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- EMDB-19177: Structure of the 55LCC ATPase complex -

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Basic information

Entry
Database: EMDB / ID: EMD-19177
TitleStructure of the 55LCC ATPase complex
Map dataSharpened lid and ATPase composite map (after local refinement)
Sample
  • Complex: Structure of the 55LCC ATPase complex
    • Protein or peptide: ATPase family gene 2 protein homolog A
    • Protein or peptide: ATPase family gene 2 protein homolog B
    • Protein or peptide: cDNA FLJ55172
    • Protein or peptide: Cyclin-dependent kinase 2-interacting protein
KeywordsDNA replication / AAA+ ATPases / proteostasis / DNA BINDING PROTEIN
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)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.5 Å
AuthorsFoglizzo M / Degtjarik O / Zeqiraj E
Funding support United Kingdom, 3 items
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, 2023-
Header (metadata) releaseMar 27, 2024-
Map releaseMar 27, 2024-
UpdateApr 10, 2024-
Current statusApr 10, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_19177.png

Downloads & links

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Map

FileDownload / File: emd_19177.map.gz / Format: CCP4 / Size: 209.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSharpened lid and ATPase composite map (after local refinement)
Voxel sizeX=Y=Z: 0.74 Å
Density
Contour LevelBy AUTHOR: 0.131
Minimum - Maximum-0.41347167 - 0.78523266
Average (Standard dev.)0.0036330218 (±0.029292637)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions380380380
Spacing380380380
CellA=B=C: 281.2 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_19177_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Unsharpened ATPase map (after local refinement)

Fileemd_19177_additional_1.map
AnnotationUnsharpened ATPase map (after local refinement)
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Unsharpened lid map (after local refinement)

Fileemd_19177_additional_2.map
AnnotationUnsharpened lid map (after local refinement)
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Sharpened full map (before local refinement)

Fileemd_19177_additional_3.map
AnnotationSharpened full map (before local refinement)
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Unsharpened full map (before local refinement)

Fileemd_19177_additional_4.map
AnnotationUnsharpened full map (before local refinement)
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map A

Fileemd_19177_half_map_1.map
AnnotationHalf map A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map B

Fileemd_19177_half_map_2.map
AnnotationHalf map B
Projections & Slices
AxesZYX

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Sample components

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Entire : Structure of the 55LCC ATPase complex

EntireName: Structure of the 55LCC ATPase complex
Components
  • Complex: Structure of the 55LCC ATPase complex
    • Protein or peptide: ATPase family gene 2 protein homolog A
    • Protein or peptide: ATPase family gene 2 protein homolog B
    • Protein or peptide: cDNA FLJ55172
    • Protein or peptide: Cyclin-dependent kinase 2-interacting protein

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Supramolecule #1: Structure of the 55LCC ATPase complex

SupramoleculeName: Structure of the 55LCC ATPase complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 649 KDa

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Macromolecule #1: ATPase family gene 2 protein homolog A

