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- PDB-6u8c: Crystal structure of an engineered ultra-high affinity Fab-Protei... -

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Basic information

Entry
Database: PDB / ID: 6u8c
TitleCrystal structure of an engineered ultra-high affinity Fab-Protein G complex
Components
  • Antibody heavy chain Fab
  • Antibody light chain Fab
  • Protein G
KeywordsIMMUNE SYSTEM / engineered Fab / protein G / high affinity
Function / homology
Function and homology information


IgG binding / extracellular region
Similarity search - Function
IgG-binding B / B domain / M protein-type anchor domain / GA-like domain / GA-like domain / Ubiquitin-like (UB roll) - #10 / Immunoglobulin/albumin-binding domain superfamily / YSIRK Gram-positive signal peptide / LPXTG cell wall anchor motif / Gram-positive cocci surface proteins LPxTG motif profile. ...IgG-binding B / B domain / M protein-type anchor domain / GA-like domain / GA-like domain / Ubiquitin-like (UB roll) - #10 / Immunoglobulin/albumin-binding domain superfamily / YSIRK Gram-positive signal peptide / LPXTG cell wall anchor motif / Gram-positive cocci surface proteins LPxTG motif profile. / LPXTG cell wall anchor domain / Ubiquitin-like (UB roll) / Immunoglobulins / Roll / Immunoglobulin-like / Sandwich / Mainly Beta / Alpha Beta
Similarity search - Domain/homology
Immunoglobulin G-binding protein G
Similarity search - Component
Biological speciesStreptococcus sp. 'group G' (bacteria)
Homo sapiens (human)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.61 Å
AuthorsSlezak, T. / Filippova, E.V. / Davydova, E.K. / Kossiakoff, A.A.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of Dental and Craniofacial Research (NIH/NIDCR)GM117372 United States
CitationJournal: Protein Sci. / Year: 2020
Title: An engineered ultra-high affinity Fab-Protein G pair enables a modular antibody platform with multifunctional capability.
Authors: Slezak, T. / Bailey, L.J. / Jaskolowski, M. / Nahotko, D.A. / Filippova, E.V. / Davydova, E.K. / Kossiakoff, A.A.
History
DepositionSep 4, 2019Deposition site: RCSB / Processing site: RCSB
Revision 1.0Oct 23, 2019Provider: repository / Type: Initial release
Revision 1.1Oct 30, 2019Group: Data collection / Database references / Category: citation / Item: _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.2Dec 11, 2019Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.3Jan 1, 2020Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.year
Revision 1.4Oct 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

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Protein G
B: Protein G
C: Antibody light chain Fab
D: Antibody heavy chain Fab
L: Antibody light chain Fab
H: Antibody heavy chain Fab


Theoretical massNumber of molelcules
Total (without water)112,0446
Polymers112,0446
Non-polymers00
Water2,288127
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)75.130, 75.130, 340.050
Angle α, β, γ (deg.)90.000, 90.000, 120.000
Int Tables number154
Space group name H-MP3221
Components on special symmetry positions
IDModelComponents
11L-330-

HOH

21H-322-

HOH

Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
11chain A
21(chain B and resid 5 through 61)
12(chain C and (resid 8 through 24 or resid 26...
22(chain L and (resid 8 through 24 or resid 26 through 56 or resid 58 through 214))
13(chain D and (resid 4 through 102 or resid 116 through 231))
23(chain H and (resid 4 through 102 or resid 116 through 144 or resid 151 through 231))

NCS domain segments:
Dom-IDComponent-IDEns-IDBeg auth comp-IDBeg label comp-IDEnd auth comp-IDEnd label comp-IDSelection detailsAuth asym-IDLabel asym-IDAuth seq-IDLabel seq-ID
111VALVALGLUGLUchain AAA5 - 614 - 60
211VALVALGLUGLU(chain B and resid 5 through 61)BB5 - 614 - 60
112SERSERCYSCYS(chain C and (resid 8 through 24 or resid 26...CC8 - 248 - 24
122ALAALATYRTYR(chain C and (resid 8 through 24 or resid 26...CC26 - 5626 - 56
132GLYGLYGLYGLY(chain C and (resid 8 through 24 or resid 26...CC58 - 6558 - 65
142GLYGLYCYSCYS(chain C and (resid 8 through 24 or resid 26...CC69 - 21469 - 214
212SERSERCYSCYS(chain L and (resid 8 through 24 or resid 26 through 56 or resid 58 through 214))LE8 - 248 - 24
222ALAALATYRTYR(chain L and (resid 8 through 24 or resid 26 through 56 or resid 58 through 214))LE26 - 5626 - 56
232GLYGLYCYSCYS(chain L and (resid 8 through 24 or resid 26 through 56 or resid 58 through 214))LE58 - 21458 - 214
113GLUGLUGLUGLU(chain D and (resid 4 through 102 or resid 116 through 231))DD4 - 1024 - 102
123GLYGLYLYSLYS(chain D and (resid 4 through 102 or resid 116 through 231))DD116 - 231116 - 231
213GLUGLUGLUGLU(chain H and (resid 4 through 102 or resid 116 through 144 or resid 151 through 231))HF4 - 1024 - 102
223GLYGLYSERSER(chain H and (resid 4 through 102 or resid 116 through 144 or resid 151 through 231))HF116 - 144116 - 144
233GLYGLYLYSLYS(chain H and (resid 4 through 102 or resid 116 through 144 or resid 151 through 231))HF151 - 231151 - 231

