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- PDB-1d8h: X-RAY CRYSTAL STRUCTURE OF YEAST RNA TRIPHOSPHATASE IN COMPLEX WI... -

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Basic information

Entry
Database: PDB / ID: 1d8h
TitleX-RAY CRYSTAL STRUCTURE OF YEAST RNA TRIPHOSPHATASE IN COMPLEX WITH SULFATE AND MANGANESE IONS.
ComponentsmRNA TRIPHOSPHATASE CET1
KeywordsHYDROLASE / RNA TRIPHOSPHATASE / BETA SUBUNIT / POLYNUCLEOTIDE 5'-TRIPHOSPHATASE / mRNA PROCESSING / mRNA CAPPING / NUCLEAR PROTEIN BETA BARREL / CATALYTIC DOMAIN / DIMER / MANGANESE-SULFATE COMPLEX
Function / homology
Function and homology information


polynucleotide 5' dephosphorylation / mRNA capping enzyme complex / mRNA 5'-triphosphate monophosphatase activity / mRNA 5'-phosphatase / polynucleotide 5'-phosphatase activity / 7-methylguanosine mRNA capping / positive regulation of transcription elongation by RNA polymerase II / positive regulation of protein localization to nucleus
Similarity search - Function
mRNA triphosphatase Cet1-like / RNA 5'-triphosphatase Cet1/Ctl1 / mRNA capping enzyme, beta chain / mRNA triphosphatase Cet1-like / mRNA triphosphatase Cet1-like superfamily / mRNA Triphosphatase Cet1; Chain A / CYTH-like domain superfamily / Alpha-Beta Barrel / Alpha Beta
Similarity search - Domain/homology
: / mRNA-capping enzyme subunit beta
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
MethodX-RAY DIFFRACTION / SYNCHROTRON / Resolution: 2 Å
AuthorsLima, C.D. / Wang, L.K. / Shuman, S.
CitationJournal: Cell(Cambridge,Mass.) / Year: 1999
Title: Structure and mechanism of yeast RNA triphosphatase: an essential component of the mRNA capping apparatus.
Authors: Lima, C.D. / Wang, L.K. / Shuman, S.
History
DepositionOct 24, 1999Deposition site: RCSB / Processing site: RCSB
Revision 1.0Nov 29, 1999Provider: repository / Type: Initial release
Revision 1.1Oct 16, 2007Group: Version format compliance
Revision 1.2Jul 13, 2011Group: Derived calculations / Version format compliance
Revision 1.3Feb 7, 2024Group: Data collection / Database references / Derived calculations
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_struct_conn_angle / struct_conn / struct_ref_seq_dif / struct_site
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _pdbx_struct_conn_angle.ptnr1_auth_comp_id / _pdbx_struct_conn_angle.ptnr1_auth_seq_id / _pdbx_struct_conn_angle.ptnr1_label_asym_id / _pdbx_struct_conn_angle.ptnr1_label_atom_id / _pdbx_struct_conn_angle.ptnr1_label_comp_id / _pdbx_struct_conn_angle.ptnr1_label_seq_id / _pdbx_struct_conn_angle.ptnr3_auth_comp_id / _pdbx_struct_conn_angle.ptnr3_auth_seq_id / _pdbx_struct_conn_angle.ptnr3_label_asym_id / _pdbx_struct_conn_angle.ptnr3_label_atom_id / _pdbx_struct_conn_angle.ptnr3_label_comp_id / _pdbx_struct_conn_angle.ptnr3_label_seq_id / _pdbx_struct_conn_angle.value / _struct_conn.pdbx_dist_value / _struct_conn.ptnr1_auth_asym_id / _struct_conn.ptnr1_auth_comp_id / _struct_conn.ptnr1_auth_seq_id / _struct_conn.ptnr1_label_asym_id / _struct_conn.ptnr1_label_atom_id / _struct_conn.ptnr1_label_comp_id / _struct_conn.ptnr1_label_seq_id / _struct_conn.ptnr2_auth_asym_id / _struct_conn.ptnr2_auth_comp_id / _struct_conn.ptnr2_auth_seq_id / _struct_conn.ptnr2_label_asym_id / _struct_conn.ptnr2_label_atom_id / _struct_conn.ptnr2_label_comp_id / _struct_conn.ptnr2_label_seq_id / _struct_ref_seq_dif.details / _struct_site.pdbx_auth_asym_id / _struct_site.pdbx_auth_comp_id / _struct_site.pdbx_auth_seq_id

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: mRNA TRIPHOSPHATASE CET1
B: mRNA TRIPHOSPHATASE CET1
C: mRNA TRIPHOSPHATASE CET1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)107,5279
Polymers107,0743
Non-polymers4536
Water12,052669
1
A: mRNA TRIPHOSPHATASE CET1
B: mRNA TRIPHOSPHATASE CET1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)71,6856
Polymers71,3832
Non-polymers3024
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area5120 Å2
ΔGint-70 kcal/mol
Surface area27670 Å2
MethodPISA
2
C: mRNA TRIPHOSPHATASE CET1
hetero molecules

C: mRNA TRIPHOSPHATASE CET1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)71,6856
Polymers71,3832
Non-polymers3024
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation2_557-x,y,-z+21
Buried area5190 Å2
ΔGint-73 kcal/mol
Surface area27690 Å2
MethodPISA
Unit cell
Length a, b, c (Å)116.436, 118.723, 85.138
Angle α, β, γ (deg.)90.00, 108.06, 90.00
Int Tables number5
Space group name H-MC121

