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- PDB-8rhn: Structure of the 55LCC ATPase complex -

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Basic information

Entry
Database: PDB / ID: 8rhn
TitleStructure of the 55LCC ATPase complex
Components
  • ATPase family gene 2 protein homolog A
  • ATPase family gene 2 protein homolog B
  • Cyclin-dependent kinase 2-interacting protein
  • cDNA FLJ55172
KeywordsDNA BINDING PROTEIN / DNA replication / AAA+ ATPases / proteostasis
Function / homology
Function and homology information


preribosome binding / non-chaperonin molecular chaperone ATPase / ribosomal large subunit biogenesis / brain development / spindle / spermatogenesis / DNA replication / cell differentiation / cell cycle / cell division ...preribosome binding / non-chaperonin molecular chaperone ATPase / ribosomal large subunit biogenesis / brain development / spindle / spermatogenesis / DNA replication / cell differentiation / cell cycle / cell division / DNA repair / ATP hydrolysis activity / mitochondrion / ATP binding / nucleus / cytoplasm
Similarity search - Function
Ribosome biogenesis protein C1orf109-like / Ribosome biogenesis protein C1orf109-like / Cyclin-dependent kinase 2-interacting protein / Aspartate decarboxylase-like domain superfamily / 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 ...Ribosome biogenesis protein C1orf109-like / Ribosome biogenesis protein C1orf109-like / Cyclin-dependent kinase 2-interacting protein / Aspartate decarboxylase-like domain superfamily / 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
cDNA FLJ55172 / ATPase family gene 2 protein homolog A / ATPase family gene 2 protein homolog B / Cyclin-dependent kinase 2-interacting protein
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.5 Å
AuthorsFoglizzo, M. / Degtjarik, O. / Zeqiraj, E.
Funding support United Kingdom, 3items
OrganizationGrant numberCountry
Medical Research Council (MRC, United Kingdom)MR/T029471/1 United Kingdom
Wellcome Trust222531/Z/21/Z United Kingdom
Wellcome Trust221524/Z/20/Z United Kingdom
CitationJournal: Cell / Year: 2024
Title: The SPATA5-SPATA5L1 ATPase complex directs replisome proteostasis to ensure genome integrity.
Authors: Vidhya Krishnamoorthy / Martina Foglizzo / Robert L Dilley / Angela Wu / Arindam Datta / Parul Dutta / Lisa J Campbell / Oksana Degtjarik / Laura J Musgrove / Antonio N Calabrese / Elton ...Authors: Vidhya Krishnamoorthy / Martina Foglizzo / Robert L Dilley / Angela Wu / Arindam Datta / Parul Dutta / Lisa J Campbell / Oksana Degtjarik / Laura J Musgrove / Antonio N Calabrese / Elton Zeqiraj / Roger A Greenberg /
Abstract: Ubiquitin-dependent unfolding of the CMG helicase by VCP/p97 is required to terminate DNA replication. Other replisome components are not processed in the same fashion, suggesting that additional ...Ubiquitin-dependent unfolding of the CMG helicase by VCP/p97 is required to terminate DNA replication. Other replisome components are not processed in the same fashion, suggesting that additional mechanisms underlie replication protein turnover. Here, we identify replisome factor interactions with a protein complex composed of AAA+ ATPases SPATA5-SPATA5L1 together with heterodimeric partners C1orf109-CINP (55LCC). An integrative structural biology approach revealed a molecular architecture of SPATA5-SPATA5L1 N-terminal domains interacting with C1orf109-CINP to form a funnel-like structure above a cylindrically shaped ATPase motor. Deficiency in the 55LCC complex elicited ubiquitin-independent proteotoxicity, replication stress, and severe chromosome instability. 55LCC showed ATPase activity that was specifically enhanced by replication fork DNA and was coupled to cysteine protease-dependent cleavage of replisome substrates in response to replication fork damage. These findings define 55LCC-mediated proteostasis as critical for replication fork progression and genome stability and provide a rationale for pathogenic variants seen in associated human neurodevelopmental disorders.
History
DepositionDec 15, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Mar 27, 2024Provider: repository / Type: Initial release
Revision 1.1Apr 10, 2024Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID
Revision 1.2May 8, 2024Group: Database references / Category: citation / Item: _citation.journal_volume / _citation.page_first

