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- PDB-6or5: Full-length S. pombe Mdn1 in the presence of AMPPNP (ring region) -

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Entry
Database: PDB / ID: 6or5
TitleFull-length S. pombe Mdn1 in the presence of AMPPNP (ring region)
ComponentsMidasin
KeywordsMOTOR PROTEIN / Ribosome biogenesis / AAA protein / Mechanochemical enzyme / Ribosome assembly factor
Function / homology
Function and homology information


preribosome, large subunit precursor / rRNA processing / ribosomal large subunit assembly / ATPase activity / nucleolus / calcium ion binding / nucleoplasm / ATP binding
ATPase, dynein-related, AAA domain / Midasin, AAA lid domain 7 / VWFA domain profile. / Midasin AAA lid domain / Midasin AAA lid domain / AAA domain (dynein-related subfamily) / von Willebrand factor, type A / AAA+ ATPase domain / Midasin AAA lid domain 5 / Midasin ...ATPase, dynein-related, AAA domain / Midasin, AAA lid domain 7 / VWFA domain profile. / Midasin AAA lid domain / Midasin AAA lid domain / AAA domain (dynein-related subfamily) / von Willebrand factor, type A / AAA+ ATPase domain / Midasin AAA lid domain 5 / Midasin / P-loop containing nucleoside triphosphate hydrolase / von Willebrand factor A-like domain superfamily
Midasin
Biological speciesSchizosaccharomyces pombe (fission yeast)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4 Å
AuthorsChen, Z. / Suzuki, H. / Wang, A.C. / DiMaio, F. / Walz, T. / Kapoor, T.M.
Funding supportUnited States , 1件
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical SciencesGM98579United States
CitationJournal: Cell / Year: 2018
Title: Structural Insights into Mdn1, an Essential AAA Protein Required for Ribosome Biogenesis.
Authors: Zhen Chen / Hiroshi Suzuki / Yuki Kobayashi / Ashley C Wang / Frank DiMaio / Shigehiro A Kawashima / Thomas Walz / Tarun M Kapoor /
Abstract: Mdn1 is an essential AAA (ATPase associated with various activities) protein that removes assembly factors from distinct precursors of the ribosomal 60S subunit. However, Mdn1's large size (∼5,000 ...Mdn1 is an essential AAA (ATPase associated with various activities) protein that removes assembly factors from distinct precursors of the ribosomal 60S subunit. However, Mdn1's large size (∼5,000 amino acid [aa]) and its limited homology to other well-studied proteins have restricted our understanding of its remodeling function. Here, we present structures for S. pombe Mdn1 in the presence of AMPPNP at up to ∼4 Å or ATP plus Rbin-1, a chemical inhibitor, at ∼8 Å resolution. These data reveal that Mdn1's MIDAS domain is tethered to its ring-shaped AAA domain through an ∼20 nm long structured linker and a flexible ∼500 aa Asp/Glu-rich motif. We find that the MIDAS domain, which also binds other ribosome-assembly factors, docks onto the AAA ring in a nucleotide state-specific manner. Together, our findings reveal how conformational changes in the AAA ring can be directly transmitted to the MIDAS domain and thereby drive the targeted release of assembly factors from ribosomal 60S-subunit precursors.
Validation Report
SummaryFull reportAbout validation report
DateDeposition: Apr 29, 2019 / Release: May 29, 2019
RevisionDateData content typeProviderType
1.0May 29, 2019Structure modelrepositoryInitial release

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Assembly

Deposited unit
A: Midasin
hetero molecules


Theoretical massNumber of molelcules
Total (without water)540,9126
Polymers538,3811
Non-polymers2,5315
Water0
1


TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein/peptide Midasin / Dynein-related AAA-ATPase mdn1 / MIDAS-containing protein


Mass: 538381.125 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Schizosaccharomyces pombe (fission yeast)
Gene: mdn1, SPCC737.08 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: O94248
#2: Chemical
ChemComp-ANP / PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER


Mass: 506.196 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: C10H17N6O12P3 / Comment: AMP-PNP (energy-carrying molecule analogue) *YM

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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: N-terminal density map of the full-length Mdn1 in the presence of AMPPNP
Type: COMPLEX / Entity ID: 1 / Source: RECOMBINANT
Molecular weightValue: 0.54 MDa / Experimental value: NO
Source (natural)Organism: Schizosaccharomyces pombe (fission yeast)
Source (recombinant)Organism: Trichoplusia ni (cabbage looper)
Buffer solutionpH: 7.5
SpecimenConc.: 0.15 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 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 magnification: 22500 X / Nominal defocus max: 3000 nm / Nominal defocus min: 1400 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µns
Specimen holderCryogen: NITROGEN / Model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 15 sec. / Electron dose: 88.8 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of real images: 8446
Image scansMovie frames/image: 50 / Used frames/image: 1-50

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Processing

EM software
IDNameVersionCategory
1Gautomatch0.53particle selection
2SerialEMimage acquisition
4CTFFIND4.1.5CTF correction
7Cootmodel fitting
9RELION2.0.3initial Euler assignment
10FREALIGNfinal Euler assignment
11RELION2.0.3classification
12FREALIGN3D reconstruction
13PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 313336 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL

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