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- PDB-6w9g: Crystal Structure of the Fab fragment of humanized 5c8 antibody c... -

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Basic information

Entry
Database: PDB / ID: 6w9g
TitleCrystal Structure of the Fab fragment of humanized 5c8 antibody containing the fluorescent non-canonical amino acid L-(7-hydroxycoumarin-4-yl)ethylglycine in complex with CD40L at pH 6.8
Components
  • 5c8* Fab (heavy chain)
  • 5c8* Fab (light chain)
  • CD40 ligand
KeywordsIMMUNE SYSTEM / Immunoglobulin / Cytokine / L-(7-hydroxycoumarin-4-yl)ethylglycine
Function / homology
Function and homology information


CD40 receptor binding / CD40 signaling pathway / TNF receptor superfamily (TNFSF) members mediating non-canonical NF-kB pathway / positive regulation of endothelial cell apoptotic process / isotype switching / tumor necrosis factor receptor binding / regulation of immunoglobulin production / leukocyte cell-cell adhesion / positive regulation of interleukin-4 production / B cell proliferation ...CD40 receptor binding / CD40 signaling pathway / TNF receptor superfamily (TNFSF) members mediating non-canonical NF-kB pathway / positive regulation of endothelial cell apoptotic process / isotype switching / tumor necrosis factor receptor binding / regulation of immunoglobulin production / leukocyte cell-cell adhesion / positive regulation of interleukin-4 production / B cell proliferation / positive regulation of interleukin-10 production / positive regulation of T cell proliferation / T cell costimulation / positive regulation of interleukin-12 production / protein serine/threonine kinase activator activity / B cell differentiation / cytokine activity / TNFR2 non-canonical NF-kB pathway / integrin-mediated signaling pathway / platelet activation / Immunoregulatory interactions between a Lymphoid and a non-Lymphoid cell / integrin binding / positive regulation of NF-kappaB transcription factor activity / inflammatory response / external side of plasma membrane / negative regulation of apoptotic process / Golgi apparatus / cell surface / extracellular space / membrane / plasma membrane
Similarity search - Function
CD40 ligand / Tumour necrosis factor, conserved site / Tumor necrosis factor (TNF) homology domain (THD) signature. / Tumour necrosis factor family. / TNF(Tumour Necrosis Factor) family / Tumour necrosis factor domain / Tumor necrosis factor (TNF) homology domain (THD) profile. / Tumour necrosis factor-like domain superfamily
Similarity search - Domain/homology
Chem-ETE / DI(HYDROXYETHYL)ETHER / trimethylamine oxide / CD40 ligand
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / molecular replacement / Resolution: 1.82 Å
AuthorsHenderson, J.N. / Simmons, C.R. / Mills, J.H.
CitationJournal: Biochemistry / Year: 2020
Title: Structural Insights into How Protein Environments Tune the Spectroscopic Properties of a Noncanonical Amino Acid Fluorophore.
Authors: Henderson, J.N. / Simmons, C.R. / Fahmi, N.E. / Jeffs, J.W. / Borges, C.R. / Mills, J.H.
History
DepositionMar 23, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Dec 23, 2020Provider: repository / Type: Initial release
Revision 1.1Oct 18, 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
Revision 1.2Nov 15, 2023Group: Data collection / Derived calculations / Category: chem_comp_atom / chem_comp_bond / struct_conn
Item: _chem_comp_atom.atom_id / _chem_comp_bond.atom_id_1 ..._chem_comp_atom.atom_id / _chem_comp_bond.atom_id_1 / _chem_comp_bond.atom_id_2 / _struct_conn.pdbx_leaving_atom_flag

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: CD40 ligand
B: CD40 ligand
C: CD40 ligand
H: 5c8* Fab (heavy chain)
L: 5c8* Fab (light chain)
K: 5c8* Fab (heavy chain)
M: 5c8* Fab (light chain)
X: 5c8* Fab (heavy chain)
Y: 5c8* Fab (light chain)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)198,46847
Polymers191,7169
Non-polymers6,75238
Water22,3031238
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)226.677, 131.200, 97.560
Angle α, β, γ (deg.)90.000, 108.820, 90.000
Int Tables number5
Space group name H-MC121
Components on special symmetry positions
IDModelComponents
11L-560-

