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- PDB-7nku: diazaborine bound Drg1(AFG2) -

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Basic information

Entry
Database: PDB / ID: 7nku
Titlediazaborine bound Drg1(AFG2)
ComponentsATPase family gene 2 protein
KeywordsRIBOSOME / DRG1 / AFG2 / ribosome maturation / AAA protein / diazaborine / inhibitor
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 / Chem-TDB / ATPase family gene 2 protein
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsPrattes, M. / 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, 2021Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jun 23, 2021Provider: repository / Type: Initial release

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Structure visualization

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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
hetero molecules


Theoretical massNumber of molelcules
Total (without water)517,01624
Polymers509,1046
Non-polymers7,91218
Water0
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area42830 Å2
ΔGint-213 kcal/mol
Surface area131790 Å2
MethodPISA

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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 (strain ATCC 204508 / S288c) (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-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.: 12 / Source method: obtained synthetically / Formula: C10H16N5O12P3S / Comment: ATP-gamma-S, energy-carrying molecule analogue*YM
#3: Chemical
ChemComp-TDB / 6-METHYL-2(PROPANE-1-SULFONYL)-2H-THIENO[3,2-D][1,2,3]DIAZABORININ-1-OL / Diazaborine


Mass: 272.152 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: C9H13BN2O3S2 / Feature type: SUBJECT OF INVESTIGATION
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 (AFG2) bound to diazaborine / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.6
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMHEPES-KOH1
2150 mMpotassium acetateKOAc1
31 mMmagnesium acetateMg(OAc)21
40.005 %Tween-201
51 mMDithiotreitolDTT1
62 mMATPyS1
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
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 FIELDBright-field microscopy
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 name: GIF Bioquantum

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Processing

SoftwareName: PHENIX / Version: 1.18.2_3874: / Classification: refinement
EM software
IDNameVersionCategory
2SerialEMimage acquisition
4cryoSPARCCTF correction
7UCSF Chimeramodel fitting
8Coot0.9model fitting
10cryoSPARCinitial Euler assignment
11cryoSPARCfinal Euler assignment
12RELIONclassification
13cryoSPARC3D reconstruction
14Rosettamodel refinement
15PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1152861
SymmetryPoint symmetry: C6 (6 fold cyclic)
3D reconstructionResolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 237914 / Algorithm: FOURIER SPACE / Details: polished with deepenhancer / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL / Space: REAL / Target criteria: correlation coefficient
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00625794
ELECTRON MICROSCOPYf_angle_d0.82535004
ELECTRON MICROSCOPYf_dihedral_angle_d11.6263642
ELECTRON MICROSCOPYf_chiral_restr0.0524026
ELECTRON MICROSCOPYf_plane_restr0.0064488

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