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- EMDB-1949: Human dynamin 1 deltaPRD polymer stabilized with GMPPCP -

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Basic information

Entry
Database: EMDB / ID: EMD-1949
TitleHuman dynamin 1 deltaPRD polymer stabilized with GMPPCP
Map dataThree-dimensional volume of deltaPRD human dynamin 1 polymer
Sample
  • Sample: GMPPCP-stablized human dynamin 1 delta PRD polymer
  • Protein or peptide: Human dynamin 1 delta PRD
  • Protein or peptide: Human dynamin 1 pleckstrin homology domain
  • Protein or peptide: Interferon-induced GTP-binding protein Mx1
Keywordsdynamin / endocytosis / GTP hydrolysis / membrane remodeling
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 / interleukin-27-mediated signaling pathway / dynamin GTPase / chromaffin granule / regulation of vesicle size / Toll Like Receptor 4 (TLR4) Cascade / photoreceptor ribbon synapse ...clathrin coat assembly involved in endocytosis / vesicle scission / synaptic vesicle budding from presynaptic endocytic zone membrane / presynaptic endocytic zone membrane / interleukin-27-mediated signaling pathway / dynamin GTPase / chromaffin granule / regulation of vesicle size / Toll Like Receptor 4 (TLR4) Cascade / photoreceptor ribbon synapse / Retrograde neurotrophin signalling / Formation of annular gap junctions / endosome organization / Gap junction degradation / membrane coat / response to type I interferon / negative regulation of viral genome replication / Recycling pathway of L1 / phosphatidylinositol-3,4,5-trisphosphate binding / antiviral innate immune response / synaptic vesicle endocytosis / endocytic vesicle / EPH-ephrin mediated repulsion of cells / clathrin-coated pit / phosphatidylinositol-4,5-bisphosphate binding / MHC class II antigen presentation / receptor-mediated endocytosis / photoreceptor inner segment / cell projection / response to virus / modulation of chemical synaptic transmission / protein homooligomerization / defense response / receptor internalization / ISG15 antiviral mechanism / endocytosis / GDP binding / : / Interferon alpha/beta signaling / presynapse / Clathrin-mediated endocytosis / microtubule binding / protein homotetramerization / defense response to virus / nuclear membrane / microtubule / innate immune response / GTPase activity / glutamatergic synapse / apoptotic process / GTP binding / endoplasmic reticulum membrane / protein kinase binding / perinuclear region of cytoplasm / signal transduction / protein homodimerization activity / RNA binding / extracellular exosome / membrane / identical protein binding / nucleus / plasma membrane / cytosol / 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
Interferon-induced GTP-binding protein Mx1 / Dynamin-1
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodhelical reconstruction / cryo EM / Resolution: 12.2 Å
AuthorsChappie JS / Mears JA / Fang S / Leonard M / Schmid SL / Milligan RA / Hinshaw JE / Dyda F
CitationJournal: Cell / Year: 2011
Title: A pseudoatomic model of the dynamin polymer identifies a hydrolysis-dependent powerstroke.
Authors: Joshua S Chappie / Jason A Mears / Shunming Fang / Marilyn Leonard / Sandra L Schmid / Ronald A Milligan / Jenny E Hinshaw / Fred Dyda /
Abstract: The GTPase dynamin catalyzes membrane fission by forming a collar around the necks of clathrin-coated pits, but the specific structural interactions and conformational changes that drive this process ...The GTPase dynamin catalyzes membrane fission by forming a collar around the necks of clathrin-coated pits, but the specific structural interactions and conformational changes that drive this process remain a mystery. We present the GMPPCP-bound structures of the truncated human dynamin 1 helical polymer at 12.2 Å and a fusion protein, GG, linking human dynamin 1's catalytic G domain to its GTPase effector domain (GED) at 2.2 Å. The structures reveal the position and connectivity of dynamin fragments in the assembled structure, showing that G domain dimers only form between tetramers in sequential rungs of the dynamin helix. Using chemical crosslinking, we demonstrate that dynamin tetramers are made of two dimers, in which the G domain of one molecule interacts in trans with the GED of another. Structural comparison of GG(GMPPCP) to the GG transition-state complex identifies a hydrolysis-dependent powerstroke that may play a role in membrane-remodeling events necessary for fission.
History
DepositionAug 22, 2011-
Header (metadata) releaseOct 6, 2011-
Map releaseOct 6, 2011-
UpdateOct 6, 2011-
Current statusOct 6, 2011Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 227
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 227
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-3zys
  • Surface level: 227
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-3zys
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_1949.tif

Downloads & links

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Map

FileDownload / File: emd_1949.map.gz / Format: CCP4 / Size: 29.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationThree-dimensional volume of deltaPRD human dynamin 1 polymer
Voxel sizeX=Y=Z: 2.26 Å
Density
Contour LevelBy AUTHOR: 227.0 / Movie #1: 227
Minimum - Maximum-749.477999999999952 - 817.053999999999974
Average (Standard dev.)37.000599999999999 (±200.730999999999995)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-100-100-99
Dimensions200200200
Spacing200200200
CellA=B=C: 452 Å
α=β=γ: 90 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.262.262.26
M x/y/z200200200
origin x/y/z0.0000.0000.000
length x/y/z452.000452.000452.000
α/β/γ90.00090.00090.000
start NX/NY/NZ-56-56-55
NX/NY/NZ112112112
MAP C/R/S123
start NC/NR/NS-100-100-99
NC/NR/NS200200200
D min/max/mean-749.478817.05437.001

