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- PDB-6rla: Structure of the dynein-2 complex; motor domains -

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Basic information

Entry
Database: PDB / ID: 6rla
TitleStructure of the dynein-2 complex; motor domains
ComponentsO6-alkylguanine-DNA alkyltransferase mutant,DYNC2H1 variant protein
KeywordsMOTOR PROTEIN / dynein / cilia / intraflagellar transport / complex
Function / homology
Function and homology information


organelle organization / methylated-DNA-[protein]-cysteine S-methyltransferase / methylated-DNA-[protein]-cysteine S-methyltransferase activity / DNA modification / cell projection organization / dynein complex / minus-end-directed microtubule motor activity / dynein light intermediate chain binding / dynein intermediate chain binding / microtubule-based movement ...organelle organization / methylated-DNA-[protein]-cysteine S-methyltransferase / methylated-DNA-[protein]-cysteine S-methyltransferase activity / DNA modification / cell projection organization / dynein complex / minus-end-directed microtubule motor activity / dynein light intermediate chain binding / dynein intermediate chain binding / microtubule-based movement / cilium / methylation / microtubule / DNA repair / ATP hydrolysis activity / DNA binding / ATP binding / metal ion binding / nucleus / plasma membrane / cytoplasm
Similarity search - Function
: / Cytoplasmic dynein 2 heavy chain 1, AAA+ ATPase domain / Methylguanine DNA methyltransferase, ribonuclease-like domain / 6-O-methylguanine DNA methyltransferase, ribonuclease-like domain / Methylated DNA-protein cysteine methyltransferase domain superfamily / Methylated-DNA-[protein]-cysteine S-methyltransferase, active site / Methylated-DNA--protein-cysteine methyltransferase active site. / Methylated-DNA-[protein]-cysteine S-methyltransferase, DNA binding / Methylated DNA-protein cysteine methyltransferase, DNA binding domain / 6-O-methylguanine DNA methyltransferase, DNA binding domain ...: / Cytoplasmic dynein 2 heavy chain 1, AAA+ ATPase domain / Methylguanine DNA methyltransferase, ribonuclease-like domain / 6-O-methylguanine DNA methyltransferase, ribonuclease-like domain / Methylated DNA-protein cysteine methyltransferase domain superfamily / Methylated-DNA-[protein]-cysteine S-methyltransferase, active site / Methylated-DNA--protein-cysteine methyltransferase active site. / Methylated-DNA-[protein]-cysteine S-methyltransferase, DNA binding / Methylated DNA-protein cysteine methyltransferase, DNA binding domain / 6-O-methylguanine DNA methyltransferase, DNA binding domain / Dynein heavy chain, C-terminal domain / Dynein heavy chain, C-terminal domain, barrel region / Dynein heavy chain C-terminal domain / P-loop containing dynein motor region / Dynein heavy chain, tail / Dynein heavy chain, N-terminal region 1 / Dynein heavy chain / Dynein heavy chain region D6 P-loop domain / Dynein heavy chain, linker / Dynein heavy chain, AAA module D4 / Dynein heavy chain, coiled coil stalk / Dynein heavy chain, hydrolytic ATP-binding dynein motor region / Dynein heavy chain, ATP-binding dynein motor region / Dynein heavy chain AAA lid domain / Dynein heavy chain AAA lid domain superfamily / Dynein heavy chain, domain 2, N-terminal / Dynein heavy chain, linker, subdomain 3 / Dynein heavy chain, AAA1 domain, small subdomain / Dynein heavy chain region D6 P-loop domain / Dynein heavy chain, N-terminal region 2 / Hydrolytic ATP binding site of dynein motor region / Microtubule-binding stalk of dynein motor / P-loop containing dynein motor region D4 / ATP-binding dynein motor region / Dynein heavy chain AAA lid domain / Winged helix-like DNA-binding domain superfamily / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
ADENOSINE-5'-DIPHOSPHATE / ADENOSINE-5'-TRIPHOSPHATE / Cytoplasmic dynein 2 heavy chain 1 / Methylated-DNA--protein-cysteine methyltransferase
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.9 Å
AuthorsToropova, K. / Zalyte, R. / Mukhopadhyay, A.G. / Mladenov, M. / Carter, A.P. / Roberts, A.J.
Funding support United Kingdom, 8items
OrganizationGrant numberCountry
Wellcome Trust104196/Z/14/Z United Kingdom
Royal Society104196/Z/14/Z United Kingdom
Wellcome TrustWT100387 United Kingdom
Biotechnology and Biological Sciences Research CouncilBB/P008348/1 United Kingdom
Royal SocietyRG170260 United Kingdom
Medical Research Council (United Kingdom)MC_UP_A025_1011 United Kingdom
Wellcome Trust079605/Z/06/Z United Kingdom
Biotechnology and Biological Sciences Research CouncilBB/L014211/1 United Kingdom
CitationJournal: Nat Struct Mol Biol / Year: 2019
Title: Structure of the dynein-2 complex and its assembly with intraflagellar transport trains.
Authors: Katerina Toropova / Ruta Zalyte / Aakash G Mukhopadhyay / Miroslav Mladenov / Andrew P Carter / Anthony J Roberts /
Abstract: Dynein-2 assembles with polymeric intraflagellar transport (IFT) trains to form a transport machinery that is crucial for cilia biogenesis and signaling. Here we recombinantly expressed the ~1.4-MDa ...Dynein-2 assembles with polymeric intraflagellar transport (IFT) trains to form a transport machinery that is crucial for cilia biogenesis and signaling. Here we recombinantly expressed the ~1.4-MDa human dynein-2 complex and solved its cryo-EM structure to near-atomic resolution. The two identical copies of the dynein-2 heavy chain are contorted into different conformations by a WDR60-WDR34 heterodimer and a block of two RB and six LC8 light chains. One heavy chain is steered into a zig-zag conformation, which matches the periodicity of the anterograde IFT-B train. Contacts between adjacent dyneins along the train indicate a cooperative mode of assembly. Removal of the WDR60-WDR34-light chain subcomplex renders dynein-2 monomeric and relieves autoinhibition of its motility. Our results converge on a model in which an unusual stoichiometry of non-motor subunits controls dynein-2 assembly, asymmetry, and activity, giving mechanistic insight into the interaction of dynein-2 with IFT trains and the origin of diverse functions in the dynein family.
History
DepositionMay 1, 2019Deposition site: PDBE / Processing site: PDBE
Revision 1.0Aug 28, 2019Provider: repository / Type: Initial release
Revision 1.1Sep 4, 2019Group: Data collection / Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_PubMed / _citation.title ..._citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _citation_author.name
Revision 1.2Sep 18, 2019Group: Data collection / Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID

