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- PDB-9hb4: Hexameric RuvBL1/RuvBL2 bound to SPAG1 C-ter -

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Basic information

Entry
Database: PDB / ID: 9hb4
TitleHexameric RuvBL1/RuvBL2 bound to SPAG1 C-ter
Components
  • RuvB-like 1
  • RuvB-like 2
  • Sperm-associated antigen 1
KeywordsCHAPERONE / AAA+ ATPase / R2SP Complex / R2TP-Like complex / PAQosome / Molecular Motor
Function / homology
Function and homology information


axonemal dynein complex assembly / promoter-enhancer loop anchoring activity / telomerase RNA localization to Cajal body / regulation of DNA strand elongation / positive regulation of telomere maintenance in response to DNA damage / establishment of protein localization to chromatin / R2TP complex / dynein axonemal particle / RPAP3/R2TP/prefoldin-like complex / Swr1 complex ...axonemal dynein complex assembly / promoter-enhancer loop anchoring activity / telomerase RNA localization to Cajal body / regulation of DNA strand elongation / positive regulation of telomere maintenance in response to DNA damage / establishment of protein localization to chromatin / R2TP complex / dynein axonemal particle / RPAP3/R2TP/prefoldin-like complex / Swr1 complex / Ino80 complex / regulation of double-strand break repair / box C/D snoRNP assembly / regulation of chromosome organization / NuA4 histone acetyltransferase complex / regulation of DNA replication / TFIID-class transcription factor complex binding / MLL1 complex / regulation of embryonic development / Telomere Extension By Telomerase / protein folding chaperone complex / single fertilization / regulation of DNA repair / RNA polymerase II core promoter sequence-specific DNA binding / positive regulation of double-strand break repair via homologous recombination / Deposition of new CENPA-containing nucleosomes at the centromere / TBP-class protein binding / DNA helicase activity / telomere maintenance / positive regulation of DNA repair / cellular response to estradiol stimulus / euchromatin / negative regulation of canonical Wnt signaling pathway / Formation of the beta-catenin:TCF transactivating complex / ADP binding / chromatin DNA binding / beta-catenin binding / DNA Damage Recognition in GG-NER / nuclear matrix / cellular response to UV / unfolded protein binding / transcription corepressor activity / UCH proteinases / positive regulation of canonical Wnt signaling pathway / nucleosome / HATs acetylate histones / protein folding / ATPase binding / DNA recombination / regulation of apoptotic process / spermatogenesis / DNA helicase / transcription coactivator activity / regulation of cell cycle / Ub-specific processing proteases / protein stabilization / cilium / nuclear speck / ciliary basal body / RNA polymerase II cis-regulatory region sequence-specific DNA binding / cadherin binding / chromatin remodeling / ribonucleoprotein complex / cell division / hydrolase activity / DNA repair / regulation of transcription by RNA polymerase II / centrosome / regulation of DNA-templated transcription / positive regulation of DNA-templated transcription / GTP binding / protein homodimerization activity / positive regulation of transcription by RNA polymerase II / ATP hydrolysis activity / extracellular exosome / nucleoplasm / ATP binding / membrane / identical protein binding / nucleus / cytoplasm / cytosol
Similarity search - Function
: / RNA-polymerase II-associated protein 3-like, C-terminal domain / Potential Monad-binding region of RPAP3 / RuvB-like / RuvB-like, AAA-lid domain / RuvBL1/2, DNA/RNA binding domain / TIP49 P-loop domain / TIP49 AAA-lid domain / TIP49, P-loop domain / Tetratricopeptide repeat ...: / RNA-polymerase II-associated protein 3-like, C-terminal domain / Potential Monad-binding region of RPAP3 / RuvB-like / RuvB-like, AAA-lid domain / RuvBL1/2, DNA/RNA binding domain / TIP49 P-loop domain / TIP49 AAA-lid domain / TIP49, P-loop domain / Tetratricopeptide repeat / Tetratricopeptide repeat / TPR repeat region circular profile. / TPR repeat profile. / Tetratricopeptide repeats / Tetratricopeptide repeat / Tetratricopeptide-like helical 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 / Sperm-associated antigen 1 / RuvB-like 2 / RuvB-like 1
