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- EMDB-8959: afTMEM16 reconstituted in nanodiscs in the presence of Ca2+ and c... -

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Basic information

Entry
Database: EMDB / ID: EMD-8959
TitleafTMEM16 reconstituted in nanodiscs in the presence of Ca2+ and ceramide 24:0
Map dataafTMEM16 in the presence of Ca2+ and ceramide 24:0
Sample
  • Complex: afTMEM16 reconstituted in nanodiscs in the presence of Ca2+ and ceramide 24:0
    • Protein or peptide: Plasma membrane channel protein (Aqy1), putative
  • Ligand: CALCIUM ION
  • Ligand: DODECANE
  • Ligand: DECANE
  • Ligand: Octadecane
  • Ligand: HEXANE
Keywordsscramblase / Ca2+-activated / membrane-reorganization / LIPID TRANSPORT
Function / homology
Function and homology information


phospholipid scramblase activity / cortical endoplasmic reticulum / phospholipid translocation / chloride channel activity / voltage-gated calcium channel activity / chloride transmembrane transport / monoatomic ion transmembrane transport / membrane
Similarity search - Function
: / Alpha-beta plait domain in TMEM16 lipid scramblase / Anoctamin / : / Calcium-activated chloride channel
Similarity search - Domain/homology
Plasma membrane channel protein (Aqy1), putative
Similarity search - Component
Biological speciesAspergillus fumigatus (mold) / Neosartorya fumigata (strain ATCC MYA-4609 / Af293 / CBS 101355 / FGSC A1100) (mold)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.59 Å
AuthorsFalzone ME / Accardi A
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)5R01GM106717 United States
CitationJournal: Elife / Year: 2019
Title: Structural basis of Ca-dependent activation and lipid transport by a TMEM16 scramblase.
Authors: Maria E Falzone / Jan Rheinberger / Byoung-Cheol Lee / Thasin Peyear / Linda Sasset / Ashleigh M Raczkowski / Edward T Eng / Annarita Di Lorenzo / Olaf S Andersen / Crina M Nimigean / Alessio Accardi /
Abstract: The lipid distribution of plasma membranes of eukaryotic cells is asymmetric and phospholipid scramblases disrupt this asymmetry by mediating the rapid, nonselective transport of lipids down their ...The lipid distribution of plasma membranes of eukaryotic cells is asymmetric and phospholipid scramblases disrupt this asymmetry by mediating the rapid, nonselective transport of lipids down their concentration gradients. As a result, phosphatidylserine is exposed to the outer leaflet of membrane, an important step in extracellular signaling networks controlling processes such as apoptosis, blood coagulation, membrane fusion and repair. Several TMEM16 family members have been identified as Ca-activated scramblases, but the mechanisms underlying their Ca-dependent gating and their effects on the surrounding lipid bilayer remain poorly understood. Here, we describe three high-resolution cryo-electron microscopy structures of a fungal scramblase from , afTMEM16, reconstituted in lipid nanodiscs. These structures reveal that Ca-dependent activation of the scramblase entails global rearrangement of the transmembrane and cytosolic domains. These structures, together with functional experiments, suggest that activation of the protein thins the membrane near the transport pathway to facilitate rapid transbilayer lipid movement.
History
DepositionJul 10, 2018-
Header (metadata) releaseAug 8, 2018-
Map releaseFeb 6, 2019-
UpdateMar 13, 2024-
Current statusMar 13, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0474
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.0474
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6e1o
  • Surface level: 0.0474
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_8959.png

Downloads & links

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Map

FileDownload / File: emd_8959.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationafTMEM16 in the presence of Ca2+ and ceramide 24:0
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.1 Å/pix.
x 256 pix.
= 280.602 Å		1.1 Å/pix.
x 256 pix.
= 280.602 Å		1.1 Å/pix.
x 256 pix.
= 280.602 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.0961 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0474 / Movie #1: 0.0474
Minimum - Maximum-0.0850799 - 0.16190724
Average (Standard dev.)-0.00022660791 (±0.006968032)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 280.6016 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.09610156251.09610156251.0961015625
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z280.602280.602280.602
α/β/γ90.00090.00090.000
start NX/NY/NZ-16-63-38
NX/NY/NZ727975
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-0.0850.162-0.000

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Supplemental data

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Mask #1

Fileemd_8959_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : afTMEM16 reconstituted in nanodiscs in the presence of Ca2+ and c...

EntireName: afTMEM16 reconstituted in nanodiscs in the presence of Ca2+ and ceramide 24:0
Components
  • Complex: afTMEM16 reconstituted in nanodiscs in the presence of Ca2+ and ceramide 24:0
    • Protein or peptide: Plasma membrane channel protein (Aqy1), putative
  • Ligand: CALCIUM ION
  • Ligand: DODECANE
  • Ligand: DECANE
  • Ligand: Octadecane
  • Ligand: HEXANE

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Supramolecule #1: afTMEM16 reconstituted in nanodiscs in the presence of Ca2+ and c...

