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- PDB-2itb: CRYSTAL STRUCTURE OF A PUTATIVE TRNA-(MS(2)IO(6)A)-HYDROXYLASE (P... -

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Basic information

Entry
Database: PDB / ID: 2itb
TitleCRYSTAL STRUCTURE OF A PUTATIVE TRNA-(MS(2)IO(6)A)-HYDROXYLASE (PP_2188) FROM PSEUDOMONAS PUTIDA KT2440 AT 2.05 A RESOLUTION
ComponentsTRNA-(Ms(2)io(6)a)-hydroxylase, putative
KeywordsOXIDOREDUCTASE / PUTATIVE ATTH / STRUCTURAL GENOMICS / JOINT CENTER FOR STRUCTURAL GENOMICS / JCSG / PROTEIN STRUCTURE INITIATIVE / PSI-2
Function / homology
Function and homology information


tRNA-(2-methylthio-N-6-(cis-hydroxy)isopentenyl adenosine)-hydroxylase activity / metal ion binding
Similarity search - Function
tRNA-hydroxylase MiaE / tRNA-(MS[2]IO[6]A)-hydroxylase (MiaE) / Ferritin, core subunit, four-helix bundle / Ferritin / Ferritin-like / Ferritin-like superfamily / Up-down Bundle / Mainly Alpha
Similarity search - Domain/homology
: / PEROXIDE ION / Unknown ligand / Putative tRNA-(Ms(2)io(6)a)-hydroxylase
Similarity search - Component
Biological speciesPseudomonas putida (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MAD / Resolution: 2.05 Å
AuthorsJoint Center for Structural Genomics (JCSG)
CitationJournal: To be published
Title: Crystal structure of putative tRNA-(ms(2)io(6)a)-hydroxylase (NP_744337.1) from Pseudomonas Putida KT2440 at 2.05 A resolution
Authors: Joint Center for Structural Genomics (JCSG)
History
DepositionOct 19, 2006Deposition site: RCSB / Processing site: RCSB
Revision 1.0Nov 7, 2006Provider: repository / Type: Initial release
Revision 1.1Oct 16, 2007Group: Version format compliance
Revision 1.2Jul 13, 2011Group: Advisory / Source and taxonomy / Version format compliance
Revision 1.3Oct 18, 2017Group: Refinement description / Category: software / Item: _software.classification / _software.name
Revision 1.4Jan 25, 2023Group: Database references / Derived calculations
Category: database_2 / pdbx_struct_conn_angle ...database_2 / pdbx_struct_conn_angle / struct_conn / struct_conn_type / 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.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.value / _struct_conn.conn_type_id / _struct_conn.id / _struct_conn.pdbx_dist_value / _struct_conn.pdbx_leaving_atom_flag / _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_conn_type.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: TRNA-(Ms(2)io(6)a)-hydroxylase, putative
B: TRNA-(Ms(2)io(6)a)-hydroxylase, putative
hetero molecules


Theoretical massNumber of molelcules
Total (without water)48,02325
Polymers46,7122
Non-polymers1,31123
Water3,999222
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)61.274, 70.215, 84.285
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number19
Space group name H-MP212121
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
11A
21B

NCS domain segments:

Component-ID: 1 / Ens-ID: 1 / Beg label comp-ID: ILE / End label comp-ID: GLN / Refine code: 4 / Auth seq-ID: 4 - 201 / Label seq-ID: 5 - 202

Dom-IDAuth asym-IDLabel asym-ID
1AA
2BB

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Components

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Protein , 1 types, 2 molecules AB

#1: Protein TRNA-(Ms(2)io(6)a)-hydroxylase, putative


Mass: 23356.146 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pseudomonas putida (bacteria) / Strain: KT2440 / Gene: NP_744337.1 / Production host: Escherichia coli (E. coli) / References: UniProt: Q88KV1

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Non-polymers , 5 types, 245 molecules

#2: Chemical
ChemComp-FE / FE (III) ION


Mass: 55.845 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Fe
#3: Chemical ChemComp-PER / PEROXIDE ION


Mass: 31.999 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: O2
#4: Chemical
ChemComp-EDO / 1,2-ETHANEDIOL / ETHYLENE GLYCOL


Mass: 62.068 Da / Num. of mol.: 17 / Source method: obtained synthetically / Formula: C2H6O2
#5: Chemical ChemComp-UNL / UNKNOWN LIGAND


Num. of mol.: 1 / Source method: obtained synthetically
#6: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 222 / Source method: isolated from a natural source / Formula: H2O

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Details

Sequence detailsTHE CONSTRUCT WAS EXPRESSED WITH A PURIFICATION TAG MGSDKIHHHHHHENLYFQG. THE TAG WAS REMOVED WITH ...THE CONSTRUCT WAS EXPRESSED WITH A PURIFICATION TAG MGSDKIHHHHHHENLYFQG. THE TAG WAS REMOVED WITH TEV PROTEASE LEAVING ONLY A GLYCINE (0) FOLLOWED BY THE TARGET SEQUENCE.

