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- EMDB-12448: diazaborine bound Drg1(AFG2) -

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Basic information

Entry
Database: EMDB / ID: EMD-12448
Titlediazaborine bound Drg1(AFG2)
Map data
Sample
  • Complex: Drg1 (AFG2) bound to diazaborine
    • Protein or peptide: ATPase family gene 2 protein
  • Ligand: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER
  • Ligand: 6-METHYL-2(PROPANE-1-SULFONYL)-2H-THIENO[3,2-D][1,2,3]DIAZABORININ-1-OL
Function / homology
Function and homology information


mitotic spindle disassembly / VCP-NPL4-UFD1 AAA ATPase complex / protein hexamerization / retrograde protein transport, ER to cytosol / non-chaperonin molecular chaperone ATPase / preribosome, large subunit precursor / autophagosome maturation / polyubiquitin modification-dependent protein binding / ribosomal large subunit biogenesis / response to xenobiotic stimulus ...mitotic spindle disassembly / VCP-NPL4-UFD1 AAA ATPase complex / protein hexamerization / retrograde protein transport, ER to cytosol / non-chaperonin molecular chaperone ATPase / preribosome, large subunit precursor / autophagosome maturation / polyubiquitin modification-dependent protein binding / ribosomal large subunit biogenesis / response to xenobiotic stimulus / ATP hydrolysis activity / ATP binding / nucleus / cytoplasm / cytosol
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 (strain ATCC 204508 / S288c) (yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsPrattes M / Hodirnau VV / Grishkovskaya I / Bergler H / Haselbach D
CitationJournal: Nat Commun / Year: 2021
Title: Structural basis for inhibition of the AAA-ATPase Drg1 by diazaborine.
Authors: Michael Prattes / Irina Grishkovskaya / Victor-Valentin Hodirnau / Ingrid Rössler / Isabella Klein / Christina Hetzmannseder / Gertrude Zisser / Christian C Gruber / Karl Gruber / David ...Authors: Michael Prattes / Irina Grishkovskaya / Victor-Valentin Hodirnau / Ingrid Rössler / Isabella Klein / Christina Hetzmannseder / Gertrude Zisser / Christian C Gruber / Karl Gruber / David Haselbach / Helmut Bergler /
Abstract: The hexameric AAA-ATPase Drg1 is a key factor in eukaryotic ribosome biogenesis and initiates cytoplasmic maturation of the large ribosomal subunit by releasing the shuttling maturation factor Rlp24. ...The hexameric AAA-ATPase Drg1 is a key factor in eukaryotic ribosome biogenesis and initiates cytoplasmic maturation of the large ribosomal subunit by releasing the shuttling maturation factor Rlp24. Drg1 monomers contain two AAA-domains (D1 and D2) that act in a concerted manner. Rlp24 release is inhibited by the drug diazaborine which blocks ATP hydrolysis in D2. The mode of inhibition was unknown. Here we show the first cryo-EM structure of Drg1 revealing the inhibitory mechanism. Diazaborine forms a covalent bond to the 2'-OH of the nucleotide in D2, explaining its specificity for this site. As a consequence, the D2 domain is locked in a rigid, inactive state, stalling the whole Drg1 hexamer. Resistance mechanisms identified include abolished drug binding and altered positioning of the nucleotide. Our results suggest nucleotide-modifying compounds as potential novel inhibitors for AAA-ATPases.
History
DepositionFeb 19, 2021-
Header (metadata) releaseJun 23, 2021-
Map releaseJun 23, 2021-
UpdateJun 23, 2021-
Current statusJun 23, 2021Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.105
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.105
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7nku
  • Surface level: 0.105
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-7nku
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_12448.png

Downloads & links

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Map

FileDownload / File: emd_12448.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.06 Å
Density
Contour LevelBy AUTHOR: 0.105 / Movie #1: 0.105
Minimum - Maximum-0.0017472899 - 1.7435274
Average (Standard dev.)0.0036650223 (±0.038571537)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 271.36 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.061.061.06
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z271.360271.360271.360
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-0.0021.7440.004

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Supplemental data

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Additional map: #2

Fileemd_12448_additional_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: #1

Fileemd_12448_additional_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Drg1 (AFG2) bound to diazaborine

EntireName: Drg1 (AFG2) bound to diazaborine
Components
  • Complex: Drg1 (AFG2) bound to diazaborine
    • Protein or peptide: ATPase family gene 2 protein
  • Ligand: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER
  • Ligand: 6-METHYL-2(PROPANE-1-SULFONYL)-2H-THIENO[3,2-D][1,2,3]DIAZABORININ-1-OL

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Supramolecule #1: Drg1 (AFG2) bound to diazaborine

SupramoleculeName: Drg1 (AFG2) bound to diazaborine / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Recombinant expressionOrganism: Escherichia coli (E. coli)

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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 (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c
Molecular weightTheoretical: 84.850719 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
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

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Macromolecule #2: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER

MacromoleculeName: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / type: ligand / ID: 2 / Number of copies: 12 / 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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Macromolecule #3: 6-METHYL-2(PROPANE-1-SULFONYL)-2H-THIENO[3,2-D][1,2,3]DIAZABORINI...

MacromoleculeName: 6-METHYL-2(PROPANE-1-SULFONYL)-2H-THIENO[3,2-D][1,2,3]DIAZABORININ-1-OL
type: ligand / ID: 3 / Number of copies: 6 / Formula: TDB
Molecular weightTheoretical: 272.152 Da
Chemical component information

ChemComp-TDB:
6-METHYL-2(PROPANE-1-SULFONYL)-2H-THIENO[3,2-D][1,2,3]DIAZABORININ-1-OL

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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
Component:
ConcentrationNameFormula
20.0 mMHEPES-KOH
150.0 mMpotassium acetateKOAc
1.0 mMmagnesium acetateMg(OAc)2
0.005 %Tween-20
1.0 mMDithiotreitolDTT
2.0 mMATPyS
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF Bioquantum
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 opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD
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: 1152861
CTF correctionSoftware - Name: cryoSPARC
Final reconstructionApplied symmetry - Point group: C6 (6 fold cyclic) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Details: polished with deepenhancer / Number images used: 237914
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final 3D classificationNumber classes: 4 / Software - Name: RELION

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Atomic model buiding 1

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

PDB-7nku:
diazaborine bound Drg1(AFG2)

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