HOH

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Components

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Antibody , 2 types, 6 molecules HKXLMY

#2: Antibody 5c8* Fab (heavy chain)


Mass: 24078.885 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Escherichia coli (E. coli)
#3: Antibody 5c8* Fab (light chain)


Mass: 24013.574 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Escherichia coli (E. coli)

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Protein / Sugars , 2 types, 6 molecules ABC

#1: Protein CD40 ligand / CD40-L / T-cell antigen Gp39 / TNF-related activation protein / TRAP / Tumor necrosis factor ligand ...CD40-L / T-cell antigen Gp39 / TNF-related activation protein / TRAP / Tumor necrosis factor ligand superfamily member 5


Mass: 15812.817 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: CD40LG, CD40L, TNFSF5, TRAP / Production host: Komagataella phaffii GS115 (fungus) / References: UniProt: P29965
#4: Polysaccharide alpha-D-mannopyranose-(1-2)-alpha-D-mannopyranose-(1-6)-alpha-D-mannopyranose-(1-6)-beta-D- ...alpha-D-mannopyranose-(1-2)-alpha-D-mannopyranose-(1-6)-alpha-D-mannopyranose-(1-6)-beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 1072.964 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DManpa1-2DManpa1-6DManpa1-6DManpb1-4DGlcpNAcb1-4DGlcpNAcb1-Glycam Condensed SequenceGMML 1.0
WURCS=2.0/3,6,5/[a2122h-1b_1-5_2*NCC/3=O][a1122h-1b_1-5][a1122h-1a_1-5]/1-1-2-3-3-3/a4-b1_b4-c1_c6-d1_d6-e1_e2-f1WURCSPDB2Glycan 1.1.0
[]{[(4+1)][b-D-GlcpNAc]{}}LINUCSPDB-CARE

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Non-polymers , 6 types, 1273 molecules

#5: Chemical
ChemComp-PEG / DI(HYDROXYETHYL)ETHER


Mass: 106.120 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: C4H10O3
#6: Chemical ChemComp-TRS / 2-AMINO-2-HYDROXYMETHYL-PROPANE-1,3-DIOL / TRIS BUFFER


Mass: 122.143 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C4H12NO3 / Comment: pH buffer*YM
#7: Chemical...
ChemComp-TMO / trimethylamine oxide


Mass: 75.110 Da / Num. of mol.: 21 / Source method: obtained synthetically / Formula: C3H9NO
#8: Chemical ChemComp-ETE / 2-{2-[2-2-(METHOXY-ETHOXY)-ETHOXY]-ETHOXY}-ETHANOL


Mass: 208.252 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C9H20O5
#9: Chemical ChemComp-1PE / PENTAETHYLENE GLYCOL / PEG400


Mass: 238.278 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H22O6 / Comment: precipitant*YM
#10: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 1238 / Source method: isolated from a natural source / Formula: H2O

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Details

Has ligand of interestY

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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: 3.58 Å3/Da / Density % sol: 65.65 %
Crystal growTemperature: 295 K / Method: vapor diffusion, sitting drop
Details: 20% PEG MME 2000, 0.1 M Trimethylamine N-oxide, 0.1 M Tris-HCl pH 7.5

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: SSRL / Beamline: BL9-2 / Wavelength: 0.97946 Å
DetectorType: DECTRIS PILATUS3 6M / Detector: PIXEL / Date: Dec 5, 2018
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.97946 Å / Relative weight: 1
ReflectionResolution: 1.82→92.34 Å / Num. obs: 233196 / % possible obs: 97.2 % / Redundancy: 4 % / CC1/2: 0.997 / Rmerge(I) obs: 0.095 / Rpim(I) all: 0.054 / Rrim(I) all: 0.11 / Net I/σ(I): 9.4 / Num. measured all: 940304 / Scaling rejects: 84
Reflection shell