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.56 Å
AuthorsSanto, P.E. / Plisson-Chastang, C.
Funding support France, Portugal, 7items
OrganizationGrant numberCountry
Centre National de la Recherche Scientifique (CNRS)ANR-16-CE11-0032-04 France
Centre National de la Recherche Scientifique (CNRS)ANR-23-CE12-0022 YMCR France
Centre National de la Recherche Scientifique (CNRS)ANR-23-CE44-0035 NAProt-XLMS France
Fundacao para a Ciencia e a TecnologiaUIDB/04462/2020 Portugal
Fundacao para a Ciencia e a TecnologiaUIDP/04462/2020 Portugal
Other governmentProFI - ANR-10-INBS-08-03
Other governmentLS4FUTURE - LA/P/0087/2020
CitationJournal: Nat Commun / Year: 2026
Title: An integrative structural biology approach reveals the dynamic organization of the R2SP quaternary chaperone complex.
Authors: Paulo E Santo / Marie-Eve Chagot / Hugo Gizardin-Fredon / Marie Ley / Thomas Chenuel / Evolène Deslignière / Laura Plassart / Ana C F Paiva / Pedro M F Sousa / Edouard Bertrand / Bruno ...Authors: Paulo E Santo / Marie-Eve Chagot / Hugo Gizardin-Fredon / Marie Ley / Thomas Chenuel / Evolène Deslignière / Laura Plassart / Ana C F Paiva / Pedro M F Sousa / Edouard Bertrand / Bruno Charpentier / Céline Verheggen / Marc Quinternet / Philippe Meyer / Tiago M Bandeiras / Sarah Cianférani / Célia Plisson-Chastang / Xavier Manival /
Abstract: R2SP belongs to the R2TP-like quaternary chaperone family and consists of RUVBL1/RUVBL2 AAA+ ATPases, which powers the machinery, and SPAG1 and PIH1D2 adapter proteins that engage specific clients to ...R2SP belongs to the R2TP-like quaternary chaperone family and consists of RUVBL1/RUVBL2 AAA+ ATPases, which powers the machinery, and SPAG1 and PIH1D2 adapter proteins that engage specific clients to promote their quaternary assembly. However, little is known about the structure of R2SP and the precise mode of action of these R2TP-like complexes. Here, we combined biochemical (ATPase and fluorescence polarization assays) and structural approaches (NMR, structural mass spectrometry, cryo-EM) to investigate the 3D organization of the R2SP complex, its mode of assembly and ATPase activity. Together with our binding, mutational and kinetic studies, these results led us to propose a model in which SPAG1 and PIH1D2 bind and cooperatively engage RUVBL1/RUVBL2 to produce R2SP. This reveals a 3D structure close to the canonical R2TP complex but also highlights differences in RUVBL1/RUVBL2 ATPase activity, as well as the cooperative binding of SPAG1 and PIH1D2 to this catalytic core that may explain functional difference between the two systems.
History
DepositionNov 5, 2024Deposition site: PDBE / Processing site: PDBE
Revision 1.0Nov 19, 2025Provider: repository / Type: Initial release
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: RuvB-like 1
B: RuvB-like 1
C: RuvB-like 1
D: RuvB-like 2
E: RuvB-like 2
F: RuvB-like 2
G: Sperm-associated antigen 1
H: Sperm-associated antigen 1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)514,71714
Polymers512,1538
Non-polymers2,5636
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 RuvB-like 1 / 49 kDa TATA box-binding protein-interacting protein / 49 kDa TBP-interacting protein / 54 kDa ...49 kDa TATA box-binding protein-interacting protein / 49 kDa TBP-interacting protein / 54 kDa erythrocyte cytosolic protein / ECP-54 / INO80 complex subunit H / Nuclear matrix protein 238 / NMP 238 / Pontin 52 / TIP49a / TIP60-associated protein 54-alpha / TAP54-alpha


