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- PDB-6dlu: Cryo-EM of the GMPPCP-bound human dynamin-1 polymer assembled on ... -

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Basic information

Entry
Database: PDB / ID: 6dlu
TitleCryo-EM of the GMPPCP-bound human dynamin-1 polymer assembled on the membrane in the constricted state
ComponentsDynamin-1
KeywordsENDOCYTOSIS / HYDROLASE / dynamin family / gtpase / pleckstrin homology domain / membrane protein
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 / chromaffin granule / regulation of vesicle size / photoreceptor ribbon synapse / Toll Like Receptor 4 (TLR4) Cascade / 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 / chromaffin granule / regulation of vesicle size / photoreceptor ribbon synapse / Toll Like Receptor 4 (TLR4) Cascade / Retrograde neurotrophin signalling / Formation of annular gap junctions / endosome organization / Gap junction degradation / 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 / cell projection / modulation of chemical synaptic transmission / protein homooligomerization / receptor internalization / endocytosis / GDP binding / : / presynapse / Clathrin-mediated endocytosis / microtubule binding / protein homotetramerization / 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
PHOSPHOMETHYLPHOSPHONIC ACID GUANYLATE ESTER / Dynamin-1
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 3.75 Å
AuthorsKong, L. / Wang, H. / Fang, S. / Canagarajah, B. / Kehr, A.D. / Rice, W.J. / Hinshaw, J.E.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM103310 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)S10 486 OD019994-01 United States
CitationJournal: Nature / Year: 2018
Title: Cryo-EM of the dynamin polymer assembled on lipid membrane.
Authors: Leopold Kong / Kem A Sochacki / Huaibin Wang / Shunming Fang / Bertram Canagarajah / Andrew D Kehr / William J Rice / Marie-Paule Strub / Justin W Taraska / Jenny E Hinshaw /
Abstract: Membrane fission is a fundamental process in the regulation and remodelling of cell membranes. Dynamin, a large GTPase, mediates membrane fission by assembling around, constricting and cleaving the ...Membrane fission is a fundamental process in the regulation and remodelling of cell membranes. Dynamin, a large GTPase, mediates membrane fission by assembling around, constricting and cleaving the necks of budding vesicles. Here we report a 3.75 Å resolution cryo-electron microscopy structure of the membrane-associated helical polymer of human dynamin-1 in the GMPPCP-bound state. The structure defines the helical symmetry of the dynamin polymer and the positions of its oligomeric interfaces, which were validated by cell-based endocytosis assays. Compared to the lipid-free tetramer form, membrane-associated dynamin binds to the lipid bilayer with its pleckstrin homology domain (PHD) and self-assembles across the helical rungs via its guanine nucleotide-binding (GTPase) domain. Notably, interaction with the membrane and helical assembly are accommodated by a severely bent bundle signalling element (BSE), which connects the GTPase domain to the rest of the protein. The BSE conformation is asymmetric across the inter-rung GTPase interface, and is unique compared to all known nucleotide-bound states of dynamin. The structure suggests that the BSE bends as a result of forces generated from the GTPase dimer interaction that are transferred across the stalk to the PHD and lipid membrane. Mutations that disrupted the BSE kink impaired endocytosis. We also report a 10.1 Å resolution cryo-electron microscopy map of a super-constricted dynamin polymer showing localized conformational changes at the BSE and GTPase domains, induced by GTP hydrolysis, that drive membrane constriction. Together, our results provide a structural basis for the mechanism of action of dynamin on the lipid membrane.
History
DepositionJun 2, 2018Deposition site: RCSB / Processing site: RCSB
Revision 1.0Aug 1, 2018Provider: repository / Type: Initial release
Revision 1.1Aug 15, 2018Group: Data collection / Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.name
Revision 1.2Aug 22, 2018Group: Data collection / Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.3Jan 8, 2020Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization

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Assembly

Deposited unit
B: Dynamin-1
P: Dynamin-1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)172,8096
Polymers171,7182
Non-polymers1,0914
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: assay for oligomerization
TypeNameSymmetry operationNumber
identity operation1_5551
SymmetryHelical symmetry: (Circular symmetry: 1 / Dyad axis: no / N subunits divisor: 1 / Num. of operations: 17 / Rise per n subunits: 6.35 Å / Rotation per n subunits: 23.68 °)

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Components

#1: Protein Dynamin-1 /


Mass: 85859.148 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: DNM1, DNM / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q05193, dynamin GTPase
#2: Chemical ChemComp-GCP / PHOSPHOMETHYLPHOSPHONIC ACID GUANYLATE ESTER


Mass: 521.208 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C11H18N5O13P3 / Comment: GMP-PCP, energy-carrying molecule analogue*YM
#3: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg

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Experimental details

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Experiment

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

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Sample preparation

ComponentName: GMPPCP-bound Human Dynamin-1 helical polymer encasing a phosphatidylserine lipid membrane bilayer tube
Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 98 kDa/nm / Experimental value: YES
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Trichoplusia ni (cabbage looper)
Buffer solutionpH: 7.2
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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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 FIELDBright-field microscopy
Image recordingElectron dose: 67 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

EM softwareName: RELION / Version: 2.0.6 / Category: 3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Helical symmertyAngular rotation/subunit: 23.68 ° / Axial rise/subunit: 6.35 Å / Axial symmetry: C1
3D reconstructionResolution: 3.75 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 452959 / Symmetry type: HELICAL

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