NCS ensembles :
ID
1
2
3

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Components

#1: Protein Protein G /


Mass: 7359.124 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Streptococcus sp. 'group G' (bacteria) / Gene: spg / Plasmid: pHFT2 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P19909*PLUS
#2: Antibody Antibody light chain Fab


Mass: 23180.799 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Plasmid: pSFV4 / Production host: Escherichia coli BL21 (bacteria)
#3: Antibody Antibody heavy chain Fab


Mass: 25482.318 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Plasmid: pSFV4 / Production host: Escherichia coli BL21 (bacteria)
#4: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 127 / 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: 2.47 Å3/Da / Density % sol: 50.2 %
Crystal growTemperature: 293 K / Method: vapor diffusion, hanging drop / pH: 5
Details: 0.1 M Magnesium Chloride, 0.1 M Sodium Acetate, 15% PEG4000

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: APS / Beamline: 23-ID-D / Wavelength: 1.03322 Å
DetectorType: DECTRIS PILATUS3 6M / Detector: PIXEL / Date: Nov 3, 2018 / Details: 3.0 undulator
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.03322 Å / Relative weight: 1
Reflection twinOperator: -h,-k,l / Fraction: 0.5
ReflectionResolution: 2.61→48.58 Å / Num. obs: 33384 / % possible obs: 95 % / Redundancy: 7.6 % / CC1/2: 0.99 / Rpim(I) all: 0.089 / Net I/σ(I): 8.5
Reflection shellResolution: 2.61→2.73 Å / Redundancy: 6.4 % / Num. unique obs: 3910 / Rpim(I) all: 0.72 / % possible all: 93.5

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Processing

Software
NameVersionClassification
PHENIX1.16_3549refinement
PDB_EXTRACT3.25data extraction
XDSdata reduction
Aimlessdata scaling
BALBESphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 2HFF,1IGD

2hff

1igd


Resolution: 2.61→47.014 Å / Cross valid method: THROUGHOUT
RfactorNum. reflection% reflection
Rfree0.2501 1877 5.64 %
Rwork0.1917 --
obs0.2018 33295 95 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å
Displacement parametersBiso max: 281.39 Å2 / Biso mean: 49.8039 Å2 / Biso min: 20.06 Å2
Refinement stepCycle: final / Resolution: 2.61→47.014 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms7147 0 0 131 7278
Biso mean---35.05 -
Num. residues----944
Refine LS restraints NCS
Ens-IDDom-IDAuth asym-IDNumberRefine-IDRmsType
11A338X-RAY DIFFRACTION5.267TORSIONAL
12B338X-RAY DIFFRACTION5.267TORSIONAL
21C1170X-RAY DIFFRACTION5.267TORSIONAL
22L1170X-RAY DIFFRACTION5.267TORSIONAL
31D1242X-RAY DIFFRACTION5.267TORSIONAL
32H1242X-RAY DIFFRACTION5.267TORSIONAL
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Rfactor Rfree error: 0

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection Rwork% reflection obs (%)
2.6111-2.68170.36071260.3028234188
2.6817-2.76050.32081210.285231589
2.7605-2.84950.34651250.2767226085
2.8495-2.95130.31691110.2463235289
2.9513-3.06930.34591440.2314234489
3.0693-3.20880.30771480.2054236388
3.2088-3.37770.2811610.197236889
3.3777-3.58890.24731320.1848242991
3.5889-3.86540.24041470.1906241090
3.8654-4.25310.24511570.1745242190
4.2531-4.86580.2121510.1509248791
4.8658-6.11970.20871290.1651256392
6.1197-27.92380.25821680.2263275594
Refinement TLS params.Method: refined / Origin x: 4.3916 Å / Origin y: 7.9103 Å / Origin z: -29.6957 Å
111213212223313233
T0.2164 Å2-0.0073 Å20.0122 Å2-0.2781 Å2-0.0242 Å2--0.2738 Å2
L0.5822 °20.2865 °20.0696 °2-0.1464 °20.0206 °2--0.2493 °2
S-0.0347 Å °0.0119 Å °-0.0758 Å °0.023 Å °0.0354 Å °-0.0426 Å °-0.0238 Å °0.0382 Å °0.0008 Å °
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection detailsAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1allA5 - 61
2X-RAY DIFFRACTION1allB4 - 61
3X-RAY DIFFRACTION1allC8 - 214
4X-RAY DIFFRACTION1allD4 - 231
5X-RAY DIFFRACTION1allL6 - 214
6X-RAY DIFFRACTION1allH4 - 231
7X-RAY DIFFRACTION1allW1 - 131

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