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- EMDB-41453: nhTMEM16 lipid scramblase in lipid nanodiscs with MSP1E3 scaffold... -

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Basic information

Entry
Database: EMDB / ID: EMD-41453
TitlenhTMEM16 lipid scramblase in lipid nanodiscs with MSP1E3 scaffold protein in the presence of Ca2+ (closed state)
Map datacomposite map, used for model building
Sample
  • Complex: Dimeric lipid scramblase nhTMEM16
    • Protein or peptide: Lipid scramblase nhTMEM16
  • Ligand: CALCIUM IONCalcium
  • Ligand: (1R)-2-{[(S)-{[(2S)-2,3-dihydroxypropyl]oxy}(hydroxy)phosphoryl]oxy}-1-[(hexadecanoyloxy)methyl]ethyl (9Z)-octadec-9-enoate
Keywordsmembrane protein / lipid scramblase / TMEM16 / LIPID TRANSPORT
Function / homology: / Alpha-beta plait domain in TMEM16 lipid scramblase / Anoctamin / : / Calcium-activated chloride channel / membrane / identical protein binding / metal ion binding / Plasma membrane channel protein
Function and homology information
Biological speciesFusarium vanettenii 77-13-4 (fungus)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.64 Å
AuthorsFeng Z / Accardi A
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM106717 United States
CitationJournal: Nat Struct Mol Biol / Year: 2024
Title: Structural basis of closed groove scrambling by a TMEM16 protein.
Authors: Zhang Feng / Omar E Alvarenga / Alessio Accardi /
Abstract: Activation of Ca-dependent TMEM16 scramblases induces phosphatidylserine externalization, a key step in multiple signaling processes. Current models suggest that the TMEM16s scramble lipids by ...Activation of Ca-dependent TMEM16 scramblases induces phosphatidylserine externalization, a key step in multiple signaling processes. Current models suggest that the TMEM16s scramble lipids by deforming the membrane near a hydrophilic groove and that Ca dependence arises from the different association of lipids with an open or closed groove. However, the molecular rearrangements underlying groove opening and how lipids reorganize outside the closed groove remain unknown. Here we directly visualize how lipids associate at the closed groove of Ca-bound fungal nhTMEM16 in nanodiscs using cryo-EM. Functional experiments pinpoint lipid-protein interaction sites critical for closed groove scrambling. Structural and functional analyses suggest groove opening entails the sequential appearance of two π-helical turns in the groove-lining TM6 helix and identify critical rearrangements. Finally, we show that the choice of scaffold protein and lipids affects the conformations of nhTMEM16 and their distribution, highlighting a key role of these factors in cryo-EM structure determination.
History
DepositionAug 3, 2023-
Header (metadata) releaseMay 8, 2024-
Map releaseMay 8, 2024-
UpdateMay 8, 2024-
Current statusMay 8, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_41453.png

Downloads & links

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Map

FileDownload / File: emd_41453.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationcomposite map, used for model building
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 256 pix.
= 211.2 Å		0.83 Å/pix.
x 256 pix.
= 211.2 Å		0.83 Å/pix.
x 256 pix.
= 211.2 Å

Surface

Projections

Slices (1/3)

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.825 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 1.1
Minimum - Maximum-6.858558 - 17.543334999999999
Average (Standard dev.)0.056860726 (±0.50779617)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 211.2 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: unsharpened map, used to assist model building

Fileemd_41453_additional_1.map
Annotationunsharpened map, used to assist model building
Projections & Slices
AxesZYX

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Histogram

Histogram (log scale)

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Additional map: local refined monomer map, used to assist model building

Fileemd_41453_additional_2.map
Annotationlocal refined monomer map, used to assist model building
Projections & Slices
AxesZYX

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Half map: halfmap1

Fileemd_41453_half_map_1.map
Annotationhalfmap1
Projections & Slices
AxesZYX

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Histogram

Histogram (log scale)

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Half map: halfmap2

Fileemd_41453_half_map_2.map
Annotationhalfmap2
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Sample components

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Entire : Dimeric lipid scramblase nhTMEM16

EntireName: Dimeric lipid scramblase nhTMEM16
Components
  • Complex: Dimeric lipid scramblase nhTMEM16
    • Protein or peptide: Lipid scramblase nhTMEM16
  • Ligand: CALCIUM IONCalcium
  • Ligand: (1R)-2-{[(S)-{[(2S)-2,3-dihydroxypropyl]oxy}(hydroxy)phosphoryl]oxy}-1-[(hexadecanoyloxy)methyl]ethyl (9Z)-octadec-9-enoate

