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- PDB-9r90: Cryo-EM structure of human ATP citrate lyase in complex with inhi... -

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Basic information

Entry
Database: PDB / ID: 9r90
TitleCryo-EM structure of human ATP citrate lyase in complex with inhibitor EVT0185-CoA
ComponentsIsoform 2 of ATP-citrate synthase
KeywordsLYASE / ATP citrate lyase / ACL / ACLY / de novo lipogenesis / acetyl-CoA / citrate / oxaloacetate / cancer
Function / homology
Function and homology information


ATP citrate synthase / ATP citrate synthase activity / citrate metabolic process / Fatty acyl-CoA biosynthesis / acetyl-CoA biosynthetic process / ChREBP activates metabolic gene expression / coenzyme A metabolic process / oxaloacetate metabolic process / negative regulation of ferroptosis / cholesterol biosynthetic process ...ATP citrate synthase / ATP citrate synthase activity / citrate metabolic process / Fatty acyl-CoA biosynthesis / acetyl-CoA biosynthetic process / ChREBP activates metabolic gene expression / coenzyme A metabolic process / oxaloacetate metabolic process / negative regulation of ferroptosis / cholesterol biosynthetic process / lipid biosynthetic process / fatty acid biosynthetic process / azurophil granule lumen / ficolin-1-rich granule lumen / ciliary basal body / Neutrophil degranulation / extracellular exosome / extracellular region / nucleoplasm / ATP binding / metal ion binding / membrane / cytosol
Similarity search - Function
ATP-citrate synthase / : / ATP-citrate synthase ATP-grasp domain / ATP-citrate synthase, citrate-binding domain / ATP citrate lyase citrate-binding / ATP-citrate lyase/succinyl-CoA ligase, active site / ATP-citrate lyase/succinyl-CoA ligase, conserved site / ATP-citrate lyase / succinyl-CoA ligases family active site. / ATP-citrate lyase / succinyl-CoA ligases family signature 1. / Succinyl-CoA synthetase, beta subunit, conserved site ...ATP-citrate synthase / : / ATP-citrate synthase ATP-grasp domain / ATP-citrate synthase, citrate-binding domain / ATP citrate lyase citrate-binding / ATP-citrate lyase/succinyl-CoA ligase, active site / ATP-citrate lyase/succinyl-CoA ligase, conserved site / ATP-citrate lyase / succinyl-CoA ligases family active site. / ATP-citrate lyase / succinyl-CoA ligases family signature 1. / Succinyl-CoA synthetase, beta subunit, conserved site / ATP-citrate lyase / succinyl-CoA ligases family signature 3. / ATP-citrate lyase/succinyl-CoA ligase / CoA-ligase / CoA binding domain / Succinyl-CoA synthetase-like / CoA binding domain / CoA-binding / Citrate synthase / Citrate synthase-like, small alpha subdomain / Citrate synthase superfamily / Citrate synthase, C-terminal domain / NAD(P)-binding domain superfamily
Similarity search - Domain/homology
ADENOSINE-5'-DIPHOSPHATE / : / ATP-citrate synthase
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.25 Å
AuthorsVerstraete, K. / Verschueren, K. / Savvides, S.N. / Steinberg, G.R.
Funding support Belgium, 1items
OrganizationGrant numberCountry
Research Foundation - Flanders (FWO)1524918N Belgium
CitationJournal: Nature / Year: 2025
Title: ACLY inhibition promotes tumour immunity and suppresses liver cancer.
Authors: Jaya Gautam / Jianhan Wu / James S V Lally / Jamie D McNicol / Russta Fayyazi / Elham Ahmadi / Daniela Carmen Oniciu / Spencer Heaton / Roger S Newton / Sonia Rehal / Dipankar Bhattacharya / ...Authors: Jaya Gautam / Jianhan Wu / James S V Lally / Jamie D McNicol / Russta Fayyazi / Elham Ahmadi / Daniela Carmen Oniciu / Spencer Heaton / Roger S Newton / Sonia Rehal / Dipankar Bhattacharya / Fiorella Di Pastena / Binh Nguyen / Celina M Valvano / Logan K Townsend / Suhrid Banskota / Battsetseg Batchuluun / Maria Joy Therese Jabile / Alice Payne / Junfeng Lu / Eric M Desjardins / Naoto Kubota / Evangelia E Tsakiridis / Bejal Mistry / Alex Aganostopoulos / Vanessa Houde / Ann Dansercoer / Koen H G Verschueren / Savvas N Savvides / Joanne A Hammill / Ksenia Bezverbnaya / Paola Muti / Theodoros Tsakiridis / Wenting Dai / Lei Jiang / Yujin Hoshida / Mark Larché / Jonathan L Bramson / Scott L Friedman / Kenneth Verstraete / Dongdong Wang / Gregory R Steinberg /
Abstract: Immunosuppressive tumour microenvironments are common in cancers such as metabolic dysfunction-associated steatohepatitis (MASH)-driven hepatocellular carcinoma (HCC) (MASH-HCC). Although immune ...Immunosuppressive tumour microenvironments are common in cancers such as metabolic dysfunction-associated steatohepatitis (MASH)-driven hepatocellular carcinoma (HCC) (MASH-HCC). Although immune cell metabolism influences effector function, the effect of tumour metabolism on immunogenicity is less understood. ATP citrate lyase (ACLY) links substrate availability and mitochondrial metabolism with lipid biosynthesis and gene regulation. Although ACLY inhibition shows antiproliferative effects in various tumours, clinical translation has been limited by challenges in inhibitor development and compensatory metabolic pathways. Here, using a mouse model of MASH-HCC that mirrors human disease, genetic inhibition of ACLY in hepatocytes and tumours reduced neoplastic lesions by over 70%. To evaluate the therapeutic potential of this pathway, a novel small-molecule ACLY inhibitor, EVT0185 (6-[4-(5-carboxy-5-methyl-hexyl)-phenyl]-2,2-dimethylhexanoic acid), was identified via phenotypic screening. EVT0185 is converted to a CoA thioester in the liver by SLC27A2 and structural analysis by cryo-electron microscopy reveals that EVT0185-CoA directly interacts with the CoA-binding site of ACLY. Oral delivery of EVT0185 in three mouse models of MASH-HCC dramatically reduces tumour burden as monotherapy and enhances efficacy of current standards of care including tyrosine kinase inhibitors and immunotherapies. Transcriptomic and spatial profiling in mice and humans linked reduced tumour ACLY with increases in the chemokine CXCL13, tumour-infiltrating B cells and tertiary lymphoid structures. The depletion of B cells blocked the antitumour effects of ACLY inhibition. Together, these findings illustrate how targeting tumour metabolism can rewire immune function and suppress cancer progression in MASH-HCC.
History
DepositionMay 18, 2025Deposition site: PDBE / Processing site: PDBE
Revision 1.0Mar 4, 2026Provider: repository / Type: Initial release
Revision 1.0Mar 4, 2026Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Mar 4, 2026Data content type: Additional map / Part number: 1 / Data content type: Additional map / Provider: repository / Type: Initial release
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Revision 1.0Mar 4, 2026Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
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Revision 1.0Mar 4, 2026Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Mar 4, 2026Data content type: Mask / Part number: 1 / Data content type: Mask / Provider: repository / Type: Initial release
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Isoform 2 of ATP-citrate synthase
B: Isoform 2 of ATP-citrate synthase
C: Isoform 2 of ATP-citrate synthase
D: Isoform 2 of ATP-citrate synthase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)494,9106
Polymers493,3704
Non-polymers1,5392
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, SEC-MALLS experiments, crystallographic studies and cryo-EM analysis show that ACLY forms homotetrameric assemblies
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein
Isoform 2 of ATP-citrate synthase / ATP-citrate (pro-S-)-lyase / ACL / Citrate cleavage enzyme


