Extended Data Table 2. Cryo-EM data collection, refinement and validation statistics.

IκBα–UB~UBE2D crosslink (C21I C107A C111D)	UBE2D2 2x2way XL	UBE2D3 3way XL	UBE2D3 3way XL	UBE2D2 3way XL	UBE2D2 3way XL	none	none
Substrate Receptor	β-TRCP2	β-TRCP1 175-C	β-TRCP1 175-C	β-TRCP1 175-C	β-TRCP1 175-C	β-TRCP1 175-C	β-TRCP1 175-C
RBX1	WT	WT	N98R	N98R	N98R	WT	WT
NEDD8	yes	yes	yes	yes	yes	no	yes
SKP1	WT EMD- 10578	∆∆ EMD- 10579	∆∆ EMD- 10580	∆∆ EMD- 10581	∆∆ EMD- 10585 PDB 6TTU	WT EMD- 10582	WT EMD- 10583
Data collection and processing
Microscope	Krios	Arctica	Arctica	Arctica	Krios	Glacios	Glacios
Magnification	105,000	92,000	73,000	73,000	130,000	36,000	22,000
Voltage (kV)	300	200	200	200	300	200	200
Electron exposure (e–/Å2)	56	61.3	60.8	70	70.2	60	59
Defocus range (μm)	-1.2 ~ -3.6	-1.5 ~ -3.5	-1.5 ~ -3.5	-1.5 ~ -3.5	-1.2 ~ -3.3	-1.2 ~ -3.3	-1.2 ~ -3.3
Pixel size (Å)	1.34	1.612	1.997	1.997	1.06	1.181	1.885
Symmetry imposed	C2	C1	C1	C1	C1	C1	C1
Initial particle images (no.)	2,575,161	464,344	601,121	459,011	1,661,870	2,051,804	1,666,293
Final particle images (no.)	33,738	47,246	107,311	40,835	106,257	262,116	349,803
Map resolution (Å)	9.3 (0.143)	8.6 (0.143)	9.4 (0.143)	8.4 (0.143)	3.72 (0.143)	4.64 (0.143)	6.7 (0.143)
FSC threshold
Map resolution range (Å)	-	-	-	-	3.46 ~ 6.0	-	-
Refinement
Initial model used (PDB code)					1LDJ 6M90 4P5O 4V3L
Model resolution (Å)					3.7 (0.143)
FSC threshold
Map sharpening B factor (Å2)	-578.9	-1159	-1272	-983.5	-94.2	-199	-338
Model composition
Non-hydrogen atoms					13028
Protein residues					1616
Ligands					3(ZN)
B factors (Å2)
Protein					88.6
Ligand					203.8
R.m.s. deviations
Bond lengths (Å)					0.006
Bond angles (°)					0.850
Validation
MolProbity score					2.23
Clashscore					13.09
Poor rotamers (%)					0.07
Ramachandran plot
Favored (%)					91
Allowed (%)					9
Disallowed (%)					0