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- PDB-3k74: Disruption of protein dynamics by an allosteric effector antibody -

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Basic information

Entry
Database: PDB / ID: 3k74
TitleDisruption of protein dynamics by an allosteric effector antibody
Components
  • Dihydrofolate reductase
  • NanobodySingle-domain antibody
KeywordsOXIDOREDUCTASE / Immunoglobulin / protein-nanobody complex / Antibiotic resistance / Methotrexate resistance / NADP / One-carbon metabolism / Trimethoprim resistance
Function / homology
Function and homology information


methotrexate binding / dihydrofolic acid binding / response to methotrexate / dihydrofolate metabolic process / NADP+ binding / folic acid binding / folic acid metabolic process / glycine biosynthetic process / dihydrofolate reductase / dihydrofolate reductase activity ...methotrexate binding / dihydrofolic acid binding / response to methotrexate / dihydrofolate metabolic process / NADP+ binding / folic acid binding / folic acid metabolic process / glycine biosynthetic process / dihydrofolate reductase / dihydrofolate reductase activity / NADPH binding / tetrahydrofolate biosynthetic process / one-carbon metabolic process / NADP binding / response to xenobiotic stimulus / response to antibiotic / cytosol
Similarity search - Function
Dihydrofolate Reductase, subunit A / Dihydrofolate Reductase, subunit A / Dihydrofolate reductase / Dihydrofolate reductase conserved site / Dihydrofolate reductase (DHFR) domain signature. / Dihydrofolate reductase domain / Dihydrofolate reductase / Dihydrofolate reductase (DHFR) domain profile. / Dihydrofolate reductase-like domain superfamily / Immunoglobulins ...Dihydrofolate Reductase, subunit A / Dihydrofolate Reductase, subunit A / Dihydrofolate reductase / Dihydrofolate reductase conserved site / Dihydrofolate reductase (DHFR) domain signature. / Dihydrofolate reductase domain / Dihydrofolate reductase / Dihydrofolate reductase (DHFR) domain profile. / Dihydrofolate reductase-like domain superfamily / Immunoglobulins / Immunoglobulin-like / Sandwich / 3-Layer(aba) Sandwich / Mainly Beta / Alpha Beta
Similarity search - Domain/homology
Dihydrofolate reductase
Similarity search - Component
Biological speciesEscherichia coli K-12 (bacteria)
Lama glama (llama)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.95 Å
AuthorsOyen, D. / Srinivasan, V. / Steyaert, J. / Barlow, J.
CitationJournal: J.Mol.Biol. / Year: 2011
Title: Constraining enzyme conformational change by an antibody leads to hyperbolic inhibition.
Authors: Oyen, D. / Srinivasan, V. / Steyaert, J. / Barlow, J.N.
History
DepositionOct 12, 2009Deposition site: RCSB / Processing site: RCSB
Revision 1.0Oct 20, 2010Provider: repository / Type: Initial release
Revision 1.1Jul 13, 2011Group: Version format compliance
Revision 1.2Sep 4, 2013Group: Database references
Revision 1.3Sep 18, 2013Group: Derived calculations
Revision 1.4Nov 1, 2017Group: Refinement description / Category: software
Revision 1.5Sep 6, 2023Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Dihydrofolate reductase
B: Nanobody


Theoretical massNumber of molelcules
Total (without water)30,5702
Polymers30,5702
Non-polymers00
Water2,594144
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)55.300, 62.322, 102.848
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number19
Space group name H-MP212121

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Components

#1: Protein Dihydrofolate reductase /


Mass: 18019.340 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli K-12 (bacteria) / Strain: RT500 / Gene: b0048, DHFR, folA, JW0047, tmrA / Plasmid: pET22 / Production host: Escherichia coli (E. coli) / Strain (production host): BL21(DE3) / References: UniProt: P0ABQ4, dihydrofolate reductase
#2: Antibody Nanobody / Single-domain antibody


Mass: 12551.153 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Lama glama (llama) / Gene: Lama / Plasmid: pHen6 / Production host: Escherichia coli (E. coli) / Strain (production host): WK6
#3: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 144 / 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: 2.9 Å3/Da / Density % sol: 57.57 %
Crystal growTemperature: 298 K / Method: vapor diffusion / pH: 8.5
Details: 40% v/v PEG300, 5% w/v PEG1000, 100 mM Tris-HCl, pH 8.5, VAPOR DIFFUSION, temperature 298K

