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- PDB-6uuw: Structure of human ATP citrate lyase E599Q mutant in complex with... -

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Entry
Database: PDB / ID: 6uuw
TitleStructure of human ATP citrate lyase E599Q mutant in complex with Mg2+, citrate, ATP and CoA
ComponentsATP-citrate synthase
KeywordsLYASE
Function / homology
Function and homology information


ATP citrate synthase / ATP citrate synthase activity / Fatty acyl-CoA biosynthesis / citrate metabolic process / ChREBP activates metabolic gene expression / acetyl-CoA biosynthetic process / oxaloacetate metabolic process / coenzyme A metabolic process / lipid biosynthetic process / cholesterol biosynthetic process ...ATP citrate synthase / ATP citrate synthase activity / Fatty acyl-CoA biosynthesis / citrate metabolic process / ChREBP activates metabolic gene expression / acetyl-CoA biosynthetic process / oxaloacetate metabolic process / coenzyme A metabolic process / lipid biosynthetic process / cholesterol biosynthetic process / tricarboxylic acid cycle / fatty acid biosynthetic process / azurophil granule lumen / ficolin-1-rich granule lumen / Neutrophil degranulation / extracellular exosome / extracellular region / nucleoplasm / ATP binding / membrane / metal ion binding / cytosol
Similarity search - Function
ATP-citrate synthase / 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 ...ATP-citrate synthase / 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 / COENZYME A / Unknown ligand / Chem-Y2A / ATP-citrate synthase
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.85 Å
AuthorsWei, X. / Marmorstein, R.
CitationJournal: Nat Struct Mol Biol / Year: 2020
Title: Molecular basis for acetyl-CoA production by ATP-citrate lyase.
Authors: Xuepeng Wei / Kollin Schultz / Gleb A Bazilevsky / Austin Vogt / Ronen Marmorstein /
Abstract: ATP-citrate lyase (ACLY) synthesizes cytosolic acetyl coenzyme A (acetyl-CoA), a fundamental cellular building block. Accordingly, aberrant ACLY activity is observed in many diseases. Here we report ...ATP-citrate lyase (ACLY) synthesizes cytosolic acetyl coenzyme A (acetyl-CoA), a fundamental cellular building block. Accordingly, aberrant ACLY activity is observed in many diseases. Here we report cryo-EM structures of human ACLY, alone or bound to substrates or products. ACLY forms a homotetramer with a rigid citrate synthase homology (CSH) module, flanked by four flexible acetyl-CoA synthetase homology (ASH) domains; CoA is bound at the CSH-ASH interface in mutually exclusive productive or unproductive conformations. The structure of a catalytic mutant of ACLY in the presence of ATP, citrate and CoA substrates reveals a phospho-citryl-CoA intermediate in the ASH domain. ACLY with acetyl-CoA and oxaloacetate products shows the products bound in the ASH domain, with an additional oxaloacetate in the CSH domain, which could function in ACLY autoinhibition. These structures, which are supported by biochemical and biophysical data, challenge previous proposals of the ACLY catalytic mechanism and suggest additional therapeutic possibilities for ACLY-associated metabolic disorders.
History
DepositionNov 1, 2019Deposition site: RCSB / Processing site: RCSB
Revision 1.0Dec 25, 2019Provider: repository / Type: Initial release
Revision 1.1Jan 1, 2020Group: Database references / Structure summary / Category: audit_author / citation / citation_author
Item: _audit_author.name / _citation.pdbx_database_id_PubMed ..._audit_author.name / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID
Revision 1.2Jan 22, 2020Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.year / _citation_author.identifier_ORCID
Revision 2.0Feb 19, 2020Group: Advisory / Atomic model ...Advisory / Atomic model / Data collection / Database references / Derived calculations / Non-polymer description / Structure summary
Category: atom_site / atom_type ...atom_site / atom_type / chem_comp / citation / em_software / entity / pdbx_entity_nonpoly / pdbx_nonpoly_scheme / pdbx_struct_assembly_gen / pdbx_struct_assembly_prop / pdbx_struct_sheet_hbond / pdbx_unobs_or_zero_occ_atoms / pdbx_validate_close_contact / pdbx_validate_torsion / struct_asym / struct_conf / struct_mon_prot_cis / struct_sheet / struct_sheet_order / struct_sheet_range / struct_site / struct_site_gen
Item: _chem_comp.formula / _chem_comp.formula_weight ..._chem_comp.formula / _chem_comp.formula_weight / _chem_comp.id / _chem_comp.mon_nstd_flag / _chem_comp.name / _chem_comp.type / _citation.title / _em_software.category / _em_software.fitting_id / _em_software.imaging_id / _em_software.name / _em_software.version / _pdbx_struct_assembly_gen.asym_id_list / _pdbx_struct_assembly_prop.value / _struct_mon_prot_cis.pdbx_omega_angle / _struct_site.pdbx_num_residues
Description: Ligand identity / Provider: author / Type: Coordinate replacement
Revision 3.0Aug 11, 2021Group: Advisory / Atomic model ...Advisory / Atomic model / Author supporting evidence / Data collection / Database references / Derived calculations / Non-polymer description / Refinement description / Structure summary
Category: atom_site / chem_comp ...atom_site / chem_comp / database_2 / em_software / entity / pdbx_entity_instance_feature / pdbx_entity_nonpoly / pdbx_nonpoly_scheme / pdbx_struct_assembly_gen / pdbx_struct_assembly_prop / pdbx_struct_sheet_hbond / pdbx_validate_close_contact / pdbx_validate_torsion / refine_ls_restr / software / struct_asym / struct_conf / struct_mon_prot_cis / struct_sheet / struct_sheet_order / struct_sheet_range / struct_site / struct_site_gen
Item: _chem_comp.formula / _chem_comp.formula_weight ..._chem_comp.formula / _chem_comp.formula_weight / _chem_comp.id / _chem_comp.mon_nstd_flag / _chem_comp.name / _chem_comp.type / _database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_software.category / _pdbx_entity_instance_feature.auth_comp_id / _pdbx_entity_instance_feature.comp_id / _pdbx_struct_assembly_gen.asym_id_list / _pdbx_struct_assembly_prop.value / _struct_mon_prot_cis.pdbx_omega_angle
Description: Ligand identity / Provider: author / Type: Coordinate replacement

