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Basic information

Entry
Database: PDB / ID: 3tht
TitleCrystal structure and RNA binding properties of the RRM/AlkB domains in human ABH8, an enzyme catalyzing tRNA hypermodification, Northeast Structural Genomics Consortium Target HR5601B
ComponentsAlkylated DNA repair protein alkB homolog 8
KeywordsOXIDOREDUCTASE / Structural Genomics / PSI-Biology / Northeast Structural Genomics Consortium / NESG / Alpha-beta two domain protein containing a zinc structure motif / tRNA modifying enzyme
Function / homology
Function and homology information


tRNA (carboxymethyluridine34-5-O)-methyltransferase / tRNA (5-carboxymethyluridine(34)-5-O)-methyltransferase activity / tRNA (uridine) methyltransferase activity / tRNA wobble uridine modification / tRNA modification in the nucleus and cytosol / tRNA methylation / 2-oxoglutarate-dependent dioxygenase activity / S-adenosylmethionine-dependent methyltransferase activity / tRNA binding / iron ion binding ...tRNA (carboxymethyluridine34-5-O)-methyltransferase / tRNA (5-carboxymethyluridine(34)-5-O)-methyltransferase activity / tRNA (uridine) methyltransferase activity / tRNA wobble uridine modification / tRNA modification in the nucleus and cytosol / tRNA methylation / 2-oxoglutarate-dependent dioxygenase activity / S-adenosylmethionine-dependent methyltransferase activity / tRNA binding / iron ion binding / DNA damage response / zinc ion binding / nucleoplasm / nucleus / cytosol / cytoplasm
Similarity search - Function
Jelly Rolls - #1520 / Alkylated DNA repair protein AlkB, homologue 8, N-terminal / ALKBH8, RNA recognition motif / Alkylated DNA repair protein alkB homolog 8, N-terminal / : / Methyltransferase type 11 / Methyltransferase domain / Alpha-ketoglutarate-dependent dioxygenase AlkB-like / 2OG-Fe(II) oxygenase superfamily / Oxoglutarate/iron-dependent dioxygenase ...Jelly Rolls - #1520 / Alkylated DNA repair protein AlkB, homologue 8, N-terminal / ALKBH8, RNA recognition motif / Alkylated DNA repair protein alkB homolog 8, N-terminal / : / Methyltransferase type 11 / Methyltransferase domain / Alpha-ketoglutarate-dependent dioxygenase AlkB-like / 2OG-Fe(II) oxygenase superfamily / Oxoglutarate/iron-dependent dioxygenase / Fe(2+) 2-oxoglutarate dioxygenase domain profile. / RRM (RNA recognition motif) domain / RNA recognition motif / RNA recognition motif domain / RNA-binding domain superfamily / Nucleotide-binding alpha-beta plait domain superfamily / Alpha-Beta Plaits / Jelly Rolls / S-adenosyl-L-methionine-dependent methyltransferase superfamily / Sandwich / 2-Layer Sandwich / Mainly Beta / Alpha Beta
Similarity search - Domain/homology
2-OXOGLUTARIC ACID / : / tRNA (carboxymethyluridine(34)-5-O)-methyltransferase ALKBH8
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 3.01 Å
AuthorsPastore, C. / Topalidou, I. / Forouhar, F. / Yan, A.C. / Levy, M. / Hunt, J.F. / Northeast Structural Genomics Consortium (NESG)
CitationJournal: J.Biol.Chem. / Year: 2012
Title: Crystal structure and RNA binding properties of the RNA recognition motif (RRM) and AlkB domains in human AlkB homolog 8 (ABH8), an enzyme catalyzing tRNA hypermodification.
Authors: Pastore, C. / Topalidou, I. / Forouhar, F. / Yan, A.C. / Levy, M. / Hunt, J.F.
History
DepositionAug 19, 2011Deposition site: RCSB / Processing site: RCSB
Revision 1.0Nov 2, 2011Provider: repository / Type: Initial release
Revision 1.1Oct 10, 2012Group: Database references
Revision 1.2Oct 25, 2017Group: Author supporting evidence / Category: pdbx_struct_assembly_auth_evidence
Revision 1.3Sep 13, 2023Group: Data collection / Database references ...Data collection / Database references / Derived calculations / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model / 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_asym_id / _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.ptnr2_auth_asym_id / _pdbx_struct_conn_angle.ptnr2_auth_comp_id / _pdbx_struct_conn_angle.ptnr2_auth_seq_id / _pdbx_struct_conn_angle.ptnr2_label_asym_id / _pdbx_struct_conn_angle.ptnr2_label_atom_id / _pdbx_struct_conn_angle.ptnr2_label_comp_id / _pdbx_struct_conn_angle.ptnr3_auth_asym_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_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: Alkylated DNA repair protein alkB homolog 8
B: Alkylated DNA repair protein alkB homolog 8
C: Alkylated DNA repair protein alkB homolog 8
D: Alkylated DNA repair protein alkB homolog 8
hetero molecules


