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- PDB-7ttr: Skd3_ATPyS_FITC-casein Hexamer, AAA+ only -

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Basic information

Entry
Database: PDB / ID: 7ttr
TitleSkd3_ATPyS_FITC-casein Hexamer, AAA+ only
Components
  • Beta-caseinCSN2
  • Caseinolytic peptidase B protein homolog
KeywordsCHAPERONE / cryoEM / AAA+
Function / homology
Function and homology information


granulocyte differentiation / RIG-I signaling pathway / ATP-dependent protein disaggregase activity / Hydrolases; Acting on acid anhydrides; In phosphorus-containing anhydrides / antiviral innate immune response / mitochondrial intermembrane space / cellular response to heat / ATP hydrolysis activity / mitochondrion / extracellular space ...granulocyte differentiation / RIG-I signaling pathway / ATP-dependent protein disaggregase activity / Hydrolases; Acting on acid anhydrides; In phosphorus-containing anhydrides / antiviral innate immune response / mitochondrial intermembrane space / cellular response to heat / ATP hydrolysis activity / mitochondrion / extracellular space / extracellular region / ATP binding / cytoplasm
Similarity search - Function
Casein, beta / Casein, alpha/beta / Casein / Casein alpha/beta, conserved site / Caseins alpha/beta signature. / ClpA/B family / Clp ATPase, C-terminal / AAA domain (Cdc48 subfamily) / C-terminal, D2-small domain, of ClpB protein / C-terminal, D2-small domain, of ClpB protein ...Casein, beta / Casein, alpha/beta / Casein / Casein alpha/beta, conserved site / Caseins alpha/beta signature. / ClpA/B family / Clp ATPase, C-terminal / AAA domain (Cdc48 subfamily) / C-terminal, D2-small domain, of ClpB protein / C-terminal, D2-small domain, of ClpB protein / Ankyrin repeats (3 copies) / Ankyrin repeat profile. / ATPase, AAA-type, core / Ankyrin repeat region circular profile. / ankyrin repeats / Ankyrin repeat / Ankyrin repeat-containing domain superfamily / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
ADENOSINE-5'-DIPHOSPHATE / PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / Mitochondrial disaggregase / Beta-casein
Similarity search - Component
Biological speciesHomo sapiens (human)
Bos taurus (cattle)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.96 Å
AuthorsRizo, A.N.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM138690 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM099836 United States
CitationJournal: Cell Rep / Year: 2022
Title: Unique structural features govern the activity of a human mitochondrial AAA+ disaggregase, Skd3.
Authors: Ryan R Cupo / Alexandrea N Rizo / Gabriel A Braun / Eric Tse / Edward Chuang / Kushol Gupta / Daniel R Southworth / James Shorter /
Abstract: The AAA+ protein, Skd3 (human CLPB), solubilizes proteins in the mitochondrial intermembrane space, which is critical for human health. Skd3 variants with defective protein-disaggregase activity ...The AAA+ protein, Skd3 (human CLPB), solubilizes proteins in the mitochondrial intermembrane space, which is critical for human health. Skd3 variants with defective protein-disaggregase activity cause severe congenital neutropenia (SCN) and 3-methylglutaconic aciduria type 7 (MGCA7). How Skd3 disaggregates proteins remains poorly understood. Here, we report a high-resolution structure of a Skd3-substrate complex. Skd3 adopts a spiral hexameric arrangement that engages substrate via pore-loop interactions in the nucleotide-binding domain (NBD). Substrate-bound Skd3 hexamers stack head-to-head via unique, adaptable ankyrin-repeat domain (ANK)-mediated interactions to form dodecamers. Deleting the ANK linker region reduces dodecamerization and disaggregase activity. We elucidate apomorphic features of the Skd3 NBD and C-terminal domain that regulate disaggregase activity. We also define how Skd3 subunits collaborate to disaggregate proteins. Importantly, SCN-linked subunits sharply inhibit disaggregase activity, whereas MGCA7-linked subunits do not. These advances illuminate Skd3 structure and mechanism, explain SCN and MGCA7 inheritance patterns, and suggest therapeutic strategies.
History
DepositionFeb 1, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 28, 2022Provider: repository / Type: Initial release
Revision 1.1Oct 12, 2022Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.year

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Caseinolytic peptidase B protein homolog
B: Caseinolytic peptidase B protein homolog
C: Caseinolytic peptidase B protein homolog
D: Caseinolytic peptidase B protein homolog
E: Caseinolytic peptidase B protein homolog
F: Caseinolytic peptidase B protein homolog
P: Beta-casein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)424,06716
Polymers421,4507
Non-polymers2,6179
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
Caseinolytic peptidase B protein homolog / Suppressor of potassium transport defect 3


Mass: 66115.047 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: CLPB, HSP78, SKD3 / Production host: Escherichia coli BL21(DE3) (bacteria)
References: UniProt: Q9H078, Hydrolases; Acting on acid anhydrides; In phosphorus-containing anhydrides
#2: Protein Beta-casein / CSN2


Mass: 24759.648 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Bos taurus (cattle) / References: UniProt: T1T0C1
#3: Chemical ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE / Adenosine diphosphate


Mass: 427.201 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Feature type: SUBJECT OF INVESTIGATION / Comment: ADP, energy-carrying molecule*YM
#4: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
#5: 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.: 4 / Source method: obtained synthetically / Formula: C10H16N5O12P3S / Feature type: SUBJECT OF INVESTIGATION / Comment: ATP-gamma-S, energy-carrying molecule analogue*YM
Has ligand of interestY
Sequence detailsThe residues that were modeled in the casein were modeled as UNK due to the fact that they could ...The residues that were modeled in the casein were modeled as UNK due to the fact that they could not be identified. The actual sequence of the casein is MKVLILACLVALALARELEELNVPGEIVESLSSSEESITRINKKIEKFQSEEQQQTEDEL QDKIHPFAQTQSLVYPFPGPIPNSLPQNIPPLTQTPVVVPPFLQPEVMGVSKVKGAMAPK HKEMPFPKYPVEPLTESQSLTLTDVENLHLPLPLLQSWMHQPHQPLPPTVMFPPQSVLSL SQSKVLPVPQKAVPYPQRDMPIQAFLLYQEPVLGPVRGPFPIIV

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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: Skd3 complex bound to FITC-casein / Type: COMPLEX / Entity ID: #1-#2 / Source: MULTIPLE SOURCES
Molecular weight
IDEntity assembly-IDExperimental value
11NO
21NO
31NO
Buffer solutionpH: 8
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 %

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Electron microscopy imaging

MicroscopyModel: FEI TITAN
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 1500 nm / Nominal defocus min: 800 nm
Image recordingElectron dose: 68 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.19.1_4122: / Classification: refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.96 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 358000 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00222004
ELECTRON MICROSCOPYf_angle_d0.41629671
ELECTRON MICROSCOPYf_dihedral_angle_d4.0932947
ELECTRON MICROSCOPYf_chiral_restr0.0343247
ELECTRON MICROSCOPYf_plane_restr0.0023864

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