MacromoleculeName: ATPase family gene 2 protein homolog A / type: protein_or_peptide / ID: 1 / Number of copies: 8 / Enantiomer: LEVO / EC number: non-chaperonin molecular chaperone ATPase
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 101.307883 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MSYYHHHHHH DYDIPTTENL YFQGAMGMSS KKNRKRLNQS AENGSSLPSA ASSCAEARAP SAGSDFAATS GTLTVTNLLE KVDDKIPKT FQNSLIHLGL NTMKSANICI GRPVLLTSLN GKQEVYTAWP MAGFPGGKVG LSEMAQKNVG VRPGDAIQVQ P LVGAVLQA ...String:
MSYYHHHHHH DYDIPTTENL YFQGAMGMSS KKNRKRLNQS AENGSSLPSA ASSCAEARAP SAGSDFAATS GTLTVTNLLE KVDDKIPKT FQNSLIHLGL NTMKSANICI GRPVLLTSLN GKQEVYTAWP MAGFPGGKVG LSEMAQKNVG VRPGDAIQVQ P LVGAVLQA EEMDVALSDK DMEINEEELT GCILRKLDGK IVLPGNFLYC TFYGRPYKLQ VLRVKGADGM ILGGPQSDSD TD AQRMAFE QSSMETSSLE LSLQLSQLDL EDTQIPTSRS TPYKPIDDRI TNKASDVLLD VTQSPGDGSG LMLEEVTGLK CNF ESAREG NEQLTEEERL LKFSIGAKCN TDTFYFISST TRVNFTEIDK NSKEQDNQFK VTYDMIGGLS SQLKAIREII ELPL KQPEL FKSYGIPAPR GVLLYGPPGT GKTMIARAVA NEVGAYVSVI NGPEIISKFY GETEAKLRQI FAEATLRHPS IIFID ELDA LCPKREGAQN EVEKRVVASL LTLMDGIGSE VSEGQVLVLG ATNRPHALDA ALRRPGRFDK EIEIGVPNAQ DRLDIL QKL LRRVPHLLTE AELLQLANSA HGYVGADLKV LCNEAGLCAL RRILKKQPNL PDVKVAGLVK ITLKDFLQAM NDIRPSA MR EIAIDVPNVS WSDIGGLESI KLKLEQAVEW PLKHPESFIR MGIQPPKGVL LYGPPGCSKT MIAKALANES GLNFLAIK G PELMNKYVGE SERAVRETFR KARAVAPSII FFDELDALAV ERGSSLGAGN VADRVLAQLL TEMDGIEQLK DVTILAATN RPDRIDKALM RPGRIDRIIY VPLPDAATRR EIFKLQFHSM PVSNEVDLDE LILQTDAYSG AEIVAVCREA ALLALEEDIQ ANLIMKRHF TQALSTVTPR IPESLRRFYE DYQEKSGLHT L

UniProtKB: ATPase family gene 2 protein homolog A

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Macromolecule #2: ATPase family gene 2 protein homolog B

MacromoleculeName: ATPase family gene 2 protein homolog B / type: protein_or_peptide / ID: 2 / Number of copies: 4 / Enantiomer: LEVO / EC number: non-chaperonin molecular chaperone ATPase
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 83.362836 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MDYKDDDDKG GGSENLYFQG AGSTMAPDSD PFPEGPLLKL LPLDARDRGT QRCRLGPAAL HALGARLGSA VKISLPDGGS CLCTAWPRR DGADGFVQLD PLCASPGAAV GASRSRRSLS LNRLLLVPCP PLRRVAVWPV LRERAGAPGA RNTAAVLEAA Q ELLRNRPI ...String:
MDYKDDDDKG GGSENLYFQG AGSTMAPDSD PFPEGPLLKL LPLDARDRGT QRCRLGPAAL HALGARLGSA VKISLPDGGS CLCTAWPRR DGADGFVQLD PLCASPGAAV GASRSRRSLS LNRLLLVPCP PLRRVAVWPV LRERAGAPGA RNTAAVLEAA Q ELLRNRPI SLGHVVVAPP GAPGLVAALH IVGGTPSPDP AGLVTPRTRV SLGGEPPSEA QPQPEVPLGG LSEAADSLRE LL RLPLRYP RALTALGLAV PRGVLLAGPP GVGKTQLVRA VAREAGAELL AVSAPALQGS RPGETEENVR RVFQRARELA SRG PSLLFL DEMDALCPQR GSRAPESRVV AQVLTLLDGA SGDREVVVVG ATNRPDALDP ALRRPGRFDR EVVIGTPTLK QRKE ILQVI TSKMPISSHV DLGLLAEMTV GYVGADLTAL CREAAMHALL HSEKNQDNPV IDEIDFLEAF KNIQPSSFRS VIGLM DIKP VDWEEIGGLE DVKLKLKQSI EWPLKFPWEF VRMGLTQPKG VLLYGPPGCA KTTLVRALAT SCHCSFVSVS GADLFS PFV GDSEKVLSQI FRQARASTPA ILFLDEIDSI LGARSASKTG CDVQERVLSV LLNELDGVGL KTIERRGSKS SQQEFQE VF NRSVMIIAAT NRPDVLDTAL LRPGRLDKII YIPPPDHKGR LSILKVCTKT MPIGPDVSLE NLAAETCFFS GADLRNLC T EAALLALQEN GLDATTVKQE HFLKSLKTVK PSLSCKDLAL YENLFKKEGF SNVEGI

