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- PDB-9hdd: Sla2 C-terminal region (Residues 560-968) (REND and THATCH domains) -

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Basic information

Entry
Database: PDB / ID: 9hdd
TitleSla2 C-terminal region (Residues 560-968) (REND and THATCH domains)
ComponentsProtein SLA2
KeywordsENDOCYTOSIS / Actin binding / membrane trafficking
Function / homology
Function and homology information


actin cortical patch assembly / clathrin light chain binding / incipient cellular bud site / negative regulation of Arp2/3 complex-mediated actin nucleation / cellular bud tip / actin cortical patch / clathrin coat assembly / clathrin adaptor activity / cellular bud neck / mating projection tip ...actin cortical patch assembly / clathrin light chain binding / incipient cellular bud site / negative regulation of Arp2/3 complex-mediated actin nucleation / cellular bud tip / actin cortical patch / clathrin coat assembly / clathrin adaptor activity / cellular bud neck / mating projection tip / phosphatidylinositol-3,4-bisphosphate binding / phosphatidylinositol-3,5-bisphosphate binding / clathrin-coated vesicle / cortical actin cytoskeleton / actin filament organization / endocytosis / actin filament binding / plasma membrane
Similarity search - Function
Sla2 family / AP180 N-terminal homology (ANTH) domain / ANTH domain / Epsin N-terminal homology (ENTH) domain / ENTH domain profile. / ENTH domain / I/LWEQ domain / I/LWEQ domain superfamily / I/LWEQ domain / I/LWEQ domain profile. ...Sla2 family / AP180 N-terminal homology (ANTH) domain / ANTH domain / Epsin N-terminal homology (ENTH) domain / ENTH domain profile. / ENTH domain / I/LWEQ domain / I/LWEQ domain superfamily / I/LWEQ domain / I/LWEQ domain profile. / I/LWEQ domain / ENTH/VHS
Similarity search - Domain/homology
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.62 Å
AuthorsDraper-Barr, G. / Gustavsson, E. / Landau, M. / Garcia-Alai, M.M.
Funding support Germany, 1items
OrganizationGrant numberCountry
EIPOD fellowship under Marie Sklodowska-Curie Actions COFUND664726 Germany
CitationJournal: Structure / Year: 2025
Title: Sla2 is a core interaction hub for clathrin light chain and the Pan1/End3/Sla1 complex.
Authors: George Draper-Barr / Lucas A Defelipe / David Ruiz-Carrillo / Emil Gustavsson / Meytal Landau / Maria García-Alai /
Abstract: The interaction network of Sla2, a vital endocytic mid-coat adaptor protein, undergoes constant rearrangement. Sla2 serves as a scaffold linking the membrane to the actin cytoskeleton, with its role ...The interaction network of Sla2, a vital endocytic mid-coat adaptor protein, undergoes constant rearrangement. Sla2 serves as a scaffold linking the membrane to the actin cytoskeleton, with its role modulated by the clathrin light chain (CLC), which inhibits Sla2's function under certain conditions. We show that Sla2 has two independent binding sites for CLC: one previously described in homologs of fungi (Sla2) and metazoa (Hip1R), and a second found only in Fungi. We present the structural model of the Sla2 actin-binding domains in the context of regulatory structural domains by cryoelectron microscopy. We provide an interaction map of Sla2 and the regulatory proteins Sla1 and Pan1, predicted by AI modeling and confirmed by molecular biophysics techniques. Pan1 may compete with CLC for the conserved Sla2-binding site. These results enhance the mapping of crucial interactions at endocytic checkpoints and highlight the divergence between Metazoa and Fungi in this vital process.
History
DepositionNov 12, 2024Deposition site: PDBE / Processing site: PDBE
Revision 1.0May 21, 2025Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Protein SLA2
B: Protein SLA2


Theoretical massNumber of molelcules
Total (without water)139,3572
Polymers139,3572
Non-polymers00
Water00
1


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

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Components

#1: Protein Protein SLA2 / Transmembrane protein MOP2


Mass: 69678.672 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Details: Complete expression construct was residues 351-968 of ScSla2. Only residues 560-968 were able to be modelled into the electron density due to the inherent flexibility of the residues 351-559 ...Details: Complete expression construct was residues 351-968 of ScSla2. Only residues 560-968 were able to be modelled into the electron density due to the inherent flexibility of the residues 351-559 that form the coiled-coil.
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: SLA2, END4, MOP2, UFG1, YNL243W, N1102 / Plasmid: pnEA-vHGST
Details (production host): 6xHis-GST-'TEVcleavagesite'-ProteinOfInterest
Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P33338
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: ScSla2 residues 351-968 / Type: COMPLEX
Details: ScSla2:351-968 forms a dimer through the coiled-coil and REND domain between residues 351-735.
Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.14 MDa / Experimental value: YES
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Source (recombinant)Organism: Escherichia coli (E. coli) / Strain: BL21 DE3 / Plasmid: pnEA
Buffer solutionpH: 8
Details: 0.03 M HEPES pH 8 0.15 M NaCl 0.5 mM TCEP 0.1 um filtered buffer and degassed for one hour at room temperature
Buffer component
IDConc.NameFormulaBuffer-ID
10.03 M2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acidHEPES1
20.15 MSodium ChlorideNaCl1
30.5 mMtris(2-carboxyethyl)phosphineTCEP1
SpecimenConc.: 2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: monodisperse dimers of the ScSla2:351-968 construct
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R2/2
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE-PROPANE / Humidity: 95 % / Chamber temperature: 279 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: SPOT SCAN
Electron lensMode: BRIGHT FIELD / Nominal magnification: 120000 X / Nominal defocus max: 2000 nm / Nominal defocus min: 500 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 45 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1

