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- PDB-9eq2: Arabidopsis thaliana R2T complex -

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Basic information

Entry
Database: PDB / ID: 9eq2
TitleArabidopsis thaliana R2T complex
Components
  • At1g56440
  • RuvB-like helicase
  • RuvB-like protein 1
KeywordsCHAPERONE / RUVBL1 / RUVBL2 / R2TP / HSP90
Function / homology
Function and homology information


meristem development / regulation of defense response to fungus / chromatin extrusion motor activity / ATP-dependent H2AZ histone chaperone activity / ATP-dependent H3-H4 histone complex chaperone activity / cohesin loader activity / flower development / Swr1 complex / DNA clamp loader activity / plastid ...meristem development / regulation of defense response to fungus / chromatin extrusion motor activity / ATP-dependent H2AZ histone chaperone activity / ATP-dependent H3-H4 histone complex chaperone activity / cohesin loader activity / flower development / Swr1 complex / DNA clamp loader activity / plastid / DNA helicase / forked DNA-dependent helicase activity / single-stranded 3'-5' DNA helicase activity / four-way junction helicase activity / double-stranded DNA helicase activity / cell differentiation / DNA repair / nucleolus / ATP hydrolysis activity / ATP binding / nucleus
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 / TPR repeat profile. ...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 / 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 / At1g56440 / RuvB-like helicase / RuvB-like protein 1
Similarity search - Component
Biological speciesArabidopsis thaliana (thale cress)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.68 Å
AuthorsLopez-Perrote, A. / Llorca, O.
Funding support Spain, 1items
OrganizationGrant numberCountry
Agencia Estatal de Investigacion (AEI)PID2020-114429RB-I00 Spain
CitationJournal: Structure / Year: 2025
Title: The structure of the R2T complex reveals a different architecture from the related HSP90 cochaperone R2TP.
Authors: Alberto Palacios-Abella / Andrés López-Perrote / Jasminka Boskovic / Sandra Fonseca / Cristina Úrbez / Vicente Rubio / Oscar Llorca / David Alabadí /
Abstract: The R2TP complex is a specialized HSP90 cochaperone essential for the maturation of macromolecular complexes such as RNAPII and TORC1. R2TP is formed by a hetero-hexameric ring of AAA-ATPases RuvBL1 ...The R2TP complex is a specialized HSP90 cochaperone essential for the maturation of macromolecular complexes such as RNAPII and TORC1. R2TP is formed by a hetero-hexameric ring of AAA-ATPases RuvBL1 and RuvBL2, which interact with RPAP3 and PIH1D1. Several R2TP-like complexes have been described, but these are less well characterized. Here, we identified, characterized and determined the cryo-electron microscopy (cryo-EM) structure of R2T from Arabidopsis thaliana, which lacks PIH1D1 and is probably the only form of the complex in seed plants. In contrast to R2TP, R2T is organized as two rings of AtRuvBL1-AtRuvBL2a interacting back-to-back, with one AtRPAP3 anchored per ring. AtRPAP3 has no effect on the ATPase activity of AtRuvBL1-AtRuvBL2a and binds with a different stoichiometry than in human R2TP. We show that the interaction of AtRPAP3 with AtRuvBL2a and AtHSP90 occurs via a conserved mechanism. However, the distinct architectures of R2T and R2TP suggest differences in their functions and mechanisms.
History
DepositionMar 20, 2024Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jan 29, 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 protein 1
D: RuvB-like helicase
C: RuvB-like protein 1
B: RuvB-like protein 1
F: RuvB-like helicase
E: RuvB-like helicase
G: At1g56440
hetero molecules


Theoretical massNumber of molelcules
Total (without water)383,70210
Polymers382,4217
Non-polymers1,2823
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 protein 1 / 49 kDa TATA box-binding protein-interacting protein homolog / AtTIP49a / Ruv DNA-helicase-like protein


Mass: 53750.754 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Details: Protein sequence contains a N-terminal 10xHistidine tag and TEV protease cleavage site that do not affect protein activity nor structure
Source: (gene. exp.) Arabidopsis thaliana (thale cress) / Gene: RIN1, RVB1, TIP49a, At5g22330 / Production host: Escherichia coli (E. coli) / References: UniProt: Q9FMR9, DNA helicase
#2: Protein RuvB-like helicase


Mass: 54007.848 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Details: Protein sequence contains a stretch of 18 residues extra at the N-terminus due to cloning design that does not affect protein activity nor structure
Source: (gene. exp.) Arabidopsis thaliana (thale cress) / Gene: ISE4, At5g67630, K9I9.20, K9I9_20 / Production host: Escherichia coli (E. coli) / References: UniProt: Q9FJW0, DNA helicase
#3: Protein At1g56440 / Tetratricopeptide repeat (TPR)-like superfamily protein


Mass: 59144.980 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: Protein sequence contains a N-terminal His-tag followed by an IF2D1 tag and a TEV protease site, and C-terminal Strep-II tag with HRV 3C protease site that do not affect protein activity nor structure
Source: (gene. exp.) Arabidopsis thaliana (thale cress)
Gene: TPR5, tetratricopeptide repeat 5, At1g56440, F13N6.2, F13N6_2
Production host: Escherichia coli (E. coli) / References: UniProt: Q5XF05
#4: Chemical ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE


Mass: 427.201 Da / Num. of mol.: 3 / 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

ComponentName: AtR2T complex / Type: COMPLEX / Details: AtRuvBL1-AtRuvBL2a in complex with AtRPAP3 / Entity ID: #1-#3 / Source: RECOMBINANT
Molecular weightValue: 0.7 MDa / Experimental value: NO
Source (natural)Organism: Arabidopsis thaliana (thale cress)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
125 mMN-(2-Hydroxyethyl)piperazine-N-(2-ethanesulfonic acid)HEPES1
2300 mMSodium chlorideNaCl1
31 mM1,4-DithiothreitolDTT1
SpecimenConc.: 0.8 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R0.6/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 90 % / 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 FIELD / Nominal magnification: 130000 X / Nominal defocus max: 3000 nm / Nominal defocus min: 1500 nm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 50.12 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k)

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Processing

EM software
IDNameVersionCategory
1RELION4particle selection
4CTFFIND4.1CTF correction
7UCSF ChimeraXmodel fitting
9cryoSPARCv4.3.1initial Euler assignment
10RELION4final Euler assignment
11RELION4classification
12RELION43D reconstruction
13ISOLDE1.7model refinement
14PHENIX1.20.1-4487model refinement
15Coot0.9.8.5model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 2334954
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.68 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 185042 / Algorithm: BACK PROJECTION / Symmetry type: POINT
Atomic model buildingProtocol: BACKBONE TRACE / Space: REAL
Atomic model buildingSource name: AlphaFold / Type: in silico model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00614810
ELECTRON MICROSCOPYf_angle_d0.97719997
ELECTRON MICROSCOPYf_dihedral_angle_d7.7342053
ELECTRON MICROSCOPYf_chiral_restr0.052388
ELECTRON MICROSCOPYf_plane_restr0.0072549

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