EMDB-3401: Electron cryo-microscopy of CSN-SCF-N8 complex

Basic information

• Entry: Database: EMDB / ID: EMD-3401
• Title: Electron cryo-microscopy of CSN-SCF-N8 complex
• Map data: reconstruction of CSN5H138A-N8_SCF/Skp2

Sample

• Sample: CSN5H138A-N8_SCF/Skp2
• Protein or peptide: x 14 types

• Keywords: COP9 signalosome / Cullin-RING ligase / SCF / deneddylation

Function / homology

Function:

COP9 signalosome assembly / trophectodermal cell proliferation / mitotic cell cycle phase transition / macrophage migration inhibitory factor binding / positive regulation of protein polyubiquitination / regulation of IRE1-mediated unfolded protein response / exosomal secretion / Parkin-FBXW7-Cul1 ubiquitin ligase complex / GTPase inhibitor activity / deNEDDylase activity / F-box domain binding / protein deneddylation / regulation of protein neddylation / eukaryotic translation initiation factor 3 complex / Aberrant regulation of mitotic exit in cancer due to RB1 defects / activation of NF-kappaB-inducing kinase activity / PcG protein complex / cullin-RING-type E3 NEDD8 transferase / NEDD8 transferase activity / COP9 signalosome / cullin-RING ubiquitin ligase complex / cellular response to chemical stress / positive regulation of ubiquitin protein ligase activity / Cul7-RING ubiquitin ligase complex / ubiquitin-dependent protein catabolic process via the C-end degron rule pathway / maintenance of protein location in nucleus / Loss of Function of FBXW7 in Cancer and NOTCH1 Signaling / positive regulation of protein autoubiquitination / cyclin-dependent protein serine/threonine kinase activator activity / protein neddylation / regulation of JNK cascade / Hydrolases; Acting on peptide bonds (peptidases) / metal-dependent deubiquitinase activity / NEDD8 ligase activity / negative regulation of response to oxidative stress / RHOBTB1 GTPase cycle / Cul5-RING ubiquitin ligase complex / regulation of DNA damage response, signal transduction by p53 class mediator / SCF ubiquitin ligase complex / inner cell mass cell proliferation / Cul2-RING ubiquitin ligase complex / Cul4A-RING E3 ubiquitin ligase complex / ubiquitin-ubiquitin ligase activity / negative regulation of type I interferon production / SCF-dependent proteasomal ubiquitin-dependent protein catabolic process / Cul3-RING ubiquitin ligase complex / positive regulation of intracellular estrogen receptor signaling pathway / Cul4B-RING E3 ubiquitin ligase complex / ubiquitin ligase complex scaffold activity / negative regulation of mitophagy / Prolactin receptor signaling / skeletal muscle cell differentiation / TGF-beta receptor signaling activates SMADs / cullin family protein binding / regulation of postsynapse assembly / regulation of proteolysis / response to light stimulus / protein K63-linked ubiquitination / anatomical structure morphogenesis / protein monoubiquitination / ubiquitin-like ligase-substrate adaptor activity / positive regulation of double-strand break repair via homologous recombination / protein K48-linked ubiquitination / Nuclear events stimulated by ALK signaling in cancer / cyclin-dependent protein kinase holoenzyme complex / JNK cascade / translation initiation factor activity / regulation of cellular response to insulin stimulus / positive regulation of TORC1 signaling / negative regulation of insulin receptor signaling pathway / post-translational protein modification / intrinsic apoptotic signaling pathway / regulation of mitotic cell cycle / T cell activation / positive regulation of DNA-binding transcription factor activity / Regulation of BACH1 activity / MAP3K8 (TPL2)-dependent MAPK1/3 activation / molecular function activator activity / ubiquitin binding / animal organ morphogenesis / SCF-beta-TrCP mediated degradation of Emi1 / NIK-->noncanonical NF-kB signaling / Vpu mediated degradation of CD4 / cellular response to amino acid stimulus / Degradation of DVL / Dectin-1 mediated noncanonical NF-kB signaling / Activation of NF-kappaB in B cells / Degradation of GLI1 by the proteasome / Recognition of DNA damage by PCNA-containing replication complex / GSK3B and BTRC:CUL1-mediated-degradation of NFE2L2 / negative regulation of canonical Wnt signaling pathway / Iron uptake and transport / Negative regulation of NOTCH4 signaling / Vif-mediated degradation of APOBEC3G / Hedgehog 'on' state / Degradation of GLI2 by the proteasome / GLI3 is processed to GLI3R by the proteasome / FBXL7 down-regulates AURKA during mitotic entry and in early mitosis / APC/C:Cdh1 mediated degradation of Cdc20 and other APC/C:Cdh1 targeted proteins in late mitosis/early G1 / DNA Damage Recognition in GG-NER

