASE RBX1 / type: protein_or_peptide / ID: 11 / Name.synonym: RBX1 / Number of copies: 1 / Recombinant expression: Yes
• Source (natural): Organism: Homo sapiens (human)
• Molecular weight: Theoretical: 11.8 KDa
• Recombinant expression: Organism: Trichoplusia ni (cabbage looper)
• Sequence: UniProtKB: E3 ubiquitin-protein ligase RBX1

Macromolecule #12: NEDD8

• Macromolecule: Name: NEDD8 / type: protein_or_peptide / ID: 12 / Name.synonym: NEDD8 / Number of copies: 1 / Recombinant expression: Yes
• Source (natural): Organism: Homo sapiens (human)
• Molecular weight: Theoretical: 8.9 KDa
• Recombinant expression: Organism: Escherichia coli (E. coli)
• Sequence: UniProtKB: Ubiquitin-like protein NEDD8

Macromolecule #13: DNA DAMAGE-BINDING PROTEIN 2

• Macromolecule: Name: DNA DAMAGE-BINDING PROTEIN 2 / type: protein_or_peptide / ID: 13 / Name.synonym: DDB2 / Number of copies: 1 / Recombinant expression: Yes
• Source (natural): Organism: Danio rerio (zebrafish)
• Molecular weight: Theoretical: 39.9 KDa
• Recombinant expression: Organism: Trichoplusia ni (cabbage looper)
• Sequence: UniProtKB: DNA damage-binding protein 2

Experimental details

Structure determination

• Method: cryo EM
• Processing: single particle reconstruction
• Aggregation state: particle

Sample preparation

• Concentration: 0.05 mg/mL
• Buffer: pH: 7.4 / Details: 50mM HEPES, 200mM NaCl, 0.25mM TCEP
• Grid: Details: Quantifoil holey carbon grids (R2/2, Cu 400 mesh) coated with an additional thin amorphous carbon layer.
• Vitrification: Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 93 K / Instrument: LEICA EM GP / Method: Blot for 1.5 seconds before plunging

Electron microscopy

• Microscope: FEI TITAN KRIOS
• Date: Apr 1, 2015
• Image recording: Category: CCD / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Number real images: 1427 ; Details: Every image is the average of 38 frames recorded by the direct electron detector
• Electron beam: Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
• Electron optics: Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 6.0 µm / Nominal defocus min: 2.0 µm
• Sample stage: Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
• Experimental equipment: Model: Titan Krios / Image courtesy: FEI Company

Image processing

• Final reconstruction: Applied symmetry - Point group: C₁ (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 8.3 Å / Resolution method: OTHER / Software - Name: EMAN2, RELION / Number images used: 16090

Atomic model buiding 1

Initial model

PDB ID:

4d10

• Refinement: Space: REAL / Protocol: FLEXIBLE FIT

Atomic model buiding 2

Initial model

PDB ID:

4d18

• Refinement: Space: REAL / Protocol: FLEXIBLE FIT

Atomic model buiding 3

Initial model

PDB ID:

4a0k

• Refinement: Space: REAL / Protocol: FLEXIBLE FIT