Cryo-EM structure of human ATP citrate lyase in complex with inhibitor EVT0185-CoA

2026-03-04 REL PDBe 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 INITIAL_RELEASE REPOSITORY INITIAL_RELEASE REPOSITORY INITIAL_RELEASE REPOSITORY INITIAL_RELEASE REPOSITORY INITIAL_RELEASE REPOSITORY PDBe PDBe 2025-05-18 2026-03-04 2026-03-04 2026-03-04 Research Foundation - Flanders (FWO) 1524918N Belgium Cryo-EM structure of human ATP citrate lyase in complex with inhibitor EVT0185-CoA Verstraete K Verschueren K Savvides SN Steinberg GR ATP citrate lyase, ACL, ACLY, de novo lipogenesis, acetyl-CoA, citrate, oxaloacetate, cancer, LYASE Gautam J Wu J Lally JSV McNicol JD Fayyazi R Ahmadi E Oniciu DC Heaton S Newton RS Rehal S Bhattacharya D Di Pastena F Nguyen B Valvano CM Townsend LK Banskota S Batchuluun B Jabile MJT Payne A Lu J Desjardins EM Kubota N Tsakiridis EE Mistry B Aganostopoulos A Houde V Dansercoer A Verschueren KHG Savvides SN Hammill JA Bezverbnaya K Muti P Tsakiridis T Dai W Jiang L Hoshida Y Larche M Bramson JL Friedman SL Verstraete K Wang D Steinberg GR ACLY inhibition promotes tumour immunity and suppresses liver cancer. Nature UK 645 507 517 2025 40739358 doi:10.1038/s41586-025-09297-0 1476-4687 0006 NATUAS 9r90 FULLOVERLAP EMDB EMD-53847 associated EM volume

Human ATP citrate lyase (homotetramer) Human ATP citrate lyase (homotetramer) 0 1

Human ACLY was purified by IMAC and size-exclusion chromatography (SEC) using HiLoad 16/600 Superdex 200 and Superose 6 (Increase) columns with 20 mM HEPES, pH 7.4, 150 mM NaCl as a running buffer. Top fractions from the final SEC elution peak were pooled and concentrated to 10 mg/mL, aliquoted, flash frozen and stored at -80 C freezer till further use.

Homo sapiens Cytoplasm 0.486 Isoform 2 of ATP-citrate synthase Homo sapiens 0.12334257

cDNA encoding full-length human ACLY (hACLY, Uniprot ID P53396-2) was prepared from poly A+ RNA from liver and cloned into the pTrcHis2 vector, in frame with a C-terminal Myc- and His-tag, resulting in pTrcHis2-hACLY (LMBP 11277). https://doi.org/10.1038/s41586-019-1095-5

4 Escherichia coli BL21(DE3) LEVO MSAKAISEQTGKELLYKFICTTSAIQNRFKYARVTPDTDWARLLQDHPWLLSQNLVVKPDQLIKRRGKLGLVGVNLTLDG VKSWLKPRLGQEATVGKATGFLKNFLIEPFVPHSQAEEFYVCIYATREGDYVLFHHEGGVDVGDVDAKAQKLLVGVDEKL NPEDIKKHLLVHAPEDKKEILASFISGLFNFYEDLYFTYLEINPLVVTKDGVYVLDLAAKVDATADYICKVKWGDIEFPP PFGREAYPEEAYIADLDAKSGASLKLTLLNPKGRIWTMVAGGGASVVYSDTICDLGGVNELANYGEYSGAPSEQQTYDYA KTILSLMTREKHPDGKILIIGGSIANFTNVAATFKGIVRAIRDYQGPLKEHEVTIFVRRGGPNYQEGLRVMGEVGKTTGI PIHVFGTETHMTAIVGMALGHRPIPNQPPTAAHTANFLLNASGSTSTPAPSRTASFSESRADEVAPAKKAKPAMPQGKST TLFSRHTKAIVWGMQTRAVQGMLDFDYVCSRDEPSVAAMVYPFTGDHKQKFYWGHKEILIPVFKNMADAMRKHPEVDVLI NFASLRSAYDSTMETMNYAQIRTIAIIAEGIPEALTRKLIKKADQKGVTIIGPATVGGIKPGCFKIGNTGGMLDNILASK LYRPGSVAYVSRSGGMSNELNNIISRTTDGVYEGVAIGGDRYPGSTFMDHVLRYQDTPGVKMIVVLGEIGGTEEYKICRG IKEGRLTKPIVCWCIGTCATMFSSEVQFGHAGACANQASETAVAKNQALKEAGVFVPRSFDELGEIIQSVYEDLVANGVI VPAQEVPPPTVPMDYSWARELGLIRKPASFMTSICDERGQELIYAGMPITEVFKEEMGIGGVLGLLWFQKRLPKYSCQFI EMCLMVTADHGPAVSGAHNTIICARAGKDLVSSLTSGLLTIGDRFGGALDAAAKMFSKAFDSGIIPMEFVNKMKKEGKLI MGIGHRVKSINNPDMRVQILKDYVRQHFPATPLLDYALEVEKITTSKKPNLILNVDGLIGVAFVDMLRNCGSFTREEADE YIDIGALNGIFVLGRSMGFIGHYLDQKRLKQGLYRHPWDDISYVLPEHMSMKGEFEAYVEQKLISEEDLNSAVDHHHHHH P53396 2.3.3.8 ADENOSINE-5'-DIPHOSPHATE 0.000427201 1 ADP 6-[4-[6-[2-[3-[[4-[[[(2~{R},3~{S},4~{R},5~{R})-5-(6-aminopurin-9-yl)-4-oxidanyl-3-phosphonooxy-oxolan-2-yl]methoxy-oxidanyl-phosphoryl]oxy-oxidanyl-phosphoryl]oxy-3,3-dimethyl-2-oxidanyl-butanoyl]amino]propanoylamino]ethylsulfanyl]-5,5-dimethyl-6-oxidanylidene-hexyl]phenyl]-2,2-dimethyl-hexanoic acid 0.0011120219999999998 1 W1K singleParticle particle 10 7.4 20.0 C8H18N2O4S HEPES 150.0 NaCl sodium chloride 0.5 C32H58N2O8S CHAPSO 1.0 C10H16N5O13P3 ATP 2.0 Mg Mg2+ 4.0 C43H68N7O19P3S EVT0185-CoA

