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- PDB-7sox: Cryo-electron tomography structure of membrane-bound EHD4 complex -

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Basic information

Entry
Database: PDB / ID: 7sox
TitleCryo-electron tomography structure of membrane-bound EHD4 complex
ComponentsEH domain-containing protein 4
KeywordsLIPID BINDING PROTEIN / EHD ATPases / dynamin family / oligomerization / membrane remodeling / membrane binding site
Function / homology
Function and homology information


pinocytosis / endocytic recycling / cell migration involved in sprouting angiogenesis / regulation of endocytosis / adherens junction / cellular response to growth factor stimulus / protein homooligomerization / recycling endosome membrane / early endosome membrane / protein-macromolecule adaptor activity ...pinocytosis / endocytic recycling / cell migration involved in sprouting angiogenesis / regulation of endocytosis / adherens junction / cellular response to growth factor stimulus / protein homooligomerization / recycling endosome membrane / early endosome membrane / protein-macromolecule adaptor activity / calcium ion binding / GTP binding / perinuclear region of cytoplasm / endoplasmic reticulum / ATP binding / plasma membrane
Similarity search - Function
EH domain-containing protein, N-terminal / Domain of unknown function DUF5600 / N-terminal EH-domain containing protein / Domain of unknown function (DUF5600) / EH domain / EH domain profile. / Eps15 homology domain / EH domain / Dynamin-type guanine nucleotide-binding (G) domain / Dynamin-type guanine nucleotide-binding (G) domain profile. ...EH domain-containing protein, N-terminal / Domain of unknown function DUF5600 / N-terminal EH-domain containing protein / Domain of unknown function (DUF5600) / EH domain / EH domain profile. / Eps15 homology domain / EH domain / Dynamin-type guanine nucleotide-binding (G) domain / Dynamin-type guanine nucleotide-binding (G) domain profile. / Dynamin, N-terminal / Dynamin family / EF-Hand 1, calcium-binding site / EF-hand calcium-binding domain. / EF-hand calcium-binding domain profile. / EF-hand domain / EF-hand domain pair / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
EH domain-containing protein 4
Similarity search - Component
Biological speciesMus musculus (house mouse)
MethodELECTRON MICROSCOPY / subtomogram averaging / cryo EM / Resolution: 7.6 Å
AuthorsMelo, A.A. / Noel, J.K. / Daumke, O.
Funding support Germany, 3items
OrganizationGrant numberCountry
German Research Foundation (DFG)SFB 958/A12 Germany
European Research Council (ERC)ERC-2013-CoG-616024 Germany
European Union (EU)iNEXT PID3536 VID5570 Germany
CitationJournal: Nat Commun / Year: 2022
Title: Cryo-electron tomography reveals structural insights into the membrane remodeling mode of dynamin-like EHD filaments.
Authors: Arthur A Melo / Thiemo Sprink / Jeffrey K Noel / Elena Vázquez-Sarandeses / Chris van Hoorn / Saif Mohd / Justus Loerke / Christian M T Spahn / Oliver Daumke /
Abstract: Eps15-homology domain containing proteins (EHDs) are eukaryotic, dynamin-related ATPases involved in cellular membrane trafficking. They oligomerize on membranes into filaments that induce membrane ...Eps15-homology domain containing proteins (EHDs) are eukaryotic, dynamin-related ATPases involved in cellular membrane trafficking. They oligomerize on membranes into filaments that induce membrane tubulation. While EHD crystal structures in open and closed conformations were previously reported, little structural information is available for the membrane-bound oligomeric form. Consequently, mechanistic insights into the membrane remodeling mechanism have remained sparse. Here, by using cryo-electron tomography and subtomogram averaging, we determined structures of nucleotide-bound EHD4 filaments on membrane tubes of various diameters at an average resolution of 7.6 Å. Assembly of EHD4 is mediated via interfaces in the G-domain and the helical domain. The oligomerized EHD4 structure resembles the closed conformation, where the tips of the helical domains protrude into the membrane. The variation in filament geometry and tube radius suggests a spontaneous filament curvature of approximately 1/70 nm. Combining the available structural and functional data, we suggest a model for EHD-mediated membrane remodeling.
History
DepositionNov 1, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Nov 9, 2022Provider: repository / Type: Initial release
Revision 1.1Jan 25, 2023Group: Database references / Refinement description
Category: citation / citation_author / pdbx_initial_refinement_model
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year
Revision 1.2Jun 5, 2024Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_3d_fitting_list / struct_ncs_dom_lim
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 / _struct_ncs_dom_lim.beg_auth_asym_id / _struct_ncs_dom_lim.beg_auth_comp_id / _struct_ncs_dom_lim.beg_auth_seq_id / _struct_ncs_dom_lim.end_auth_asym_id / _struct_ncs_dom_lim.end_auth_comp_id / _struct_ncs_dom_lim.end_auth_seq_id

