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- PDB-4j8t: Engineered Digoxigenin binder DIG10.2 -

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Basic information

Entry
Database: PDB / ID: 4j8t
TitleEngineered Digoxigenin binder DIG10.2
ComponentsEngineered Digoxigenin binder protein DIG10.2
KeywordsDIGOXIGENIN BINDING PROTEIN / engineered / computationally designed / Digoxigenin-binding
Function / homologySnoaL-like domain / SnoaL-like domain / Nuclear Transport Factor 2; Chain: A, - #50 / NTF2-like domain superfamily / Nuclear Transport Factor 2; Chain: A, / Roll / Alpha Beta / DIGOXIGENIN / Uncharacterized PhzA/B-like protein PA3332
Function and homology information
Biological speciesPseudomonas aeruginosa (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / molecular replacement / Resolution: 2.05 Å
AuthorsStoddard, B.L. / Doyle, L.A.
CitationJournal: Nature / Year: 2013
Title: Computational design of ligand-binding proteins with high affinity and selectivity.
Authors: Tinberg, C.E. / Khare, S.D. / Dou, J. / Doyle, L. / Nelson, J.W. / Schena, A. / Jankowski, W. / Kalodimos, C.G. / Johnsson, K. / Stoddard, B.L. / Baker, D.
History
DepositionFeb 14, 2013Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jun 26, 2013Provider: repository / Type: Initial release
Revision 1.1Aug 14, 2013Group: Database references
Revision 1.2Sep 18, 2013Group: Database references
Revision 1.3Sep 25, 2013Group: Database references
Revision 1.4Nov 15, 2017Group: Refinement description / Category: software
Revision 1.5Jul 17, 2019Group: Data collection / Refinement description / Category: software
Item: _software.classification / _software.contact_author ..._software.classification / _software.contact_author / _software.contact_author_email / _software.location / _software.name / _software.type / _software.version
Revision 1.6Feb 28, 2024Group: 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 / struct_ncs_dom_lim / 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 / _struct_ncs_dom_lim.beg_auth_comp_id / _struct_ncs_dom_lim.beg_label_asym_id / _struct_ncs_dom_lim.beg_label_comp_id / _struct_ncs_dom_lim.beg_label_seq_id / _struct_ncs_dom_lim.end_auth_comp_id / _struct_ncs_dom_lim.end_label_asym_id / _struct_ncs_dom_lim.end_label_comp_id / _struct_ncs_dom_lim.end_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: Engineered Digoxigenin binder protein DIG10.2
B: Engineered Digoxigenin binder protein DIG10.2
C: Engineered Digoxigenin binder protein DIG10.2
D: Engineered Digoxigenin binder protein DIG10.2
hetero molecules


Theoretical massNumber of molelcules
Total (without water)65,2308
Polymers63,6684
Non-polymers1,5624
Water1,54986
1
A: Engineered Digoxigenin binder protein DIG10.2
hetero molecules


Theoretical massNumber of molelcules
Total (without water)16,3082
Polymers15,9171
Non-polymers3911
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
2
B: Engineered Digoxigenin binder protein DIG10.2
hetero molecules


Theoretical massNumber of molelcules
Total (without water)16,3082
Polymers15,9171
Non-polymers3911
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
3
C: Engineered Digoxigenin binder protein DIG10.2
hetero molecules


Theoretical massNumber of molelcules
Total (without water)16,3082
Polymers15,9171
Non-polymers3911
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
4
D: Engineered Digoxigenin binder protein DIG10.2
hetero molecules


Theoretical massNumber of molelcules
Total (without water)16,3082
Polymers15,9171
Non-polymers3911
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
5
D: Engineered Digoxigenin binder protein DIG10.2
hetero molecules

C: Engineered Digoxigenin binder protein DIG10.2
hetero molecules

A: Engineered Digoxigenin binder protein DIG10.2
B: Engineered Digoxigenin binder protein DIG10.2
hetero molecules