HBS buffer supplemented with 0.5 % CHAPSO, 1 mM Mg2ATP and 4 mM EVT0185-CoA

C-flat-1.2/1.3 COPPER 300

Grids were glow discharged with a Pelco EasiGlOW instrument using a current of 15 mA for 10s at a pressure of 0.4 mBar.

ETHANE 99 295 LEICA EM GP

Leica EM GP2.

The sample was monodisperse. For cryo-EM grid preparation, purified ACLY (10 mg/mL in HBS buffer) was supplemented with 0.5 % CHAPSO, 1 mM Mg2ATP and 4 mM EVT0185-CoA.

JEOL CRYO ARM 300 FLOOD BEAM BRIGHT FIELD FIELD EMISSION GUN 300 1.2 1.8 JEOL CRYOSPECPORTER NITROGEN In-column Omega Filter GATAN K3 (6k x 4k) 1 7263 61.8 1

Movies were processed via patch-based motion correction and CTF estimation as implemented in cryoSPARC v3.1.0.

210900

Initial high-resolution 2D classes were obtained via the Blob Picker job in cryoSPARC, followed by template-based picking and neural network-based particle picking via TOPAZ as implemented in cryoSPARC. Ensuing 2D classification, 2D class selection and removal of potential duplicate particles within a distance of 150 Angstrom resulted in a particle set of 210,900 particles. Particles were extracted with a box size of 480 pixels with 2x binning.

cryoSPARC v3.1.0 patch CTF PHASE FLIPPING AND AMPLITUDE CORRECTION 3 C1 3.25 FSC 0.143 CUT-OFF cryoSPARC v3.1.0 3D Classification

280072 MAXIMUM LIKELIHOOD cryoSPARC v3.1.0 Ab initio

Ab initio reconstruction in cryoSPARC (number of classes = 5)

MAXIMUM LIKELIHOOD cryoSPARC v3.1.0 Local refinement

Local refinement around one CCS arm and CCL module

4 93000.0 cryoSPARC v3.1.0 3D Classification

Refinement in C1 symmetry resulted in a pseudo-D2-symmetric cryo-EM volume with a golden-standard FSC0.143 resolution of 3.65 Angstrom that represented the ACLY tetramer. To possibly resolve the CCS-CCL assembly at higher resolution we applied symmetry expansion in combination with local refinement around one CCS arm and the central CCL module followed by 3D classification without alignment. 3 out of 4 classes were selected for the final reconstruction (total number of particles 280,072).

emd_53847.map.gz 1 IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) 480 480 480 0 0 0 480 480 480 345.6 345.6 345.6 90.0 90.0 90.0 X Y Z -0.5252451 0.8031328 -0.000004346402 0.017192211 0.72 0.72 0.72 0.12 AUTHOR ::::EMDATABANK.org::::EMD-53847:::: Sharpened cryo-EM map following local refinement in combination with symmetry expansion (D2) 6HXH PDB experimental model FLEXIBLE FIT

