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- EMDB-46644: Cryo-EM structure of a trapped ARIH1-diUB-CRL2-KLHDC10 complex - ... -

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Basic information

Entry
Database: EMDB / ID: EMD-46644
TitleCryo-EM structure of a trapped ARIH1-diUB-CRL2-KLHDC10 complex - consensus map
Map data
Sample
  • Complex: Trapped ARIH1-diUB-CRL2-KLHDC10 complex
    • Protein or peptide: KLHDC10
    • Protein or peptide: EloB
    • Protein or peptide: EloC
    • Protein or peptide: CUL2
    • Protein or peptide: RBX1
    • Protein or peptide: ARIH1
    • Protein or peptide: UB
    • Protein or peptide: UB
    • Protein or peptide: NEDD8
KeywordsKLHDC10 / ubiquitin / E3 / Cullin-RING / C-Degron / LIGASE
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsChittori S / Scott DC / Schulman BA
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Cancer Institute (NIH/NCI)5R01CA247365 United States
CitationJournal: Nat Commun / Year: 2024
Title: Structural basis for C-degron selectivity across KLHDCX family E3 ubiquitin ligases.
Authors: Daniel C Scott / Sagar Chittori / Nicholas Purser / Moeko T King / Samuel A Maiwald / Kelly Churion / Amanda Nourse / Chan Lee / Joao A Paulo / Darcie J Miller / Stephen J Elledge / J Wade ...Authors: Daniel C Scott / Sagar Chittori / Nicholas Purser / Moeko T King / Samuel A Maiwald / Kelly Churion / Amanda Nourse / Chan Lee / Joao A Paulo / Darcie J Miller / Stephen J Elledge / J Wade Harper / Gary Kleiger / Brenda A Schulman /
Abstract: Specificity of the ubiquitin-proteasome system depends on E3 ligase-substrate interactions. Many such pairings depend on E3 ligases binding to peptide-like sequences - termed N- or C-degrons - at the ...Specificity of the ubiquitin-proteasome system depends on E3 ligase-substrate interactions. Many such pairings depend on E3 ligases binding to peptide-like sequences - termed N- or C-degrons - at the termini of substrates. However, our knowledge of structural features distinguishing closely related C-degron substrate-E3 pairings is limited. Here, by systematically comparing ubiquitylation activities towards a suite of common model substrates, and defining interactions by biochemistry, crystallography, and cryo-EM, we reveal principles of C-degron recognition across the KLHDCX family of Cullin-RING ligases (CRLs). First, a motif common across these E3 ligases anchors a substrate's C-terminus. However, distinct locations of this C-terminus anchor motif in different blades of the KLHDC2, KLHDC3, and KLHDC10 β-propellers establishes distinct relative positioning and molecular environments for substrate C-termini. Second, our structural data show KLHDC3 has a pre-formed pocket establishing preference for an Arg or Gln preceding a C-terminal Gly, whereas conformational malleability contributes to KLHDC10's recognition of varying features adjacent to substrate C-termini. Finally, additional non-consensus interactions, mediated by C-degron binding grooves and/or by distal propeller surfaces and substrate globular domains, can substantially impact substrate binding and ubiquitylatability. Overall, the data reveal combinatorial mechanisms determining specificity and plasticity of substrate recognition by KLDCX-family C-degron E3 ligases.
History
DepositionAug 19, 2024-
Header (metadata) releaseNov 20, 2024-
Map releaseNov 20, 2024-
UpdateFeb 5, 2025-
Current statusFeb 5, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_46644.png

Downloads & links

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Map

FileDownload / File: emd_46644.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.3 Å/pix.
x 360 pix.
= 466.92 Å		1.3 Å/pix.
x 360 pix.
= 466.92 Å		1.3 Å/pix.
x 360 pix.
= 466.92 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.297 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.109
Minimum - Maximum-0.4246339 - 0.99982005
Average (Standard dev.)-0.0004077808 (±0.013843748)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions360360360
Spacing360360360
CellA=B=C: 466.92 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_46644_half_map_1.map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_46644_half_map_2.map
Projections & Slices
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Histogram (log scale)

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Sample components

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Entire : Trapped ARIH1-diUB-CRL2-KLHDC10 complex

