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- EMDB-19860: Focused map 5 - K48-linked ubiquitin chain formation with a culli... -

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Entry
Database: EMDB / ID: EMD-19860
TitleFocused map 5 - K48-linked ubiquitin chain formation with a cullin-RING E3 ligase & Cdc34: NEDD8-CUL2-RBX1-ELOB/C-FEM1C with trapped UBE2R2~donor UB~acceptor UB-SIL1 peptide
Map dataDeepEMhancer map
Sample
  • Complex: K48-linked ubiquitin chain formation with a cullin-RING E3 ligase and CDC34:NEDD8-CUL2-RBX1-ELOB/C-FEM1C with trapped UBE2R2-donor UB-acceptor UB-SIL1 peptide
    • Complex: RING E3 ligase (RBX1) and Cullin-2 (CUL2)
      • Protein or peptide: E3 ubiquitin-protein ligase RBX1
      • Protein or peptide: Cullin-2CUL2
    • Complex: CDC34, NEDD8, FEM1C with UBE2R2-donor UB-acceptor UB-SIL1 peptide and K48-linked ubiquitin chain
      • Protein or peptide: Ubiquitin-conjugating enzyme E2 R2
      • Protein or peptide: Polyubiquitin-C,Nucleotide exchange factor SIL1
      • Protein or peptide: Protein fem-1 homolog C
      • Protein or peptide: Ubiquitin
KeywordsCUL2 / FEM1C / ELOBC / SIL1 / Ubiquitin / Ubiquitin Ligase / Ubiquitin chain formation / Poliubiquitylation / LIGASE
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.94 Å
AuthorsLiwocha J / Prabu JR / Kleiger G / Schulman BA
Funding support Germany, 1 items
OrganizationGrant numberCountry
Max Planck Society Germany
CitationJournal: Nat Struct Mol Biol / Year: 2024
Title: Mechanism of millisecond Lys48-linked poly-ubiquitin chain formation by cullin-RING ligases.
Authors: Joanna Liwocha / Jerry Li / Nicholas Purser / Chutima Rattanasopa / Samuel Maiwald / David T Krist / Daniel C Scott / Barbara Steigenberger / J Rajan Prabu / Brenda A Schulman / Gary Kleiger /
Abstract: E3 ubiquitin ligases, in collaboration with E2 ubiquitin-conjugating enzymes, modify proteins with poly-ubiquitin chains. Cullin-RING ligase (CRL) E3s use Cdc34/UBE2R-family E2s to build Lys48-linked ...E3 ubiquitin ligases, in collaboration with E2 ubiquitin-conjugating enzymes, modify proteins with poly-ubiquitin chains. Cullin-RING ligase (CRL) E3s use Cdc34/UBE2R-family E2s to build Lys48-linked poly-ubiquitin chains to control an enormous swath of eukaryotic biology. Yet the molecular mechanisms underlying this exceptional linkage specificity and millisecond kinetics of poly-ubiquitylation remain unclear. Here we obtain cryogenic-electron microscopy (cryo-EM) structures that provide pertinent insight into how such poly-ubiquitin chains are forged. The CRL RING domain not only activates the E2-bound ubiquitin but also shapes the conformation of a distinctive UBE2R2 loop, positioning both the ubiquitin to be transferred and the substrate-linked acceptor ubiquitin within the active site. The structures also reveal how the ubiquitin-like protein NEDD8 uniquely activates CRLs during chain formation. NEDD8 releases the RING domain from the CRL, but unlike previous CRL-E2 structures, does not contact UBE2R2. These findings suggest how poly-ubiquitylation may be accomplished by many E2s and E3s.
History
DepositionMar 14, 2024-
Header (metadata) releaseApr 10, 2024-
Map releaseApr 10, 2024-
UpdateApr 10, 2024-
Current statusApr 10, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_19860.png

Downloads & links

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Map

FileDownload / File: emd_19860.map.gz / Format: CCP4 / Size: 209.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationDeepEMhancer map
Voxel sizeX=Y=Z: 0.8512 Å
Density
Contour LevelBy AUTHOR: 0.095
Minimum - Maximum-0.026746325 - 2.0967648
Average (Standard dev.)0.00052016356 (±0.017764872)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions380380380
Spacing380380380
CellA=B=C: 323.456 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_19860_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: #1

Fileemd_19860_additional_1.map
Projections & Slices
AxesZYX

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Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_19860_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_19860_half_map_2.map
Projections & Slices
AxesZYX

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Histogram (log scale)

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Sample components

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Entire : K48-linked ubiquitin chain formation with a cullin-RING E3 ligase...