Theoretical massNumber of molelcules
Total (without water)158,50516
Polymers157,4394
Non-polymers1,06612
Water1,802100
1
A: Alkylated DNA repair protein alkB homolog 8
hetero molecules


Theoretical massNumber of molelcules
Total (without water)39,6264
Polymers39,3601
Non-polymers2663
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
2
B: Alkylated DNA repair protein alkB homolog 8
hetero molecules


Theoretical massNumber of molelcules
Total (without water)39,6264
Polymers39,3601
Non-polymers2663
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
3
C: Alkylated DNA repair protein alkB homolog 8
hetero molecules


Theoretical massNumber of molelcules
Total (without water)39,6264
Polymers39,3601
Non-polymers2663
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
4
D: Alkylated DNA repair protein alkB homolog 8
hetero molecules


Theoretical massNumber of molelcules
Total (without water)39,6264
Polymers39,3601
Non-polymers2663
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
5
A: Alkylated DNA repair protein alkB homolog 8
B: Alkylated DNA repair protein alkB homolog 8
hetero molecules

A: Alkylated DNA repair protein alkB homolog 8
B: Alkylated DNA repair protein alkB homolog 8
hetero molecules


Theoretical massNumber of molelcules
Total (without water)158,50516
Polymers157,4394
Non-polymers1,06612
Water724
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation2_556-x,y,-z+11
Buried area10140 Å2
ΔGint-56 kcal/mol
Surface area55220 Å2
MethodPISA
6
A: Alkylated DNA repair protein alkB homolog 8
B: Alkylated DNA repair protein alkB homolog 8
hetero molecules


Theoretical massNumber of molelcules
Total (without water)79,2528
Polymers78,7192
Non-polymers5336
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area3040 Å2
ΔGint-18 kcal/mol
Surface area29640 Å2
MethodPISA
7
C: Alkylated DNA repair protein alkB homolog 8
D: Alkylated DNA repair protein alkB homolog 8
hetero molecules

C: Alkylated DNA repair protein alkB homolog 8
D: Alkylated DNA repair protein alkB homolog 8
hetero molecules


Theoretical massNumber of molelcules
Total (without water)158,50516
Polymers157,4394
Non-polymers1,06612
Water724
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation2_455-x-1,y,-z1
Buried area9620 Å2
ΔGint-46 kcal/mol
Surface area53600 Å2
MethodPISA
8
C: Alkylated DNA repair protein alkB homolog 8
D: Alkylated DNA repair protein alkB homolog 8
hetero molecules


Theoretical massNumber of molelcules
Total (without water)79,2528
Polymers78,7192
Non-polymers5336
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area2950 Å2
ΔGint-16 kcal/mol
Surface area28650 Å2
MethodPISA
Unit cell
Length a, b, c (Å)150.166, 73.279, 149.814
Angle α, β, γ (deg.)90.00, 112.73, 90.00
Int Tables number5
Space group name H-MC121
DetailsThe light scattering data of this construct without C-tag shows mainly monomeric protein in the solution, while the same construct with C-tag appears to have population of monomer and tetrameric species. Consistent with the latter observation, the crystal structure reveals that a few residues of the C-tag play an important role in protein tetramerization, which is possibly not a physiological phenomenon

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Components

#1: Protein
Alkylated DNA repair protein alkB homolog 8 / Probable alpha-ketoglutarate-dependent dioxygenase ABH8


