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- PDB-7ykt: Cryo-EM structure of Drg1 hexamer in helical state treated with A... -

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Basic information

Entry
Database: PDB / ID: 7ykt
TitleCryo-EM structure of Drg1 hexamer in helical state treated with ADP/AMPPNP/benzo-diazaborine
ComponentsATPase family gene 2 protein
KeywordsRIBOSOMAL PROTEIN / Involved in maturation of pre-60S ribosomal particles
Function / homology
Function and homology information


protein hexamerization / non-chaperonin molecular chaperone ATPase / preribosome, large subunit precursor / ribosomal large subunit biogenesis / response to xenobiotic stimulus / ATP hydrolysis activity / ATP binding / cytoplasm
Similarity search - Function
: / AAA ATPase, AAA+ lid domain / AAA+ lid domain / ATPase, AAA-type, conserved site / AAA-protein family signature. / ATPase family associated with various cellular activities (AAA) / ATPase, AAA-type, core / 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 / ATPase family gene 2 protein
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 5.9 Å
AuthorsMa, C.Y. / Wu, D.M. / Chen, Q. / Gao, N.
Funding support China, 1items
OrganizationGrant numberCountry
National Science Foundation (NSF, China) China
CitationJournal: Nat Commun / Year: 2022
Title: Structural dynamics of AAA + ATPase Drg1 and mechanism of benzo-diazaborine inhibition.
Authors: Chengying Ma / Damu Wu / Qian Chen / Ning Gao /
Abstract: The type II AAA + ATPase Drg1 is a ribosome assembly factor, functioning to release Rlp24 from the pre-60S particle just exported from nucleus, and its activity in can be inhibited by a drug ...The type II AAA + ATPase Drg1 is a ribosome assembly factor, functioning to release Rlp24 from the pre-60S particle just exported from nucleus, and its activity in can be inhibited by a drug molecule diazaborine. However, molecular mechanisms of Drg1-mediated Rlp24 removal and diazaborine-mediated inhibition are not fully understood. Here, we report Drg1 structures in different nucleotide-binding and benzo-diazaborine treated states. Drg1 hexamers transits between two extreme conformations (planar or helical arrangement of protomers). By forming covalent adducts with ATP molecules in both ATPase domain, benzo-diazaborine locks Drg1 hexamers in a symmetric and non-productive conformation to inhibits both inter-protomer and inter-ring communication of Drg1 hexamers. We also obtained a substrate-engaged mutant Drg1 structure, in which conserved pore-loops form a spiral staircase to interact with the polypeptide through a sequence-independent manner. Structure-based mutagenesis data highlight the functional importance of the pore-loop, the D1-D2 linker and the inter-subunit signaling motif of Drg1, which share similar regulatory mechanisms with p97. Our results suggest that Drg1 may function as an unfoldase that threads a substrate protein within the pre-60S particle.
History
DepositionJul 23, 2022Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Dec 14, 2022Provider: repository / Type: Initial release
Revision 1.1Jul 3, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
F: ATPase family gene 2 protein
A: ATPase family gene 2 protein
B: ATPase family gene 2 protein
C: ATPase family gene 2 protein
D: ATPase family gene 2 protein
E: ATPase family gene 2 protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)513,42915
Polymers509,1046
Non-polymers4,3259
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
ATPase family gene 2 protein / Diazaborine resistance gene 1 protein


Mass: 84850.719 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Strain: ATCC 204508 / S288c / Gene: AFG2, DRG1, YLR397C, L8084.16 / Production host: Escherichia coli (E. coli)
References: UniProt: P32794, non-chaperonin molecular chaperone ATPase
#2: Chemical
ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE


Mass: 507.181 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: C10H16N5O13P3 / Feature type: SUBJECT OF INVESTIGATION / Comment: ATP, energy-carrying molecule*YM
#3: Chemical ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE


Mass: 427.201 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Feature type: SUBJECT OF INVESTIGATION / Comment: ADP, energy-carrying molecule*YM
Has ligand of interestY

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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: Drg1 hexamer / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 8
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 FIELD / Nominal defocus max: 2500 nm / Nominal defocus min: 1000 nm
Image recordingElectron dose: 8 e/Å2 / Film or detector model: DIRECT ELECTRON DE-16 (4k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.19.2_4158: / Classification: refinement
CTF correctionType: NONE
3D reconstructionResolution: 5.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 88912 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00234548
ELECTRON MICROSCOPYf_angle_d0.63646825
ELECTRON MICROSCOPYf_dihedral_angle_d4.6234769
ELECTRON MICROSCOPYf_chiral_restr0.0455431
ELECTRON MICROSCOPYf_plane_restr0.0046066

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