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- PDB-7asv: Crystal structure of tWHD2 of Rpc5 subunit of human RNA Polymerase III -

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Basic information

Entry
Database: PDB / ID: 7asv
TitleCrystal structure of tWHD2 of Rpc5 subunit of human RNA Polymerase III
ComponentsDNA-directed RNA polymerase III subunit RPC5
KeywordsTRANSCRIPTION / RNA Pol III / RNA Polymerase III / Rpc5
Function / homology
Function and homology information


RNA Polymerase III Chain Elongation / RNA Polymerase III Transcription Termination / Cytosolic sensors of pathogen-associated DNA / RNA Polymerase III Transcription Initiation From Type 1 Promoter / RNA Polymerase III Transcription Initiation From Type 2 Promoter / RNA Polymerase III Transcription Initiation From Type 3 Promoter / RNA Polymerase III Abortive And Retractive Initiation / RNA polymerase III complex / tRNA transcription by RNA polymerase III / defense response to virus ...RNA Polymerase III Chain Elongation / RNA Polymerase III Transcription Termination / Cytosolic sensors of pathogen-associated DNA / RNA Polymerase III Transcription Initiation From Type 1 Promoter / RNA Polymerase III Transcription Initiation From Type 2 Promoter / RNA Polymerase III Transcription Initiation From Type 3 Promoter / RNA Polymerase III Abortive And Retractive Initiation / RNA polymerase III complex / tRNA transcription by RNA polymerase III / defense response to virus / innate immune response / nucleoplasm / cytosol
Similarity search - Function
DNA-directed RNA polymerase III subunit RPC5, C-terminal / DNA-directed RNA polymerase III subunit RPC5 C-terminal / DNA-directed RNA polymerase III subunit Rpc5 / RPC5 protein
Similarity search - Domain/homology
ACETATE ION / DNA-directed RNA polymerase III subunit RPC5
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MAD / Resolution: 1.55 Å
AuthorsVannini, A. / Abascal-Palacios, G. / Ramsay, E.P.
Funding support United Kingdom, European Union, 3items
OrganizationGrant numberCountry
Cancer Research UKCR-UK C47547/A21536 United Kingdom
Wellcome Trust200818/Z/16/Z United Kingdom
H2020 Marie Curie Actions of the European Commission655238European Union
CitationJournal: Nat Commun / Year: 2020
Title: Structure of human RNA polymerase III.
Authors: Ewan Phillip Ramsay / Guillermo Abascal-Palacios / Julia L Daiß / Helen King / Jerome Gouge / Michael Pilsl / Fabienne Beuron / Edward Morris / Philip Gunkel / Christoph Engel / Alessandro Vannini /
Abstract: In eukaryotes, RNA Polymerase (Pol) III is specialized for the transcription of tRNAs and other short, untranslated RNAs. Pol III is a determinant of cellular growth and lifespan across eukaryotes. ...In eukaryotes, RNA Polymerase (Pol) III is specialized for the transcription of tRNAs and other short, untranslated RNAs. Pol III is a determinant of cellular growth and lifespan across eukaryotes. Upregulation of Pol III transcription is observed in cancer and causative Pol III mutations have been described in neurodevelopmental disorders and hypersensitivity to viral infection. Here, we report a cryo-EM reconstruction at 4.0 Å of human Pol III, allowing mapping and rationalization of reported genetic mutations. Mutations causing neurodevelopmental defects cluster in hotspots affecting Pol III stability and/or biogenesis, whereas mutations affecting viral sensing are located in proximity to DNA binding regions, suggesting an impairment of Pol III cytosolic viral DNA-sensing. Integrating x-ray crystallography and SAXS, we also describe the structure of the higher eukaryote specific RPC5 C-terminal extension. Surprisingly, experiments in living cells highlight a role for this module in the assembly and stability of human Pol III.
History
DepositionOct 28, 2020Deposition site: PDBE / Processing site: PDBE
Revision 1.0Dec 30, 2020Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
B: DNA-directed RNA polymerase III subunit RPC5
A: DNA-directed RNA polymerase III subunit RPC5
hetero molecules


Theoretical massNumber of molelcules
Total (without water)162,2684
Polymers162,1502
Non-polymers1182
Water9,332518
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area3410 Å2
ΔGint-20 kcal/mol
Surface area17040 Å2
MethodPISA
Unit cell
Length a, b, c (Å)41.110, 75.760, 62.400
Angle α, β, γ (deg.)90.000, 103.570, 90.000
Int Tables number4
Space group name H-MP1211
Space group name HallP2yb
Symmetry operation#1: x,y,z
#2: -x,y+1/2,-z

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Components

#1: Protein DNA-directed RNA polymerase III subunit RPC5 / RNA polymerase III subunit C5 / DNA-directed RNA polymerase III 80 kDa polypeptide


Mass: 81075.016 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: POLR3E, KIAA1452 / Production host: Escherichia coli (E. coli) / References: UniProt: Q9NVU0
#2: Chemical ChemComp-ACT / ACETATE ION


Mass: 59.044 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C2H3O2
#3: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 518 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestN

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

Crystal growTemperature: 277 K / Method: vapor diffusion, hanging drop
Details: 3.2-3.8 M Ammonium Acetate and 100 mM Bis-Tris Propane pH 6.5-7

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Data collection

Diffraction
IDMean temperature (K)Crystal-IDSerial crystal experiment
11001N
21001Y
Diffraction source
SourceSiteBeamlineIDWavelength (Å)
SYNCHROTRONDiamond I0310.97942
SYNCHROTRONDiamond I0320.97942, 0.97957, 0.97174
Detector
TypeIDDetectorDate
DECTRIS PILATUS3 6M1PIXELMay 15, 2015
DECTRIS PILATUS3 6M2PIXELMay 15, 2015
Radiation
IDProtocolMonochromatic (M) / Laue (L)Scattering typeWavelength-ID
1SINGLE WAVELENGTHMx-ray1
2MADMx-ray2
Radiation wavelength
IDWavelength (Å)Relative weight
10.979421
20.979571
30.971741
ReflectionResolution: 1.48→30.33 Å / Num. obs: 59533 / % possible obs: 96.7 % / Redundancy: 6.1 % / Rmerge(I) obs: 0.075 / Net I/σ(I): 11.8
Reflection shellResolution: 1.48→1.52 Å / Rmerge(I) obs: 0.735 / Num. unique obs: 3246
Serial crystallography sample deliveryMethod: fixed target

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Processing

Software
NameVersionClassification
PHENIXphenix-1.18.1-3865refinement
xia20.3.8.0data reduction
xia20.3.8.0data scaling
PHENIXphenix-1.18.1-3865phasing
RefinementMethod to determine structure: MAD / Resolution: 1.55→30.33 Å / Cross valid method: FREE R-VALUE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflection
Rfree0.21 --
Rwork0.18 --
obs-59533 96.1 %
Displacement parametersBiso mean: 26.32 Å2
Refinement stepCycle: LAST / Resolution: 1.55→30.33 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms2460 0 8 518 2986
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.01142591
X-RAY DIFFRACTIONf_angle_d1.14653505
X-RAY DIFFRACTIONf_chiral_restr0.0528388
X-RAY DIFFRACTIONf_plane_restr0.0083456
X-RAY DIFFRACTIONf_dihedral_angle_d15.6049987

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