Domain/homology:

COP9 signalosome, subunit CSN8 / 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 complex subunit 3, N-terminal helical repeats / COP9 signalosome subunit 6 / : / COP9 signalosome complex subunit 1, C-terminal helix / Cop9 signalosome subunit 5 C-terminal domain / COP9 signalosome complex subunit 4, helix turn helix domain / Cop9 signalosome subunit 5 C-terminal domain / CSN4/RPN5/eIF3a helix turn helix domain / Cyclin-dependent kinase, regulatory subunit / Cyclin-dependent kinase, regulatory subunit superfamily / Cyclin-dependent kinase regulatory subunit / Cyclin-dependent kinases regulatory subunits signature 1. / Cyclin-dependent kinases regulatory subunits signature 2. / Cyclin-dependent kinase regulatory subunit / Nedd8-like ubiquitin / Eukaryotic translation initiation factor 3 subunit M eIF3m/COP9 signalosome complex subunit 7 COPS7 / Leucine-rich repeat, cysteine-containing subtype / Leucine-rich repeat - CC (cysteine-containing) subfamily / A Receptor for Ubiquitination Targets / F-box domain profile. / F-box-like domain superfamily / F-box-like / SKP1 component, dimerisation / S-phase kinase-associated protein 1 / SKP1-like, dimerisation domain superfamily / Skp1 family, dimerisation domain / F-box domain / Zinc finger, RING-H2-type / RING-H2 zinc finger domain / : / : / : / PSMD12/CSN4, N-terminal / Cullin protein neddylation domain / 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, conserved site / Cullin family signature. / Cullin repeat-like-containing domain superfamily / Cullin protein, neddylation domain / Cullin / Cullin protein neddylation domain / PCI/PINT associated module / Cullin / CSN8/PSMD8/EIF3K / CSN8/PSMD8/EIF3K family / Rpn11/EIF3F, C-terminal / Maintenance of mitochondrial structure and function / Cullin, N-terminal / Cullin homology domain / Cullin homology domain superfamily / Cullin family / Cullin family profile. / : / motif in proteasome subunits, Int-6, Nip-1 and TRIP-15 / PCI domain / Proteasome component (PCI) domain / PCI domain profile. / S-phase kinase-associated protein 1-like / SKP1 component, POZ domain / Skp1 family, tetramerisation domain / Found in Skp1 protein family / JAB1/Mov34/MPN/PAD-1 ubiquitin protease / JAB/MPN domain / JAB1/MPN/MOV34 metalloenzyme domain / MPN domain / MPN domain profile. / SKP1/BTB/POZ domain superfamily / Leucine-rich repeat domain superfamily / Zinc finger RING-type profile. / Zinc finger, RING-type / : / Ubiquitin domain signature. / Ubiquitin conserved site / Ubiquitin domain / Ubiquitin family / Ubiquitin homologues / Tetratricopeptide-like helical domain superfamily / Ubiquitin domain profile. / Ubiquitin-like domain / Zinc finger, RING/FYVE/PHD-type / Ubiquitin-like domain superfamily / Winged helix DNA-binding domain superfamily / Winged helix-like DNA-binding domain superfamily

Component:

Cyclin-dependent kinases regulatory subunit 1 / COP9 signalosome complex subunit 2 / E3 ubiquitin-protein ligase RBX1 / S-phase kinase-associated protein 1 / COP9 signalosome complex subunit 1 / S-phase kinase-associated protein 2 / Cullin-1 / Ubiquitin-like protein NEDD8 / COP9 signalosome complex subunit 6 / COP9 signalosome complex subunit 5 / COP9 signalosome complex subunit 8 / COP9 signalosome complex subunit 4 / COP9 signalosome complex subunit 7b / COP9 signalosome complex subunit 3

