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- PDB-9qls: Human dysferlin (1-2017) in the lipid-free, Ca2+-bound state -

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Basic information

Entry
Database: PDB / ID: 9qls
TitleHuman dysferlin (1-2017) in the lipid-free, Ca2+-bound state
ComponentsDysferlin
KeywordsMEMBRANE PROTEIN / Ferlins / dysferlin / multi-C2 domains / lipid nanodisc
Function / homology
Function and homology information


monocyte activation involved in immune response / regulation of neurotransmitter secretion / calcium-dependent phospholipid binding / macrophage activation involved in immune response / negative regulation of phagocytosis / endocytic vesicle / Smooth Muscle Contraction / T-tubule / cytoplasmic vesicle membrane / sarcolemma ...monocyte activation involved in immune response / regulation of neurotransmitter secretion / calcium-dependent phospholipid binding / macrophage activation involved in immune response / negative regulation of phagocytosis / endocytic vesicle / Smooth Muscle Contraction / T-tubule / cytoplasmic vesicle membrane / sarcolemma / phospholipid binding / centriolar satellite / synaptic vesicle membrane / late endosome / early endosome / endosome / calcium ion binding / extracellular exosome / plasma membrane
Similarity search - Function
Ferlin A-domain / FerA (NUC095) domain / FerA / Ferlin B-domain / FerIin domain / Ferlin, C-terminal domain / Ferlin, second C2 domain / Ferlin family / Ferlin, third C2 domain / Ferlin, fourth C2 domain ...Ferlin A-domain / FerA (NUC095) domain / FerA / Ferlin B-domain / FerIin domain / Ferlin, C-terminal domain / Ferlin, second C2 domain / Ferlin family / Ferlin, third C2 domain / Ferlin, fourth C2 domain / Ferlin, fifth C2 domain / Ferlin, sixth C2 domain / Ferlin, first C2 domain / : / FerB (NUC096) domain / FerI (NUC094) domain / Ferlin C-terminus / Ferlin dsRNA-binding domain-like domain / FerB / FerI / Peroxin/Ferlin domain / Dysferlin domain, N-terminal region. / Dysferlin domain, C-terminal region. / Protein kinase C conserved region 2 (CalB) / C2 domain / C2 domain / C2 domain profile. / C2 domain superfamily
Similarity search - Domain/homology
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.54 Å
AuthorsCretu, C. / Moser, T.
Funding support Germany, 1items
OrganizationGrant numberCountry
German Research Foundation (DFG)CR 937/2-1 and EXC 2067/1-390729940 Germany
CitationJournal: EMBO J / Year: 2025
Title: Structural insights into lipid membrane binding by human ferlins.
Authors: Constantin Cretu / Aleksandar Chernev / Csaba Zoltán Kibédi Szabó / Vladimir Pena / Henning Urlaub / Tobias Moser / Julia Preobraschenski /
Abstract: Ferlins are ancient membrane proteins with a unique architecture, and play central roles in crucial processes that involve Ca-dependent vesicle fusion. Despite their links to multiple human diseases ...Ferlins are ancient membrane proteins with a unique architecture, and play central roles in crucial processes that involve Ca-dependent vesicle fusion. Despite their links to multiple human diseases and numerous functional studies, a mechanistic understanding of how these multi-C domain-containing proteins interact with lipid membranes to promote membrane remodelling and fusion is currently lacking. Here we obtain near-complete cryo-electron microscopy structures of human myoferlin and dysferlin in their Ca- and lipid-bound states. We show that ferlins adopt compact, ring-like tertiary structures upon membrane binding. The top arch of the ferlin ring, composed of the CC-CD region, is rigid and exhibits only little variability across the observed functional states. In contrast, the N-terminal CB and the C-terminal CF-CG domains cycle between alternative conformations and, in response to Ca, close the ferlin ring, promoting tight interaction with the target membrane. Probing key domain interfaces validates the observed architecture, and informs a model of how ferlins engage lipid bilayers in a Ca-dependent manner. This work reveals the general principles of human ferlin structures and provides a framework for future analyses of ferlin-dependent cellular functions and disease mechanisms.
History
DepositionMar 21, 2025Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jun 4, 2025Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Dysferlin
hetero molecules


