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- PDB-3er8: Crystal structure of the heterodimeric vaccinia virus mRNA polyad... -

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Basic information

Entry
Database: PDB / ID: 3er8
TitleCrystal structure of the heterodimeric vaccinia virus mRNA polyadenylate polymerase complex with two fragments of RNA
Components
  • Cap-specific mRNA (nucleoside-2'-O-)-methyltransferase
  • Poly(A) polymerase catalytic subunit
  • RNA/DNA chimera 5'-D(CP*CP*)R(UP*UP*)D(C)-3'
  • RNA/DNA chimera 5'-D(P*CP*)R(UP*U)-3'
  • RNA/DNA chimera 5'-R(P*UP*UP*)D(C)-3'
KeywordsTranscription / Transferase/DNA / RNA / Polyadenylate polymerase / translocation / single tranded RNA poly(A) polymerase / RNA protein complex / processivity / heterodimer / nucleotidyltransferase / poxvirus / Methyltransferase / mRNA capping / mRNA processing / S-adenosyl-L-methionine / Transferase / RNA COMPLEX / Transferase-DNA
Function / homology
Function and homology information


regulation of mRNA 3'-end processing / polynucleotide adenylyltransferase / poly(A) RNA polymerase activity / 7-methylguanosine mRNA capping / virion component / mRNA processing / methyltransferase cap1 / methylation / methyltransferase cap1 activity / RNA binding ...regulation of mRNA 3'-end processing / polynucleotide adenylyltransferase / poly(A) RNA polymerase activity / 7-methylguanosine mRNA capping / virion component / mRNA processing / methyltransferase cap1 / methylation / methyltransferase cap1 activity / RNA binding / ATP binding / metal ion binding
Similarity search - Function
Poxvirus poly(A) polymerase, N domain / Poxvirus poly(A) polymerase, nucleotidyltransferase domain / Poly(A) polymerase catalytic subunit, Poxviridae / Poxvirus poly(A) polymerase nucleotidyltransferase domain / Poxvirus poly(A) polymerase, catalytic subunit, C-terminal / Poxvirus poly(A) polymerase, catalytic subunit, N-terminal / Poly(A) polymerase catalytic subunit superfamily / Poxvirus poly(A) polymerase, catalytic subunit, N-terminal domain superfamily / Poxvirus poly(A) polymerase, nucleotidyltransferase domain superfamily / Poxvirus poly(A) polymerase nucleotidyltransferase domain ...Poxvirus poly(A) polymerase, N domain / Poxvirus poly(A) polymerase, nucleotidyltransferase domain / Poly(A) polymerase catalytic subunit, Poxviridae / Poxvirus poly(A) polymerase nucleotidyltransferase domain / Poxvirus poly(A) polymerase, catalytic subunit, C-terminal / Poxvirus poly(A) polymerase, catalytic subunit, N-terminal / Poly(A) polymerase catalytic subunit superfamily / Poxvirus poly(A) polymerase, catalytic subunit, N-terminal domain superfamily / Poxvirus poly(A) polymerase, nucleotidyltransferase domain superfamily / Poxvirus poly(A) polymerase nucleotidyltransferase domain / Poxvirus poly(A) polymerase C-terminal domain / Poxvirus poly(A) polymerase N-terminal domain / mRNA methyltransferase-like / Poxvirus/kinetoplastid-type cap-specific nucleoside 2-O-methyltransferase / Poxvirus cap-specific nucleoside 2-O-methyltransferase / Poly A polymerase regulatory subunit / Poxvirus/kinetoplastid-type ribose 2'-O-methyltransferase (EC 2.1.1.57) family profile. / Substrate Binding Domain Of Dnak; Chain:A; Domain 2 / Beta Polymerase; domain 2 / Vaccinia Virus protein VP39 / S-adenosyl-L-methionine-dependent methyltransferase superfamily / Up-down Bundle / Rossmann fold / 2-Layer Sandwich / 3-Layer(aba) Sandwich / Mainly Alpha / Alpha Beta
Similarity search - Domain/homology
DNA/RNA hybrid / Cap-specific mRNA (nucleoside-2'-O-)-methyltransferase / Poly(A) polymerase catalytic subunit
Similarity search - Component
Biological speciesvaccinia virus WR
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 3.18 Å
AuthorsLi, C. / Li, H. / Zhou, S. / Poulos, T.L. / Gershon, P.D.
Citation
Journal: Structure / Year: 2009
Title: Polymerase Translocation with Respect to Single-Stranded Nucleic Acid: Looping or Wrapping of Primer around a Poly(A) Polymerase
Authors: Li, C. / Li, H. / Zhou, S. / Sun, E. / Yoshizawa, J. / Poulos, T.L. / Gershon, P.D.
#1: Journal: Mol.Cell / Year: 2006
Title: Crystal structure of the vaccinia virus polyadenylate polymerase heterodimer: insight into ATP selecttivity and processivity
Authors: Moure, C.M. / Bowman, B.R. / Gershon, P.D. / Quiocho, F.A.
History
DepositionOct 1, 2008Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jun 16, 2009Provider: repository / Type: Initial release
Revision 1.1Jul 13, 2011Group: Version format compliance
Revision 1.2Sep 18, 2013Group: Derived calculations
Revision 1.3Jan 24, 2018Group: Structure summary / Category: audit_author / Item: _audit_author.name
Revision 1.4Oct 20, 2021Group: Database references / Category: database_2 / struct_ref_seq_dif
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession / _struct_ref_seq_dif.details
Revision 1.5Dec 27, 2023Group: Data collection / Category: chem_comp_atom / chem_comp_bond