• Biological species: Homo sapiens (human)
• Method: single particle reconstruction / cryo EM / Resolution: 7.2 Å
• Authors: Mosadeghi R / Reichermeier KM / Winkler M / Schreiber A / Reitsma JM / Zhang Y / Stengel F / Cao J / Kim M / Sweredoski MJ / Hess S / Leitner A / Aebersold R / Peter M / Deshaies RJ / Enchev RI
• Citation: Journal: Elife / Year: 2016; Title: Structural and kinetic analysis of the COP9-Signalosome activation and the cullin-RING ubiquitin ligase deneddylation cycle.; Authors: Ruzbeh Mosadeghi / Kurt M Reichermeier / Martin Winkler / Anne Schreiber / Justin M Reitsma / Yaru Zhang / Florian Stengel / Junyue Cao / Minsoo Kim / Michael J Sweredoski / Sonja Hess / Alexander Leitner / Ruedi Aebersold / Matthias Peter / Raymond J Deshaies / Radoslav I Enchev / ; Abstract: The COP9-Signalosome (CSN) regulates cullin-RING ubiquitin ligase (CRL) activity and assembly by cleaving Nedd8 from cullins. Free CSN is autoinhibited, and it remains unclear how it becomes activated. We combine structural and kinetic analyses to identify mechanisms that contribute to CSN activation and Nedd8 deconjugation. Both CSN and neddylated substrate undergo large conformational changes upon binding, with important roles played by the N-terminal domains of Csn2 and Csn4 and the RING domain of Rbx1 in enabling formation of a high affinity, fully active complex. The RING domain is crucial for deneddylation, and works in part through conformational changes involving insert-2 of Csn6. Nedd8 deconjugation and re-engagement of the active site zinc by the autoinhibitory Csn5 glutamate-104 diminish affinity for Cul1/Rbx1 by ~100-fold, resulting in its rapid ejection from the active site. Together, these mechanisms enable a dynamic deneddylation-disassembly cycle that promotes rapid remodeling of the cellular CRL network.

History

Deposition	Mar 29, 2016	-
Header (metadata) release	Apr 6, 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_3401.map.gz / Format: CCP4 / Size: 39.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
• Annotation: reconstruction of CSN5H138A-N8_SCF/Skp2
• Voxel size: X=Y=Z: 1.74 Å

Density

Contour Level	By AUTHOR: 0.065 / Movie #1: 0.065
Minimum - Maximum	-0.08129014 - 0.22193521
Average (Standard dev.)	0.00045421 (±0.01186982)

• Symmetry: Space group: 1

Details

EMDB XML:

Map geometry	Axis order X Y Z Origin 0 0 0 Dimensions 220 220 220 Spacing 220 220 220
Cell	A=B=C: 382.8 Å α=β=γ: 90.0 °

CCP4 map header:

mode	Image stored as Reals
Å/pix. X/Y/Z	1.74	1.74	1.74
M x/y/z	220	220	220
origin x/y/z	0.000	0.000	0.000
length x/y/z	382.800	382.800	382.800
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	0	0	0
NC/NR/NS	220	220	220
D min/max/mean	-0.081	0.222	0.000

Supplemental data

Sample components

Entire : CSN5H138A-N8_SCF/Skp2

• Entire: Name: CSN5H138A-N8_SCF/Skp2

Components

• Sample: CSN5H138A-N8_SCF/Skp2
• Protein or peptide: Csn1
• Protein or peptide: Csn2
• Protein or peptide: Csn3
• Protein or peptide: Csn4
• Protein or peptide: Csn5
• Protein or peptide: Csn6
• Protein or peptide: Csn7b
• Protein or peptide: Csn8
• Protein or peptide: Cul1
• Protein or peptide: Rbx1
• Protein or peptide: Nedd8
• Protein or peptide: Skp1
• Protein or peptide: Skp2
• Protein or peptide: Cks1

Supramolecule #1000: CSN5H138A-N8_SCF/Skp2

• Supramolecule: Name: CSN5H138A-N8_SCF/Skp2 / type: sample / ID: 1000 / Oligomeric state: one CSN monomer and one SCF monomer / Number unique components: 14
• Molecular weight: Theoretical: 500 KDa

Macromolecule #1: Csn1

• Macromolecule: Name: Csn1 / type: protein_or_peptide / ID: 1 / Name.synonym: Cops1 / Number of copies: 1 / Oligomeric state: monomer (part of the CSN) / Recombinant expression: Yes
• Source (natural): Organism: Homo sapiens (human) / synonym: Human
• Molecular weight: Theoretical: 55 KDa
• Recombinant expression: Organism: High Five insect cells / Recombinant strain: Trichoplusia ni / Recombinant cell: BTI-TN-5B1-4 / Recombinant plasmid: multibac system
• Sequence: UniProtKB: COP9 signalosome complex subunit 1