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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: 1.94 Å3/Da / Density % sol: 36.6 %
Crystal growTemperature: 293 K
Details: 30.0% Ethylene-Glycol, 0.1M NaAcetate, VAPOR DIFFUSION, SITTING DROP, NANODROP, temperature 293K

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: ALS / Beamline: 8.2.2 / Wavelength: 0.9740, 0.9799, 0.9798
DetectorType: ADSC QUANTUM 315 / Detector: CCD / Date: Aug 30, 2006
RadiationMonochromator: Double Crystal Si(111) / Protocol: MAD / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelength
IDWavelength (Å)Relative weight
10.9741
20.97991
30.97981
ReflectionResolution: 2→28.796 Å / Num. obs: 23439 / % possible obs: 99.9 % / Redundancy: 3.4 % / Rmerge(I) obs: 0.102 / Rsym value: 0.102 / Net I/σ(I): 6.6
Reflection shell

Diffraction-ID: 1

Resolution (Å)Redundancy (%)Rmerge(I) obsMean I/σ(I) obsNum. measured allNum. unique allRsym value% possible all
2.05-2.13.50.8170.9593516900.81799.9
2.1-2.163.50.6331.2591016870.633100
2.16-2.223.50.5041.5559916040.50499.9
2.22-2.293.50.4121.9544715610.41299.9
2.29-2.373.50.3742.1537115360.37499.9
2.37-2.453.50.3022.6512714680.302100
2.45-2.543.50.2493.1496814260.24999.8
2.54-2.653.50.2053.7482713940.205100
2.65-2.763.40.1654.6455613210.16599.9
2.76-2.93.40.1485.2435912680.14899.8
2.9-3.063.40.1146.6416912200.11499.9
3.06-3.243.40.0898.3387111410.089100
3.24-3.473.40.07110.1364510750.07199.9
3.47-3.743.30.05811.9337010130.05899.7
3.74-4.13.20.04614.529969360.04699.9
4.1-4.583.10.04215.126198460.04299.7
4.58-5.293.20.0451424777720.04599.6
5.29-6.483.40.0513.322486570.05100
6.48-9.173.30.03815.617005200.03899.8
9.17-28.82.90.03315.28843040.03396.7