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Assembly

Deposited unit
A: O6-alkylguanine-DNA alkyltransferase mutant,DYNC2H1 variant protein
B: O6-alkylguanine-DNA alkyltransferase mutant,DYNC2H1 variant protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)1,034,07212
Polymers1,030,4462
Non-polymers3,62610
Water0
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area9360 Å2
ΔGint-60 kcal/mol
Surface area263330 Å2
MethodPISA

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Components

#1: Protein O6-alkylguanine-DNA alkyltransferase mutant,DYNC2H1 variant protein


Mass: 515223.031 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: E5BBQ0, UniProt: B0I1S0
#2: Chemical
ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE / Adenosine diphosphate


Mass: 427.201 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Comment: ADP, energy-carrying molecule*YM
#3: Chemical ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE / Adenosine triphosphate


Mass: 507.181 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C10H16N5O13P3 / Comment: ATP, energy-carrying molecule*YM
#4: Chemical ChemComp-MG / MAGNESIUM ION


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

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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: Dynein-2 complex; motor domains / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm)
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER
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 FIELDBright-field microscopy / Nominal magnification: 105000 X / Nominal defocus max: 3500 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 49.6 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k)
Details: Average electron dose per image (e-/A2) for additional datasets was 46.8 and 45.4
EM imaging opticsEnergyfilter slit width: 20 eV

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Processing

EM software
IDNameVersionCategory
1Gautomatchparticle selection
2EPUimage acquisition
4GctfCTF correction
5RELION2.1CTF correction
11RELION2.1initial Euler assignment
12RELION2.1final Euler assignment
14RELION2.13D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 57265 / Symmetry type: POINT

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