Mass: 123342.570 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Details: 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, ...Details: 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
Source: (gene. exp.) Homo sapiens (human) / Gene: ACLY / Plasmid: pTrcHis2-hACLY
Details (production host): Human ACLY in frame with a C-terminal Myc- and His-tag (LMBP 11277)
Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P53396, ATP citrate synthase
#2: Chemical ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE


Mass: 427.201 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Feature type: SUBJECT OF INVESTIGATION / Comment: ADP, energy-carrying molecule*YM
#3: Chemical ChemComp-W1K / 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


Mass: 1112.022 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C43H68N7O19P3S
Has ligand of interestY
Has protein modificationN

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Human ATP citrate lyase (homotetramer) / Type: COMPLEX
Details: 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 ...Details: 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.
Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.486 MDa / Experimental value: YES
Source (natural)Organism: Homo sapiens (human) / Cellular location: Cytoplasm
Source (recombinant)Organism: Escherichia coli (E. coli) / Plasmid: pTrcHis2-hACLY
Buffer solutionpH: 7.4
Details: HBS buffer supplemented with 0.5 % CHAPSO, 1 mM Mg2ATP and 4 mM EVT0185-CoA
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMHEPESC8H18N2O4S1
2150 mMsodium chlorideNaCl1
30.5 % (w/v)CHAPSOC32H58N2O8S1
41 mMATPC10H16N5O13P31
52 mMMg2+Mg1
64 mMEVT0185-CoAC43H68N7O19P3S1
SpecimenConc.: 10 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: 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.
Specimen supportDetails: Grids were glow discharged with a Pelco EasiGlOW instrument using a current of 15 mA for 10s at a pressure of 0.4 mBar.
Grid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: C-flat-1.2/1.3
VitrificationInstrument: LEICA EM GP / Cryogen name: ETHANE / Humidity: 99 % / Chamber temperature: 295 K / Details: Leica EM GP2

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Electron microscopy imaging

MicroscopyModel: JEOL CRYO ARM 300
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 1800 nm / Nominal defocus min: 1200 nm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: JEOL CRYOSPECPORTER
Image recordingElectron dose: 61.8 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 7263
EM imaging opticsEnergyfilter name: In-column Omega Filter

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Processing

EM software
IDNameVersionCategoryDetails
1cryoSPARCv3.1.0particle selection
2SerialEMimage acquisition
4cryoSPARCv3.1.0CTF correctionpatch CTF
7UCSF Chimera1.17model fitting
9PHENIX1.20.1model refinement
10cryoSPARCv3.1.0initial Euler assignmentAb initio
11cryoSPARCv3.1.0final Euler assignmentLocal refinement
12cryoSPARCv3.1.0classification3D Classification
13cryoSPARCv3.1.03D reconstruction3D Classification
Image processingDetails: Movies were processed via patch-based motion correction and CTF estimation as implemented in cryoSPARC v3.1.0.
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 210900
Details: 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 ...Details: 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.
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.25 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 280072
Details: 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 ...Details: 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.
Num. of class averages: 3 / Symmetry type: POINT
Atomic model buildingB value: 138.8 / Protocol: FLEXIBLE FIT / Space: REAL
Details: 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 ...Details: 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).
Atomic model buildingPDB-ID: 6HXH

6hxh


Accession code: 6HXH / Source name: PDB / Type: experimental model
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 56.36 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.002514339
ELECTRON MICROSCOPYf_angle_d0.559619373
ELECTRON MICROSCOPYf_chiral_restr0.04142149
ELECTRON MICROSCOPYf_plane_restr0.00432483
ELECTRON MICROSCOPYf_dihedral_angle_d4.66811973

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