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: EH domain-containing protein 4
B: EH domain-containing protein 4
C: EH domain-containing protein 4
D: EH domain-containing protein 4
E: EH domain-containing protein 4
F: EH domain-containing protein 4
G: EH domain-containing protein 4
H: EH domain-containing protein 4
I: EH domain-containing protein 4
J: EH domain-containing protein 4
K: EH domain-containing protein 4
L: EH domain-containing protein 4
M: EH domain-containing protein 4
N: EH domain-containing protein 4
O: EH domain-containing protein 4
P: EH domain-containing protein 4


Theoretical massNumber of molelcules
Total (without water)938,78716
Polymers938,78716
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
d_1ens_1chain "A"
d_2ens_1chain "B"
d_3ens_1chain "C"
d_4ens_1chain "D"
d_5ens_1chain "E"
d_6ens_1chain "F"
d_7ens_1chain "G"
d_8ens_1chain "H"
d_9ens_1chain "I"
d_10ens_1chain "J"
d_11ens_1chain "K"
d_12ens_1chain "L"
d_13ens_1chain "M"
d_14ens_1chain "N"
d_15ens_1chain "O"
d_16ens_1chain "P"

NCS domain segments:

Component-ID: 1 / Ens-ID: ens_1 / Beg auth comp-ID: GLN / Beg label comp-ID: GLN / End auth comp-ID: LYS / End label comp-ID: LYS / Auth seq-ID: 22 - 535 / Label seq-ID: 1 - 514

Dom-IDAuth asym-IDLabel asym-ID
d_1AA
d_2BB
d_3CC
d_4DD
d_5EE
d_6FF
d_7GG
d_8HH
d_9II
d_10JJ
d_11KK
d_12LL
d_13MM
d_14NN
d_15OO
d_16PP

NCS oper:
IDCodeMatrixVector
1given(-0.999994521746, 0.00330602726823, -0.000163284092328), (-0.00328448844174, -0.997181535568, -0.0749546346638), (-0.000410625948, -0.0749536877385, 0.997186931363)273.544002566, 232.82451905, 9.19884717512
2given(0.99993552633, -0.00822999468006, -0.0078237056704), (0.00880402828004, 0.99703721782, 0.0764151514305), (0.00717162944491, -0.0764791047913, 0.997045392277)-19.9034632436, 18.7771608564, 8.19825565895
3given(-0.999995233839, 0.00256552995858, -0.00171765964133), (-0.00256355067956, -0.999996048788, -0.00115352326756), (-0.00172061225299, -0.00114911446214, 0.999997859512)252.114959235, 252.989369571, 0.381332329905
4given(0.999998605391, 0.00149526772557, -0.000743902137706), (-0.00136955194673, 0.989117262771, 0.147122958155), (0.000955794657237, -0.147121734164, 0.989117931186)-43.1649618845, 41.4077856394, 15.5644703204
5given(-0.999923967925, 0.0105818903591, -0.00633103188613), (-0.0110539479464, -0.996743816209, 0.0798722422654), (-0.00546521757234, 0.0799361523101, 0.996785003374)230.205816897, 273.073134765, -10.1293634225
6given(0.999985339139, -0.0046316636227, -0.00280520934922), (0.00515039004969, 0.973494834449, 0.228651002148), (0.00167182228208, -0.22866209785, 0.973504417051)-64.3180870754, 61.1393974929, 22.1358272808
7given(-0.999867504016, 0.0160172598066, -0.00290203409713), (-0.0149626064088, -0.974553569595, -0.223654779512), (-0.00641052439887, -0.223581724158, 0.974664105115)315.978739468, 191.605119292, 23.0206599712
8given(0.999943494771, -0.0100167554869, 0.0035597577027), (0.0102659089838, 0.996844957772, -0.0787066787285), (-0.00276014096082, 0.0787387755382, 0.99689146192)22.0055570408, -20.5528108076, -10.3248240844
9given(-0.999990484044, 0.00271869650153, 0.00341181935149), (-0.00318354580311, -0.989482315434, -0.144618852437), (0.00298276014358, -0.144628337933, 0.989481554658)294.74298869, 211.372983888, 15.3470440417
10given(0.999814389562, -0.0133497969855, -0.0138913406858), (0.0149571288062, 0.992306152334, 0.122901522931), (0.0121437524464, -0.123086485698, 0.992321644587)86.6548988507, 40.0267669448, 11.7932503793
11given(0.999648898684, 0.0260142321194, 0.00503379459694), (-0.0264824835354, 0.974674240935, 0.222055853613), (0.000870302591559, -0.222111196893, 0.975020953)59.2954623947, 64.1720029096, 21.0620292338
12given(-0.999997800742, 0.00093971473767, -0.00187495250799), (-0.000532002982541, -0.978417837653, -0.206635553413), (-0.00202866545344, -0.206634101488, 0.978416185792)190.30711266, 190.123097482, 19.7285572767
13given(-0.999966733132, 0.00327355370362, 0.00747104246189), (-0.00416572286226, -0.992433351395, -0.122713853297), (0.00701280131992, -0.122740893284, 0.992413972964)167.130221393, 211.257616311, 12.5139276294
14given(-0.999037400405, -0.0438653194904, -0.000326095142417), (0.0422417309242, -0.960002357325, -0.276787120542), (0.011828303368, -0.276534460196, 0.960931206468)215.548766477, 162.981464219, 22.5137460122
15given(0.998566599625, 0.0526421545172, -0.00967210840659), (-0.0476577633322, 0.956747432409, 0.286989700464), (0.0243615210407, -0.286117378267, 0.957884837622)39.6621226483, 87.8070588489, 22.287060085

