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- PDB-7fif: Cryo-EM structure of the hedgehog release protein Disp from water... -

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Basic information

Entry
Database: PDB / ID: 7fif
TitleCryo-EM structure of the hedgehog release protein Disp from water bear (Hypsibius dujardini)
ComponentsProtein dispatched-like protein 1
KeywordsMEMBRANE PROTEIN / cryo-EM / Dispatched / Hedgehog release / Hedgehog signaling / setrol sensing domain / water bear
Function / homologyMembrane transport protein MMPL domain / MMPL family / Sterol-sensing domain (SSD) profile. / Sterol-sensing domain / membrane => GO:0016020 / plasma membrane / Protein dispatched-like protein 1
Function and homology information
Biological speciesHypsibius dujardini (invertebrata)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 6.5 Å
AuthorsLuo, Y. / Wan, G. / Wang, Q. / Zhao, Y. / Cong, Y. / Li, D.
Funding support China, 6items
OrganizationGrant numberCountry
Chinese Academy of SciencesXDB37020204 China
Chinese Academy of SciencesXDB19020100 China
Chinese Academy of SciencesQYZDB SSW SMC037 China
National Natural Science Foundation of China (NSFC)31870726 China
National Natural Science Foundation of China (NSFC)31630047 China
National Natural Science Foundation of China (NSFC)31771610 China
CitationJournal: Front Mol Biosci / Year: 2021
Title: Architecture of Dispatched, a Transmembrane Protein Responsible for Hedgehog Release.
Authors: Yitian Luo / Guoyue Wan / Xuan Zhou / Qiuwen Wang / Yunbin Zhang / Juan Bao / Yao Cong / Yun Zhao / Dianfan Li /
Abstract: The evolutionarily conserved Hedgehog (Hh) signaling pathway is crucial for programmed cell differentiation and proliferation. Dispatched (Disp) is a 12-transmembrane protein that plays a critical ...The evolutionarily conserved Hedgehog (Hh) signaling pathway is crucial for programmed cell differentiation and proliferation. Dispatched (Disp) is a 12-transmembrane protein that plays a critical role in the Hedgehog (Hh) signaling pathway by releasing the dually lipidated ligand HhN from the membrane, a prerequisite step to the downstream signaling cascade. In this study, we focus on the Disp from water bear, a primitive animal known as the most indestructible on Earth. Using a zebrafish model, we show that the water bear homolog possesses the function of Disp. We have solved its structure to a 6.5-Å resolution using single-particle cryogenic electron microscopy. Consistent with the evolutional conservation of the pathway, the water bear Disp structure is overall similar to the previously reported structures of the fruit fly and human homologs. Although not revealing much detail at this resolution, the water bear Disp shows a different conformation compared to published structures, suggesting that they represent different functional snapshots.
History
DepositionJul 31, 2021Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Sep 8, 2021Provider: repository / Type: Initial release
Revision 1.1Feb 16, 2022Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _citation_author.name
Revision 1.2Jun 12, 2024Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_3d_fitting_list / pdbx_initial_refinement_model
Item: _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id ..._em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type

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Structure visualization

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Assembly

Deposited unit
A: Protein dispatched-like protein 1


Theoretical massNumber of molelcules
Total (without water)124,9431
Polymers124,9431
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Protein dispatched-like protein 1


Mass: 124943.414 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Hypsibius dujardini (invertebrata) / Gene: BV898_06340 / Cell line (production host): Expi293 / Production host: Homo sapiens (human) / References: UniProt: A0A1W0WWK2

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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: Dispatched / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 130 kDa/nm / Experimental value: YES
Source (natural)Organism: Hypsibius dujardini (invertebrata)
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
1150 mMsodium chlorideNaCl1
220 mM(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)HEPES1
30.1 %digitoninC56H92O291
40.5 mMEthylenediaminetetraacetic acidEDTA1
SpecimenConc.: 3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 22500 X / Nominal defocus max: 2500 nm / Nominal defocus min: 1500 nm / Calibrated defocus min: 900 nm / Calibrated defocus max: 3000 nm / Cs: 0.01 mm / C2 aperture diameter: 100 µm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 7.6 sec. / Electron dose: 60.8 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 3 / Num. of real images: 5380

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Processing

SoftwareName: PHENIX / Version: 1.18.2_3874: / Classification: refinement
EM software
IDNameVersionCategory
1RELION3particle selection
2SerialEMimage acquisition
4Gctf1.1.8CTF correction
7Cootmodel fitting
9RELION3initial Euler assignment
10RELION3final Euler assignment
11RELION3classification
12RELION33D reconstruction
13PHENIXmodel refinement
CTF correctionType: NONE
3D reconstructionResolution: 6.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 62850 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Atomic model buildingPDB-ID: 6TBU

6tbu


Accession code: 6TBU / Source name: PDB / Type: experimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.014094
ELECTRON MICROSCOPYf_angle_d1.2715709
ELECTRON MICROSCOPYf_dihedral_angle_d7.59212
ELECTRON MICROSCOPYf_chiral_restr0.042777
ELECTRON MICROSCOPYf_plane_restr0.0081617

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