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- PDB-8t6i: Structure of VHH-Fab complex with engineered Crystal Kappa region -

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Basic information

Entry
Database: PDB / ID: 8t6i
TitleStructure of VHH-Fab complex with engineered Crystal Kappa region
Components
  • Fab heavy chain
  • Fab light chain
  • VHH domain
KeywordsIMMUNE SYSTEM / NabFab / Nanobody / Antibody Fragment
Biological speciesHomo sapiens (human)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.55 Å
AuthorsFilippova, E.V. / Thompson, I. / Kossiakoff, A.A.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS) United States
CitationJournal: Protein Sci. / Year: 2024
Title: Engineered antigen-binding fragments for enhanced crystallization of antibody:antigen complexes.
Authors: Bruce, H.A. / Singer, A.U. / Filippova, E.V. / Blazer, L.L. / Adams, J.J. / Enderle, L. / Ben-David, M. / Radley, E.H. / Mao, D.Y.L. / Pau, V. / Orlicky, S. / Sicheri, F. / Kurinov, I. / ...Authors: Bruce, H.A. / Singer, A.U. / Filippova, E.V. / Blazer, L.L. / Adams, J.J. / Enderle, L. / Ben-David, M. / Radley, E.H. / Mao, D.Y.L. / Pau, V. / Orlicky, S. / Sicheri, F. / Kurinov, I. / Atwell, S. / Kossiakoff, A.A. / Sidhu, S.S.
History
DepositionJun 16, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Nov 29, 2023Provider: repository / Type: Initial release
Revision 1.1Jan 3, 2024Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.title ..._citation.journal_volume / _citation.title / _citation.year / _citation_author.name
Revision 1.2Oct 16, 2024Group: Structure summary / Category: pdbx_entry_details / pdbx_modification_feature / Item: _pdbx_entry_details.has_protein_modification

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: VHH domain
L: Fab light chain
H: Fab heavy chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)62,9734
Polymers62,8813
Non-polymers921
Water70339
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area5900 Å2
ΔGint-34 kcal/mol
Surface area24490 Å2
MethodPISA
Unit cell
Length a, b, c (Å)54.481, 74.189, 218.423
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number19
Space group name H-MP212121

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Components

#1: Antibody VHH domain


Mass: 14170.781 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Plasmid: PET26B+ / Production host: Escherichia coli BL21(DE3) (bacteria)
#2: Antibody Fab light chain


Mass: 23025.490 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: Chain contains a substitution called Crystal Kappa "QGTTS"
Source: (gene. exp.) Homo sapiens (human) / Plasmid: RH2.2 / Production host: Escherichia coli BL21(DE3) (bacteria)
#3: Antibody Fab heavy chain


Mass: 25684.463 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Plasmid: RH2.2 / Production host: Escherichia coli BL21(DE3) (bacteria)
#4: Chemical ChemComp-GOL / GLYCEROL / GLYCERIN / PROPANE-1,2,3-TRIOL


Mass: 92.094 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C3H8O3 / Feature type: SUBJECT OF INVESTIGATION
#5: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 39 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestY
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: 3.51 Å3/Da / Density % sol: 64.96 %
Crystal growTemperature: 293 K / Method: vapor diffusion, hanging drop
Details: 0.2 M Potassium citrate tribasic monohydrate, 20% PEG3350

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: APS / Beamline: 24-ID-E / Wavelength: 0.97918 Å
DetectorType: DECTRIS EIGER2 X 16M / Detector: PIXEL / Date: Oct 1, 2022
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.97918 Å / Relative weight: 1
ReflectionResolution: 2.55→74.19 Å / Num. obs: 29702 / % possible obs: 99.7 % / Redundancy: 5.6 % / CC1/2: 0.99 / Rpim(I) all: 0.03 / Net I/σ(I): 18.5
Reflection shellResolution: 2.55→2.66 Å / Redundancy: 5.8 % / Mean I/σ(I) obs: 1 / Num. unique obs: 3539 / CC1/2: 0.54 / Rpim(I) all: 0.8 / % possible all: 99.9

