Cryo-EM structure of DDB1dB:CRBN:mezigdomide:SALL4(392-449)

2026-02-04 REL RCSB INITIAL_RELEASE REPOSITORY INITIAL_RELEASE REPOSITORY INITIAL_RELEASE REPOSITORY INITIAL_RELEASE REPOSITORY INITIAL_RELEASE REPOSITORY INITIAL_RELEASE REPOSITORY INITIAL_RELEASE REPOSITORY INITIAL_RELEASE REPOSITORY INITIAL_RELEASE REPOSITORY INITIAL_RELEASE REPOSITORY RCSB RCSB 2025-03-24 2026-02-04 2026-02-04 2026-02-04 National Institutes of Health/National Cancer Institute (NIH/NCI) R01CA214608 United States Cryo-EM structure of DDB1dB:CRBN:mezigdomide:SALL4(392-449) Park J Hunkeler M Roy Burman SS Fishcer ES DDB1, cereblon, mezigdomide, SALL4, LIGASE Slabicki M Park J Nowak RP Roy Burman SS Pellman J Zou C Razumkov H Carreiro J Rastogi S Goldstein A Nagiec MM Donovan KA Che J Hunkeler M Geng Q Hsu CL Lakshminarayan M Shu C Zon RL Kozicka Z Park PMC Tsai JM Yoon H Jones LH Sperling AS Gray NS Fischer ES Ebert BL Expanding the druggable zinc-finger proteome defines properties of drug-induced degradation. Mol.Cell US 85 3184 3201.e14 2025 40845806 doi:10.1016/j.molcel.2025.07.019 1097-2765 2168 MOCEFL 9nws FULLOVERLAP EMDB EMD-49892 associated EM volume

Ternary complex of DDB1dB:CRBN:mezigdomide:SALL4(392-449) Ternary complex of DDB1dB:CRBN:mezigdomide:SALL4(392-449) 0 1 2 3 Homo sapiens Sal-like protein 4 Homo sapiens 0.011493773999999998

StrepII-Avi-TEV-SALL4(392-449)

1 Trichoplusia ni LEVO MDWSHPQFEKSAVGLNDIFEAQKIEWHEGGGGSGENLYFQGGGRGTDSSLQIHLRSHTGERPFVCSVCGHRFTTKGNLKV HFHRHPQVKANPQLFAEFQDKV Q9UJQ4 DNA damage-binding protein 1 Homo sapiens 0.09603394500000001

His6-TEV-DDB1(1-1140), with residues 396-705 replaced with a GNGNSG linker,His6-TEV-DDB1(1-1140), with residues 396-705 replaced with a GNGNSG linker

1 Trichoplusia ni LEVO MGSSHHHHHHSAVDENLYFQGGGRMSYNYVVTAQKPTAVNGCVTGHFTSAEDLNLLIAKNTRLEIYVVTAEGLRPVKEVG MYGKIAVMELFRPKGESKDLLFILTAKYNACILEYKQSGESIDIITRAHGNVQDRIGRPSETGIIGIIDPECRMIGLRLY DGLFKVIPLDRDNKELKAFNIRLEELHVIDVKFLYGCQAPTICFVYQDPQGRHVKTYEVSLREKEFNKGPWKQENVEAEA SMVIAVPEPFGGAIIIGQESITYHNGDKYLAIAPPIIKQSTIVCHNRVDPNGSRYLLGDMEGRLFMLLLEKEEQMDGTVT LKDLRVELLGETSIAECLTYLDNGVVFVGSRLGDSQLVKLNVDSNEQGSYVVAMETFTNLGPIVDMCVVDLERQGQGQLV TCSGAFKEGSLRIIRNGIGGNGNSGEIQKLHIRTVPLYESPRKICYQEVSQCFGVLSSRIEVQDTSGGTTALRPSASTQA LSSSVSSSKLFSSSTAPHETSFGEEVEVHNLLIIDQHTFEVLHAHQFLQNEYALSLVSCKLGKDPNTYFIVGTAMVYPEE AEPKQGRIVVFQYSDGKLQTVAEKEVKGAVYSMVEFNGKLLASINSTVRLYEWTTEKELRTECNHYNNIMALYLKTKGDF ILVGDLMRSVLLLAYKPMEGNFEEIARDFNPNWMSAVEILDDDNFLGAENAFNLFVCQKDSAATTDEERQHLQEVGLFHL GEFVNVFCHGSLVMQNLGETSTPTQGSVLFGTVNGMIGLVTSLSESWYNLLLDMQNRLNKVIKSVGKIEHSFWRSFHTER KTEPATGFIDGDLIESFLDISRPKMQEVVANLQYDDGSGMKREATADDLIKVVEELTRIH Q16531 Q16531 Protein cereblon Homo sapiens 0.055144594