Mass: 39359.684 Da / Num. of mol.: 4 / Fragment: RRM and AlkB domains of ABH8 / Mutation: A C-tag (ENLYFQGLEHHHHHH) was added
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: ABH8, ALKBH8 / Plasmid: pET26 / Production host: Escherichia coli (E. coli) / Strain (production host): Rosetta 2
References: UniProt: Q96BT7, Oxidoreductases; Acting on paired donors, with incorporation or reduction of molecular oxygen; With 2-oxoglutarate as one donor, and incorporation of one atom of oxygen into each donor
#2: Chemical
ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Zn
#3: Chemical
ChemComp-MN / MANGANESE (II) ION


Mass: 54.938 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Mn
#4: Chemical
ChemComp-AKG / 2-OXOGLUTARIC ACID


Mass: 146.098 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C5H6O5
#5: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 100 / Source method: isolated from a natural source / Formula: H2O

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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: 2.41 Å3/Da / Density % sol: 49.06 %
Crystal growTemperature: 293 K / Method: microbatch under oil / pH: 5.6
Details: 2.8 mM Mn2Cl and 8.6 mM 2OG and a precipitant containing 28.5-30.5% (w/v) PEG 4K, 15% (v/v) glycerol, 170 mM NH4(CH3COO), 85 mM Na-citrate, pH 5.6, microbatch under oil, temperature 293K

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: NSLS / Beamline: X4A / Wavelength: 0.97912 Å
DetectorType: ADSC QUANTUM 4 / Detector: CCD / Date: Sep 14, 2010 / Details: mirrors
RadiationMonochromator: Si 111 CHANNEL / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.97912 Å / Relative weight: 1
ReflectionResolution: 3→40 Å / Num. all: 30026 / Num. obs: 29876 / % possible obs: 99.5 % / Observed criterion σ(F): 0 / Observed criterion σ(I): 0 / Redundancy: 3.5 % / Biso Wilson estimate: 44.3 Å2 / Rmerge(I) obs: 0.082 / Rsym value: 0.071 / Net I/σ(I): 16.4
Reflection shellResolution: 3→3.11 Å / Redundancy: 3.4 % / Rmerge(I) obs: 0.304 / Mean I/σ(I) obs: 3.7 / Num. unique all: 2993 / Rsym value: 0.272 / % possible all: 98.6

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Processing

Software
NameVersionClassification
ADSCQuantumdata collection
MOLREPphasing
CNS1.2 & Xtalviewrefinement
HKL-2000data reduction
SCALEPACKdata scaling
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: PDB entry 3THP

3thp


Resolution: 3.01→39.06 Å / Rfactor Rfree error: 0.005 / Data cutoff high absF: 233864.68 / Data cutoff low absF: 0 / Isotropic thermal model: RESTRAINED / Cross valid method: THROUGHOUT / σ(F): 0 / σ(I): 0 / Stereochemistry target values: Engh & Huber
RfactorNum. reflection% reflectionSelection details
Rfree0.277 2904 10 %RANDOM
Rwork0.22 ---
all0.224 30026 --
obs0.22 29071 96.2 %-
Solvent computationSolvent model: FLAT MODEL / Bsol: 23.431 Å2 / ksol: 0.3 e/Å3
Displacement parametersBiso mean: 48.7 Å2
Baniso -1Baniso -2Baniso -3
1--10.63 Å20 Å21.64 Å2
2--15.57 Å20 Å2
3----4.93 Å2
Refine analyze
FreeObs
Luzzati coordinate error0.49 Å0.36 Å
Luzzati d res low-5 Å
Luzzati sigma a0.7 Å0.53 Å
Refinement stepCycle: LAST / Resolution: 3.01→39.06 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms9610 0 48 100 9758
Refine LS restraints
Refine-IDTypeDev ideal
X-RAY DIFFRACTIONc_bond_d0.009
X-RAY DIFFRACTIONc_angle_deg1.2
X-RAY DIFFRACTIONc_dihedral_angle_d24.8
X-RAY DIFFRACTIONc_improper_angle_d0.77
LS refinement shellResolution: 3→3.11 Å / Rfactor Rfree error: 0.028 / Total num. of bins used: 10
RfactorNum. reflection% reflection
Rfree0.44 243 10.3 %
Rwork0.343 2120 -
obs-2120 79.5 %

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