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: SOLEIL / Beamline: PROXIMA 1 / Wavelength: 0.98 Å
DetectorType: ADSC QUANTUM 210 / Detector: CCD / Date: Feb 19, 2008
RadiationMonochromator: GRAPHITE / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.98 Å / Relative weight: 1
ReflectionResolution: 1.9→30 Å / Num. obs: 28725 / % possible obs: 99.9 % / Redundancy: 7.1 % / Rmerge(I) obs: 0.102 / Χ2: 1.616 / Net I/σ(I): 12.5
Reflection shellResolution: 1.9→1.93 Å / Redundancy: 7.2 % / Rmerge(I) obs: 0.388 / Num. unique all: 1394 / Χ2: 1.298 / % possible all: 100

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Processing

Software
NameVersionClassificationNB
DENZOdata reduction
SCALEPACKdata scaling
REFMAC5.2.0019refinement
PDB_EXTRACT3.005data extraction
ADSCQuantumdata collection
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: PDB entries 1HCV, 5DFR

1hcv

5dfr


Resolution: 1.95→7.99 Å / Cor.coef. Fo:Fc: 0.955 / Cor.coef. Fo:Fc free: 0.932 / Occupancy max: 1 / Occupancy min: 1 / SU B: 7.363 / SU ML: 0.105 / Cross valid method: THROUGHOUT / σ(F): 0 / ESU R: 0.149 / ESU R Free: 0.145 / Stereochemistry target values: MAXIMUM LIKELIHOOD / Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflectionSelection details
Rfree0.24 1333 5.1 %RANDOM
Rwork0.196 ---
obs0.198 26145 99.98 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK
Displacement parametersBiso max: 78.85 Å2 / Biso mean: 33.372 Å2 / Biso min: 14.55 Å2
Baniso -1Baniso -2Baniso -3
1-0.01 Å20 Å20 Å2
2--0.43 Å20 Å2
3----0.44 Å2
Refinement stepCycle: LAST / Resolution: 1.95→7.99 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms2149 0 0 144 2293
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0180.0222203
X-RAY DIFFRACTIONr_angle_refined_deg1.7461.942991
X-RAY DIFFRACTIONr_dihedral_angle_1_deg7.6895272
X-RAY DIFFRACTIONr_dihedral_angle_2_deg35.81223.96101
X-RAY DIFFRACTIONr_dihedral_angle_3_deg14.81215360
X-RAY DIFFRACTIONr_dihedral_angle_4_deg22.8971514
X-RAY DIFFRACTIONr_chiral_restr0.120.2319
X-RAY DIFFRACTIONr_gen_planes_refined0.0090.021693
X-RAY DIFFRACTIONr_nbd_refined0.2320.2980
X-RAY DIFFRACTIONr_nbtor_refined0.3130.21502
X-RAY DIFFRACTIONr_xyhbond_nbd_refined0.2050.2152
X-RAY DIFFRACTIONr_symmetry_vdw_refined0.2730.238
X-RAY DIFFRACTIONr_symmetry_hbond_refined0.2490.28
X-RAY DIFFRACTIONr_mcbond_it1.3131.51404
X-RAY DIFFRACTIONr_mcangle_it2.03522182
X-RAY DIFFRACTIONr_scbond_it3.2183954
X-RAY DIFFRACTIONr_scangle_it5.0434.5809
LS refinement shellResolution: 1.95→1.998 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.279 102 -
Rwork0.212 1712 -
all-1814 -
obs--100 %
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)
11.5420.14460.35880.39070.3851.4724-0.0376-0.0088-0.05870.00440.0781-0.0694-0.04820.0116-0.0405-0.01590.0452-0.0052-0.1644-0.0180.048528.91330.94928.398
21.1592-0.20921.29870.6603-0.16524.54040.0264-0.0476-0.0067-0.0226-0.04730.01450.0957-0.34530.0209-0.06550.0573-0.0025-0.06060.01940.01749.55326.6826.32
Refinement TLS group
IDRefine-IDRefine TLS-IDAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1A2 - 158
2X-RAY DIFFRACTION2A164 - 274

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