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- EMDB-2701: Human dynamin 1 K44A superconstricted polymer stabilized with GTP -

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Basic information

Entry
Database: EMDB / ID: EMD-2701
TitleHuman dynamin 1 K44A superconstricted polymer stabilized with GTP
Map dataReconstruction of a dynamin mutant, K44A, bound to DOPS lipid tube
Sample
  • Sample: GTP-stablized human dynamin 1 K44A dynamin polymer bound to DOPS lipid bilayer
  • Protein or peptide: Human dynamin 1DNM1
KeywordsDynamin / endocytosis / membrane fission / GTPase / intracellular trafficking
Function / homology
Function and homology information


clathrin coat assembly involved in endocytosis / vesicle scission / synaptic vesicle budding from presynaptic endocytic zone membrane / presynaptic endocytic zone membrane / dynamin GTPase / regulation of vesicle size / photoreceptor ribbon synapse / Toll Like Receptor 4 (TLR4) Cascade / Formation of annular gap junctions / Retrograde neurotrophin signalling ...clathrin coat assembly involved in endocytosis / vesicle scission / synaptic vesicle budding from presynaptic endocytic zone membrane / presynaptic endocytic zone membrane / dynamin GTPase / regulation of vesicle size / photoreceptor ribbon synapse / Toll Like Receptor 4 (TLR4) Cascade / Formation of annular gap junctions / Retrograde neurotrophin signalling / Gap junction degradation / endosome organization / membrane coat / Recycling pathway of L1 / phosphatidylinositol-3,4,5-trisphosphate binding / synaptic vesicle endocytosis / endocytic vesicle / EPH-ephrin mediated repulsion of cells / clathrin-coated pit / phosphatidylinositol-4,5-bisphosphate binding / MHC class II antigen presentation / photoreceptor inner segment / receptor-mediated endocytosis / modulation of chemical synaptic transmission / protein homooligomerization / receptor internalization / endocytosis / GDP binding / protein self-association / presynapse / Clathrin-mediated endocytosis / protein homotetramerization / microtubule binding / microtubule / GTPase activity / glutamatergic synapse / GTP binding / protein kinase binding / protein homodimerization activity / RNA binding / extracellular exosome / membrane / identical protein binding / plasma membrane / cytoplasm
Similarity search - Function
Dynamin GTPase effector / Dynamin GTPase effector domain / Dynamin GTPase effector domain / Dynamin, GTPase region, conserved site / Dynamin-type guanine nucleotide-binding (G) domain signature. / Dynamin stalk domain / Dynamin central region / GTPase effector domain / GED domain profile. / Dynamin, GTPase domain ...Dynamin GTPase effector / Dynamin GTPase effector domain / Dynamin GTPase effector domain / Dynamin, GTPase region, conserved site / Dynamin-type guanine nucleotide-binding (G) domain signature. / Dynamin stalk domain / Dynamin central region / GTPase effector domain / GED domain profile. / Dynamin, GTPase domain / Dynamin, GTPase / Dynamin / Dynamin-type guanine nucleotide-binding (G) domain / Dynamin-type guanine nucleotide-binding (G) domain profile. / Dynamin, N-terminal / Dynamin family / PH domain / PH domain profile. / Pleckstrin homology domain. / Pleckstrin homology domain / PH-like domain superfamily / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Biological speciesHomo sapiens (human)
Methodhelical reconstruction / cryo EM / Resolution: 12.5 Å
AuthorsSundborger AC / Fang S / Heymann JA / Ray P / Chappie JS / Hinshaw JE
CitationJournal: Cell Rep / Year: 2014
Title: A dynamin mutant defines a superconstricted prefission state.
Authors: Anna C Sundborger / Shunming Fang / Jürgen A Heymann / Pampa Ray / Joshua S Chappie / Jenny E Hinshaw /
Abstract: Dynamin is a 100 kDa GTPase that organizes into helical assemblies at the base of nascent clathrin-coated vesicles. Formation of these oligomers stimulates the intrinsic GTPase activity of dynamin, ...Dynamin is a 100 kDa GTPase that organizes into helical assemblies at the base of nascent clathrin-coated vesicles. Formation of these oligomers stimulates the intrinsic GTPase activity of dynamin, which is necessary for efficient membrane fission during endocytosis. Recent evidence suggests that the transition state of dynamin's GTP hydrolysis reaction serves as a key determinant of productive fission. Here, we present the structure of a transition-state-defective dynamin mutant K44A trapped in a prefission state at 12.5 Å resolution. This structure constricts to 3.7 nm, reaching the theoretical limit required for spontaneous membrane fission. Computational docking indicates that the ground-state conformation of the dynamin polymer is sufficient to achieve this superconstricted prefission state and reveals how a two-start helical symmetry promotes the most efficient packing of dynamin tetramers around the membrane neck. These data suggest a model for the assembly and regulation of the minimal dynamin fission machine.
History
DepositionJul 8, 2014-
Header (metadata) releaseAug 6, 2014-
Map releaseAug 13, 2014-
UpdateSep 3, 2014-
Current statusSep 3, 2014Processing site: PDBe / Status: Released

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Structure visualization

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  • Surface view with section colored by density value
  • Surface level: 3
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  • Surface view colored by cylindrical radius
  • Surface level: 3
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  • Surface view with fitted model
  • Atomic models: PDB-4uud, PDB-4uuk
  • Surface level: 3
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-4uud, PDB-4uuk
  • Surface level: 3
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-4uud
  • Imaged by Jmol
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-4uuk
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Movie viewer
Structure viewerEM map:
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Supplemental images