EntireName: Trapped ARIH1-diUB-CRL2-KLHDC10 complex
Components
  • Complex: Trapped ARIH1-diUB-CRL2-KLHDC10 complex
    • Protein or peptide: KLHDC10
    • Protein or peptide: EloB
    • Protein or peptide: EloC
    • Protein or peptide: CUL2
    • Protein or peptide: RBX1
    • Protein or peptide: ARIH1
    • Protein or peptide: UB
    • Protein or peptide: UB
    • Protein or peptide: NEDD8

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Supramolecule #1: Trapped ARIH1-diUB-CRL2-KLHDC10 complex

SupramoleculeName: Trapped ARIH1-diUB-CRL2-KLHDC10 complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Molecular weightTheoretical: 270 KDa

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Macromolecule #1: KLHDC10

MacromoleculeName: KLHDC10 / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: QLNRFVQLSG RPHLPGKKKI RWDPVRRRFI QSCPIIRIPN RFLRGHRPPP ARSGHRCVAD NTNLYVFGGY NPDYDESGGP DNEDYPLFRE LWRYHFATGV WHQMGTDGYM PRELASMSLV LHGNNLLVFG GTGIPFGESN GNDVHVCNVK YKRWALLSCR GKKPSRIYGQ ...String:
QLNRFVQLSG RPHLPGKKKI RWDPVRRRFI QSCPIIRIPN RFLRGHRPPP ARSGHRCVAD NTNLYVFGGY NPDYDESGGP DNEDYPLFRE LWRYHFATGV WHQMGTDGYM PRELASMSLV LHGNNLLVFG GTGIPFGESN GNDVHVCNVK YKRWALLSCR GKKPSRIYGQ AMAIINGSLY VFGGTTGYIY STDLHKLDLN TREWTQLKPN NLSCDLPEER YRHEIAHDGQ RIYILGGGTS WTAYSLNKIH AYNLETNAWE EIATKPHEKI GFPAARRCHS CVQIKNDVFI CGGYNGEVIL GDIWKLNLQT FQWVKLPATM PEPVYFHCAA VTPAGCMYIH GGVVNIHENK RTGSLFKIWL VVPSLLELAW EKLLAAFPNL ANLSRTQLLH LGLTQGLIER LK

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Macromolecule #2: EloB

MacromoleculeName: EloB / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString:
MDVFLMIRRH KTTIFTDAKE SSTVFELKRI VEGILKRPPD EQRLYKDDQL LDDGKTLGEC GFTSQTARPQ APATVGLAFR ADDTFEALCI EPFSSPPELP DVMKPQDSGS SANEQAVQ

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Macromolecule #3: EloC

MacromoleculeName: EloC / type: protein_or_peptide / ID: 3 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString:
MDGEEKTYGG CEGPDAMYVK LISSDGHEFI VKREHALTSG TIKAMLSGPG QFAENETNEV NFREIPSHVL SKVCMYFTYK VRYTNSSTEI PEFPIAPEIA LELLMAANFL DC

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Macromolecule #4: CUL2

MacromoleculeName: CUL2 / type: protein_or_peptide / ID: 4 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: MSLKPRVVDF DETWNKLLTT IKAVVMLEYV ERATWNDRFS DIYALCVAYP EPLGERLYTE TKIFLENHVR HLHKRVLESE EQVLVMYHRY WEEYSKGADY MDCLYRYLNT QFIKKNKLTE ADLQYGYGGV DMNEPLMEIG ELALDMWRKL MVEPLQAILI RMLLREIKND ...String:
MSLKPRVVDF DETWNKLLTT IKAVVMLEYV ERATWNDRFS DIYALCVAYP EPLGERLYTE TKIFLENHVR HLHKRVLESE EQVLVMYHRY WEEYSKGADY MDCLYRYLNT QFIKKNKLTE ADLQYGYGGV DMNEPLMEIG ELALDMWRKL MVEPLQAILI RMLLREIKND RGGEDPNQKV IHGVINSFVH VEQYKKKFPL KFYQEIFESP FLTETGEYYK QEASNLLQES NCSQYMEKVL GRLKDEEIRC RKYLHPSSYT KVIHECQQRM VADHLQFLHA ECHNIIRQEK KNDMANMYVL LRAVSTGLPH MIQELQNHIH DEGLRATSNL TQENMPTLFV ESVLEVHGKF VQLINTVLNG DQHFMSALDK ALTSVVNYRE PKSVCKAPEL LAKYCDNLLK KSAKGMTENE VEDRLTSFIT VFKYIDDKDV FQKFYARMLA KRLIHGLSMS MDSEEAMINK LKQACGYEFT SKLHRMYTDM SVSADLNNKF NNFIKNQDTV IDLGISFQIY VLQAGAWPLT QAPSSTFAIP QELEKSVQMF ELFYSQHFSG RKLTWLHYLC TGEVKMNYLG KPYVAMVTTY QMAVLLAFNN SETVSYKELQ DSTQMNEKEL TKTIKSLLDV KMINHDSEKE DIDAESSFSL NMNFSSKRTK FKITTSMQKD TPQEMEQTRS AVDEDRKMYL QAAIVRIMKA RKVLRHNALI QEVISQSRAR FNPSISMIKK CIEVLIDKQY IERSQASADE YSYVA

