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- PDB-7wbb: Cryo-EM structure of substrate engaged Drg1 hexamer -

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Basic information

Entry
Database: PDB / ID: 7wbb
TitleCryo-EM structure of substrate engaged Drg1 hexamer
Components
  • AFG2 isoform 1
  • substrate
KeywordsATP-binding protein / cryo-EM structure of Drg1 / RIBOSOME
Function / homology
Function and homology information


ATP hydrolysis activity / ATP binding
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'-TRIPHOSPHATE / AFG2 isoform 1
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Escherichia coli (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.6 Å
AuthorsMa, C.Y. / Wu, D.M. / Chen, Q. / Gao, N.
Funding support1items
OrganizationGrant numberCountry
Not funded
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
DepositionDec 16, 2021Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Dec 28, 2022Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: AFG2 isoform 1
B: AFG2 isoform 1
D: AFG2 isoform 1
E: AFG2 isoform 1
F: AFG2 isoform 1
H: substrate
C: AFG2 isoform 1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)516,64718
Polymers511,0687
Non-polymers5,57911
Water0
1


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

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Components

#1: Protein
AFG2 isoform 1


Mass: 84848.742 Da / Num. of mol.: 6 / Mutation: E346Q, E617Q
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: AFG2 / Production host: Escherichia coli (E. coli) / References: UniProt: A0A6A5PRU8
#2: Protein/peptide substrate


Mass: 1975.426 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Production host: Escherichia coli (E. coli)
#3: Chemical
ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE / Adenosine triphosphate


Mass: 507.181 Da / Num. of mol.: 11 / Source method: obtained synthetically / Formula: C10H16N5O13P3 / Feature type: SUBJECT OF INVESTIGATION / Comment: ATP, 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: Double mutant Drg1 in the presence of ATP with substrate engaged
Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT
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
Specimen supportGrid type: Quantifoil R1.2/1.3
VitrificationCryogen name: ETHANE / Humidity: 100 %

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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 defocus max: 2500 nm / Nominal defocus min: 1500 nm
Image recordingElectron dose: 8 e/Å2 / Film or detector model: GATAN K2 QUANTUM (4k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.19.2_4158: / Classification: refinement
CTF correctionType: NONE
3D reconstructionResolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 152973 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00234277
ELECTRON MICROSCOPYf_angle_d0.49346481
ELECTRON MICROSCOPYf_dihedral_angle_d4.2834700
ELECTRON MICROSCOPYf_chiral_restr0.0415376
ELECTRON MICROSCOPYf_plane_restr0.0045988

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