Diffraction-ID: 1

Resolution (Å)Redundancy (%)Rmerge(I) obsNum. measured allNum. unique obsCC1/2Rpim(I) allRrim(I) allNet I/σ(I) obs% possible all
1.82-1.854.10.82147655116080.3280.4620.9461.698
9.98-92.344.50.023685015250.9930.0130.02733.999.8

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Phasing

PhasingMethod: molecular replacement
Phasing MRModel details: Phaser MODE: MR_AUTO
Highest resolutionLowest resolution
Rotation8.73 Å92.34 Å
Translation8.73 Å92.34 Å

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Processing

Software
NameVersionClassification
Aimless0.5.21data scaling
PHASER2.6.1phasing
REFMAC5.8.0135refinement
PDB_EXTRACT3.25data extraction
MOSFLMdata reduction
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 1i9r

1i9r


Resolution: 1.82→48.47 Å / Cor.coef. Fo:Fc: 0.96 / Cor.coef. Fo:Fc free: 0.948 / SU B: 4.967 / SU ML: 0.076 / SU R Cruickshank DPI: 0.1012 / Cross valid method: THROUGHOUT / σ(F): 0 / ESU R: 0.101 / ESU R Free: 0.1
Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS U VALUES : WITH TLS ADDED
RfactorNum. reflection% reflectionSelection details
Rfree0.2067 11697 5 %RANDOM
Rwork0.1789 ---
obs0.1804 221486 97.06 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å
Displacement parametersBiso max: 92.46 Å2 / Biso mean: 32.814 Å2 / Biso min: 10.3 Å2
Baniso -1Baniso -2Baniso -3
1--0.92 Å20 Å20.35 Å2
2---0.47 Å20 Å2
3---0.93 Å2
Refinement stepCycle: final / Resolution: 1.82→48.47 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms12782 0 488 1238 14508
Biso mean--56.03 35.65 -
Num. residues----1696
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0150.0213915
X-RAY DIFFRACTIONr_bond_other_d0.0020.0212751
X-RAY DIFFRACTIONr_angle_refined_deg1.8931.98719012
X-RAY DIFFRACTIONr_angle_other_deg0.989329530
X-RAY DIFFRACTIONr_dihedral_angle_1_deg8.06951775
X-RAY DIFFRACTIONr_dihedral_angle_2_deg38.32624.634533
X-RAY DIFFRACTIONr_dihedral_angle_3_deg14.512152088
X-RAY DIFFRACTIONr_dihedral_angle_4_deg20.9661547
X-RAY DIFFRACTIONr_chiral_restr0.1130.22201
X-RAY DIFFRACTIONr_gen_planes_refined0.0040.02115480
X-RAY DIFFRACTIONr_gen_planes_other0.0010.023039
LS refinement shellResolution: 1.822→1.869 Å / Rfactor Rfree error: 0 / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.303 874 -
Rwork0.298 16478 -
all-17352 -
obs--97.94 %
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)
12.1274-0.59750.56241.5765-1.10973.12610.0327-0.13390.14770.13890.0504-0.0137-0.3395-0.0301-0.0830.0439-0.00080.00410.0191-0.01910.1115-28.7691-55.986912.5883
23.1363-1.2257-2.0561.43862.91996.02890.27810.15050.5201-0.2643-0.0657-0.1729-0.4961-0.1023-0.21240.27890.0170.04840.20190.01370.5023-33.1385-37.488321.3609
32.08060.5121-1.46231.4415-0.2253.13340.0472-0.1406-0.050.04990.0168-0.10450.13190.2395-0.0640.01740.0063-0.02920.02230.00960.1318-34.6292-77.184112.843
49.1515.01856.19863.49292.7844.71840.37991.4286-1.4390.20270.2838-1.11140.22991.3727-0.66370.12280.132-0.10650.6187-0.03540.588-16.3601-82.512221.2673