EntireName: K48-linked ubiquitin chain formation with a cullin-RING E3 ligase and CDC34:NEDD8-CUL2-RBX1-ELOB/C-FEM1C with trapped UBE2R2-donor UB-acceptor UB-SIL1 peptide
Components
  • Complex: K48-linked ubiquitin chain formation with a cullin-RING E3 ligase and CDC34:NEDD8-CUL2-RBX1-ELOB/C-FEM1C with trapped UBE2R2-donor UB-acceptor UB-SIL1 peptide
    • Complex: RING E3 ligase (RBX1) and Cullin-2 (CUL2)
      • Protein or peptide: E3 ubiquitin-protein ligase RBX1
      • Protein or peptide: Cullin-2CUL2
    • Complex: CDC34, NEDD8, FEM1C with UBE2R2-donor UB-acceptor UB-SIL1 peptide and K48-linked ubiquitin chain
      • Protein or peptide: Ubiquitin-conjugating enzyme E2 R2
      • Protein or peptide: Polyubiquitin-C,Nucleotide exchange factor SIL1
      • Protein or peptide: Protein fem-1 homolog C
      • Protein or peptide: Ubiquitin

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Supramolecule #1: K48-linked ubiquitin chain formation with a cullin-RING E3 ligase...

SupramoleculeName: K48-linked ubiquitin chain formation with a cullin-RING E3 ligase and CDC34:NEDD8-CUL2-RBX1-ELOB/C-FEM1C with trapped UBE2R2-donor UB-acceptor UB-SIL1 peptide
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Molecular weightTheoretical: 0.19 kDa/nm

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Supramolecule #2: RING E3 ligase (RBX1) and Cullin-2 (CUL2)

SupramoleculeName: RING E3 ligase (RBX1) and Cullin-2 (CUL2) / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #4, #6
Source (natural)Organism: Homo sapiens (human)

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Supramolecule #3: CDC34, NEDD8, FEM1C with UBE2R2-donor UB-acceptor UB-SIL1 peptide...

SupramoleculeName: CDC34, NEDD8, FEM1C with UBE2R2-donor UB-acceptor UB-SIL1 peptide and K48-linked ubiquitin chain
type: complex / ID: 3 / Parent: 1 / Macromolecule list: #1-#3, #5
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: Ubiquitin-conjugating enzyme E2 R2

MacromoleculeName: Ubiquitin-conjugating enzyme E2 R2 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: E2 ubiquitin-conjugating enzyme
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 27.190932 KDa
Recombinant expressionOrganism: Escherichia coli BL21 (bacteria)
SequenceString: MAQQQMTSSQ KALMLELKSL QEEPVEGFRI TLVDESDLYN WEVAIFGPPN TLYEGGYFKA HIKFPIDYPY SPPTFRFLTK MWHPNIYEN GDVCISILHP PVDDPQSGEL PSERWNPTQN VRTILLSVIS LLNEPNTFSP ANVDASVMFR KWRDSKGKDK E YAEIIRKQ ...String:
MAQQQMTSSQ KALMLELKSL QEEPVEGFRI TLVDESDLYN WEVAIFGPPN TLYEGGYFKA HIKFPIDYPY SPPTFRFLTK MWHPNIYEN GDVCISILHP PVDDPQSGEL PSERWNPTQN VRTILLSVIS LLNEPNTFSP ANVDASVMFR KWRDSKGKDK E YAEIIRKQ VSATKAEAEK DGVKVPTTLA EYCIKTKVPS NDNSSDLLYD DLYDDDIDDE DEEEEDADCY DDDDSGNEES

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Macromolecule #2: Polyubiquitin-C,Nucleotide exchange factor SIL1