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Phasing

PhasingMethod: MAD

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Processing

Software
NameVersionClassificationNB
MolProbity3beta29model building
SHELXphasing
REFMAC5.2.0005refinement
SCALAdata scaling
PDB_EXTRACT2data extraction
MOSFLMdata reduction
CCP4(SCALA)data scaling
SHELXDphasing
SHARPphasing
RefinementMethod to determine structure: MAD / Resolution: 2.05→28.796 Å / Cor.coef. Fo:Fc: 0.963 / Cor.coef. Fo:Fc free: 0.937 / SU B: 9.968 / SU ML: 0.14 / TLS residual ADP flag: LIKELY RESIDUAL / Cross valid method: THROUGHOUT / σ(F): 0 / ESU R: 0.222 / ESU R Free: 0.186
Stereochemistry target values: MAXIMUM LIKELIHOOD WITH PHASES
Details: 1. HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS 2. ATOM RECORD CONTAINS RESIDUAL B FACTORS ONLY 3. SEVENTEEN ETHYLENE GLYCOL MOLECULES FROM CRYSTALLIZATION SOLUTION ARE BUILT IN THE ...Details: 1. HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS 2. ATOM RECORD CONTAINS RESIDUAL B FACTORS ONLY 3. SEVENTEEN ETHYLENE GLYCOL MOLECULES FROM CRYSTALLIZATION SOLUTION ARE BUILT IN THE MODEL. 4. A MET-INHIBITION PROTOCOL WAS USED FOR SELENOMETHIONINE INCORPORATION DURING PROTEIN EXPRESSION. THE OCCUPANCY OF THE SE ATOMS IN THE MSE RESIDUES WAS REDUCED TO 0.75 TO ACCOUNT FOR THE REDUCED SCATTERING POWER DUE TO PARTIAL S-MET INCORPORATION. 5. THE METAL IONS AT THE ACTIVE SITE WERE ASSIGNED AS IRON BASED ON AN X-RAY FLUORESCENCE SCAN AROUND FE EDGE, ANOMALOUS DIFFERENCE FOURIER PEAKS, AND COORDINATION. 6. A PEROXIDE ION (PER) IS TENTATIVELY ASSIGNED AT THE ACTIVE SITE OF CHAIN A. THE ASSIGNMENT IS BASED ON THE ENZYME FUNCTION AND THE ELECTRON DENSITY. HOWEVER, SPECTROSCOPIC DATA WERE NOT AVAILABLE. 7. IN SUBUNIT B, AN UNKNOWN LIGAND (UNL) IS BUILT IN NEAR FE BINDING SITE. THE DENSITY AT THE UNL SITE DIFFERS FROM DENSITY AT THE NCS RELATED SITE. 8. THERE IS AN UNKNOWN ELECTRON DENSITY NEAR RESIDUES CYS37 AND HIS196 ON THE PROTEIN SURFACE IN SUBUNIT A. THIS DENSITY WAS LEFT UNMODELED.
RfactorNum. reflection% reflectionSelection details
Rfree0.229 1201 5.1 %RANDOM
Rwork0.171 ---
obs0.174 23393 99.77 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK
Displacement parametersBiso mean: 30.129 Å2
Baniso -1Baniso -2Baniso -3
1-1.02 Å20 Å20 Å2
2--1.32 Å20 Å2
3----2.33 Å2
Refinement stepCycle: LAST / Resolution: 2.05→28.796 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms3063 0 78 222 3363
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0170.0213192
X-RAY DIFFRACTIONr_bond_other_d0.0020.023020
X-RAY DIFFRACTIONr_angle_refined_deg1.5991.9744295
X-RAY DIFFRACTIONr_angle_other_deg0.85236937
X-RAY DIFFRACTIONr_dihedral_angle_1_deg3.2815399
X-RAY DIFFRACTIONr_dihedral_angle_2_deg30.63722.714140
X-RAY DIFFRACTIONr_dihedral_angle_3_deg12.91915517
X-RAY DIFFRACTIONr_dihedral_angle_4_deg15.8291529
X-RAY DIFFRACTIONr_chiral_restr0.0930.2483
X-RAY DIFFRACTIONr_gen_planes_refined0.0060.023528
X-RAY DIFFRACTIONr_gen_planes_other0.0010.02650
X-RAY DIFFRACTIONr_nbd_refined0.220.3828
X-RAY DIFFRACTIONr_nbd_other0.180.33136
X-RAY DIFFRACTIONr_nbtor_refined0.1870.51589
X-RAY DIFFRACTIONr_nbtor_other0.0920.51868
X-RAY DIFFRACTIONr_xyhbond_nbd_refined0.230.5290
X-RAY DIFFRACTIONr_symmetry_vdw_refined0.1110.319
X-RAY DIFFRACTIONr_symmetry_vdw_other0.2750.388
X-RAY DIFFRACTIONr_symmetry_hbond_refined0.2510.528
X-RAY DIFFRACTIONr_mcbond_it2.13932037
X-RAY DIFFRACTIONr_mcbond_other0.6463803
X-RAY DIFFRACTIONr_mcangle_it2.70253175
X-RAY DIFFRACTIONr_scbond_it5.06981269
X-RAY DIFFRACTIONr_scangle_it6.339111119
Refine LS restraints NCS

Dom-ID: 1 / Auth asym-ID: A / Ens-ID: 1 / Number: 2916 / Refine-ID: X-RAY DIFFRACTION

TypeRms dev position (Å)Weight position
MEDIUM POSITIONAL0.490.5
MEDIUM THERMAL1.062
LS refinement shellResolution: 2.05→2.103 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.336 96 -
Rwork0.225 1591 -
obs-1687 99.76 %
Refinement TLS params.

Method: refined / Refine-ID: X-RAY DIFFRACTION

IDL11 (°2)L12 (°2)L13 (°2)L22 (°2)L23 (°2)L33 (°2)S11 (Å °)S12 (Å °)S13 (Å °)S21 (Å °)S22 (Å °)S23 (Å °)S31 (Å °)S32 (Å °)S33 (Å °)T11 (Å2)T12 (Å2)T13 (Å2)T22 (Å2)T23 (Å2)T33 (Å2)Origin x (Å)Origin y (Å)Origin z (Å)
11.5644-0.08740.85921.65830.20922.1856-0.0870.15550.1387-0.2165-0.07260.1786-0.2374-0.21830.1596-0.03550.0536-0.0422-0.0184-0.0159-0.0391-8.919344.624941.7849
20.89210.102-0.97450.35270.01461.9743-0.02220.014-0.0616-0.0193-0.0097-0.02070.04750.01330.0319-0.0871-0.00390.0074-0.1106-0.0017-0.042216.95525.604541.5361
Refinement TLS group

Refine-ID: X-RAY DIFFRACTION / Selection: ALL

IDRefine TLS-IDAuth asym-IDLabel asym-IDAuth seq-IDLabel seq-ID
11AA3 - 2014 - 202
22BB4 - 2015 - 202

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