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- EMDB-14437: Structure of substrate bound DRG1 (AFG2) -

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Basic information

Entry
Database: EMDB / ID: EMD-14437
TitleStructure of substrate bound DRG1 (AFG2)
Map data
Sample
  • Complex: AFG2 hexamer
    • Protein or peptide: ATPase family gene 2 protein
    • Protein or peptide: peptide substrate
  • Ligand: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER
KeywordsRibosome biogenesis / AAA-ATPases / substrate recognition / single particle cryo-EM / CHAPERONE
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
ATPase family gene 2 protein
Similarity search - Component
Biological speciesSaccharomyces cerevisiae S288C (yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsPrattes M / Grishkovskaya I
Funding support Austria, 1 items
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, 2022-
Header (metadata) releaseSep 21, 2022-
Map releaseSep 21, 2022-
UpdateJul 17, 2024-
Current statusJul 17, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_14437.png

Downloads & links

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Map

FileDownload / File: emd_14437.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.07 Å/pix.
x 256 pix.
= 273.92 Å		1.07 Å/pix.
x 256 pix.
= 273.92 Å		1.07 Å/pix.
x 256 pix.
= 273.92 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.07 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.02
Minimum - Maximum-0.020881018 - 2.0358553
Average (Standard dev.)0.003361057 (±0.040644433)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 273.92 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : AFG2 hexamer

EntireName: AFG2 hexamer
Components
  • Complex: AFG2 hexamer
    • Protein or peptide: ATPase family gene 2 protein
    • Protein or peptide: peptide substrate
  • Ligand: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER

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Supramolecule #1: AFG2 hexamer

SupramoleculeName: AFG2 hexamer / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Saccharomyces cerevisiae S288C (yeast)

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Macromolecule #1: ATPase family gene 2 protein

MacromoleculeName: ATPase family gene 2 protein / type: protein_or_peptide / ID: 1 / Number of copies: 6 / Enantiomer: LEVO / EC number: non-chaperonin molecular chaperone ATPase
Source (natural)Organism: Saccharomyces cerevisiae S288C (yeast) / Strain: ATCC 204508 / S288c
Molecular weightTheoretical: 84.850719 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae BY4743 (yeast)
SequenceString: MAPKSSSSGS KKKSSASSNS ADAKASKFKL PAEFITRPHP SKDHGKETCT AYIHPNVLSS LEINPGSFCT VGKIGENGIL VIARAGDEE VHPVNVITLS TTIRSVGNLI LGDRLELKKA QVQPPYATKV TVGSLQGYNI LECMEEKVIQ KLLDDSGVIM P GMIFQNLK ...String:
MAPKSSSSGS KKKSSASSNS ADAKASKFKL PAEFITRPHP SKDHGKETCT AYIHPNVLSS LEINPGSFCT VGKIGENGIL VIARAGDEE VHPVNVITLS TTIRSVGNLI LGDRLELKKA QVQPPYATKV TVGSLQGYNI LECMEEKVIQ KLLDDSGVIM P GMIFQNLK TKAGDESIDV VITDASDDSL PDVSQLDLNM DDMYGGLDNL FYLSPPFIFR KGSTHITFSK ETQANRKYNL PE PLSYAAV GGLDKEIESL KSAIEIPLHQ PTLFSSFGVS PPRGILLHGP PGTGKTMLLR VVANTSNAHV LTINGPSIVS KYL GETEAA LRDIFNEARK YQPSIIFIDE IDSIAPNRAN DDSGEVESRV VATLLTLMDG MGAAGKVVVI AATNRPNSVD PALR RPGRF DQEVEIGIPD VDARFDILTK QFSRMSSDRH VLDSEAIKYI ASKTHGYVGA DLTALCRESV MKTIQRGLGT DANID KFSL KVTLKDVESA MVDIRPSAMR EIFLEMPKVY WSDIGGQEEL KTKMKEMIQL PLEASETFAR LGISAPKGVL LYGPPG CSK TLTAKALATE SGINFLAVKG PEIFNKYVGE SERAIREIFR KARSAAPSII FFDEIDALSP DRDGSSTSAA NHVLTSL LN EIDGVEELKG VVIVAATNRP DEIDAALLRP GRLDRHIYVG PPDVNARLEI LKKCTKKFNT EESGVDLHEL ADRTEGYS G AEVVLLCQEA GLAAIMEDLD VAKVELRHFE KAFKGIARGI TPEMLSYYEE FALRSGSSS

UniProtKB: ATPase family gene 2 protein

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Macromolecule #2: peptide substrate

MacromoleculeName: peptide substrate / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae S288C (yeast)
Molecular weightTheoretical: 1.720111 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae BY4743 (yeast)
SequenceString:
(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK) (UNK)

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Macromolecule #3: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER

MacromoleculeName: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / type: ligand / ID: 3 / Number of copies: 11 / Formula: AGS
Molecular weightTheoretical: 523.247 Da
Chemical component information

ChemComp-AGS:
PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / ATP-gamma-S, energy-carrying molecule analogue*YM

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7.6
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 200 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 60.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 0.5 µm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 3148330
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

7nku

Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 114728
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. v3.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. v3.0)
Final 3D classificationSoftware - Name: cryoSPARC (ver. v3.0)
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: AB INITIO MODEL / Target criteria: correlation coefficient
Output model

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

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