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Processing

EM software
IDNameVersionCategory
1cryoSPARC4particle selection
2EPUimage acquisition
4CTFFIND4.1CTF correction
7ISOLDEmodel fitting
8UCSF ChimeraXmodel fitting
10PHENIXmodel refinement
11cryoSPARC4initial Euler assignment
12cryoSPARC4final Euler assignment
13cryoSPARC4classification
14cryoSPARC43D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 3.62 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 249299 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL / Target criteria: CC
Atomic model buildingAccession code: AF-P33338-F1 / Chain residue range: 560-968 / Details: two chains of this model / Source name: AlphaFold / Type: in silico model
RefinementResolution: 3.62→3.62 Å / Cor.coef. Fo:Fc: 0.927 / SU B: 29.55 / SU ML: 0.443 / Cross valid method: NONE / ESU R: 0.989
Stereochemistry target values: MAXIMUM LIKELIHOOD WITH PHASES
Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflection
Rwork0.37297 --
obs0.37297 37437 100 %
Solvent computationSolvent model: PARAMETERS FOR MASK CACLULATION
Displacement parametersBiso mean: 255.035 Å2
Refinement stepCycle: 1 / Total: 6296
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
ELECTRON MICROSCOPYr_bond_refined_d0.0090.0126378
ELECTRON MICROSCOPYr_bond_other_d00.0166074
ELECTRON MICROSCOPYr_angle_refined_deg1.8471.8158666
ELECTRON MICROSCOPYr_angle_other_deg0.6211.75614048
ELECTRON MICROSCOPYr_dihedral_angle_1_deg8.0465816
ELECTRON MICROSCOPYr_dihedral_angle_2_deg3.292520
ELECTRON MICROSCOPYr_dihedral_angle_3_deg13.603101158
ELECTRON MICROSCOPYr_dihedral_angle_4_deg
ELECTRON MICROSCOPYr_chiral_restr0.0850.21058
ELECTRON MICROSCOPYr_gen_planes_refined0.0060.027404
ELECTRON MICROSCOPYr_gen_planes_other0.0010.021284
ELECTRON MICROSCOPYr_nbd_refined
ELECTRON MICROSCOPYr_nbd_other
ELECTRON MICROSCOPYr_nbtor_refined
ELECTRON MICROSCOPYr_nbtor_other
ELECTRON MICROSCOPYr_xyhbond_nbd_refined
ELECTRON MICROSCOPYr_xyhbond_nbd_other
ELECTRON MICROSCOPYr_metal_ion_refined
ELECTRON MICROSCOPYr_metal_ion_other
ELECTRON MICROSCOPYr_symmetry_vdw_refined
ELECTRON MICROSCOPYr_symmetry_vdw_other
ELECTRON MICROSCOPYr_symmetry_hbond_refined
ELECTRON MICROSCOPYr_symmetry_hbond_other
ELECTRON MICROSCOPYr_symmetry_metal_ion_refined
ELECTRON MICROSCOPYr_symmetry_metal_ion_other
ELECTRON MICROSCOPYr_mcbond_it34.12825.4123270
ELECTRON MICROSCOPYr_mcbond_other34.12825.4133270
ELECTRON MICROSCOPYr_mcangle_it51.2645.4754084
ELECTRON MICROSCOPYr_mcangle_other51.25545.4854085
ELECTRON MICROSCOPYr_scbond_it3127.2353108
ELECTRON MICROSCOPYr_scbond_other31.00127.2283107
ELECTRON MICROSCOPYr_scangle_it
ELECTRON MICROSCOPYr_scangle_other49.30649.4394583
ELECTRON MICROSCOPYr_long_range_B_refined74.709280.8125075
ELECTRON MICROSCOPYr_long_range_B_other74.708280.8225076
ELECTRON MICROSCOPYr_rigid_bond_restr
ELECTRON MICROSCOPYr_sphericity_free
ELECTRON MICROSCOPYr_sphericity_bonded
LS refinement shellResolution: 3.7→3.796 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0 0 -
Rwork0.909 2786 -
obs--100 %

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