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
K: ATPase family gene 2 protein homolog A
L: ATPase family gene 2 protein homolog A
M: ATPase family gene 2 protein homolog A
N: ATPase family gene 2 protein homolog A
O: ATPase family gene 2 protein homolog B
P: ATPase family gene 2 protein homolog B
A: cDNA FLJ55172
B: cDNA FLJ55172
C: Cyclin-dependent kinase 2-interacting protein
D: Cyclin-dependent kinase 2-interacting protein
E: ATPase family gene 2 protein homolog A
F: ATPase family gene 2 protein homolog A
G: ATPase family gene 2 protein homolog A
H: ATPase family gene 2 protein homolog A
I: ATPase family gene 2 protein homolog B
J: ATPase family gene 2 protein homolog B


Theoretical massNumber of molelcules
Total (without water)1,257,71716
Polymers1,257,71716
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein
ATPase family gene 2 protein homolog A / AFG2 AAA ATPase homolog A / Ribosome biogenesis protein SPATA5 / Spermatogenesis-associated factor ...AFG2 AAA ATPase homolog A / Ribosome biogenesis protein SPATA5 / Spermatogenesis-associated factor protein / Spermatogenesis-associated protein 5


Mass: 101307.883 Da / Num. of mol.: 8
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: AFG2A, SPAF, SPATA5 / Production host: Spodoptera frugiperda (fall armyworm)
References: UniProt: Q8NB90, non-chaperonin molecular chaperone ATPase
#2: Protein
ATPase family gene 2 protein homolog B / AFG2 AAA ATPase homolog B / Ribosome biogenesis protein SPATA5L1 / Spermatogenesis-associated ...AFG2 AAA ATPase homolog B / Ribosome biogenesis protein SPATA5L1 / Spermatogenesis-associated protein 5-like protein 1


Mass: 83362.836 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: AFG2B, SPATA5L1 / Production host: Spodoptera frugiperda (fall armyworm)
References: UniProt: Q9BVQ7, non-chaperonin molecular chaperone ATPase
#3: Protein cDNA FLJ55172


Mass: 29439.279 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: B4DRQ5
#4: Protein Cyclin-dependent kinase 2-interacting protein / CDK2-interacting protein


Mass: 27462.139 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: CINP / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q9BW66

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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: Structure of the 55LCC ATPase complex / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.649 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm)
Buffer solutionpH: 7.5
Details: 25 mM Tris pH 7.5, 400 mM NaCl, 5% (v/v) glycerol, 1 mM EGTA, 2 mM ADP, 2 mM MgCl2 and 1 mM TCEP
Buffer component
IDConc.NameFormulaBuffer-ID
125 mMtris(hydroxymethyl)aminomethaneTris1
2400 mMsodium chlorideNaCl1
31 mMegtazic acidEGTA1
42 mMadenosine diphosphateADP1
52 mMmagnesium chlorideMgCl21
61 mMtris(2-carboxyethyl)phosphineTCEP1
SpecimenConc.: 3.7 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: Quantifoil R3.5/1 200-mesh grids (Quantifoil Micro Tools GmbH) were glow-discharged for 30 s at 12 mA and 0.38 mBar pressure using a PELCO easiGlow system (Ted Pella).
Grid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R3.5/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K / Details: blot force = 0 N blot time = 8 s

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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 FIELD / Nominal magnification: 165000 X / Nominal defocus max: 3100 nm / Nominal defocus min: 1200 nm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 2.99 sec. / Electron dose: 37.6 e/Å2 / Film or detector model: TFS FALCON 4i (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 27595
EM imaging opticsEnergyfilter name: TFS Selectris / Energyfilter slit width: 10 eV / Phase plate: VOLTA PHASE PLATE

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Processing

EM software
IDNameVersionCategory
1RELION4particle selection
2EPU3.3image acquisition
4RELION4CTF correction
7UCSF ChimeraX1.6.1model fitting
9RELION4initial Euler assignment
10cryoSPARC4.0.1final Euler assignment
11RELION4classification
12cryoSPARC4.0.13D reconstruction
13PHENIX1.17.1model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 4182380
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 4.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 165778 / Num. of class averages: 2 / Symmetry type: POINT
Atomic model buildingB value: 127.55 / Protocol: RIGID BODY FIT / Space: REAL
Atomic model buildingSource name: AlphaFold / Type: in silico model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00639017
ELECTRON MICROSCOPYf_angle_d0.7752843
ELECTRON MICROSCOPYf_dihedral_angle_d8.7145355
ELECTRON MICROSCOPYf_chiral_restr0.0456169
ELECTRON MICROSCOPYf_plane_restr0.0056842

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