Theoretical massNumber of molelcules
Total (without water)234,6247
Polymers234,3831
Non-polymers2406
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein Dysferlin / Dystrophy-associated fer-1-like protein / Fer-1-like protein 1


Mass: 234383.266 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: Cytosolic domain of human dysferlin (residues 1-2017), cloned in frame with an N-terminal twin-StrepII affinity tag.
Source: (gene. exp.) Homo sapiens (human) / Gene: DYSF, FER1L1 / Plasmid: pCC17
Details (production host): twin-StrepII-HRV3C-Dysferlin(1-2017)
Cell (production host): Hi5 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: O75923
#2: Chemical
ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: Ca / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY
Has protein modificationN

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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: Lipid-free human dysferlin (1-2017) in the Ca2+-bound state
Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm)
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
120 mM4-(2-Hydroxyethyl)-1-piperazine ethanesulfonic acidHEPES1
2150 mMpotassium chlorideKCl1
32.5 % (v/v)glycerolC3H8O31
41 mMDithiothreitolDTT1
51 mMcalcium chlorideCaCl21
SpecimenConc.: 1.08 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: Prior to cryo-EM grid preparation, the sample was subjected to GraFix, in the presence of 1 mM CaCl2, buffer exchanged and concentrated to A280~1.08. After the application of 3 uL sample, ...Details: Prior to cryo-EM grid preparation, the sample was subjected to GraFix, in the presence of 1 mM CaCl2, buffer exchanged and concentrated to A280~1.08. After the application of 3 uL sample, the grid was blotted for 3 s at a blot force of 5 and frozen in liquid ethane cooled by liquid nitrogen.
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: UltrAuFoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 K

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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 magnification: 165000 X / Nominal defocus max: 2300 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 3.01 sec. / Electron dose: 40.184 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 15910
Details: Two datasets were collected from the same grid: (i) Dataset 1: 8641 movies, 4582 movies were accepted after curation, total fluence on sample 40.184 e-/A2 (3.01 s per exposure) (ii) Dataset ...Details: Two datasets were collected from the same grid: (i) Dataset 1: 8641 movies, 4582 movies were accepted after curation, total fluence on sample 40.184 e-/A2 (3.01 s per exposure) (ii) Dataset 2: 7269 movies, 4727 movies were accepted after curation, total fluence on sample 40.227 e-/A2 (2.96 s per exposure)
EM imaging opticsEnergyfilter name: TFS Selectris
Details: Energy filter slit width was set to either 15 eV (Dataset 1) or 10 eV (Dataset 2)
Energyfilter slit width: 15 eV

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Processing

EM software
IDNameVersionCategory
1cryoSPARCv.4.1particle selection
2EPUimage acquisition
4cryoSPARCv.4.1CTF correction
7UCSF Chimerav.1.16model fitting
8ISOLDEmodel fitting
10cryoSPARCv.4.1initial Euler assignment
11cryoSPARCv.4.1final Euler assignment
12RELIONv.4.0classification
13cryoSPARCv.4.13D reconstruction
14PHENIX1.21.2_5419model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 2861382
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.54 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 29793 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingB value: 162.24 / Protocol: AB INITIO MODEL / Space: REAL
Atomic model buildingSource name: AlphaFold / Type: in silico model
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 173.97 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.003811379
ELECTRON MICROSCOPYf_angle_d0.915515430
ELECTRON MICROSCOPYf_chiral_restr0.05211656
ELECTRON MICROSCOPYf_plane_restr0.0081995
ELECTRON MICROSCOPYf_dihedral_angle_d6.17251506

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