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Cap-specific mRNA (nucleoside-2'-O-)-methyltransferase
B: Cap-specific mRNA (nucleoside-2'-O-)-methyltransferase
C: Poly(A) polymerase catalytic subunit
D: Poly(A) polymerase catalytic subunit
E: RNA/DNA chimera 5'-D(CP*CP*)R(UP*UP*)D(C)-3'
F: RNA/DNA chimera 5'-R(P*UP*UP*)D(C)-3'
G: RNA/DNA chimera 5'-D(P*CP*)R(UP*U)-3'
H: RNA/DNA chimera 5'-D(P*CP*)R(UP*U)-3'


Theoretical massNumber of molelcules
Total (without water)184,3318
Polymers184,3318
Non-polymers00
Water00
1
A: Cap-specific mRNA (nucleoside-2'-O-)-methyltransferase
D: Poly(A) polymerase catalytic subunit
E: RNA/DNA chimera 5'-D(CP*CP*)R(UP*UP*)D(C)-3'
G: RNA/DNA chimera 5'-D(P*CP*)R(UP*U)-3'


Theoretical massNumber of molelcules
Total (without water)92,4554
Polymers92,4554
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area5390 Å2
ΔGint-18 kcal/mol
Surface area31480 Å2
MethodPISA
2
B: Cap-specific mRNA (nucleoside-2'-O-)-methyltransferase
C: Poly(A) polymerase catalytic subunit
F: RNA/DNA chimera 5'-R(P*UP*UP*)D(C)-3'
H: RNA/DNA chimera 5'-D(P*CP*)R(UP*U)-3'


Theoretical massNumber of molelcules
Total (without water)91,8764
Polymers91,8764
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area5050 Å2
ΔGint-21 kcal/mol
Surface area31310 Å2
MethodPISA
Unit cell
Length a, b, c (Å)70.605, 77.241, 106.851
Angle α, β, γ (deg.)74.88, 74.00, 63.69
Int Tables number1
Space group name H-MP1

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Components

#1: Protein Cap-specific mRNA (nucleoside-2'-O-)-methyltransferase / E.C.2.1.1.57 / Poly(A) polymerase regulatory subunit / Poly(A) polymerase small subunit / PAP-S / VP39


Mass: 34588.961 Da / Num. of mol.: 2 / Mutation: R140A,K142A,R143A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) vaccinia virus WR / Strain: Western Reserve / WR / Gene: PAPS, VACWR095, F9 / Plasmid: pET15b / Production host: Escherichia coli (E. coli) / Strain (production host): BL21(DE3)pLyS / References: UniProt: P07617, methyltransferase cap1
#2: Protein Poly(A) polymerase catalytic subunit / E.C.2.7.7.19 / Poly(A) polymerase large subunit / PAP-L / VP55


Mass: 55574.125 Da / Num. of mol.: 2 / Mutation: L36S
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) vaccinia virus WR / Strain: Western Reserve / WR / Gene: PAPL, VACWR057, E1L / Plasmid: pET15b / Production host: Escherichia coli (E. coli) / Strain (production host): BL21(DE3)pLyS
References: UniProt: P23371, polynucleotide adenylyltransferase
#3: DNA/RNA hybrid RNA/DNA chimera 5'-D(CP*CP*)R(UP*UP*)D(C)-3'


Mass: 1434.920 Da / Num. of mol.: 1 / Source method: obtained synthetically
#4: DNA/RNA hybrid RNA/DNA chimera 5'-R(P*UP*UP*)D(C)-3'


Mass: 856.556 Da / Num. of mol.: 1 / Source method: obtained synthetically
#5: DNA/RNA hybrid RNA/DNA chimera 5'-D(P*CP*)R(UP*U)-3'


Mass: 856.556 Da / Num. of mol.: 2 / Source method: obtained synthetically

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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

