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- PDB-3egz: Crystal structure of an in vitro evolved tetracycline aptamer and... -

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Basic information

Entry
Database: PDB / ID: 3egz
TitleCrystal structure of an in vitro evolved tetracycline aptamer and artificial riboswitch
Components
  • Tetracycline aptamer and artificial riboswitch
  • U1 small nuclear ribonucleoprotein A
KeywordsRNA / tetracycline / aptamer / riboswitch / antibiotic
Function / homology
Function and homology information


U1 snRNP binding / U1 snRNP / U1 snRNA binding / U4/U6 x U5 tri-snRNP complex / mRNA Splicing - Major Pathway / spliceosomal complex / mRNA splicing, via spliceosome / DNA binding / RNA binding / nucleoplasm ...U1 snRNP binding / U1 snRNP / U1 snRNA binding / U4/U6 x U5 tri-snRNP complex / mRNA Splicing - Major Pathway / spliceosomal complex / mRNA splicing, via spliceosome / DNA binding / RNA binding / nucleoplasm / identical protein binding / nucleus
Similarity search - Function
U1 small nuclear ribonucleoprotein A, RNA recognition motif 2 / U1 small nuclear ribonucleoprotein A, RNA recognition motif 1 / RRM (RNA recognition motif) domain / RNA recognition motif / RNA recognition motif / Eukaryotic RNA Recognition Motif (RRM) profile. / RNA recognition motif domain / RNA-binding domain superfamily / Nucleotide-binding alpha-beta plait domain superfamily / Alpha-Beta Plaits ...U1 small nuclear ribonucleoprotein A, RNA recognition motif 2 / U1 small nuclear ribonucleoprotein A, RNA recognition motif 1 / RRM (RNA recognition motif) domain / RNA recognition motif / RNA recognition motif / Eukaryotic RNA Recognition Motif (RRM) profile. / RNA recognition motif domain / RNA-binding domain superfamily / Nucleotide-binding alpha-beta plait domain superfamily / Alpha-Beta Plaits / 2-Layer Sandwich / Alpha Beta
Similarity search - Domain/homology
7-CHLOROTETRACYCLINE / RNA / RNA (> 10) / U1 small nuclear ribonucleoprotein A
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MAD / Resolution: 2.2 Å
AuthorsXiao, H. / Edwards, T.E. / Ferre-D'Amare, A.R.
CitationJournal: Chem.Biol. / Year: 2008
Title: Structural basis for specific, high-affinity tetracycline binding by an in vitro evolved aptamer and artificial riboswitch
Authors: Xiao, H. / Edwards, T.E. / Ferre-D'Amare, A.R.
History
DepositionSep 11, 2008Deposition site: RCSB / Processing site: RCSB
Revision 1.0Oct 28, 2008Provider: repository / Type: Initial release
Revision 1.1Jul 13, 2011Group: Version format compliance
Revision 1.2Oct 25, 2017Group: Refinement description / Category: software
Revision 1.3Oct 20, 2021Group: Database references / Derived calculations
Category: database_2 / pdbx_struct_conn_angle ...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.ptnr2_auth_seq_id / _pdbx_struct_conn_angle.ptnr2_label_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.pdbx_leaving_atom_flag / _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_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: U1 small nuclear ribonucleoprotein A
B: Tetracycline aptamer and artificial riboswitch
hetero molecules


Theoretical massNumber of molelcules
Total (without water)33,25315
Polymers32,4822
Non-polymers77113
Water2,630146
1
A: U1 small nuclear ribonucleoprotein A
B: Tetracycline aptamer and artificial riboswitch
hetero molecules

A: U1 small nuclear ribonucleoprotein A
B: Tetracycline aptamer and artificial riboswitch
hetero molecules

A: U1 small nuclear ribonucleoprotein A
B: Tetracycline aptamer and artificial riboswitch
hetero molecules

A: U1 small nuclear ribonucleoprotein A
B: Tetracycline aptamer and artificial riboswitch
hetero molecules


Theoretical massNumber of molelcules
Total (without water)133,01260
Polymers129,9298
Non-polymers3,08252
Water1448
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation2_565-x,-y+1,z1
crystal symmetry operation3_555-y+1/2,x+1/2,z1
crystal symmetry operation4_455y-1/2,-x+1/2,z1
Buried area20160 Å2
ΔGint-383 kcal/mol
Surface area54890 Å2
MethodPISA
Unit cell
Length a, b, c (Å)120.835, 120.835, 55.279
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number90
Space group name H-MP4212
Components on special symmetry positions
IDModelComponents
11A-510-

MG

21B-505-

MG

31A-327-

HOH

41B-434-

HOH

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Components

#1: Protein U1 small nuclear ribonucleoprotein A / U1 snRNP protein A / U1A protein / U1-A


Mass: 11574.792 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: SNRPA / Plasmid: pET / Production host: Escherichia coli (E. coli) / References: UniProt: P09012
#2: RNA chain Tetracycline aptamer and artificial riboswitch


