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- EMDB-47335: Cryo-EM focused map masking butyrolactol A binding site of the C.... -

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Basic information

Entry
Database: EMDB / ID: EMD-47335
TitleCryo-EM focused map masking butyrolactol A binding site of the C. neoformans lipid flippase Apt1-Cdc50 bound with butyrolactol A in the E2P state
Map dataFocused map
Sample
  • Complex: Apt1-Cdc50 heterodimer bound with butyrolactol A
KeywordsCryptococcus neoformans / lipid flippase / P4-ATPase / Cdc50 protein / butyrolactol A / LIPID TRANSPORT
Biological speciesCryptococcus neoformans var. grubii H99 (fungus)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.93 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 potentiates caspofungin efficacy against resistant fungi via phospholipid flippase inhibition.
Authors: Xuefei Chen / H Diessel Duan / Michael J Hoy / Kalinka Koteva / Michaela Spitzer / Allison K Guitor / Emily Puumala / Aline A Fiebig / Guanggan Hu / Bonnie Yiu / Sommer Chou / Zhuyun Bian / ...Authors: Xuefei Chen / H Diessel Duan / Michael J Hoy / Kalinka Koteva / Michaela Spitzer / Allison K Guitor / Emily Puumala / Aline A Fiebig / Guanggan Hu / Bonnie Yiu / Sommer Chou / Zhuyun Bian / Yeseul Choi / Amelia Bing Ya Guo / Wenliang Wang / Sheng Sun / Nicole Robbins / Anna Floyd Averette / Michael A Cook / Ray Truant / Lesley T MacNeil / Eric D Brown / James W Kronstad / Brian K Coombes / 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 therapeutic options and rising resistance. Cryptococci are intrinsically resistant to the latest ...Fungal infections cause millions of deaths annually and are challenging to treat due to limited therapeutic options and rising 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 to rescue caspofungin fungicidal activity against H99 and identified butyrolactol A, which restores echinocandin efficacy against resistant fungal pathogens, including multidrug-resistant . Mode of action studies revealed that butyrolactol A inhibits the phospholipid flippase Apt1-Cdc50, blocking phospholipid transport. Cryo-electron microscopy analysis of the Apt1•butyrolactol A complex revealed that the flippase is trapped in a dead-end state. Apt1 inhibition disrupts membrane asymmetry, vesicular trafficking, and cytoskeletal organization, thereby enhancing echinocandin uptake and potency. This study identifies lipid 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-
UpdateFeb 5, 2025-
Current statusFeb 5, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_47335.png

Downloads & links

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Map

FileDownload / File: emd_47335.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationFocused 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 - Maximum-0.001822755 - 2.3154423
Average (Standard dev.)0.0015744044 (±0.029307295)
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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Mask #1

Fileemd_47335_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map B

Fileemd_47335_half_map_1.map
AnnotationHalf map B
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map A

Fileemd_47335_half_map_2.map
AnnotationHalf map A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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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

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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
Source (natural)Organism: Cryptococcus neoformans var. grubii H99 (fungus)
Molecular weightTheoretical: 222 KDa

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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

Startup modelType of model: INSILICO MODEL
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.93 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 169849
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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