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Structure visualization

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Assembly

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


Theoretical massNumber of molelcules
Total (without water)489,80018
Polymers483,9334
Non-polymers5,86714
Water48627
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area44240 Å2
ΔGint-223 kcal/mol
Surface area146330 Å2

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Components

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Protein , 1 types, 4 molecules ABCD

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


Mass: 120983.141 Da / Num. of mol.: 4 / Mutation: E599Q
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: ACLY
Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
References: UniProt: P53396, ATP citrate synthase

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Non-polymers , 5 types, 41 molecules

#2: Chemical
ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE / Adenosine diphosphate


Mass: 427.201 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Comment: ADP, energy-carrying molecule*YM
#3: Chemical
ChemComp-COA / COENZYME A / Coenzyme A


Mass: 767.534 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C21H36N7O16P3S
#4: Chemical
ChemComp-Y2A / (2S)-2-hydroxy-2-[2-oxo-2-(phosphonooxy)ethyl]butanedioic acid


Mass: 272.103 Da / Num. of mol.: 4 / Source method: isolated from a natural source / Formula: C6H9O10P / Feature type: SUBJECT OF INVESTIGATION
#5: Chemical ChemComp-UNL / UNKNOWN LIGAND


Num. of mol.: 2 / Source method: obtained synthetically
#6: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 27 / Source method: isolated from a natural source / Formula: H2O

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Details

Has ligand of interestY

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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: ACLY(E599Q) with Mg2+, citrate, ATP and CoA / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
1200 mMsodium chlorideNaclSodium chloride1
225 mMTris HydrochlorideTris-HclTris1
35 mMmagnesiumMg1
45 mMcitrateCitric acidcitrateCitric acid1
55 mMcoenzyme ACoA1
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: FEI TALOS ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN
Image recordingElectron dose: 40 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.19.2_4158: / Classification: refinement
EM software
IDNameVersionCategory
2SerialEMimage acquisition
4Gctf1.06CTF correction
11RELION3final Euler assignment
12RELIONclassification
CTF correctionType: NONE
SymmetryPoint symmetry: D2 (2x2 fold dihedral)
3D reconstructionResolution: 2.85 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 207653 / Symmetry type: POINT
Atomic model buildingProtocol: OTHER
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00633125
ELECTRON MICROSCOPYf_angle_d0.90644904
ELECTRON MICROSCOPYf_dihedral_angle_d8.0554560
ELECTRON MICROSCOPYf_chiral_restr0.0564980
ELECTRON MICROSCOPYf_plane_restr0.0085719

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