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Processing

Software
NameVersionClassification
PHENIX(1.16_3549: ???)refinement
XDSdata reduction
Aimlessdata scaling
PHASERphasing
PDB_EXTRACTdata extraction
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 2.55→61.368 Å / SU ML: 0.42 / Cross valid method: FREE R-VALUE / σ(F): 1.34 / Phase error: 31.52 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.2545 1412 4.78 %
Rwork0.1944 --
obs0.1972 29535 99.18 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Refinement stepCycle: LAST / Resolution: 2.55→61.368 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms4267 0 6 39 4312
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0084373
X-RAY DIFFRACTIONf_angle_d1.1015943
X-RAY DIFFRACTIONf_dihedral_angle_d4.4242576
X-RAY DIFFRACTIONf_chiral_restr0.057657
X-RAY DIFFRACTIONf_plane_restr0.006755
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.55-2.64060.44521500.38962672X-RAY DIFFRACTION97
2.6406-2.74640.36911540.32872762X-RAY DIFFRACTION100
2.7464-2.87140.31971290.28562804X-RAY DIFFRACTION100
2.8714-3.02270.34261330.27162764X-RAY DIFFRACTION100
3.0227-3.21210.32811390.23712823X-RAY DIFFRACTION100
3.2121-3.46010.29121400.21912783X-RAY DIFFRACTION100
3.4601-3.80830.25851300.21742848X-RAY DIFFRACTION100
3.8083-4.35920.20161570.16772797X-RAY DIFFRACTION99
4.3592-5.49160.20361450.15012860X-RAY DIFFRACTION99
5.4916-61.30.27181350.18223010X-RAY DIFFRACTION99
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)
10.1481-0.1277-0.05820.54920.0910.0222-0.0134-0.0685-0.13140.2292-0.4844-0.3161-0.52790.0555-0.02220.79170.194-0.07151.02440.15590.9464-1.317733.9002-0.1704
21.72640.2363-0.23450.31120.5251.4004-0.03160.41290.1265-0.10860.23650.35890.0543-0.25480.00060.45950.0706-0.08671.02810.11410.77564.331124.5892-4.08
30.4731-0.1481-0.56830.258-0.15530.4279-0.16080.38140.1123-0.1862-0.1678-0.56690.0587-0.2637-0.00270.64630.0661-0.05420.94560.10050.773313.763325.2657-5.1176
40.05380.02830.07940.01720.07720.5411-0.12281.7667-0.34190.14040.0718-0.42390.3742-0.1772-0.02380.5628-0.07410.02971.29610.01950.81128.517920.3657-10.3733
50.39110.0601-0.32070.001-0.07940.2792-0.38140.136-0.3147-0.19870.22040.0238-0.0191-1.6119-0.02980.57780.2512-0.19161.410.08390.76690.715825.1912-10.0467
60.74420.0579-0.19660.08990.15760.4521-0.24010.32830.25810.24320.2247-0.28630.0711-0.3351-0.00090.68830.022-0.06710.98010.10210.81699.99124.2614-1.5313
71.1327-0.7326-0.72050.41140.12941.15970.1172-0.1521-0.04110.52910.0272-0.2954-0.274-0.1223-0.00020.60950.0515-0.08450.8674-0.01520.773210.42627.43910.7613
80.99210.29251.52420.7451-0.97382.8830.25360.0423-0.13750.52620.0615-0.02230.4526-0.02850.00090.92850.1832-0.11630.712-0.07360.73829.24497.677829.2137
90.63470.4084-0.10080.6417-0.51380.37660.2372-0.0775-0.57840.1109-0.157-0.0299-0.1232-0.5221-0.00871.31880.056-0.15440.695-0.010.789219.12712.37453.812
102.5869-0.10280.51332.416-0.6450.3870.0179-0.3501-0.14130.6482-0.07850.3714-0.0602-0.1325-0.00021.33050.0612-0.13260.7171-0.04590.804720.7673.774258.5241
112.25520.4821.42422.0599-0.43961.2253-0.4642-0.07140.49610.48360.3426-0.0616-0.44690.3971-0.00140.78960.1561-0.04960.8118-0.03270.832110.465531.072927.9915
120.66740.68660.66150.46640.54820.644-0.3352-0.16220.84510.41950.68610.12290.0505-1.1676-0.02150.78030.23790.04190.88630.03220.9397-0.678928.267427.6027
131.73090.04231.09860.2649-0.93182.84190.00530.08070.13170.3578-0.0165-0.0127-0.2063-0.15890.00030.81180.2099-0.08570.5133-0.11630.642516.158921.841538.4129
140.64290.12090.20490.6093-0.31130.28190.13290.20560.7662-0.8063-0.3592-0.4919-0.67710.3351-0.00981.23320.0754-0.23950.66680.01920.876630.497823.064150.6725
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection details
1X-RAY DIFFRACTION1chain 'A' and (resid 1 through 7 )
2X-RAY DIFFRACTION2chain 'A' and (resid 8 through 44 )
3X-RAY DIFFRACTION3chain 'A' and (resid 45 through 70 )
4X-RAY DIFFRACTION4chain 'A' and (resid 72 through 81 )
5X-RAY DIFFRACTION5chain 'A' and (resid 82 through 91 )
6X-RAY DIFFRACTION6chain 'A' and (resid 92 through 112 )
7X-RAY DIFFRACTION7chain 'A' and (resid 113 through 137 )
8X-RAY DIFFRACTION8chain 'L' and (resid 4 through 146 )
9X-RAY DIFFRACTION9chain 'L' and (resid 147 through 168 )
10X-RAY DIFFRACTION10chain 'L' and (resid 169 through 231 )
11X-RAY DIFFRACTION11chain 'H' and (resid 1 through 44 )
12X-RAY DIFFRACTION12chain 'H' and (resid 45 through 76 )
13X-RAY DIFFRACTION13chain 'H' and (resid 77 through 219 )
14X-RAY DIFFRACTION14chain 'H' and (resid 220 through 239 )

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