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Components

#1: Protein
EH domain-containing protein 4 / PAST homolog 2 / mPAST2


Mass: 58674.195 Da / Num. of mol.: 16
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Gene: Ehd4, Past2 / Production host: Escherichia coli (E. coli) / References: UniProt: Q9EQP2

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: HELICAL ARRAY / 3D reconstruction method: subtomogram averaging

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Sample preparation

ComponentName: EHD4 with AMPPNP and liposomes / Type: ORGANELLE OR CELLULAR COMPONENT / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.59 kDa/nm / Experimental value: YES
Source (natural)Organism: Mus musculus (house mouse)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
SpecimenConc.: 0.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil
VitrificationInstrument: FEI VITROBOT MARK II / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 298 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
Image recordingElectron dose: 2.3 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 QUANTUM (4k x 4k)

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Processing

Software
NameVersionClassificationNB
phenix.real_space_refine1.19.2_4158refinement
PHENIX1.19.2_4158refinement
EM software
IDNameCategory
1Dynamovolume selection
2SerialEMimage acquisition
4CTFFINDCTF correction
7VMDmodel fitting
10Dynamofinal Euler assignment
11Dynamoclassification
12IMOD3D reconstruction
13PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING ONLY
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 7.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 23813 / Algorithm: BACK PROJECTION / Symmetry type: POINT
EM volume selectionNum. of tomograms: 56 / Num. of volumes extracted: 84000
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Atomic model buildingPDB-ID: 4CID

4cid


Accession code: 4CID / Source name: PDB / Type: experimental model
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 336.99 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.003867440
ELECTRON MICROSCOPYf_angle_d0.821891008
ELECTRON MICROSCOPYf_chiral_restr0.04799808
ELECTRON MICROSCOPYf_plane_restr0.008211888
ELECTRON MICROSCOPYf_dihedral_angle_d6.06978928
Refine LS restraints NCS
Ens-IDDom-IDAuth asym-IDRefine-IDTypeRms dev position (Å)
ens_1d_2AELECTRON MICROSCOPYNCS constraints0.000711701747557
ens_1d_3AELECTRON MICROSCOPYNCS constraints0.000710655326437
ens_1d_4AELECTRON MICROSCOPYNCS constraints0.000711374360405
ens_1d_5AELECTRON MICROSCOPYNCS constraints0.000709525471907
ens_1d_6AELECTRON MICROSCOPYNCS constraints0.000709259819366
ens_1d_7AELECTRON MICROSCOPYNCS constraints0.000708728316589
ens_1d_8AELECTRON MICROSCOPYNCS constraints0.000705692056827
ens_1d_9AELECTRON MICROSCOPYNCS constraints0.000711772418427
ens_1d_10AELECTRON MICROSCOPYNCS constraints0.000713202550048
ens_1d_11AELECTRON MICROSCOPYNCS constraints0.000715071413958
ens_1d_12AELECTRON MICROSCOPYNCS constraints0.000707757802473
ens_1d_13AELECTRON MICROSCOPYNCS constraints0.000713589131804
ens_1d_14AELECTRON MICROSCOPYNCS constraints0.00070989918752
ens_1d_15AELECTRON MICROSCOPYNCS constraints0.000706125218704
ens_1d_16AELECTRON MICROSCOPYNCS constraints0.000709164807945

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