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- EMDB-47338: Cryo-EM structure of the C. neoformans lipid flippase Apt1-Cdc50 ... -

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Basic information

Entry
Database: EMDB / ID: EMD-47338
TitleCryo-EM structure of the C. neoformans lipid flippase Apt1-Cdc50 bound with butyrolactol A in the E2P state
Map dataComposite map
Sample
  • Complex: Apt1-Cdc50 heterodimer bound with butyrolactol A
    • Protein or peptide: Transcription regulator
    • Protein or peptide: Phospholipid-transporting ATPase
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
  • Ligand: BERYLLIUM TRIFLUORIDE ION
  • Ligand: MAGNESIUM ION
  • Ligand: (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
KeywordsCryptococcus 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
Transcription regulator / Phospholipid-transporting ATPase
Similarity search - Component
Biological speciesCryptococcus neoformans var. grubii H99 (fungus)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.72 Å
AuthorsDuan HD / Li H
Funding support United States, 1 items
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, 2024-
Header (metadata) releaseFeb 5, 2025-
Map releaseFeb 5, 2025-
UpdateMay 21, 2025-
Current statusMay 21, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_47338.png

Downloads & links

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Map

FileDownload / File: emd_47338.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationComposite map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 300 pix.
= 248.4 Å		0.83 Å/pix.
x 300 pix.
= 248.4 Å		0.83 Å/pix.
x 300 pix.
= 248.4 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.828 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.01
Minimum - Maximum0.0 - 2.3154423
Average (Standard dev.)0.0015071733 (±0.0270967)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 248.40001 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : Apt1-Cdc50 heterodimer bound with butyrolactol A

EntireName: Apt1-Cdc50 heterodimer bound with butyrolactol A
Components
  • Complex: Apt1-Cdc50 heterodimer bound with butyrolactol A
    • Protein or peptide: Transcription regulator
    • Protein or peptide: Phospholipid-transporting ATPase
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
  • Ligand: BERYLLIUM TRIFLUORIDE ION
  • Ligand: MAGNESIUM ION
  • Ligand: (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

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Supramolecule #1: Apt1-Cdc50 heterodimer bound with butyrolactol A

SupramoleculeName: Apt1-Cdc50 heterodimer bound with butyrolactol A / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Cryptococcus neoformans var. grubii H99 (fungus)
Molecular weightTheoretical: 222 KDa

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Macromolecule #1: Transcription regulator

MacromoleculeName: Transcription regulator / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Cryptococcus neoformans var. grubii H99 (fungus)
Molecular weightTheoretical: 45.937961 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast)
SequenceString: MAIFNRKPKA RLDGEPAPTE KEKVKWSKRP ANTAFKQQRL KAWQPILTPK SVLPTLLIIG IIFAPIGALI VWGSGKVTTI TLDYTECDV DAPTDGSYQA MPNSAYQYDL ATSSSVSESS IASPTWTFSN DSSREVGETA RCEIEFEVPY DLGPGLFLYY K LTNYYQNH ...String:
MAIFNRKPKA RLDGEPAPTE KEKVKWSKRP ANTAFKQQRL KAWQPILTPK SVLPTLLIIG IIFAPIGALI VWGSGKVTTI TLDYTECDV DAPTDGSYQA MPNSAYQYDL ATSSSVSESS IASPTWTFSN DSSREVGETA RCEIEFEVPY DLGPGLFLYY K LTNYYQNH RRYSSSFDAT QLIGDSRSLS QINGGNCKPI TSRDGKPYYP CGLIANSLFN DTFPSVVLLN PTNGAQNQTY NF SESGIAW GGIKKNYAST LTYISPSDVL PPPNWALKYP NGYVDGFPNL REDEHFQVWM RVAALPTFRK LWARNDGEIM SQG RYRIVA NMNYPVKQFS GTKSIVISTV SWIGGKQPFL GWAYIAAAIL CVVLAVAGLI RHLVKPRKLG DMSLLSWNQP NANG LHHHH HHHHHH

UniProtKB: Transcription regulator

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Macromolecule #2: Phospholipid-transporting ATPase