CrystalDensity Matthews: 2.69 Å3/Da / Density % sol: 54.25 %
Crystal growTemperature: 295 K / Method: vapor diffusion, sitting drop / pH: 8.7
Details: proteins(4-5mg.ml) in 10mM Tris-HCl, pH8.7, 75 mM NaCl, 0.5 mM DTT, mixed with equal volume buffer which composed of 10mM Tris-HCl, pH 8.7, 15-20% PEG 4000, 5% glycerol, 0.5 mM DTT. room ...Details: proteins(4-5mg.ml) in 10mM Tris-HCl, pH8.7, 75 mM NaCl, 0.5 mM DTT, mixed with equal volume buffer which composed of 10mM Tris-HCl, pH 8.7, 15-20% PEG 4000, 5% glycerol, 0.5 mM DTT. room temperature for several days., VAPOR DIFFUSION, SITTING DROP, temperature 295K
Components of the solutions
IDNameCrystal-IDSol-ID
1Tris-HCl11
2NaCl11
3DTT11
4PEG 400011
5glycerol11
6Tris-HCl12
7DTT12
8PEG 400012
9glycerol12

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: SSRL / Beamline: BL9-2 / Wavelength: 1 Å
DetectorType: ADSC QUANTUM 315 / Detector: CCD / Date: Dec 14, 2007 / Details: mirrors
RadiationMonochromator: graphite / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1 Å / Relative weight: 1
ReflectionResolution: 3.18→50 Å / Num. obs: 30650 / % possible obs: 93.1 % / Observed criterion σ(I): -3 / Redundancy: 3.7 % / Rmerge(I) obs: 0.084 / Rsym value: 0.084 / Net I/σ(I): 14.5
Reflection shellResolution: 3.18→3.38 Å / Redundancy: 3.5 % / Rmerge(I) obs: 0.526 / Mean I/σ(I) obs: 2 / Rsym value: 0.526 / % possible all: 87.2

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Processing

Software
NameVersionClassification
ADSCQuantumdata collection
CNS1.2refinement
HKL-2000data reduction
HKL-2000data scaling
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 3.18→38.9 Å / Rfactor Rfree error: 0.007 / Data cutoff high absF: 603426.2 / Data cutoff low absF: 0 / Isotropic thermal model: RESTRAINED / Cross valid method: THROUGHOUT / σ(F): 0
Details: Residues 27-32, 142-144 in chains A & B and residues 1-11, 118-129, 150-160 in chains C & D are disordered and not included in the model.
RfactorNum. reflection% reflectionSelection details
Rfree0.285 1492 5 %RANDOM
Rwork0.243 ---
all0.243 30120 --
obs0.243 30120 93.3 %-
Solvent computationSolvent model: FLAT MODEL, BULK FLAT MODEL / Bsol: 49.8436 Å2 / ksol: 0.35 e/Å3
Displacement parametersBiso mean: 81.6 Å2
Baniso -1Baniso -2Baniso -3
1--9.77 Å23.09 Å23.33 Å2
2---9.61 Å22.5 Å2
3---19.38 Å2
Refine analyze
FreeObs
Luzzati coordinate error0.53 Å0.43 Å
Luzzati d res low-5 Å
Luzzati sigma a0.62 Å0.56 Å
Refinement stepCycle: LAST / Resolution: 3.18→38.9 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms12018 271 0 0 12289
Refine LS restraints
Refine-IDTypeDev idealDev ideal target
X-RAY DIFFRACTIONc_bond_d0.011
X-RAY DIFFRACTIONc_angle_deg1.4
X-RAY DIFFRACTIONc_dihedral_angle_d22.3
X-RAY DIFFRACTIONc_improper_angle_d1.07
X-RAY DIFFRACTIONc_mcbond_it10.291.5
X-RAY DIFFRACTIONc_mcangle_it15.972
X-RAY DIFFRACTIONc_scbond_it11.062
X-RAY DIFFRACTIONc_scangle_it16.172.5
LS refinement shellResolution: 3.18→3.38 Å / Rfactor Rfree error: 0.023 / Total num. of bins used: 6
RfactorNum. reflection% reflection
Rfree0.361 245 5.2 %
Rwork0.325 4453 -
obs--86.6 %
Xplor file
Refine-IDSerial noParam fileTopol file
X-RAY DIFFRACTION1protein_rep.param&_1_TOPOLOGY_INFILE_1
X-RAY DIFFRACTION2dna-rna_rep_1.par&_1_TOPOLOGY_INFILE_2
X-RAY DIFFRACTION3water_rep.param&_1_TOPOLOGY_INFILE_3
X-RAY DIFFRACTION4&_1_TOPOLOGY_INFILE_4
X-RAY DIFFRACTION5&_1_TOPOLOGY_INFILE_5

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