UniProtKB: ATPase family gene 2 protein homolog B

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Macromolecule #3: cDNA FLJ55172

MacromoleculeName: cDNA FLJ55172 / type: protein_or_peptide / ID: 3 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 29.439279 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MSAWSHPQFE KGGGSGGGSG GSAWSHPQFE KGAGSENLYF QGAGSDSLEF IASKLAGGGS TMTQDRPLLA VQEALKKCFP VVEEQQGLW QSALRDCQPL LSSLSNLAEQ LQAAQNLRFE DVPALRAFPD LKERLRRKQL VAGDIVLDKL GERLAILLKV R DMVSSHVE ...String:
MSAWSHPQFE KGGGSGGGSG GSAWSHPQFE KGAGSENLYF QGAGSDSLEF IASKLAGGGS TMTQDRPLLA VQEALKKCFP VVEEQQGLW QSALRDCQPL LSSLSNLAEQ LQAAQNLRFE DVPALRAFPD LKERLRRKQL VAGDIVLDKL GERLAILLKV R DMVSSHVE RVFQIYEQHA DTVGIDAVLQ PSAVSPSVAD MLEWLQDIER HYRKSYLKRK YLLSSIQWGD LANIQALPKA WD RISKDEH QDLVQDILLN VSFFLEE

UniProtKB: cDNA FLJ55172

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Macromolecule #4: Cyclin-dependent kinase 2-interacting protein

MacromoleculeName: Cyclin-dependent kinase 2-interacting protein / type: protein_or_peptide / ID: 4 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 27.462139 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MSYYHHHHHH DYDIPTTENL YFQGAMEAKT LGTVTPRKPV LSVSARKIKD NAADWHNLIL KWETLNDAGF TTANNIANLK ISLLNKDKI ELDSSSPASK ENEEKVCLEY NEELEKLCEE LQATLDGLTK IQVKMEKLSS TTKGICELEN YHYGEESKRP P LFHTWPTT ...String:
MSYYHHHHHH DYDIPTTENL YFQGAMEAKT LGTVTPRKPV LSVSARKIKD NAADWHNLIL KWETLNDAGF TTANNIANLK ISLLNKDKI ELDSSSPASK ENEEKVCLEY NEELEKLCEE LQATLDGLTK IQVKMEKLSS TTKGICELEN YHYGEESKRP P LFHTWPTT HFYEVSHKLL EMYRKELLLK RTVAKELAHT GDPDLTLSYL SMWLHQPYVE SDSRLHLESM LLETGHRAL

UniProtKB: Cyclin-dependent kinase 2-interacting protein

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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

Concentration3.7 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
25.0 mMTristris(hydroxymethyl)aminomethane
400.0 mMNaClSodium chloridesodium chloride
1.0 mMEGTAegtazic acid
2.0 mMADPAdenosine diphosphateadenosine diphosphate
2.0 mMMgCl2magnesium chloride
1.0 mMTCEPtris(2-carboxyethyl)phosphine

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
GridModel: Quantifoil R3.5/1 / Material: COPPER / Mesh: 200 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec. / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.00038 kPa
Details: 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).
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV / Details: blot force = 0 N blot time = 8 s.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 3.1 µm / Nominal defocus min: 1.2 µm / Nominal magnification: 165000
Specialist opticsPhase plate: VOLTA PHASE PLATE / Energy filter - Name: TFS Selectris / Energy filter - Slit width: 10 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: TFS FALCON 4i (4k x 4k) / Number grids imaged: 1 / Number real images: 27595 / Average exposure time: 2.99 sec. / Average electron dose: 37.6 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 4182380
Startup modelType of model: OTHER
Details: A previously determined 3D volume was used as the startup model
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 4.0)
Final 3D classificationNumber classes: 6 / Software - Name: RELION (ver. 4.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.0.1)
Final reconstructionNumber classes used: 2 / Applied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 4.5 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.0.1) / Number images used: 165778
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelChain - Source name: AlphaFold / Chain - Initial model type: in silico model
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Overall B value: 127.55
Output model

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

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