Macromolecule #2: Csn2

• Macromolecule: Name: Csn2 / type: protein_or_peptide / ID: 2 / Name.synonym: Cops2 / Number of copies: 1 / Oligomeric state: monomer (part of the CSN) / Recombinant expression: Yes
• Source (natural): Organism: Homo sapiens (human) / synonym: Human
• Molecular weight: Theoretical: 51 KDa
• Recombinant expression: Organism: High Five insect cells / Recombinant strain: Trichoplusia ni / Recombinant cell: BTI-TN-5B1-4 / Recombinant plasmid: multibac system
• Sequence: UniProtKB: COP9 signalosome complex subunit 2

Macromolecule #3: Csn3

• Macromolecule: Name: Csn3 / type: protein_or_peptide / ID: 3 / Name.synonym: Cops3 / Number of copies: 1 / Oligomeric state: monomer (part of the CSN) / Recombinant expression: Yes
• Source (natural): Organism: Homo sapiens (human) / synonym: Human
• Molecular weight: Theoretical: 47 KDa
• Recombinant expression: Organism: High Five insect cells / Recombinant strain: Trichoplusia ni / Recombinant cell: BTI-TN-5B1-4 / Recombinant plasmid: multibac system
• Sequence: UniProtKB: COP9 signalosome complex subunit 3

Macromolecule #4: Csn4

• Macromolecule: Name: Csn4 / type: protein_or_peptide / ID: 4 / Name.synonym: Cops4 / Number of copies: 1 / Oligomeric state: monomer (part of the CSN) / Recombinant expression: Yes
• Source (natural): Organism: Homo sapiens (human) / synonym: Human
• Molecular weight: Theoretical: 46 KDa
• Recombinant expression: Organism: High Five insect cells / Recombinant strain: Trichoplusia ni / Recombinant cell: BTI-TN-5B1-4 / Recombinant plasmid: multibac system
• Sequence: UniProtKB: COP9 signalosome complex subunit 4

Macromolecule #5: Csn5

• Macromolecule: Name: Csn5 / type: protein_or_peptide / ID: 5 / Name.synonym: Cops5 / Details: His6 tag on the N-terminus / Number of copies: 1 / Oligomeric state: monomer (part of the CSN) / Recombinant expression: Yes
• Source (natural): Organism: Homo sapiens (human) / synonym: Human
• Molecular weight: Theoretical: 37 KDa
• Recombinant expression: Organism: High Five insect cells / Recombinant strain: Trichoplusia ni / Recombinant cell: BTI-TN-5B1-4 / Recombinant plasmid: multibac system
• Sequence: UniProtKB: COP9 signalosome complex subunit 5

Macromolecule #6: Csn6

• Macromolecule: Name: Csn6 / type: protein_or_peptide / ID: 6 / Name.synonym: Cops6 / Number of copies: 1 / Oligomeric state: monomer (part of the CSN) / Recombinant expression: Yes
• Source (natural): Organism: Homo sapiens (human) / synonym: Human
• Molecular weight: Theoretical: 36 KDa
• Recombinant expression: Organism: High Five insect cells / Recombinant strain: Trichoplusia ni / Recombinant cell: BTI-TN-5B1-4 / Recombinant plasmid: multibac system
• Sequence: UniProtKB: COP9 signalosome complex subunit 6

Macromolecule #7: Csn7b

• Macromolecule: Name: Csn7b / type: protein_or_peptide / ID: 7 / Name.synonym: Cops7b / Number of copies: 1 / Oligomeric state: monomer (part of the CSN) / Recombinant expression: Yes
• Source (natural): Organism: Homo sapiens (human) / synonym: Human
• Molecular weight: Theoretical: 29.6 KDa
• Recombinant expression: Organism: High Five insect cells / Recombinant strain: Trichoplusia ni / Recombinant cell: BTI-TN-5B1-4 / Recombinant plasmid: multibac system
• Sequence: UniProtKB: COP9 signalosome complex subunit 7b