Downloads & links

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Map

FileDownload / File: emd_2701.map.gz / Format: CCP4 / Size: 29.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of a dynamin mutant, K44A, bound to DOPS lipid tube
Voxel sizeX=Y=Z: 2.55 Å
Density
Contour LevelBy AUTHOR: 0.6 / Movie #1: 3
Minimum - Maximum-18.064981459999998 - 18.351728439999999
Average (Standard dev.)0.36057079 (±3.62353349)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-100-100-99
Dimensions200200200
Spacing200200200
CellA=B=C: 510.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.552.552.55
M x/y/z200200200
origin x/y/z0.0000.0000.000
length x/y/z510.000510.000510.000
α/β/γ90.00090.00090.000
start NX/NY/NZ-180-180-179
NX/NY/NZ360360360
MAP C/R/S123
start NC/NR/NS-100-100-99
NC/NR/NS200200200
D min/max/mean-18.06518.3520.361

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Supplemental data

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Sample components

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Entire : GTP-stablized human dynamin 1 K44A dynamin polymer bound to DOPS ...

EntireName: GTP-stablized human dynamin 1 K44A dynamin polymer bound to DOPS lipid bilayer
Components
  • Sample: GTP-stablized human dynamin 1 K44A dynamin polymer bound to DOPS lipid bilayer
  • Protein or peptide: Human dynamin 1DNM1

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Supramolecule #1000: GTP-stablized human dynamin 1 K44A dynamin polymer bound to DOPS ...

SupramoleculeName: GTP-stablized human dynamin 1 K44A dynamin polymer bound to DOPS lipid bilayer
type: sample / ID: 1000 / Oligomeric state: helical assembly of dynamin / Number unique components: 1

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Macromolecule #1: Human dynamin 1

MacromoleculeName: Human dynamin 1 / type: protein_or_peptide / ID: 1 / Oligomeric state: helical assembly / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human
Molecular weightExperimental: 98 MDa / Theoretical: 97.3 MDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
Recombinant plasmid: pBlueBacIII baculovirus expression vector
SequenceUniProtKB: Dynamin-1

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Experimental details

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Structure determination

Methodcryo EM
Processinghelical reconstruction
Aggregation statehelical array

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Sample preparation

Concentration0.2 mg/mL
BufferpH: 7.2
Details: 20 mM Hepes, 150 mM NaCl, 1 mM EGTA, 1 mM DTT, 1 mM MgCl
GridDetails: Quantifoil R3.5/1 200 mesh Cu Holey Carbon
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 93 K / Instrument: LEICA EM GP / Method: 40 seconds pre-blot, 2 second blot before plunging

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Electron microscopy

MicroscopeFEI POLARA 300
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 49000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.26 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 49000
Sample stageSpecimen holder: Liquid Nitrogen cooled / Specimen holder model: OTHER
TemperatureMin: 93 K / Max: 95 K / Average: 94 K
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 100,000 times magnification
DateDec 17, 2012
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: NIKON SUPER COOLSCAN 9000 / Digitization - Sampling interval: 12.5 µm / Number real images: 357 / Average electron dose: 10 e/Å2 / Od range: 1.5
Tilt angle min0
Tilt angle max0
Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: each image using bsoft
Final reconstructionApplied symmetry - Helical parameters - Δz: 17.19 Å
Applied symmetry - Helical parameters - Δ&Phi: 30.59 °
Applied symmetry - Helical parameters - Axial symmetry: C1 (asymmetric)
Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 12.5 Å / Resolution method: OTHER / Software - Name: Spider
Details: A total of 7525 helical segments were incorporated by the IHRSR algorithm into the final reconstruction after 50 cycles
DetailsThe particles were aligned using IHRSR

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Atomic model buiding 1

Initial modelPDB ID:
SoftwareName: YUP
DetailsInitial fitting was performed using GG-GMPPCP monomers (PDB: 3ZYC), dynamin middle/GED stalk monomers excised from (PDB: 3SNH), and PH domain monomers (PDB: 1DYN). All-atom structures were refined using the YUP.SCX method of the YUP software package.
RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-4uud:
Human dynamin 1 K44A superconstricted polymer stabilized with GTP

PDB-4uuk:
Human dynamin 1 K44A superconstricted polymer stabilized with GTP strand 2

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Atomic model buiding 2

Initial modelPDB ID:
SoftwareName: YUP
DetailsInitial fitting was performed using GG-GMPPCP monomers (PDB: 3ZYC), dynamin middle/GED stalk monomers excised from (PDB: 3SNH), and PH domain monomers (PDB: 1DYN). All-atom structures were refined using the YUP.SCX method of the YUP software package.
RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-4uud:
Human dynamin 1 K44A superconstricted polymer stabilized with GTP

PDB-4uuk:
Human dynamin 1 K44A superconstricted polymer stabilized with GTP strand 2

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Atomic model buiding 3

Initial modelPDB ID:
SoftwareName: YUP
DetailsInitial fitting was performed using GG-GMPPCP monomers (PDB: 3ZYC), dynamin middle/GED stalk monomers excised from (PDB: 3SNH), and PH domain monomers (PDB: 1DYN). All-atom structures were refined using the YUP.SCX method of the YUP software package.
RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-4uud:
Human dynamin 1 K44A superconstricted polymer stabilized with GTP

PDB-4uuk:
Human dynamin 1 K44A superconstricted polymer stabilized with GTP strand 2

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