Theoretical massNumber of molelcules
Total (without water)65,2308
Polymers63,6684
Non-polymers1,5624
Water724
TypeNameSymmetry operationNumber
crystal symmetry operation4_445-x-1,-y-1,z+1/21
crystal symmetry operation5_545y,-x+y-1,z+1/61
identity operation1_555x,y,z1
Buried area7200 Å2
ΔGint-37 kcal/mol
Surface area21240 Å2
MethodPISA
Unit cell
Length a, b, c (Å)74.368, 74.368, 161.097
Angle α, β, γ (deg.)90.00, 90.00, 120.00
Int Tables number170
Space group name H-MP65
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
11A
21B
12A
22C
13A
23D
14B
24C
15B
25D
16C
26D

NCS domain segments:

Component-ID: _ / Refine code: _

Dom-IDEns-IDBeg auth comp-IDBeg label comp-IDEnd auth comp-IDEnd label comp-IDAuth asym-IDLabel asym-IDAuth seq-IDLabel seq-ID
11ASNASNVALVALAA2 - 1242 - 124
21ASNASNVALVALBB2 - 1242 - 124
12METMETLEULEUAA1 - 1251 - 125
22METMETLEULEUCC1 - 1251 - 125
13METMETLEULEUAA1 - 1281 - 128
23METMETLEULEUDD1 - 1281 - 128
14ASNASNLEULEUBB2 - 1252 - 125
24ASNASNLEULEUCC2 - 1252 - 125
15ASNASNVALVALBB2 - 1242 - 124
25ASNASNVALVALDD2 - 1242 - 124
16METMETLEULEUCC1 - 1251 - 125
26METMETLEULEUDD1 - 1251 - 125

NCS ensembles :
ID
1
2
3
4
5
6

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Components

#1: Protein
Engineered Digoxigenin binder protein DIG10.2


Mass: 15917.065 Da / Num. of mol.: 4
Mutation: L7V, S10A, F34Y, A37P, W41Y, H61Y, L62M, V64I, A90H, Q99A, D117L, W119F, H124V, A127P, G130L and V131E
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pseudomonas aeruginosa (bacteria) / Strain: ATCC 15692 / PAO1 / 1C / PRS 101 / LMG 12228 / Gene: PA3332 / Plasmid: pET15 / Production host: Escherichia coli (E. coli) / Strain (production host): BL21(DE3)-RIL / References: UniProt: Q9HYR3
#2: Chemical
ChemComp-DOG / DIGOXIGENIN / 4-(3,12,14-TRIHYDROXY-10,13-DIMETHYL-HEXADECAHYDRO-CYCLOPENTA[A]PHENANTHREN-17-YL)-5H-FURAN-2-ONE


Mass: 390.513 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C23H34O5
#3: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 86 / Source method: isolated from a natural source / Formula: H2O
Sequence detailsDIG10.2 WAS COMPUTATIONALLY ENGINEERED BASED ON PRE-DEFINED CRITERIA OF AFFINITY FOR DIGOXIGENIN. ...DIG10.2 WAS COMPUTATIONALLY ENGINEERED BASED ON PRE-DEFINED CRITERIA OF AFFINITY FOR DIGOXIGENIN. THE FOLLOWING MUTATIONS TO PA3332 (PDB ID 1Z1S) WERE FOUND TO MAXIMIZE BINDING AND OPTIMIZE PROTEIN STABILITY: L7V, S10A, F34Y, A37P, W41Y, H61Y, L62M, V64I, A90H, Q99A, D117L, W119F, H124V, A127P, G130L AND V131E. TO AIDE IN CRYSTALLIZATION THE C-TERMINAL RESIDUES 132-141 WERE REMOVED AND REPLACED WITH 6X HIS-TAG.