Mass: 20907.549 Da / Num. of mol.: 1 / Source method: obtained synthetically / Details: In vitro evolved aptamer
#3: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 12 / Source method: obtained synthetically / Formula: Mg
#4: Chemical ChemComp-CTC / 7-CHLOROTETRACYCLINE / Chlortetracycline


Mass: 478.880 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C22H23ClN2O8 / Comment: antibiotic*YM
#5: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 146 / 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: 3.21 Å3/Da / Density % sol: 61.72 %
Crystal growTemperature: 296 K / Method: vapor diffusion, sitting drop / pH: 7
Details: 0.3 mM RNA, 0.33 mM selenomethionine labeled U1A-RBD double mutant, 0.5 mM chlorotetracycline, 50 mM Tris pH 7.5, 5 mM MgCl2, 0.25 mM spermine; 1uL macromolecular complex:1 uL reservoir; 50 ...Details: 0.3 mM RNA, 0.33 mM selenomethionine labeled U1A-RBD double mutant, 0.5 mM chlorotetracycline, 50 mM Tris pH 7.5, 5 mM MgCl2, 0.25 mM spermine; 1uL macromolecular complex:1 uL reservoir; 50 mM HEPES-KOH pH 7.0, 20 mM MGCl2, 12.5-15% PEG 8000; cryo 30% glycerol, 50 mM HEPES-KOH pH 7.0, 20 mM MGCl2, 15% PEG 8000, 0.5 mM spermine, 0.5 mM chlorotetracycline, VAPOR DIFFUSION, SITTING DROP, temperature 296K
Components of the solutions
IDNameCrystal-IDSol-ID
1MgCl211
2spermine11
3HEPES-KOH11
4PEG 800011
5glycerol11
6MgCl212
7PEG 800012
8HEPES-KOH12

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: ALS / Beamline: 5.0.2 / Wavelength: 0.9570,0.9797,0.9795
DetectorType: ADSC QUANTUM 315 / Detector: CCD / Date: Feb 14, 2008
RadiationMonochromator: Double crystal, Si111 / Protocol: MAD / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelength
IDWavelength (Å)Relative weight
10.9571
20.97971
30.97951
ReflectionResolution: 2.2→29.31 Å / Num. obs: 21415 / % possible obs: 99.1 % / Redundancy: 9.5 % / Rmerge(I) obs: 0.008 / Net I/σ(I): 37.6
Reflection shellResolution: 2.2→2.28 Å / Redundancy: 9.8 % / Rmerge(I) obs: 0.532 / Mean I/σ(I) obs: 3.8 / Num. unique all: 2156 / % possible all: 99.9

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Phasing

PhasingMethod: MAD

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Processing

Software
NameVersionClassificationNB
DENZOdata reduction
SCALEPACKdata scaling
SOLVEphasing
REFMAC5.4.0067refinement
PDB_EXTRACT3.006data extraction
RefinementMethod to determine structure: MAD / Resolution: 2.2→29.31 Å / Cor.coef. Fo:Fc: 0.956 / Cor.coef. Fo:Fc free: 0.937 / Occupancy max: 1 / Occupancy min: 0.25 / SU B: 5.694 / SU ML: 0.146 / Cross valid method: THROUGHOUT / σ(F): 0 / ESU R: 0.235 / ESU R Free: 0.207 / Stereochemistry target values: MAXIMUM LIKELIHOOD / Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflectionSelection details
Rfree0.26 2156 10.2 %RANDOM
Rwork0.212 ---
obs0.217 21171 98.8 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK
Displacement parametersBiso max: 98.97 Å2 / Biso mean: 50.61 Å2 / Biso min: 27.33 Å2
Baniso -1Baniso -2Baniso -3
1-1.12 Å20 Å20 Å2
2--1.12 Å20 Å2
3----2.24 Å2
Refinement stepCycle: LAST / Resolution: 2.2→29.31 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms727 1380 45 146 2298
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0130.0212321
X-RAY DIFFRACTIONr_bond_other_d0.0010.021057
X-RAY DIFFRACTIONr_angle_refined_deg1.9872.7013461
X-RAY DIFFRACTIONr_angle_other_deg1.2732668
X-RAY DIFFRACTIONr_dihedral_angle_1_deg6.36590
X-RAY DIFFRACTIONr_dihedral_angle_2_deg32.52723.63633
X-RAY DIFFRACTIONr_dihedral_angle_3_deg15.34915140
X-RAY DIFFRACTIONr_dihedral_angle_4_deg15.365155
X-RAY DIFFRACTIONr_chiral_restr0.0850.2442
X-RAY DIFFRACTIONr_gen_planes_refined0.0080.021511
X-RAY DIFFRACTIONr_gen_planes_other0.0010.02297
X-RAY DIFFRACTIONr_mcbond_it0.781.5455
X-RAY DIFFRACTIONr_mcbond_other0.1431.5181
X-RAY DIFFRACTIONr_mcangle_it1.5192735
X-RAY DIFFRACTIONr_scbond_it2.08131866
X-RAY DIFFRACTIONr_scangle_it3.2764.52726
LS refinement shellResolution: 2.198→2.255 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.434 158 -
Rwork0.335 1347 -
all-1505 -
obs--97.1 %

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