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Supramolecule #1: Dimeric lipid scramblase nhTMEM16

SupramoleculeName: Dimeric lipid scramblase nhTMEM16 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Fusarium vanettenii 77-13-4 (fungus)

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Macromolecule #1: Lipid scramblase nhTMEM16

MacromoleculeName: Lipid scramblase nhTMEM16 / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Fusarium vanettenii 77-13-4 (fungus)
Molecular weightTheoretical: 83.294062 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast)
SequenceString: GPSNLKDFSQ PGSGQESNFG VDFVIHYKVP AAERDEAEAG FVQLIRALTT VGLATEVRHG ENESLLVFVK VASPDLFAKQ VYRARLGDW LHGVRVSAPH NDIAQALQDE PVVEAERLRL IYLMITKPHN EGGAGVTPTN AKWKHVESIF PLHSHSFNKE W IKKWSSKY ...String:
GPSNLKDFSQ PGSGQESNFG VDFVIHYKVP AAERDEAEAG FVQLIRALTT VGLATEVRHG ENESLLVFVK VASPDLFAKQ VYRARLGDW LHGVRVSAPH NDIAQALQDE PVVEAERLRL IYLMITKPHN EGGAGVTPTN AKWKHVESIF PLHSHSFNKE W IKKWSSKY TLEQTDIDNI RDKFGESVAF YFAFLRSYFR FLVIPSAFGF GAWLLLGQFS YLYALLCGLW SVVFFEYWKK QE VDLAVQW GVRGVSSIQQ SRPEFEWEHE AEDPITGEPV KVYPPMKRVK TQLLQIPFAL ACVVALGALI VTCNSLEVFI NEV YSGPGK QYLGFLPTIF LVIGTPTISG VLMGAAEKLN AMENYATVDA HDAALIQKQF VLNFMTSYMA LFFTAFVYIP FGHI LHPFL NFWRATAQTL TFSEKELPTR EFQINPARIS NQMFYFTVTA QIVNFATEVV VPYIKQQAFQ KAKQLKSGSK VQEDH EEEA EFLQRVREEC TLEEYDVSGD YREMVMQFGY VAMFSVAWPL AACCFLVNNW VELRSDALKI AISSRRPIPW RTDSIG PWL TALSFLSWLG SITSSAIVYL CSNSKNGTQG EASPLKAWGL LLSILFAEHF YLVVQLAVRF VLSKLDSPGL QKERKER FQ TKKRLLQENL GQDAAEEAAA PGIEHSEKIT REALEEEARQ ASIRGHGTPE EMFWQRQRGM QETIEIGRRM IEQQLAAG K NGKKSAPAVP SEKASA

UniProtKB: Plasma membrane channel protein

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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: (1R)-2-{[(S)-{[(2S)-2,3-dihydroxypropyl]oxy}(hydroxy)phosphoryl]o...

MacromoleculeName: (1R)-2-{[(S)-{[(2S)-2,3-dihydroxypropyl]oxy}(hydroxy)phosphoryl]oxy}-1-[(hexadecanoyloxy)methyl]ethyl (9Z)-octadec-9-enoate
type: ligand / ID: 3 / Number of copies: 36 / Formula: PGW
Molecular weightTheoretical: 749.007 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

Concentration5 mg/mL
BufferpH: 8
Component:
ConcentrationNameFormula
50.0 mMTris
150.0 mMKCl
0.5 mMCaCl2
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 288 K / Instrument: FEI VITROBOT MARK IV
DetailsThis sample was monodisperse

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: OTHER / Imaging mode: OTHER / Nominal defocus max: 1.9000000000000001 µm / Nominal defocus min: 0.9 µm
Specialist opticsEnergy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number real images: 13735 / Average exposure time: 2.0 sec. / Average electron dose: 59.4 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 4574959
Startup modelType of model: NONE
Initial angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION (ver. 3.1.2)
Final 3D classificationSoftware - Name: RELION (ver. 3.1.2)
Final angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION (ver. 3.1.2)
Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 2.64 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1.2) / Number images used: 1017312
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

6qm9


Chain - Source name: PDB / Chain - Initial model type: experimental model
Output model

PDB-8toi:
nhTMEM16 lipid scramblase in lipid nanodiscs with MSP1E3 scaffold protein in the presence of Ca2+ (closed state)

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