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- PDB-9dzu: Cryo-EM structure of the C. neoformans lipid flippase Apt1-Cdc50 ... -

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Basic information

Entry
Database: PDB / ID: 9dzu
TitleCryo-EM structure of the C. neoformans lipid flippase Apt1-Cdc50 bound with butyrolactol A in the E2P state
Components
  • Phospholipid-transporting ATPase
  • Transcription regulator
KeywordsTRANSLOCASE/INHIBITOR / Cryptococcus neoformans / lipid flippase / P4-ATPase / Cdc50 protein / butyrolactol A / TRANSLOCASE-INHIBITOR complex
Function / homology
Function and homology information


phosphatidylethanolamine flippase activity / P-type phospholipid transporter / phospholipid translocation / magnesium ion binding / endoplasmic reticulum / Golgi apparatus / ATP hydrolysis activity / ATP binding / plasma membrane
Similarity search - Function
CDC50/LEM3 family / LEM3 (ligand-effect modulator 3) family / CDC50 family / P-type ATPase, subfamily IV / P-type ATPase, C-terminal / P-type ATPase, N-terminal / Phospholipid-translocating ATPase N-terminal / Phospholipid-translocating P-type ATPase C-terminal / Cation transport ATPase (P-type) / P-type ATPase, haloacid dehalogenase domain / P-type ATPase, phosphorylation site ...CDC50/LEM3 family / LEM3 (ligand-effect modulator 3) family / CDC50 family / P-type ATPase, subfamily IV / P-type ATPase, C-terminal / P-type ATPase, N-terminal / Phospholipid-translocating ATPase N-terminal / Phospholipid-translocating P-type ATPase C-terminal / Cation transport ATPase (P-type) / P-type ATPase, haloacid dehalogenase domain / P-type ATPase, phosphorylation site / P-type ATPase, cytoplasmic domain N / E1-E2 ATPases phosphorylation site. / P-type ATPase, A domain superfamily / P-type ATPase / P-type ATPase, transmembrane domain superfamily / HAD superfamily / HAD-like superfamily
Similarity search - Domain/homology
: / BERYLLIUM TRIFLUORIDE ION / Transcription regulator / Phospholipid-transporting ATPase
Similarity search - Component
Biological speciesCryptococcus neoformans var. grubii H99 (fungus)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.72 Å
AuthorsDuan, H.D. / Li, H.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Cancer Institute (NIH/NCI)CA231466 United States
CitationJournal: bioRxiv / Year: 2025
Title: Butyrolactol A is a phospholipid flippase inhibitor that potentiates the bioactivity of caspofungin against resistant fungi.
Authors: Xuefei Chen / H Diessel Duan / Michael J Hoy / Kalinka Koteva / Michaela Spitzer / Allison K Guitor / Emily Puumala / Guanggan Hu / Bonnie Yiu / Sommer Chou / Zhuyun Bian / Amelia Bing Ya ...Authors: Xuefei Chen / H Diessel Duan / Michael J Hoy / Kalinka Koteva / Michaela Spitzer / Allison K Guitor / Emily Puumala / Guanggan Hu / Bonnie Yiu / Sommer Chou / Zhuyun Bian / Amelia Bing Ya Guo / Sheng Sun / Nicole Robbins / Michael A Cook / Ray Truant / Lesley T MacNeil / Eric D Brown / James W Kronstad / Leah E Cowen / Joseph Heitman / Huilin Li / Gerard D Wright
Abstract: Fungal infections cause millions of deaths annually and are challenging to treat due to limited antifungal options and increasing drug resistance. Cryptococci are intrinsically resistant to the ...Fungal infections cause millions of deaths annually and are challenging to treat due to limited antifungal options and increasing drug resistance. Cryptococci are intrinsically resistant to the latest generation of antifungals, echinocandins, while , a notorious global threat, is also increasingly resistant. We performed a natural product extract screen for rescue of the activity of the echinocandin caspofungin against H99, identifying butyrolactol A, which restores echinocandin efficacy against resistant fungal pathogens, including . Mode of action studies revealed that butyrolactol A inhibits the phospholipid flippase Apt1-Cdc50, blocking phospholipid transport. Cryoelectron-microscopy analysis of the Apt1●butyrolactol A complex revealed that the flippase is locked in a dead-end state. Apt1 inhibition disrupts membrane asymmetry, vesicular trafficking, and cytoskeletal organization, thereby enhancing echinocandin uptake and potency. This study identifies flippases as promising antifungal targets and demonstrates the potential of revisiting natural products to expand the antifungal arsenal and combat resistance.
History
DepositionOct 17, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Feb 5, 2025Provider: repository / Type: Initial release
Revision 1.0Feb 5, 2025Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Feb 5, 2025Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0Feb 5, 2025Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Feb 5, 2025Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release
Revision 1.1May 21, 2025Group: Data collection / Derived calculations / Structure summary
Category: chem_comp / chem_comp_atom ...chem_comp / chem_comp_atom / chem_comp_bond / em_admin / entity / pdbx_entity_nonpoly
Item: _chem_comp.name / _chem_comp_atom.atom_id ..._chem_comp.name / _chem_comp_atom.atom_id / _chem_comp_bond.atom_id_1 / _chem_comp_bond.atom_id_2 / _chem_comp_bond.pdbx_stereo_config / _chem_comp_bond.value_order / _em_admin.last_update / _entity.pdbx_description / _pdbx_entity_nonpoly.name
Revision 1.1May 21, 2025Data content type: EM metadata / Data content type: EM metadata / EM metadata / Group: Experimental summary / Structure summary / Data content type: EM metadata / EM metadata / Category: em_admin / entity / Data content type: EM metadata / EM metadata / EM metadata
Item: _chem_comp.name / _em_admin.last_update / _entity.pdbx_description