51.083-0.0280.66872.40981.192.77980.0631-0.1084-0.08920.14720.03410.17650.1399-0.303-0.09720.0144-0.00780.01420.04730.03280.1291-50.1981-61.566212.8557
65.6258.189-3.513.4899-3.51563.7696-0.33040.23440.3425-0.84060.12830.9061-0.1522-0.36240.20210.18360.01750.02140.20210.04960.4371-63.7745-74.727121.3976
74.8203-0.7688-0.6581.3388-0.57522.6389-0.0678-0.7886-0.16590.15880.00220.01210.13060.40560.06560.04610.0194-0.01640.3790.03910.0588-6.5942-68.133131.8946
86.39410.692-1.82671.193-0.88231.9105-0.0625-0.8056-0.18580.28330.2303-0.1851-0.1488-0.1539-0.16780.24310.1841-0.02220.6750.10930.243426.2984-79.595837.8317
94.0619-0.1217-0.21990.81350.00981.0575-0.0205-0.31820.08710.02180.0457-0.0281-0.00260.0821-0.02520.0052-0.0015-0.0170.0991-0.00470.06084.4405-64.106912.519
101.706-0.59380.48743.4172-2.24124.5971-0.0985-0.6395-0.67390.02210.23880.01280.7345-0.1852-0.14020.19240.0640.00810.40540.22710.390827.2262-89.085224.6319
112.84981.8097-0.50783.0540.21992.10040.1716-0.19840.21770.4252-0.22520.2530.1379-0.19520.05360.1331-0.09170.02950.0932-0.0410.0831-56.6152-90.378531.8215
122.20.57940.33864.72352.17393.20320.2384-0.3893-0.00290.3417-0.14610.20950.2521-0.0002-0.09240.1953-0.16520.08450.34-0.08710.1218-83.4947-112.515937.9263
131.60551.2438-0.10092.84670.14471.1860.0669-0.0353-0.04640.0909-0.1013-0.06320.0434-0.04070.03440.0264-0.0247-0.01120.02620.00070.0629-58.2196-102.082712.4423
143.787-0.4435-1.7811.88321.44893.74590.157-0.24180.3560.0779-0.21480.2941-0.2693-0.53250.05770.1261-0.05050.04560.26-0.10350.2102-91.6083-108.939623.8224
150.8801-0.84780.40524.2548-0.16312.016-0.2755-0.17490.06750.5680.2222-0.0959-0.2840.0620.05330.24460.124-0.00980.1123-0.00040.0624-49.9801-36.005931.9711
163.6014-2.3552.15194.4994-1.93382.5821-0.1918-0.44570.20690.92960.3172-0.0145-0.0789-0.2333-0.12530.54880.0677-0.08940.251-0.05810.2153-55.2098-1.718137.9479
171.6191-1.118-0.02363.1695-0.01421.1026-0.0828-0.0541-0.02980.14360.0740.1462-0.0616-0.03120.00880.05140.0371-0.0030.03120.01240.0767-59.4608-28.631312.6266
182.67571.06791.81373.50271.05423.6068-0.0867-0.08160.21620.46450.076-0.5358-0.23320.46010.01070.25920.0161-0.15090.1572-0.04270.2972-48.59343.649723.8001
Refinement TLS group
IDRefine-IDRefine TLS-IDAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1A119 - 261
2X-RAY DIFFRACTION2A301 - 306
3X-RAY DIFFRACTION3B119 - 261
4X-RAY DIFFRACTION4B301 - 306
5X-RAY DIFFRACTION5C119 - 261
6X-RAY DIFFRACTION6C301 - 306
7X-RAY DIFFRACTION7H1 - 117
8X-RAY DIFFRACTION8H118 - 218
9X-RAY DIFFRACTION9L1 - 114
10X-RAY DIFFRACTION10L115 - 215
11X-RAY DIFFRACTION11K1 - 117
12X-RAY DIFFRACTION12K118 - 219
13X-RAY DIFFRACTION13M1 - 114
14X-RAY DIFFRACTION14M115 - 216
15X-RAY DIFFRACTION15X1 - 117
16X-RAY DIFFRACTION16X118 - 218
17X-RAY DIFFRACTION17Y1 - 114
18X-RAY DIFFRACTION18Y115 - 216

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