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- PDB-7z11: Structure of substrate bound DRG1 (AFG2) -

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Basic information

Entry
Database: PDB / ID: 7z11
TitleStructure of substrate bound DRG1 (AFG2)
Components
  • ATPase family gene 2 protein
  • peptide substrate
KeywordsCHAPERONE / Ribosome biogenesis / AAA-ATPases / substrate recognition / single particle cryo-EM
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
PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / ATPase family gene 2 protein
Similarity search - Component
Biological speciesSaccharomyces cerevisiae S288C (yeast)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsPrattes, M. / Grishkovskaya, I. / Bergler, H. / Haselbach, D.
Funding support Austria, 1items
OrganizationGrant numberCountry
Austrian Science Fund Austria
CitationJournal: Nat Struct Mol Biol / Year: 2022
Title: Visualizing maturation factor extraction from the nascent ribosome by the AAA-ATPase Drg1.
Authors: Michael Prattes / Irina Grishkovskaya / Victor-Valentin Hodirnau / Christina Hetzmannseder / Gertrude Zisser / Carolin Sailer / Vasileios Kargas / Mathias Loibl / Magdalena Gerhalter / Lisa ...Authors: Michael Prattes / Irina Grishkovskaya / Victor-Valentin Hodirnau / Christina Hetzmannseder / Gertrude Zisser / Carolin Sailer / Vasileios Kargas / Mathias Loibl / Magdalena Gerhalter / Lisa Kofler / Alan J Warren / Florian Stengel / David Haselbach / Helmut Bergler /
Abstract: The AAA-ATPase Drg1 is a key factor in eukaryotic ribosome biogenesis that initiates cytoplasmic maturation of the large ribosomal subunit. Drg1 releases the shuttling maturation factor Rlp24 from ...The AAA-ATPase Drg1 is a key factor in eukaryotic ribosome biogenesis that initiates cytoplasmic maturation of the large ribosomal subunit. Drg1 releases the shuttling maturation factor Rlp24 from pre-60S particles shortly after nuclear export, a strict requirement for downstream maturation. The molecular mechanism of release remained elusive. Here, we report a series of cryo-EM structures that captured the extraction of Rlp24 from pre-60S particles by Saccharomyces cerevisiae Drg1. These structures reveal that Arx1 and the eukaryote-specific rRNA expansion segment ES27 form a joint docking platform that positions Drg1 for efficient extraction of Rlp24 from the pre-ribosome. The tips of the Drg1 N domains thereby guide the Rlp24 C terminus into the central pore of the Drg1 hexamer, enabling extraction by a hand-over-hand translocation mechanism. Our results uncover substrate recognition and processing by Drg1 step by step and provide a comprehensive mechanistic picture of the conserved modus operandi of AAA-ATPases.
History
DepositionFeb 24, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0Sep 21, 2022Provider: repository / Type: Initial release
Revision 1.1Oct 5, 2022Group: 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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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
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
F: ATPase family gene 2 protein
G: peptide substrate
hetero molecules


Theoretical massNumber of molelcules
Total (without water)516,58018
Polymers510,8247
Non-polymers5,75611
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
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 S288C (yeast) / Strain: ATCC 204508 / S288c / Gene: AFG2, DRG1, YLR397C, L8084.16 / Production host: Saccharomyces cerevisiae BY4743 (yeast)
References: UniProt: P32794, non-chaperonin molecular chaperone ATPase
#2: Protein/peptide peptide substrate


Mass: 1720.111 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae S288C (yeast) / Production host: Saccharomyces cerevisiae BY4743 (yeast)
#3: Chemical
ChemComp-AGS / PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / ATP-GAMMA-S / ADENOSINE 5'-(3-THIOTRIPHOSPHATE) / ADENOSINE 5'-(GAMMA-THIOTRIPHOSPHATE) / ADENOSINE-5'-DIPHOSPHATE MONOTHIOPHOSPHATE


Mass: 523.247 Da / Num. of mol.: 11 / Source method: obtained synthetically / Formula: C10H16N5O12P3S / Feature type: SUBJECT OF INVESTIGATION / Comment: ATP-gamma-S, energy-carrying molecule analogue*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: AFG2 hexamer / Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT
Source (natural)Organism: Saccharomyces cerevisiae S288C (yeast)
Source (recombinant)Organism: Saccharomyces cerevisiae BY4743 (yeast)
Buffer solutionpH: 7.6
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 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 defocus max: 2500 nm / Nominal defocus min: 500 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 60 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)
EM imaging opticsEnergyfilter slit width: 20 eV

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Processing

SoftwareName: PHENIX / Version: 1.18.2_3874: / Classification: refinement
EM software
IDNameVersionCategory
1cryoSPARCv3.0particle selection
2SerialEMimage acquisition
4cryoSPARCv3.0CTF correction
9cryoSPARCv3.0initial Euler assignment
10cryoSPARCv3.0final Euler assignment
11cryoSPARCv3.0classification
13Rosettamodel refinement
14PHENIXv1.18.2-3874model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 3148330
3D reconstructionResolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 114728 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL / Space: REAL / Target criteria: correlation coefficient
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00434688
ELECTRON MICROSCOPYf_angle_d0.80547038
ELECTRON MICROSCOPYf_dihedral_angle_d16.70713053
ELECTRON MICROSCOPYf_chiral_restr0.0555455
ELECTRON MICROSCOPYf_plane_restr0.0066063

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