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Supplemental data

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Sample components

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Entire : GMPPCP-stablized human dynamin 1 delta PRD polymer

EntireName: GMPPCP-stablized human dynamin 1 delta PRD polymer
Components
  • Sample: GMPPCP-stablized human dynamin 1 delta PRD polymer
  • Protein or peptide: Human dynamin 1 delta PRD
  • Protein or peptide: Human dynamin 1 pleckstrin homology domain
  • Protein or peptide: Interferon-induced GTP-binding protein Mx1

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Supramolecule #1000: GMPPCP-stablized human dynamin 1 delta PRD polymer

SupramoleculeName: GMPPCP-stablized human dynamin 1 delta PRD polymer / type: sample / ID: 1000 / Oligomeric state: Helical assembly of dynamin tetramers / Number unique components: 3

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Macromolecule #1: Human dynamin 1 delta PRD

MacromoleculeName: Human dynamin 1 delta PRD / type: protein_or_peptide / ID: 1 / Name.synonym: Human dynamin 1 delta PRD / Details: contains bound GMPPCP / Oligomeric state: Dimer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human / Location in cell: Plasma membrane and cytosol
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria) / Recombinant plasmid: PMALC2XP5D

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Macromolecule #2: Human dynamin 1 pleckstrin homology domain

MacromoleculeName: Human dynamin 1 pleckstrin homology domain / type: protein_or_peptide / ID: 2 / Name.synonym: Human dynamin 1 pleckstrin homology domain / Oligomeric state: Dimer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human / Location in cell: Plasma membrane and cytosol
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria) / Recombinant plasmid: PSKB-LNB

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Macromolecule #3: Interferon-induced GTP-binding protein Mx1

MacromoleculeName: Interferon-induced GTP-binding protein Mx1 / type: protein_or_peptide / ID: 3 / Name.synonym: Interferon-induced GTP-binding protein Mx1 / Oligomeric state: Dimer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant plasmid: PET11A

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Experimental details

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Structure determination

Methodcryo EM
Processinghelical reconstruction
Aggregation statefilament

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Sample preparation

Concentration0.5 mg/mL
BufferpH: 7.5
Details: 20 mM Hepes pH 7.5, 50 mM NaCl, 2 mM EGTA, 4 mM MgCl2, 1 mM DTT, 1 mg/ml 0.4 um 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS) liposomes, 2 mM GMPPCP
GridDetails: 400 mesh C-flat grids
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 93 K / Instrument: HOMEMADE PLUNGER / Details: Vitrification instrument: Manual
Method: Absorbed samples to grids, blotted, washed with 20 mM Hepes pH 7.5, blotted and plunged.

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Electron microscopy

MicroscopeFEI TECNAI 20
Electron beamAcceleration voltage: 120 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 50000
Sample stageSpecimen holder: Gatan 626 side entry cryo-stage / Specimen holder model: GATAN LIQUID NITROGEN
TemperatureMin: 93 K / Max: 95 K
Alignment procedureLegacy - Astigmatism: objective lens astigmatism was corrected at 100,000X magnification
Image recordingCategory: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Average electron dose: 10 e/Å2 / Details: Images were collected on CCD
Tilt angle min0
Tilt angle max0

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Image processing

CTF correctionDetails: Each image using ACE2
Final reconstructionApplied symmetry - Helical parameters - Δz: 7.52 Å
Applied symmetry - Helical parameters - Δ&Phi: 27.3 °
Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 12.2 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: Spider
Details: A total of 4,814 helical segments were incorporated by the IHRSR algorithm into the final reconstruction after 50 cycles

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Atomic model buiding 1

Initial modelPDB ID:

3zyc


Chain - Chain ID: A
SoftwareName: YUP
DetailsPDBEntryID_givenInChain. Protocol: Manual and Flexible fitting. Models were initially placed manually and initial positions were refined using the YUP.SCX method of the YUP software package
RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-3zys:
Human dynamin 1 deltaPRD polymer stabilized with GMPPCP

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Atomic model buiding 2

Initial modelPDB ID:

3ljb


Chain - Chain ID: A
SoftwareName: YUP
DetailsPDBEntryID_givenInChain. Protocol: Manual and Flexible fitting. Models were initially placed manually and initial positions were refined using the YUP.SCX method of the YUP software package
RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-3zys:
Human dynamin 1 deltaPRD polymer stabilized with GMPPCP

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Atomic model buiding 3

Initial modelPDB ID:

1dyn


Chain - Chain ID: A
SoftwareName: YUP
DetailsPDBEntryID_givenInChain. Protocol: Manual and Flexible fitting. Models were initially placed manually and initial positions were refined using the YUP.SCX method of the YUP software package
RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-3zys:
Human dynamin 1 deltaPRD polymer stabilized with GMPPCP

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