MacromoleculeName: Phospholipid-transporting ATPase / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO / EC number: P-type phospholipid transporter
Source (natural)Organism: Cryptococcus neoformans var. grubii H99 (fungus)
Molecular weightTheoretical: 176.534609 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast)
SequenceString: MGASKPPLVP RSKKHNPSWL DRNIVKPLES LAPSKLFARR RSPPVPRSVF INEPLPSEYY DKKGKILRAH HFATNQNVTS KYTVITFIP KNLFEQFRRV ANCFFLAISI LQFFPKFSTI SPGLVILPLI IVLAITALKD GYEDIKRHQA DHRTNHAIVH V LGGQDYTN ...String:
MGASKPPLVP RSKKHNPSWL DRNIVKPLES LAPSKLFARR RSPPVPRSVF INEPLPSEYY DKKGKILRAH HFATNQNVTS KYTVITFIP KNLFEQFRRV ANCFFLAISI LQFFPKFSTI SPGLVILPLI IVLAITALKD GYEDIKRHQA DHRTNHAIVH V LGGQDYTN QNPMASKDKT FIPAIPLPKR RSKKAKKAEE EAALNMQGRS SSTENFAAEP VPGAEPRGQD ELQRMRSQVS NW DEDPEAG DSPGELGWHR TIWEDVKVGD FVKIYENEQF PADIVICATS EEEDVAYIET KNLDGETNLK SRNGVPGLSH LNT AEACAK AHLCIDLDAP ESNMFRLNGA VINLEEYDED EQHPIHPITL ETTMLRGCVL KNTAWVIGII VYTGEDTKII RNAG ATPSK RSKVEKQMNP QVIINLVILA AIAVVCAIVD HVNEVEWDRQ QAYWMLFADT SGDNPNINGL VTFANAFITF QNIVP ISLY ISIEAVRTIQ AAFIYWDRDI KYKKDGVTTR TTARSWNLSD DLGQIEYIFS DKTGTLTQNA MIFRQCSVGG KIYTGD GLP PSHPTITHQH QPPPVHQHDD QDDPIAKSAS ESDDSDPKKI STEDPDEIKV TLPKEVLATF HDAELDKDLE AHDSEQS RI LHGFFAVLGL CHTVLAAETE PGVIEYKAQS PDEAALVQSA ADVGFVFRGR DHNILRMSTP FSDVSDEYEL LHVLEFNS A RKRMSVILRK LDEDGRIFLL CKGADNVIFE RLTKDSNQRE MREKTDQDLQ YFASEGLRTL CLAYRILDPQ VYEQWAKEY HNATVALQDR EERIESVSSS IERDLILLGA TAIEDKLQDG VPDTISDLKR AGIKVWVATG DKLETAVAIG YTTNLLTKDT NLIVVREGR HSIGDQLREA LEEFFGEDAG LRTTLSRIDS RRNSMDPPRL TRVNTGVRSL VGRDNGTRPG GFSLVIEGHA L AHCFDDEE TEALLLALST RCNTVICCRV SPLQKAQIVH LIKDNLGVMC LAIGDGANDV SMIQAADVGV GISGEEGLQA VN SSDYAIA QFRYLKRLLL VHGHWSYFRN SSMILNFFYK NIIGIGVLFW FMIYCGWSTT YVFAYVYLLF WNVFWTLVPV IAI GLFDRN IDDETLMALP ELYRASREGK YFGLMRFAYY IFEGVYQSAV IYFFLNYTYV TTTARGDGYD VYMYEMSTTQ AIGA VMVAN LFSGLNIDAW TGWVWFAIWF GPFLIWVFTA VYSVIPPSSF YTGVYGNDVF LFRSAAYWFG WPFVTIIALL PRYLI KTFR QNIFPNDVDT MRLVRKYHPE VDLYNHPMLG GKLAPKKDED ESDYGEEPFD GPEGRRSSIK MANLRHSHGA FGRGDQ AGD MELGMGRKSL GNRPGLRSSM DSSRFGIHSG ARGSTVDMST GLEQPPSRGF GFTMEEGGVA IQRMQSRLSQ TSSHASR SR WPRFNNNSSS SHPFETKPPS SMSKLRSRAG SILTRKRADT TDTRNSDDKS LSSPVKTGFF GRHMPGQNHG QHEGRSMG T PLKSETGRGD NWEEEELEDE SLGRGFGVGQ NMAPPEIPRM DYKDDDDKI

UniProtKB: Phospholipid-transporting ATPase

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Macromolecule #5: 2-acetamido-2-deoxy-beta-D-glucopyranose

MacromoleculeName: 2-acetamido-2-deoxy-beta-D-glucopyranose / type: ligand / ID: 5 / Number of copies: 1 / Formula: NAG
Molecular weightTheoretical: 221.208 Da
Chemical component information

ChemComp-NAG:
2-acetamido-2-deoxy-beta-D-glucopyranose

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Macromolecule #6: BERYLLIUM TRIFLUORIDE ION

MacromoleculeName: BERYLLIUM TRIFLUORIDE ION / type: ligand / ID: 6 / Number of copies: 1 / Formula: BEF
Molecular weightTheoretical: 66.007 Da
Chemical component information

ChemComp-BEF:
BERYLLIUM TRIFLUORIDE ION

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Macromolecule #7: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 7 / Number of copies: 1 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #8: (3R,4R,5S)-3,4-dihydroxy-5-[(1R,2R,3S,4S,5R,6R,8E,10E,14E,16Z)-1,...

MacromoleculeName: (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
type: ligand / ID: 8 / Number of copies: 1 / Formula: A1BD6
Molecular weightTheoretical: 526.659 Da

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7.4
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 60.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 1.7 µm / Nominal defocus min: 1.3 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: INSILICO MODEL
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.72 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 639772
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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