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- PDB-6dn0: Retrofitted antibodies with stabilizing mutations: Herceptin scFv... -

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Basic information

Entry
Database: PDB / ID: 6dn0
TitleRetrofitted antibodies with stabilizing mutations: Herceptin scFv mutant with VH K30D and VL S52D.
Components
  • Human Variable Heavy Chain of Herceptin containing VH mutation K30D
  • Human Variable Light Chain of Herceptin containing VL mutation S52D
KeywordsIMMUNE SYSTEM / Herceptin / retrofit / stabilizing mutation
Function / homologyFORMIC ACID
Function and homology information
Biological speciesHomo sapiens (human)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / molecular replacement / Resolution: 2 Å
AuthorsLangley, D.B. / Roome, B. / Christ, D.
Funding support Australia, 2items
OrganizationGrant numberCountry
National Health and Medical Research Council (NHMRC, Australia)1113904 Australia
Australian Research Council (ARC)160104915 Australia
CitationJournal: To Be Published
Title: Retrofitting antibodies with stabilizing mutations
Authors: Roome, B. / Langley, D.B. / Rouet, R. / Christ, D.
History
DepositionJun 5, 2018Deposition site: RCSB / Processing site: RCSB
SupersessionJun 12, 2019ID: 4X4Y
Revision 1.0Jun 12, 2019Provider: repository / Type: Initial release
Revision 1.1Jan 1, 2020Group: Author supporting evidence / Category: pdbx_audit_support
Item: _pdbx_audit_support.country / _pdbx_audit_support.funding_organization
Revision 1.2Oct 11, 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 / struct_ncs_dom_lim
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
Revision 1.3Oct 23, 2024Group: Structure summary / Category: pdbx_entry_details / pdbx_modification_feature

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Human Variable Heavy Chain of Herceptin containing VH mutation K30D
B: Human Variable Light Chain of Herceptin containing VL mutation S52D
C: Human Variable Heavy Chain of Herceptin containing VH mutation K30D
D: Human Variable Light Chain of Herceptin containing VL mutation S52D
hetero molecules


Theoretical massNumber of molelcules
Total (without water)51,6858
Polymers51,5014
Non-polymers1844
Water1,13563
1
A: Human Variable Heavy Chain of Herceptin containing VH mutation K30D
B: Human Variable Light Chain of Herceptin containing VL mutation S52D
hetero molecules


Theoretical massNumber of molelcules
Total (without water)25,8885
Polymers25,7502
Non-polymers1383
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area2020 Å2
ΔGint-11 kcal/mol
Surface area10070 Å2
MethodPISA
2
C: Human Variable Heavy Chain of Herceptin containing VH mutation K30D
D: Human Variable Light Chain of Herceptin containing VL mutation S52D
hetero molecules


Theoretical massNumber of molelcules
Total (without water)25,7963
Polymers25,7502
Non-polymers461
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area1620 Å2
ΔGint-10 kcal/mol
Surface area9990 Å2
MethodPISA
Unit cell
Length a, b, c (Å)91.981, 91.981, 114.677
Angle α, β, γ (deg.)90.000, 90.000, 120.000
Int Tables number152
Space group name H-MP3121
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
11A
21C
12B
22D

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
11GLUGLUSERSERAA1 - 1191 - 119
21GLUGLUSERSERCC1 - 1191 - 119
12ASPASPILEILEBB1 - 1061 - 106
22ASPASPILEILEDD1 - 1061 - 106

NCS ensembles :
ID
1
2

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Components

#1: Antibody Human Variable Heavy Chain of Herceptin containing VH mutation K30D


Mass: 14145.466 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Escherichia coli (E. coli) / Strain (production host): BL21 Gold (DE3)
#2: Antibody Human Variable Light Chain of Herceptin containing VL mutation S52D


Mass: 11604.841 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Plasmid: pET12a / Production host: Escherichia coli (E. coli) / Strain (production host): BL21 Gold (DE3)
#3: Chemical
ChemComp-FMT / FORMIC ACID


Mass: 46.025 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: CH2O2
#4: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 63 / Source method: isolated from a natural source / Formula: H2O
Has protein modificationY

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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: 2.84 Å3/Da / Density % sol: 56.76 % / Description: hexagonal rods
Crystal growTemperature: 293 K / Method: vapor diffusion, sitting drop / pH: 4.6
Details: Equal volumes of protein (8.0 mg/mL in 25 mM Tris (pH 8.0)) were combined with an equal volume of well solution (3.5 M sodium formate, 100 mM sodium acetate (pH 4.6)