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Components

#1: Protein mRNA TRIPHOSPHATASE CET1


Mass: 35691.465 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Plasmid details: T7 PROMOTER / Plasmid: PET16B / Species (production host): Escherichia coli / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21 (DE3) / References: UniProt: O13297, polynucleotide 5'-phosphatase
#2: Chemical ChemComp-MN / MANGANESE (II) ION


Mass: 54.938 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: Mn
#3: Chemical ChemComp-SO4 / SULFATE ION / Sulfate


Mass: 96.063 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: SO4
#4: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 669 / Source method: isolated from a natural source / Formula: H2O
Compound detailsRNA triphosphatase is an essential mRNA processing enzyme that catalyzes the first step in 5' cap ...RNA triphosphatase is an essential mRNA processing enzyme that catalyzes the first step in 5' cap formation. The 2.05 A crystal structure of yeast RNA triphosphatase Cet1p reveals a novel active site fold whereby an 8-strand beta barrel forms a topologically closed triphosphatase tunnel. Interactions of a sulfate bound in the center of the tunnel with a divalent cation and basic amino acids projecting into the tunnel suggest a catalytic mechanism that is supported by mutational data. Discrete surface domains are responsible for Cet1p homodimerization and Cet1p-binding to the guanylyltransferase component of the yeast capping apparatus. The structure and mechanism of the fungal RNA triphosphatases are completely different from those of the mammalian mRNA capping enzymes.
Sequence detailsTHE EXPRESSION CONSTRUCTS USED TO PRODUCE THE RECOMBINANT PROTEIN INCLUDED AN N-TERMINAL HIS TAG ...THE EXPRESSION CONSTRUCTS USED TO PRODUCE THE RECOMBINANT PROTEIN INCLUDED AN N-TERMINAL HIS TAG AND RESIDUES 241 TO 539 OF THE S. CEREVISIAE CET1P PROTEIN. TWO N-TERMINAL AMINO ACIDS (HIS A 239 AND MET A 240) ARE FROM HIS-TAG USED FOR AFFINITY PURIFICATION

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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: 2.61 Å3/Da / Density % sol: 52.9 %
Crystal growTemperature: 295 K / Method: vapor diffusion, hanging drop / pH: 6.34
Details: 0.1M MES, 38% SATURATED (NH4)2SO4, 5MM DTT STABILIZED IN 2.5M AMSO4 + BUFFER, 200MM MNCL2 IN STABILIZATION CONDITION FOR MANGANESE COMPLEX, pH 6.34, VAPOR DIFFUSION, HANGING DROP, temperature 295K
Crystal grow
*PLUS
Temperature: 22 ℃
Details: drop solution was mixed with an equal volume of reservoir solution
Components of the solutions
*PLUS
IDConc.Common nameCrystal-IDSol-ID
128 mg/mlprotein1drop
20.1 MMES1reservoir
338 %satammonium sulfate1reservoir
45 mMdithiothreitol1reservoir

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: NSLS / Beamline: X4A / Wavelength: 1.2141
DetectorType: ADSC QUANTUM 4 / Detector: CCD / Date: Aug 8, 1999
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.2141 Å / Relative weight: 1
ReflectionResolution: 2→25 Å / Num. all: 72277 / Num. obs: 63710 / % possible obs: 88.1 % / Observed criterion σ(F): 0 / Observed criterion σ(I): 0 / Redundancy: 2.3 % / Biso Wilson estimate: 22.199 Å2 / Rmerge(I) obs: 0.066 / Net I/σ(I): 14.6
Reflection shellResolution: 2→2.07 Å / Redundancy: 1.5 % / Rmerge(I) obs: 0.261 / % possible all: 72.2
Reflection shell
*PLUS
% possible obs: 72.2 %

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Processing

Software
NameClassification
AMoREphasing
REFMACrefinement
DENZOdata reduction
SCALEPACKdata scaling
RefinementResolution: 2→25 Å / σ(F): 0 / σ(I): 0 / Stereochemistry target values: ENGH & HUBER
Details: RESTRAINED MAXIMUM LIKELIHOOD FOR FS CONJUGATE DIRECTION EXPERIMENTAL SIGMAS USED FOR WEIGHTING
RfactorNum. reflection% reflectionSelection details
Rfree0.31 3225 -RANDOM
Rwork0.229 ---
all-72277 --
obs-63710 88.1 %-
Refinement stepCycle: LAST / Resolution: 2→25 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms6939 0 18 669 7626
Refine LS restraints
Refine-IDTypeDev ideal
X-RAY DIFFRACTIONp_bond_d0.033
X-RAY DIFFRACTIONp_angle_d2.7
X-RAY DIFFRACTIONp_angle_deg
X-RAY DIFFRACTIONp_planar_d
X-RAY DIFFRACTIONp_hb_or_metal_coord
X-RAY DIFFRACTIONp_mcbond_it
X-RAY DIFFRACTIONp_mcangle_it
X-RAY DIFFRACTIONp_scbond_it
X-RAY DIFFRACTIONp_scangle_it
X-RAY DIFFRACTIONp_plane_restr
X-RAY DIFFRACTIONp_chiral_restr
X-RAY DIFFRACTIONp_singtor_nbd
X-RAY DIFFRACTIONp_multtor_nbd
X-RAY DIFFRACTIONp_xhyhbond_nbd
X-RAY DIFFRACTIONp_xyhbond_nbd
X-RAY DIFFRACTIONp_planar_tor
X-RAY DIFFRACTIONp_staggered_tor
X-RAY DIFFRACTIONp_orthonormal_tor
X-RAY DIFFRACTIONp_transverse_tor
X-RAY DIFFRACTIONp_special_tor

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