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Macromolecule #5: RBX1

MacromoleculeName: RBX1 / type: protein_or_peptide / ID: 5 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString:
MDVDTPSGTN SGAGKKRFEV KKWNAVALWA WDIVVDNCAI CRNHIMDLCI ECQANQASAT SEECTVAWGV CNHAFHFHCI SRWLKTRQVC PLDNREWEFQ KYGH

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Macromolecule #6: ARIH1

MacromoleculeName: ARIH1 / type: protein_or_peptide / ID: 6 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MDSDEGYNYE FDEDEECSEE DSGAEEEEDE DDDEPDDDTL DLGEVELVEP GLGVGGERDG LLCGETGGGG GSALGPGGGG GGGGGGGGGG PGHEQEEDYR YEVLTAEQIL QHMVECIREV NEVIQNPATI TRILLSHFNW DKEKLMERYF DGNLEKLFAE CHVINPSKKS ...String:
MDSDEGYNYE FDEDEECSEE DSGAEEEEDE DDDEPDDDTL DLGEVELVEP GLGVGGERDG LLCGETGGGG GSALGPGGGG GGGGGGGGGG PGHEQEEDYR YEVLTAEQIL QHMVECIREV NEVIQNPATI TRILLSHFNW DKEKLMERYF DGNLEKLFAE CHVINPSKKS RTRQMNTRSS AQDMPCQICY LNYPNSYFTG LECGHKFCMQ CWSEYLTTKI MEEGMGQTIS CPAHGCDILV DDNTVMRLIT DSKVKLKYQH LITNSFVECN RLLKWCPAPD CHHVVKVQYP DAKPVRCKCG RQFCFNCGEN WHDPVKCKWL KKWIKKCDDD SETSNWIAAN TKECPKCHVT IEKDGGCNHM VCRNQNCKAE FCWVCLGPWE PHGSAWYNCN RYNEDDAKAA RDAQERSRAA LQRYLFYCNR YMNHMQSLRF EHKLYAQVKQ KMEEMQQHNM SWIEVQFLKK AVDVLCQCRA TLMYTYVFAF YLKKNNQSII FENNQADLEN ATEVLSGYLE RDISQDSLQD IKQKVQDKYR YCESRRRVLL QHVHEGYEKD LWEYIED

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Macromolecule #7: UB

MacromoleculeName: UB / type: protein_or_peptide / ID: 7 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MQIFVKTLTG KTITLEVEPS DTIENVKAKI QDKEGIPPDQ QRLIFAGCQL EDGRTLSDYN IQKESTLHLV LRLRGG

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Macromolecule #8: UB

MacromoleculeName: UB / type: protein_or_peptide / ID: 8 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MQIFVKTLTG KTITLEVEPS DTIENVKAKI QDKEGIPPDQ QRLIFAGKQL EDGRTLSDYN IQKESTLHLV LRLRG

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Macromolecule #9: NEDD8

MacromoleculeName: NEDD8 / type: protein_or_peptide / ID: 9 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MLIKVKTLTG KEIEIDIEPT DKVERIKERV EEKEGIPPQQ QRLIYSGKQM NDEKTAADYK ILGGSVLHLV LALRGG

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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

Concentration0.8 mg/mL
BufferpH: 7.5
VitrificationCryogen name: ETHANE / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number real images: 13395 / Average electron dose: 40.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.25 µm / Nominal defocus min: 0.6 µm / Nominal magnification: 130000
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: INSILICO MODEL
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 234648
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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