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: Australian Synchrotron / Beamline: MX2 / Wavelength: 0.9537 Å
DetectorType: ADSC QUANTUM 315r / Detector: CCD / Date: Aug 13, 2016
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9537 Å / Relative weight: 1
ReflectionResolution: 2→39.83 Å / Num. obs: 38572 / % possible obs: 99.9 % / Redundancy: 11.9 % / CC1/2: 0.999 / Rmerge(I) obs: 0.101 / Rpim(I) all: 0.031 / Rrim(I) all: 0.106 / Net I/σ(I): 14.6 / Num. measured all: 458269 / Scaling rejects: 95
Reflection shellResolution: 2→2.05 Å / Redundancy: 11.7 % / Rmerge(I) obs: 1.018 / Num. unique obs: 2773 / CC1/2: 0.855 / Rpim(I) all: 0.304 / Rrim(I) all: 1.064 / % possible all: 98.8

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Phasing

PhasingMethod: molecular replacement
Phasing MRModel details: Phaser MODE: MR_AUTO
Highest resolutionLowest resolution
Rotation6.16 Å39.83 Å
Translation6.16 Å39.83 Å

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Processing

Software
NameVersionClassification
MOSFLMdata reduction
Aimless0.5.17data scaling
PHASER2.5.7phasing
REFMAC5.8.0222refinement
PDB_EXTRACT3.24data extraction
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 4x4x

4x4x


Resolution: 2→39.83 Å / Cor.coef. Fo:Fc: 0.958 / Cor.coef. Fo:Fc free: 0.945 / SU B: 9.929 / SU ML: 0.127 / SU R Cruickshank DPI: 0.1637 / Cross valid method: THROUGHOUT / σ(F): 0 / ESU R: 0.164 / ESU R Free: 0.147
Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS U VALUES : WITH TLS ADDED
RfactorNum. reflection% reflectionSelection details
Rfree0.2338 1925 5 %RANDOM
Rwork0.2048 ---
obs0.2063 36495 99.93 %-
Solvent computationIon probe radii: 0.7 Å / Shrinkage radii: 0.7 Å / VDW probe radii: 1 Å
Displacement parametersBiso max: 98.12 Å2 / Biso mean: 45.853 Å2 / Biso min: 23.28 Å2
Baniso -1Baniso -2Baniso -3
1-0.99 Å20.49 Å20 Å2
2--0.99 Å20 Å2
3----3.2 Å2
Refinement stepCycle: final / Resolution: 2→39.83 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms3389 0 12 63 3464
Biso mean--56.22 42.51 -
Num. residues----447
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.010.0143500
X-RAY DIFFRACTIONr_bond_other_d0.0010.0172968
X-RAY DIFFRACTIONr_angle_refined_deg1.3221.6614762
X-RAY DIFFRACTIONr_angle_other_deg0.8861.6426939
X-RAY DIFFRACTIONr_dihedral_angle_1_deg7.4265446
X-RAY DIFFRACTIONr_dihedral_angle_2_deg30.21521.726168
X-RAY DIFFRACTIONr_dihedral_angle_3_deg13.75515513
X-RAY DIFFRACTIONr_dihedral_angle_4_deg17.9761521
X-RAY DIFFRACTIONr_chiral_restr0.0630.2466
X-RAY DIFFRACTIONr_gen_planes_refined0.0070.024007
X-RAY DIFFRACTIONr_gen_planes_other0.0010.02697
Refine LS restraints NCS

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

Ens-IDDom-IDAuth asym-IDNumberRms dev position (Å)
11A33610.14
12C33610.14
21B28690.12
22D28690.12
LS refinement shellResolution: 2→2.052 Å / Rfactor Rfree error: 0 / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.265 148 -
Rwork0.265 2646 -
all-2794 -
obs--99.75 %
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)
12.72110.7819-0.45761.38530.41243.7769-0.17540.06780.0759-0.12430.1869-0.0915-0.27680.2138-0.01160.0399-0.0304-0.00210.0389-0.01620.030330.3333-16.984115.8307
23.6573-0.27880.6042.95451.3134.77310.14890.1211-0.2316-0.1552-0.0150.03060.03840.113-0.13390.0986-0.08060.04630.0979-0.07070.114727.1226-30.2321-1.0752
34.09721.8435-1.34394.0123-1.31254.7596-0.0307-0.4592-0.1856-0.083-0.2273-0.19650.09320.49640.2580.01370.02180.02220.09470.05270.042162.4098-15.09831.545
44.008-0.5035-0.31823.313-0.70175.2446-0.1222-0.35860.00650.5335-0.1403-0.32550.03240.69960.26240.19130.0858-0.00440.37280.18560.202566.9853-24.564420.9771
Refinement TLS group
IDRefine-IDRefine TLS-IDAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1A1 - 119
2X-RAY DIFFRACTION2B1 - 106
3X-RAY DIFFRACTION3C1 - 119
4X-RAY DIFFRACTION4D1 - 106

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