Mass: 50296.914 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: RUVBL1, INO80H, NMP238, TIP49, TIP49A / Production host: Escherichia coli (E. coli) / References: UniProt: Q9Y265, DNA helicase
#2: Protein RuvB-like 2 / 48 kDa TATA box-binding protein-interacting protein / 48 kDa TBP-interacting protein / 51 kDa ...48 kDa TATA box-binding protein-interacting protein / 48 kDa TBP-interacting protein / 51 kDa erythrocyte cytosolic protein / ECP-51 / INO80 complex subunit J / Repressing pontin 52 / Reptin 52 / TIP49b / TIP60-associated protein 54-beta / TAP54-beta


Mass: 51222.465 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: RUVBL2, INO80J, TIP48, TIP49B, CGI-46 / Production host: Escherichia coli (E. coli) / References: UniProt: Q9Y230, DNA helicase
#3: Protein Sperm-associated antigen 1 / HSD-3.8 / Infertility-related sperm protein Spag-1


Mass: 103797.664 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Details: Truncated Construct from 622-926 / Source: (gene. exp.) Homo sapiens (human) / Gene: SPAG1 / Production host: Escherichia coli (E. coli) / References: UniProt: Q07617
#4: Chemical
ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE


Mass: 427.201 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Feature type: SUBJECT OF INVESTIGATION / Comment: ADP, energy-carrying molecule*YM
Has ligand of interestY
Has protein modificationN

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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

Component
IDNameTypeDetailsEntity IDParent-IDSource
1Hexameric RuvBL1/RuvBL2 complex bound to C-terminal Reagion of SPAG1COMPLEXCo-expressed RuvBL1/2 complex incubated with co-expressed SPAG1/PIH1D2 complex#1-#30RECOMBINANT
2Components that form the hexameric RingCOMPLEXRuvBL1 (Chains A-C), amino acids 125-235, and RuvBL2 (Chain E), amino acids 133-238, are present in this model, although the final PostProcess map lacks sufficient density. These reagions were rigid fitted in the Refine Map (Reference models 2C9O for RuvBL1 and 6H7X for RuvBL2).#1-#21RECOMBINANT
Molecular weight
IDEntity assembly-IDValue (°)Experimental value
110.338 MDaNO
22
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
21Homo sapiens (human)9606
32Homo sapiens (human)9606
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
21Escherichia coli (E. coli)562
32Escherichia coli (E. coli)562
Buffer solutionpH: 8 / Details: 20 mM HEPES, 170 mM NaCL, 2mM MgCl2, 0.5 mM TCEP
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMHEPESC8H18N2O4S1
2170 mMSodium ClorideNaCl1
30.5 mMTCEPC9H15O6P1
42 mMMagnesium ChlorideMgCl21
SpecimenConc.: 0.45 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: Sample was monodisperse
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R2/1
VitrificationInstrument: LEICA EM GP / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 292 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 130000 X / Nominal defocus max: 2000 nm / Nominal defocus min: 900 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 51.92 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 16000 / Details: 40 frames
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV

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Processing

EM software
IDNameVersionCategory
1RELION3particle selection
2SerialEMimage acquisition
4GctfCTF correction
7UCSF Chimera1.17model fitting
9PHENIX1.21model refinement
10RELION3initial Euler assignment
11RELION3final Euler assignment
12RELION3classification
13RELION33D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 2158039
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.56 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 167033 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingB value: 126 / Protocol: RIGID BODY FIT / Space: REAL / Target criteria: Cross-correlation coefficient
Details: Initial Local fitting was done using chimera, and PHENIX was used for flexible fitting
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00320097
ELECTRON MICROSCOPYf_angle_d0.70827109
ELECTRON MICROSCOPYf_dihedral_angle_d7.9322802
ELECTRON MICROSCOPYf_chiral_restr0.0443197
ELECTRON MICROSCOPYf_plane_restr0.0043443

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