FLAG-Spy-TEV-CRBN(1-442)

1 Trichoplusia ni LEVO MDYKDDDDKSAVDENLYFQGGGRGGSAHIVMVDAYKPTKGGSGMAGEGDQQDAAHNMGNHLPLLPAESEEEDEMEVEDQD SKEAKKPNIINFDTSLPTSHTYLGADMEEFHGRTLHDDDSCQVIPVLPQVMMILIPGQTLPLQLFHPQEVSMVRNLIQKD RTFAVLAYSNVQEREAQFGTTAEIYAYREEQDFGIEIVKVKAIGRQRFKVLELRTQSDGIQQAKVQILPECVLPSTMSAV QLESLNKCQIFPSKPVSREDQCSYKWWQKYQKRKFHCANLTSWPRWLYSLYDAETLMDRIKKQLREWDENLKDDSLPSNP IDFSYRVAACLPIDDVLRIQLLKIGSAIQRLRCELDIMNKCTSLCCKQCQETEITTKNEIFSLSLCGPMAAYVNPHGYVH ETLTVYKACNLNLIGRPSTEHSWFPGYAWTVAQCKICASHIGWKFTATKKDMSPQKFWGLTRSALLPTIPDTEDEISPDK VILCL Q96SW2 ZINC ION 6.5409e-05 2 ZN Mezigdomide 0.00056761 1 QFC singleParticle particle 7.4 50.0 C8H18N2O4S/NaOH HEPES/NaOH pH 7.4 150.0 NaCl NaCl 3.0 C9H15O6P TCEP

50 mM HEPES/NaOH pH 7.4, 150 mM NaCl, 3 mM TCEP

Quantifoil R0.6/1 GOLD 300 GOLD HOLEY 50.0 GLOW DISCHARGE 120 AIR 0.039 ETHANE 90 283 LEICA EM GP

UltrAuFoil R 0.6/1 300 mesh grids were glow-discharged for 2 min at 20 mA and 39 Pa, pre-incubated with 4 uL of 10 uM FLAG-IKZF1(140-196;Q146A/G151N) for 1 min, and blotted from behind for 4 s. 4 uL of the sample was then applied to the grid. Grids were vitrified using an EM GP plunge freezer operated at 90% humidity and 10 C with 0 s pre-blot, 4 s blot, and 0 s post-blot..

15 uM His6-DDB1dB:FLAG-Spy-CRBN, 150 uM mezigdomide, and 30 uM StrepII-Avi-SALL4(392-449) were mixed in dilution buffer (50 mM HEPES/NaOH pH 7.4, 150 mM NaCl, 3 mM TCEP) and incubated for 1 hr. Additional dilution buffer was added to the mixture to achieve final concentrations of 1.5 uM DDB1dB:CRBN, 15.0 uM mezigdomide, and 3.0 uM SALL4(392-449), and 4 uL of the diluted mixture were applied to the grid.