Following 3D classification, local refinement resulted in a cryo-EM volume with a golden standard FSC0.143-resolution of 3.25 Angstrom in which the atomic models for the CSS and CSH modules (extracted from pdb 6xhx) were fitted using Chimera and real-space refined in Phenix using reference restraints to the starting model. Cryo-EM map regions representing ligands in the ATP-grasp fold domain of the CCS module and in the CoA-binding domain were of the CSH module modelled as Mg.ADP and the adenosine 3'-phosphate 5'-diphosphate moiety of bound EVT0185-CoA, respectively. Restraints for EVT0185-CoA were generated via de Grade Web Server (https://grade.globalphasing.org).

REAL 138.800000000000011 emd_53847_msk_1.map emd_53847_additional_7.map.gz 1 IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) 480 480 480 0 0 0 480 480 480 345.6 345.6 345.6 90.0 90.0 90.0 X Y Z 0.0 1.0 0.008616063 0.08737143 0.72 0.72 0.72 AUTHOR ::::EMDATABANK.org::::EMD-53847:::: Mask for FSC calculation following local refinement in combination with symmetry expansion (D2) emd_53847_additional_6.map.gz 1 IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) 480 480 480 0 0 0 480 480 480 345.6 345.6 345.6 90.0 90.0 90.0 X Y Z -0.14968647 0.30559665 -0.000004346397 0.008566276 0.72 0.72 0.72 AUTHOR ::::EMDATABANK.org::::EMD-53847:::: Non-sharpened cryo-EM map following local refinement in combination with symmetry expansion (D2) emd_53847_additional_5.map.gz 1 IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) 240 240 240 0 0 0 240 240 240 345.6 345.6 345.6 90.0 90.0 90.0 X Y Z -0.3469539 0.64339757 0.00070891646 0.025207931 1.44 1.44 1.44 AUTHOR ::::EMDATABANK.org::::EMD-53847:::: Half-map A following non-uniform refinement in C1 symmetry emd_53847_additional_4.map.gz 1 IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) 240 240 240 0 0 0 240 240 240 345.6 345.6 345.6 90.0 90.0 90.0 X Y Z -0.2574019 0.52792734 0.000089852605 0.021058239 1.44 1.44 1.44 AUTHOR ::::EMDATABANK.org::::EMD-53847:::: Non-sharpened cryo-EM map in C1 symmetry emd_53847_additional_3.map.gz 1 IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) 240 240 240 0 0 0 240 240 240 345.6 345.6 345.6 90.0 90.0 90.0 X Y Z 0.0 1.0 0.016952502 0.11945023 1.44 1.44 1.44 AUTHOR ::::EMDATABANK.org::::EMD-53847:::: Mask for FSC calculation in C1 symmetry emd_53847_additional_2.map.gz 1 IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) 240 240 240 0 0 0 240 240 240 345.6 345.6 345.6 90.0 90.0 90.0 X Y Z -0.60786045 1.0209568 0.00008975549 0.026414867 1.44 1.44 1.44 AUTHOR ::::EMDATABANK.org::::EMD-53847:::: Sharpened cryo-EM map in C1 symmetry emd_53847_additional_1.map.gz 1 IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) 240 240 240 0 0 0 240 240 240 345.6 345.6 345.6 90.0 90.0 90.0 X Y Z -0.31573707 0.65492207 0.0007193519 0.025130836 1.44 1.44 1.44 AUTHOR ::::EMDATABANK.org::::EMD-53847:::: Half-map B following non-uniform refinement in C1 symmetry emd_53847_half_map_2.map.gz 1 IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) 480 480 480 0 0 0 480 480 480 345.6 345.6 345.6 90.0 90.0 90.0 X Y Z -0.63795733 0.6373587 0.00032751038 0.07377217 0.72 0.72 0.72 AUTHOR ::::EMDATABANK.org::::EMD-53847:::: Half-map B following local refinement in combination with symmetry expansion (D2) emd_53847_half_map_1.map.gz 1 IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) 480 480 480 0 0 0 480 480 480 345.6 345.6 345.6 90.0 90.0 90.0 X Y Z -0.73737365 0.6122084 0.00032545757 0.073702626 0.72 0.72 0.72 AUTHOR ::::EMDATABANK.org::::EMD-53847:::: Half-map A following local refinement in combination with symmetry expansion (D2)