Macromolecule #8: Csn8

• Macromolecule: Name: Csn8 / type: protein_or_peptide / ID: 8 / Name.synonym: Cops8 / Number of copies: 1 / Oligomeric state: monomer (part of the CSN) / Recombinant expression: Yes
• Source (natural): Organism: Homo sapiens (human) / synonym: Human
• Molecular weight: Theoretical: 23 KDa
• Recombinant expression: Organism: High Five insect cells / Recombinant strain: Trichoplusia ni / Recombinant cell: BTI-TN-5B1-4 / Recombinant plasmid: multibac system
• Sequence: UniProtKB: COP9 signalosome complex subunit 8

Macromolecule #9: Cul1

• Macromolecule: Name: Cul1 / type: protein_or_peptide / ID: 9 / Details: StrepII2x tag on the N-terminus / Number of copies: 1 / Oligomeric state: monomer (part of SCF) / Recombinant expression: Yes
• Source (natural): Organism: Homo sapiens (human) / synonym: Human
• Molecular weight: Theoretical: 89 KDa
• Recombinant expression: Organism: High Five insect cells / Recombinant strain: Trichoplusia ni / Recombinant cell: BTI-TN-5B1-4 / Recombinant plasmid: multibac system
• Sequence: UniProtKB: Cullin-1

Macromolecule #10: Rbx1

• Macromolecule: Name: Rbx1 / type: protein_or_peptide / ID: 10 / Name.synonym: Roc1 / Number of copies: 1 / Oligomeric state: monomer (part of SCF) / Recombinant expression: Yes
• Source (natural): Organism: Homo sapiens (human) / synonym: Human
• Molecular weight: Theoretical: 12 KDa
• Recombinant expression: Organism: High Five insect cells / Recombinant strain: Trichoplusia ni / Recombinant cell: BTI-TN-5B1-4 / Recombinant plasmid: multibac system
• Sequence: UniProtKB: E3 ubiquitin-protein ligase RBX1

Macromolecule #11: Nedd8

• Macromolecule: Name: Nedd8 / type: protein_or_peptide / ID: 11 / Number of copies: 1 / Oligomeric state: monomer (part of SCF) / Recombinant expression: Yes
• Source (natural): Organism: Homo sapiens (human) / synonym: Human
• Molecular weight: Theoretical: 9 KDa
• Recombinant expression: Organism: Escherichia coli (E. coli) / Recombinant strain: BL21 / Recombinant plasmid: see publication
• Sequence: UniProtKB: Ubiquitin-like protein NEDD8

Macromolecule #12: Skp1

• Macromolecule: Name: Skp1 / type: protein_or_peptide / ID: 12 ; Details: see Enchev et al 2012 Cell Reports for details of the used construct; Number of copies: 1 / Oligomeric state: monomer (part of SCF) / Recombinant expression: Yes
• Source (natural): Organism: Homo sapiens (human) / synonym: Human
• Recombinant expression: Organism: Escherichia coli (E. coli) / Recombinant strain: BL21 / Recombinant plasmid: see publication
• Sequence: UniProtKB: S-phase kinase-associated protein 1

Macromolecule #13: Skp2

• Macromolecule: Name: Skp2 / type: protein_or_peptide / ID: 13 ; Details: see Enchev et al 2012 Cell Reports for details of the used construct; Number of copies: 1 / Oligomeric state: monomer (part of SCF) / Recombinant expression: Yes
• Source (natural): Organism: Homo sapiens (human) / synonym: Human
• Recombinant expression: Organism: Escherichia coli (E. coli) / Recombinant strain: BL21 / Recombinant plasmid: see publication
• Sequence: UniProtKB: S-phase kinase-associated protein 2

Macromolecule #14: Cks1

• Macromolecule: Name: Cks1 / type: protein_or_peptide / ID: 14 ; Details: see Enchev et al 2012 Cell Reports for details of the used construct; Number of copies: 1 / Oligomeric state: monomer (part of SCF) / Recombinant expression: Yes
• Source (natural): Organism: Homo sapiens (human) / synonym: Human
• Molecular weight: Theoretical: 9.6 KDa
• Recombinant expression: Organism: Escherichia coli (E. coli) / Recombinant strain: BL21 / Recombinant plasmid: see publication
• Sequence: UniProtKB: Cyclin-dependent kinases regulatory subunit 1