TFS TALOS FLOOD BEAM BRIGHT FIELD FIELD EMISSION GUN 200 50.0 2.7 0.8 2.0 36000.0 FEI TITAN KRIOS AUTOGRID HOLDER NITROGEN GATAN K3 (6k x 4k) 1 3024 4.8 51.7

One movie was recorded per hole with nine holes per stage position

1 3386439

Particles were picked using Topaz (v0.2.5) trained on templates from on-the-fly 2D classification

cryoSPARC 4.2.0 PHASE FLIPPING AND AMPLITUDE CORRECTION

Ab-initio reconstruction

C1 FOURIER SPACE 2.7 FSC 0.143 CUT-OFF cryoSPARC 4.2.0

Particles were re-extracted at 1.10 A/pixel, and subsequently processed by reference-based motion correction, local CTF refinement (per-particle defocus), and global CTF refinement (per-group beam tilt and trefoil). Homogeneous refinement of these processed particles followed by local refinement with a mask encompassing the full particle yielded a final reconstruction at 2.7 A. A local refinement using a mask encompassing the CRBN:drug:ZF portion further improved drug and ZF density.

539282 MAXIMUM LIKELIHOOD cryoSPARC 4.2.0 MAXIMUM LIKELIHOOD cryoSPARC 4.2.0 cryoSPARC 4.2.0

Unmasked 3D classification into 20 classes with an online expectation maximization (O-EM) learning rate of 0.2 yielded 4 populated classes and 16 empty classes. Particles from 2 classes with the most defined ZF density were retained for homogeneous refinement, leading to a reconstruction at 3.1 A.

emd_49892.map.gz 1 IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) 256 256 256 0 0 0 256 256 256 281.6 281.6 281.6 90.0 90.0 90.0 X Y Z -0.119554825 0.32096857 -0.00016000419 0.0076824697 1.1 1.1 1.1 0.06 AUTHOR ::::EMDATABANK.org::::EMD-49892:::: Main map, unsharpened 8TNQ A PDB experimental model 8TNQ B PDB experimental model 8U17 C PDB experimental model 8D7Z D PDB experimental model

Sharpened and unsharpened maps processed by cryoSPARC, in addition to maps post-processed with DeepEMhancer (v0.16), were used for model building. Models of DDB1dB:CRBN (PDB: 8TNQ) and SALL4(ZF3) (PDB: 8U17) were fit into the density as individual chains using ChimeraX (v1.6.1). Mezigdomide from a model of DDB1:CRBN:mezigdomide:IKZF1(ZF1-3) (PDB: 8D7Z) was added after aligning both models on CRBN, and residues outside of SALL4(ZF3) were built using Coot (v0.9.8). A ligand restraint file for mezigdomide was generated using the Grade2 (v1.6.0), and the entire model was then relaxed into the density using Rosetta (v3.13) followed by manual adjustment in Coot. The model was prepared for refinement using phenix.ready_set (v1.21-5207) and refined using phenix.real_space_refine (v1.21-5207).

REAL emd_49892_msk_1.map emd_49892_additional_1.map.gz 1 IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) 256 256 256 0 0 0 256 256 256 281.6 281.6 281.6 90.0 90.0 90.0 X Y Z -1.0424782 1.5860618 -0.00016000419 0.034364816 1.1 1.1 1.1 AUTHOR ::::EMDATABANK.org::::EMD-49892:::: Primary map, auto-sharpened with cryoSPARC emd_49892_additional_3.map.gz 1 IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) 256 256 256 0 0 0 256 256 256 281.6 281.6 281.6 90.0 90.0 90.0 X Y Z -0.0017151292 1.7894357 0.0008431415 0.021034872 1.1 1.1 1.1 AUTHOR ::::EMDATABANK.org::::EMD-49892:::: Main map, post-processed with DeepEMhancer emd_49892_half_map_1.map.gz 1 IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) 256 256 256 0 0 0 256 256 256 281.6 281.6 281.6 90.0 90.0 90.0 X Y Z -0.12436197 0.3251408 0.00013461465 0.012087659 1.1 1.1 1.1 AUTHOR ::::EMDATABANK.org::::EMD-49892:::: Main map, half-map A emd_49892_half_map_2.map.gz 1 IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) 256 256 256 0 0 0 256 256 256 281.6 281.6 281.6 90.0 90.0 90.0 X Y Z -0.12219713 0.3201263 0.00013453358 0.012100086 1.1 1.1 1.1 AUTHOR ::::EMDATABANK.org::::EMD-49892:::: Main map, half-map B