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
B: Transcription regulator
A: Phospholipid-transporting ATPase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)225,2339
Polymers222,4732
Non-polymers2,7607
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Protein , 2 types, 2 molecules BA

#1: Protein Transcription regulator


Mass: 45937.961 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Cryptococcus neoformans var. grubii H99 (fungus)
Gene: CNAG_06465 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: J9VW44
#2: Protein Phospholipid-transporting ATPase


Mass: 176534.609 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Cryptococcus neoformans var. grubii H99 (fungus)
Gene: CNAG_06469 / Production host: Saccharomyces cerevisiae (brewer's yeast)
References: UniProt: J9VZ19, P-type phospholipid transporter

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Sugars , 3 types, 4 molecules

#3: Polysaccharide 2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 424.401 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DGlcpNAcb1-4DGlcpNAcb1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/1,2,1/[a2122h-1b_1-5_2*NCC/3=O]/1-1/a4-b1WURCSPDB2Glycan 1.1.0
[][D-1-deoxy-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{}}LINUCSPDB-CARE
#4: Polysaccharide alpha-D-mannopyranose-(1-4)-alpha-D-mannopyranose-(1-6)-[alpha-D-mannopyranose-(1-3)]beta-D- ...alpha-D-mannopyranose-(1-4)-alpha-D-mannopyranose-(1-6)-[alpha-D-mannopyranose-(1-3)]beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 1072.964 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DManpa1-4DManpa1-6[DManpa1-3]DManpb1-4DGlcpNAcb1-4DGlcpNAcb1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/3,6,5/[a2122h-1b_1-5_2*NCC/3=O][a1122h-1b_1-5][a1122h-1a_1-5]/1-1-2-3-3-3/a4-b1_b4-c1_c3-d1_c6-e1_e4-f1WURCSPDB2Glycan 1.1.0
[][D-1-deoxy-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-Manp]{[(3+1)][a-D-Manp]{}[(6+1)][a-D-Manp]{[(4+1)][a-D-Manp]{}}}}}LINUCSPDB-CARE
#5: Sugar ChemComp-NAG / 2-acetamido-2-deoxy-beta-D-glucopyranose / N-acetyl-beta-D-glucosamine / 2-acetamido-2-deoxy-beta-D-glucose / 2-acetamido-2-deoxy-D-glucose / 2-acetamido-2-deoxy-glucose / N-ACETYL-D-GLUCOSAMINE


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C8H15NO6
IdentifierTypeProgram
DGlcpNAcbCONDENSED IUPAC CARBOHYDRATE SYMBOLGMML 1.0
N-acetyl-b-D-glucopyranosamineCOMMON NAMEGMML 1.0
b-D-GlcpNAcIUPAC CARBOHYDRATE SYMBOLPDB-CARE 1.0
GlcNAcSNFG CARBOHYDRATE SYMBOLGMML 1.0

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Non-polymers , 3 types, 3 molecules

#6: Chemical ChemComp-BEF / BERYLLIUM TRIFLUORIDE ION


Mass: 66.007 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: BeF3
#7: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg
#8: Chemical ChemComp-A1BD6 / (3R,4R,5S)-3,4-dihydroxy-5-[(1R,2R,3S,4S,5R,6R,8E,10E,14E,16Z)-1,2,3,4,5-pentahydroxy-6,20,20-trimethylhenicosa-8,10,14,16-tetraen-1-yl]oxolan-2-one


Mass: 526.659 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C28H46O9 / Feature type: SUBJECT OF INVESTIGATION

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Details

Has ligand of interestY
Has protein modificationY

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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: Apt1-Cdc50 heterodimer bound with butyrolactol A / Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT
Molecular weightValue: 0.222 MDa / Experimental value: NO
Source (natural)Organism: Cryptococcus neoformans var. grubii H99 (fungus)
Source (recombinant)Organism: Saccharomyces cerevisiae (brewer's yeast)
Buffer solutionpH: 7.4
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: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 1700 nm / Nominal defocus min: 1300 nm
Image recordingElectron dose: 60 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

EM softwareName: PHENIX / Version: 1.20.1_4487: / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.72 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 639772 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00312056
ELECTRON MICROSCOPYf_angle_d0.48416393
ELECTRON MICROSCOPYf_dihedral_angle_d11.9654349
ELECTRON MICROSCOPYf_chiral_restr0.0421867
ELECTRON MICROSCOPYf_plane_restr0.0042075

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