MacromoleculeName: Polyubiquitin-C,Nucleotide exchange factor SIL1 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 79.226711 KDa
Recombinant expressionOrganism: Escherichia coli BL21 (bacteria)
SequenceString: MQIFVKTLTG KTITLEVEPS DTIENVKAKI QDKEGIPPDQ QRLIFAGCQL EDGRTLSDYN IQKESTLHLV LRLRGGMQIF VKTLTGKTI TLEVEPSDTI ENVKAKIQDK EGIPPDQQRL IFAGKQLEDG RTLSDYNIQK ESTLHLVLRL RGGMQIFVKT L TGKTITLE ...String:
MQIFVKTLTG KTITLEVEPS DTIENVKAKI QDKEGIPPDQ QRLIFAGCQL EDGRTLSDYN IQKESTLHLV LRLRGGMQIF VKTLTGKTI TLEVEPSDTI ENVKAKIQDK EGIPPDQQRL IFAGKQLEDG RTLSDYNIQK ESTLHLVLRL RGGMQIFVKT L TGKTITLE VEPSDTIENV KAKIQDKEGI PPDQQRLIFA GKQLEDGRTL SDYNIQKEST LHLVLRLRGG MQIFVKTLTG KT ITLEVEP SDTIENVKAK IQDKEGIPPD QQRLIFAGKQ LEDGRTLSDY NIQKESTLHL VLRLRGGMQI FVKTLTGKTI TLE VEPSDT IENVKAKIQD KEGIPPDQQR LIFAGKQLED GRTLSDYNIQ KESTLHLVLR LRGGMQIFVK TLTGKTITLE VEPS DTIEN VKAKIQDKEG IPPDQQRLIF AGKQLEDGRT LSDYNIQKES TLHLVLRLRG GMQIFVKTLT GKTITLEVEP SDTIE NVKA KIQDKEGIPP DQQRLIFAGK QLEDGRTLSD YNIQKESTLH LVLRLRGGMQ IFVKTLTGKT ITLEVEPSDT IENVKA KIQ DKEGIPPDQQ RLIFAGKQLE DGRTLSDYNI QKESTLHLVL RLRGGMQIFV KTLTGKTITL EVEPSDTIEN VKAKIQD KE GIPPDQQRLI FAGKQLEDGR TLSDYNIQKE STLHLVLRLR GGVEGYFQEL LGSVNPTQGR AR

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Macromolecule #3: Protein fem-1 homolog C

MacromoleculeName: Protein fem-1 homolog C / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 68.767312 KDa
Recombinant expressionOrganism: Escherichia coli BL21 (bacteria)
SequenceString: MDLKTAVFNA ARDGKLRLLT KLLASKSKEE VSSLISEKTN GATPLLMAAR YGHLDMVEFL LEQCSASIEV GGSVNFDGET IEGAPPLWA ASAAGHLKVV QSLLNHGASV NNTTLTNSTP LRAACFDGHL EIVKYLVEHK ADLEVSNRHG HTCLMISCYK G HKEIAQYL ...String:
MDLKTAVFNA ARDGKLRLLT KLLASKSKEE VSSLISEKTN GATPLLMAAR YGHLDMVEFL LEQCSASIEV GGSVNFDGET IEGAPPLWA ASAAGHLKVV QSLLNHGASV NNTTLTNSTP LRAACFDGHL EIVKYLVEHK ADLEVSNRHG HTCLMISCYK G HKEIAQYL LEKGADVNRK SVKGNTALHD CAESGSLDIM KMLLMYCAKM EKDGYGMTPL LSASVTGHTN IVDFLTHHAQ TS KTERINA LELLGATFVD KKRDLLGALK YWKKAMNMRY SDRTNIISKP VPQTLIMAYD YAKEVNSAEE LEGLIADPDE MRM QALLIR ERILGPSHPD TSYYIRYRGA VYADSGNFKR CINLWKYALD MQQSNLDPLS PMTASSLLSF AELFSFMLQD RAKG LLGTT VTFDDLMGIL CKSVLEIERA IKQTQCPADP LQLNKALSII LHLICLLEKV PCTLEQDHFK KQTIYRFLKL HPRGK NNFS PLHLAVDKNT TCVGRYPVCK FPSLQVTAIL IECGADVNVR DSDDNSPLHI AALNNHPDIM NLLIKSGAHF DATNLH KQT ASDLLDEKEI AKNLIQPINH TTLQCLAARV IVNHRIYYKG HIPEKLETFV SLHR

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Macromolecule #4: E3 ubiquitin-protein ligase RBX1

MacromoleculeName: E3 ubiquitin-protein ligase RBX1 / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO / EC number: RING-type E3 ubiquitin transferase
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 12.289977 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString:
MAAAMDVDTP SGTNSGAGKK RFEVKKWNAV ALWAWDIVVD NCAICRNHIM DLCIECQANQ ASATSEECTV AWGVCNHAFH FHCISRWLK TRQVCPLDNR EWEFQKYGH