SupramoleculeName: afTMEM16 reconstituted in nanodiscs in the presence of Ca2+ and ceramide 24:0
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Aspergillus fumigatus (mold)
Molecular weightTheoretical: 168 kDa/nm

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Macromolecule #1: Plasma membrane channel protein (Aqy1), putative

MacromoleculeName: Plasma membrane channel protein (Aqy1), putative / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Neosartorya fumigata (strain ATCC MYA-4609 / Af293 / CBS 101355 / FGSC A1100) (mold)
Strain: ATCC MYA-4609 / Af293 / CBS 101355 / FGSC A1100
Molecular weightTheoretical: 84.616859 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast)
SequenceString: MAFNPAPKAV QENHHVDYVI RFNYGDIDTP EAIKKFEVLL LELSEVGLQT EVRQGDENSL FVFVRAASKK KLKRAVYQSR VRDWLYGVR NTEPEPASSA KPQSEAERLL VIYHLITVPK AEGGAGITPR HGEWKNVDAI FPLHDEETNR QCMREWSKKT F LSTEDLDR ...String:
MAFNPAPKAV QENHHVDYVI RFNYGDIDTP EAIKKFEVLL LELSEVGLQT EVRQGDENSL FVFVRAASKK KLKRAVYQSR VRDWLYGVR NTEPEPASSA KPQSEAERLL VIYHLITVPK AEGGAGITPR HGEWKNVDAI FPLHDEETNR QCMREWSKKT F LSTEDLDR IRNTFGEHVG FYFAFLQSYF RFLMFPAAFG FSCWLLLGSF SIIYTVVNCL WCIVFIEYWK RQEEDLSCRW QT KGVSAVH EKRAEFKPEK EIRDESTGEV RGVFPATKRM YRQLLQVPFA LLAAVALGAI IATCFAIEIF ISEVYNGPLK GYL VFIPTI LVSALIPTMS AVLLTVATKL NDYENYETQD AYKVALTQKI FVVNFITSYL PIILTAFVYV PFASRIVPYL DVFH LTVRP FVSKEHAIKA RTEFSINPDR LRKQVIYFTV TAQIVGFALE TIVPFVKQRV FREYKEYTKK QHAKAEPGNG AGEKK TVSL GDDEDEARFL TRVRNEAELE DYDVTDDLRE MCIQFGYLAL FSPVWPLVPV SFLINNWVEL RSDFFKICVE CKRPWP QRA DTIGPWLDSL GFLSWVGSIT SSALVYMFSN GHEGPNGEPT TIRCWALLLT IFFSEHLYLI VRYAVRSALA KLEPPNT RR ERIERFMMRK RYLDTVLSAE SDDDADEVKG VVSSIPPSEI TRESLEQDAR DWSKQGTDPT ERFWMRQRGW KESAEVGL S LITKAKGDET KKQQ

UniProtKB: Plasma membrane channel protein (Aqy1), putative

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Macromolecule #2: CALCIUM ION

MacromoleculeName: CALCIUM ION / type: ligand / ID: 2 / Number of copies: 4 / Formula: CA
Molecular weightTheoretical: 40.078 Da

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Macromolecule #3: DODECANE

MacromoleculeName: DODECANE / type: ligand / ID: 3 / Number of copies: 8 / Formula: D12
Molecular weightTheoretical: 170.335 Da
Chemical component information

ChemComp-D12:
DODECANE

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Macromolecule #4: DECANE

MacromoleculeName: DECANE / type: ligand / ID: 4 / Number of copies: 2 / Formula: D10
Molecular weightTheoretical: 142.282 Da
Chemical component information

ChemComp-D10:
DECANE

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Macromolecule #5: Octadecane

MacromoleculeName: Octadecane / type: ligand / ID: 5 / Number of copies: 2 / Formula: 8K6
Molecular weightTheoretical: 254.494 Da
Chemical component information

ChemComp-8K6:
Octadecane

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Macromolecule #6: HEXANE

MacromoleculeName: HEXANE / type: ligand / ID: 6 / Number of copies: 2 / Formula: HEX
Molecular weightTheoretical: 86.175 Da
Chemical component information

ChemComp-HEX:
HEXANE

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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

Concentration7 mg/mL
BufferpH: 8
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 288 K / Instrument: FEI VITROBOT MARK IV
DetailsafTMEM16 reconstituted in nanodiscs in the absence of Ca2+

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Number grids imaged: 1 / Average electron dose: 62.75 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: OTHER / Imaging mode: BRIGHT FIELD
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

4wis

Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.59 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 24602
Initial angle assignmentType: OTHER
Final angle assignmentType: ANGULAR RECONSTITUTION
FSC plot (resolution estimation)

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