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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: 2.02 Å3/Da / Density % sol: 39.1 %
Crystal growTemperature: 298 K / Method: vapor diffusion, hanging drop / pH: 5.5
Details: 0.1M Acetate pH5.5, 1.5% MPD, 2.5M NaCl, 12% PEG1500, VAPOR DIFFUSION, HANGING DROP, temperature 298K

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: ALS / Beamline: 5.0.2 / Wavelength: 1.5998 Å
DetectorType: ADSC QUANTUM 315r / Detector: CCD / Date: Dec 13, 2012 / Details: mirrors
RadiationMonochromator: Double-crystal / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.5998 Å / Relative weight: 1
ReflectionResolution: 2.05→50 Å / Num. obs: 30506 / % possible obs: 96.5 % / Redundancy: 15.8 % / Rmerge(I) obs: 0.101 / Net I/σ(I): 16.2
Reflection shell
Resolution (Å)Redundancy (%)Rmerge(I) obsDiffraction-ID% possible all
2.05-2.124.80.336172
2.12-2.217.70.3193
2.21-2.3112.90.291199.9
2.31-2.4317.30.2591100
2.43-2.5818.30.191100
2.58-2.7818.70.1521100
2.78-3.0618.80.1221100
3.06-3.5118.80.11100
3.51-4.4218.70.091100
4.42-5018.50.069199.9

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Phasing

PhasingMethod: molecular replacement
Phasing MRModel details: Phaser MODE: MR_AUTO
Highest resolutionLowest resolution
Rotation2.05 Å41.24 Å
Translation2.05 Å41.24 Å

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Processing

Software
NameVersionClassificationNB
PHENIX1.7_650refinement
SCALEPACKdata scaling
PHASER2.5.1phasing
REFMACrefinement
PDB_EXTRACT3.11data extraction
HKL-3000data reduction
HKL-3000data scaling
DENZOdata reduction
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 2.05→41.244 Å / Cor.coef. Fo:Fc: 0.943 / Cor.coef. Fo:Fc free: 0.936 / Occupancy max: 1 / Occupancy min: 1 / SU ML: 0.28 / SU R Cruickshank DPI: 0.2422 / σ(F): 1.37 / Phase error: 25.9 / Stereochemistry target values: ML
Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS U VALUES : REFINED INDIVIDUALLY
RfactorNum. reflection% reflection
Rfree0.2463 1529 5.03 %
Rwork0.2108 --
obs0.2126 30423 96.43 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL / Bsol: 48.919 Å2 / ksol: 0.378 e/Å3
Displacement parameters
Baniso -1Baniso -2Baniso -3
1-0.0557 Å20 Å2-0 Å2
2--0.0557 Å20 Å2
3----0.1113 Å2
Refinement stepCycle: LAST / Resolution: 2.05→41.244 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms3693 0 112 86 3891
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0083935
X-RAY DIFFRACTIONf_angle_d1.1535410
X-RAY DIFFRACTIONf_dihedral_angle_d11.841311
X-RAY DIFFRACTIONf_chiral_restr0.152612
X-RAY DIFFRACTIONf_plane_restr0.005663
Refine LS restraints NCS

Refine-ID: X-RAY DIFFRACTION / Type: interatomic distance / Weight position: 0.05

Ens-IDDom-IDAuth asym-IDNumberRms dev position (Å)
11A53190.25
12B53190.25
21A53760.25
22C53760.25
31A59830.17
32D59830.17
41B58930.17
42C58930.17
51B52330.24
52D52330.24
61C53070.24
62D53070.24
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.05-2.11620.3227900.27771954X-RAY DIFFRACTION71
2.1162-2.19180.28551360.22992462X-RAY DIFFRACTION91
2.1918-2.27950.25631170.22172699X-RAY DIFFRACTION100
2.2795-2.38330.34021590.23152719X-RAY DIFFRACTION100
2.3833-2.50890.30381480.21912703X-RAY DIFFRACTION100
2.5089-2.66610.29821440.22272721X-RAY DIFFRACTION100
2.6661-2.87190.28711630.22952717X-RAY DIFFRACTION100
2.8719-3.16080.25861360.22442708X-RAY DIFFRACTION100
3.1608-3.61790.21661380.20342747X-RAY DIFFRACTION100
3.6179-4.55730.19521450.17852731X-RAY DIFFRACTION100
4.5573-41.25210.22761530.20912733X-RAY DIFFRACTION99

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