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Macromolecule #5: Ubiquitin

MacromoleculeName: Ubiquitin / type: protein_or_peptide / ID: 5 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 77.120398 KDa
Recombinant expressionOrganism: Escherichia coli BL21 (bacteria)
SequenceString: MQIFVKTLTG KTITLEVEPS DTIENVKAKI QDKEGIPPDQ QRLIFAGCQL EDGRTLSDYN IQKESTLHLV LRLRGGMQIF VKTLTGKTI TLEVEPSDTI ENVKAKIQDK EGIPPDQQRL IFAGKQLEDG RTLSDYNIQK ESTLHLVLRL RGGMQIFVKT L TGKTITLE ...String:
MQIFVKTLTG KTITLEVEPS DTIENVKAKI QDKEGIPPDQ QRLIFAGCQL EDGRTLSDYN IQKESTLHLV LRLRGGMQIF VKTLTGKTI TLEVEPSDTI ENVKAKIQDK EGIPPDQQRL IFAGKQLEDG RTLSDYNIQK ESTLHLVLRL RGGMQIFVKT L TGKTITLE VEPSDTIENV KAKIQDKEGI PPDQQRLIFA GKQLEDGRTL SDYNIQKEST LHLVLRLRGG MQIFVKTLTG KT ITLEVEP SDTIENVKAK IQDKEGIPPD QQRLIFAGKQ LEDGRTLSDY NIQKESTLHL VLRLRGGMQI FVKTLTGKTI TLE VEPSDT IENVKAKIQD KEGIPPDQQR LIFAGKQLED GRTLSDYNIQ KESTLHLVLR LRGGMQIFVK TLTGKTITLE VEPS DTIEN VKAKIQDKEG IPPDQQRLIF AGKQLEDGRT LSDYNIQKES TLHLVLRLRG GMQIFVKTLT GKTITLEVEP SDTIE NVKA KIQDKEGIPP DQQRLIFAGK QLEDGRTLSD YNIQKESTLH LVLRLRGGMQ IFVKTLTGKT ITLEVEPSDT IENVKA KIQ DKEGIPPDQQ RLIFAGKQLE DGRTLSDYNI QKESTLHLVL RLRGGMQIFV KTLTGKTITL EVEPSDTIEN VKAKIQD KE GIPPDQQRLI FAGKQLEDGR TLSDYNIQKE STLHLVLRLR GGV

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Macromolecule #6: Cullin-2

MacromoleculeName: Cullin-2 / type: protein_or_peptide / ID: 6 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 87.09893 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: MSLKPRVVDF DETWNKLLTT IKAVVMLEYV ERATWNDRFS DIYALCVAYP EPLGERLYTE TKIFLENHVR HLHKRVLESE EQVLVMYHR YWEEYSKGAD YMDCLYRYLN TQFIKKNKLT EADLQYGYGG VDMNEPLMEI GELALDMWRK LMVEPLQAIL I RMLLREIK ...String:
MSLKPRVVDF DETWNKLLTT IKAVVMLEYV ERATWNDRFS DIYALCVAYP EPLGERLYTE TKIFLENHVR HLHKRVLESE EQVLVMYHR YWEEYSKGAD YMDCLYRYLN TQFIKKNKLT EADLQYGYGG VDMNEPLMEI GELALDMWRK LMVEPLQAIL I RMLLREIK NDRGGEDPNQ KVIHGVINSF VHVEQYKKKF PLKFYQEIFE SPFLTETGEY YKQEASNLLQ ESNCSQYMEK VL GRLKDEE IRCRKYLHPS SYTKVIHECQ QRMVADHLQF LHAECHNIIR QEKKNDMANM YVLLRAVSTG LPHMIQELQN HIH DEGLRA TSNLTQENMP TLFVESVLEV HGKFVQLINT VLNGDQHFMS ALDKALTSVV NYREPKSVCK APELLAKYCD NLLK KSAKG MTENEVEDRL TSFITVFKYI DDKDVFQKFY ARMLAKRLIH GLSMSMDSEE AMINKLKQAC GYEFTSKLHR MYTDM SVSA DLNNKFNNFI KNQDTVIDLG ISFQIYVLQA GAWPLTQAPS STFAIPQELE KSVQMFELFY SQHFSGRKLT WLHYLC TGE VKMNYLGKPY VAMVTTYQMA VLLAFNNSET VSYKELQDST QMNEKELTKT IKSLLDVKMI NHDSEKEDID AESSFSL NM NFSSKRTKFK ITTSMQKDTP QEMEQTRSAV DEDRKMYLQA AIVRIMKARK VLRHNALIQE VISQSRARFN PSISMIKK C IEVLIDKQYI ERSQASADEY SYVA

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7.5
VitrificationCryogen name: ETHANE-PROPANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2.6 µm / Nominal defocus min: 0.6 µm
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 60.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

5n4w

Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.94 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 55024
FSC plot (resolution estimation)

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