Experimental details

Structure determination

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

Sample preparation

• Concentration: 0.1 mg/mL
• Buffer: pH: 7.6 / Details: 15 mM HEPES, pH 7.6, 100 mM NaCl, 0.5 mM DTT
• Grid: Details: Quantifoil grids (R1.2/1.3 Cu 400 mesh), freshly coated with an extra layer of thin carbon and glow-discharged for 2 min at 50 mA and 0.2 mbar vacuum
• Vitrification: Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 120 K / Instrument: HOMEMADE PLUNGER; Method: the sample was diluted to 0.1 mg/ml and 2 ul were applied to Quantifoil grids (R1.2/1.3 Cu 400 mesh), freshly coated with an extra layer of thin carbon and glow-discharged for 2 min at 50 mA and 0.2 mbar vacuum. The grids were manually blotted to produce a thin sample film and plunge-frozen into liquid ethane

Electron microscopy

• Microscope: FEI TITAN KRIOS
• Temperature: Min: 90 K / Max: 110 K / Average: 100 K
• Alignment procedure: Legacy - Astigmatism: Objective lens astigmatism was corrected manually at a nominal magnification of 100,000x
• Date: May 12, 2015
• Image recording: Category: CCD / Film or detector model: FEI FALCON II (4k x 4k) / Average electron dose: 25 e/Ų; Details: Microgrpahs were the average of six frames recorded by the direct electron detector. All micrographs were initially visually inspected and only those with appropriate ice thickness as well as Thon rings in their power spectra showing regularity and extending to 6 A or beyond were used for subsequent analysis.
• Electron beam: Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
• Electron optics: Calibrated magnification: 80460 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 5.0 µm / Nominal defocus min: 2.5 µm / Nominal magnification: 60000
• Sample stage: Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
• Experimental equipment: Model: Titan Krios / Image courtesy: FEI Company

Image processing

• Details: CTF-estimation and subsequent correction were performed using RELION (Scheres, 2012) and CTFFIND3 (Mindell and Grigorieff, 2003). All micrographs were initially visually inspected and only those with appropriate ice thickness as well as Thon rings in their power spectra showing regularity and extending to 6 A or beyond were used for subsequent analysis. In order to generate 2D references for automated particle selection, ~ 4,000 single particles were manually picked and subjected to 2D classification in RELION. Six well-defined 2D class averages were selected, low-pass filtered to 35 A to prevent reference bias, and used as references. Approximately 150,000 single particles were automatically selected and subjected to reference-free 2D and 3D classification, in order to de-select the particles, which resulted in poorly defined or noisy averages. Approximately half of these single particles resulted in a well-defined 3D class average, which resembled the previously published negative stain EM map of the same complex (Enchev et al., 2012). This dataset was subject to 3D auto-refinement in RELION, using a version low-pass filtered to 50 A as an initial reference. The converged map was further post-processed in RELION, using MTF-correction, FSC-weighting and a soft spherical mask with a 5-pixel fall-off.
• CTF correction: Details: micrographs
• Final reconstruction: Applied symmetry - Point group: C₁ (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 7.2 Å / Resolution method: OTHER / Software - Name: relion, CTFFIND3 / Number images used: 75000

Atomic model buiding 1

Initial model

PDB ID:

1ldk

Chain - #0 - Chain ID: A / Chain - #1 - Chain ID: B / Chain - #2 - Chain ID: C / Chain - #3 - Chain ID: D / Chain - #4 - Chain ID: E

• Software: Name: Chmiera
• Details: some domains were fitted separately to account for conformational rearrangements, see publication for details
• Refinement: Space: REAL / Protocol: RIGID BODY FIT / Target criteria: overlap

Atomic model buiding 2

Initial model

PDB ID:

4d10

Chain - #0 - Chain ID: A / Chain - #1 - Chain ID: B / Chain - #2 - Chain ID: C / Chain - #3 - Chain ID: D / Chain - #4 - Chain ID: E / Chain - #5 - Chain ID: F / Chain - #6 - Chain ID: H

• Software: Name: Chmiera
• Details: Csn7b was modeled using Csn7a as a template on the Phyre2 server; some domains were fitted separately to account for conformational rearrangements, see publication for details
• Refinement: Space: REAL / Protocol: RIGID BODY FIT / Target criteria: overlap

Atomic model buiding 3

Initial model

PDB ID:

1fqv

Chain - #0 - Chain ID: A / Chain - #1 - Chain ID: B

• Software: Name: Chmiera
• Refinement: Space: REAL / Protocol: RIGID BODY FIT / Target criteria: overlap