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- PDB-7sgm: Crystal structure of a Fab variant containing a fluorescent nonca... -

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Basic information

Entry
Database: PDB / ID: 7sgm
TitleCrystal structure of a Fab variant containing a fluorescent noncanonical amino acid with blocked excited state proton transfer and in complex with its antigen, CD40L
Components
  • 5c8* Fab heavy chain
  • 5c8* Fab light chain
  • CD40 ligand
KeywordsCYTOKINE/IMMUNE SYSTEM / Fragment antigen binding (Fab) / noncanonical amino acid / 7-hydroxycoumarin / IMMUNE SYSTEM / CYTOKINE-IMMUNE SYSTEM complex
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
DI(HYDROXYETHYL)ETHER / trimethylamine oxide / CD40 ligand
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2 Å
AuthorsHenderson, J.N. / Mills, J.H. / Simmons, C.R.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01 GM136996 United States
CitationJournal: J.Mol.Biol. / Year: 2022
Title: Structural Basis for Blocked Excited State Proton Transfer in a Fluorescent, Photoacidic Non-Canonical Amino Acid-Containing Antibody Fragment.
Authors: Henderson, J.N. / Simmons, C.R. / Mills, J.H.
History
DepositionOct 6, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Feb 2, 2022Provider: repository / Type: Initial release
Revision 1.1Apr 27, 2022Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.title / _citation_author.name
Revision 1.2Oct 18, 2023Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond / pdbx_initial_refinement_model
Revision 1.3Nov 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)193,88729
Polymers191,4979
Non-polymers2,39120
Water22,9871276
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)226.847, 131.252, 97.296
Angle α, β, γ (deg.)90.000, 109.100, 90.000
Int Tables number5
Space group name H-MC121
Components on special symmetry positions
IDModelComponents
11M-567-

HOH

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Components

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Antibody , 2 types, 6 molecules HKXLMY

#2: Antibody 5c8* Fab heavy chain


Mass: 24005.830 Da / Num. of mol.: 3 / Mutation: W47L
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: Pichia (fungus) / References: UniProt: P29965
#4: Sugar ChemComp-NAG / 2-acetamido-2-deoxy-beta-D-glucopyranose / N-acetyl-beta-D-glucosamine / 2-acetamido-2-deoxy-beta-D-glucose / 2-acetamido-2-deoxy-D-glucose / 2-acetamido-2-deoxy-glucose / N-ACETYL-D-GLUCOSAMINE


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Formula: C8H15NO6
IdentifierTypeProgram
DGlcpNAcbCONDENSED IUPAC CARBOHYDRATE SYMBOLGMML 1.0
N-acetyl-b-D-glucopyranosamineCOMMON NAMEGMML 1.0
b-D-GlcpNAcIUPAC CARBOHYDRATE SYMBOLPDB-CARE 1.0
GlcNAcSNFG CARBOHYDRATE SYMBOLGMML 1.0

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Non-polymers , 4 types, 1293 molecules

#5: Chemical ChemComp-TRS / 2-AMINO-2-HYDROXYMETHYL-PROPANE-1,3-DIOL / TRIS BUFFER


Mass: 122.143 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C4H12NO3 / Comment: pH buffer*YM
#6: Chemical
ChemComp-PEG / DI(HYDROXYETHYL)ETHER


Mass: 106.120 Da / Num. of mol.: 13 / Source method: obtained synthetically / Formula: C4H10O3
#7: Chemical ChemComp-TMO / trimethylamine oxide


Mass: 75.110 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C3H9NO
#8: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 1276 / 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: 65.6 %
Crystal growTemperature: 295 K / Method: vapor diffusion, sitting drop
Details: 16-24% PEG MME 2000, 0.1 M Trimethylamine N-oxide, 0.1 M Tris-HCl pH 7.5-9.0

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: ALS / Beamline: 8.2.2 / Wavelength: 0.98 Å
DetectorType: ADSC QUANTUM 315r / Detector: CCD / Date: May 17, 2017
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.98 Å / Relative weight: 1
ReflectionResolution: 2→50 Å / Num. obs: 175991 / % possible obs: 97.8 % / Redundancy: 3.5 % / Rmerge(I) obs: 0.171 / Rpim(I) all: 0.104 / Rrim(I) all: 0.2 / Χ2: 1.362 / Net I/σ(I): 5.9 / Num. measured all: 611818
Reflection shell

Diffraction-ID: 1

Resolution (Å)Redundancy (%)Rmerge(I) obsNum. unique obsCC1/2Rpim(I) allRrim(I) allΧ2% possible all
2-2.033.30.55187680.7020.3530.6560.71197.4
2.03-2.073.30.50187370.7180.320.5960.77597
2.07-2.113.30.43386550.7860.2760.5150.78796.6
2.11-2.153.30.39786580.8250.2530.4720.80296.7
2.15-2.23.30.36986680.840.2340.4380.84196.5
2.2-2.253.30.33985580.8670.2140.4020.89695.5
2.25-2.313.40.32686040.8680.2060.3870.92395.9
2.31-2.373.40.29885750.8930.1870.3520.90995.6
2.37-2.443.40.27786110.9080.1730.3280.93796
2.44-2.523.40.25986500.9120.1610.3051.04896.2
2.52-2.613.40.23887320.9290.1470.281.08797
2.61-2.713.50.21287550.910.1310.251.23997.7
2.71-2.843.50.18988800.9520.1160.2221.35398.5
2.84-2.993.60.17288840.9560.1050.2021.61599.1
2.99-3.173.60.15589630.9590.0940.1822.0399.5
3.17-3.423.70.14689900.9610.0880.172.55199.8
3.42-3.763.70.14290360.9590.0860.1672.338100
3.76-4.313.70.13390130.9660.080.1552.168100
4.31-5.433.70.12390820.9680.0750.1441.835100
5.43-503.80.11891720.9730.070.1371.519100

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Processing

Software
NameVersionClassification
REFMAC5.8.0267refinement
HKL-2000data scaling
PDB_EXTRACT3.27data extraction
HKL-2000data reduction
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 6w9g

6w9g


Resolution: 2→48.42 Å / Cor.coef. Fo:Fc: 0.942 / Cor.coef. Fo:Fc free: 0.92 / SU B: 7.474 / SU ML: 0.108 / Cross valid method: THROUGHOUT / σ(F): 0 / ESU R: 0.143 / ESU R Free: 0.137 / Stereochemistry target values: MAXIMUM LIKELIHOOD
Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS U VALUES : WITH TLS ADDED
RfactorNum. reflection% reflectionSelection details
Rfree0.2281 8919 5.1 %RANDOM
Rwork0.1927 ---
obs0.1945 167072 97.61 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK
Displacement parametersBiso max: 86.53 Å2 / Biso mean: 23.84 Å2 / Biso min: 0.5 Å2
Baniso -1Baniso -2Baniso -3
1--0.09 Å20 Å20.04 Å2
2--0.02 Å20 Å2
3---0.03 Å2
Refinement stepCycle: final / Resolution: 2→48.42 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms12733 0 151 1276 14160
Biso mean--51.56 29.53 -
Num. residues----1693
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0130.01313406
X-RAY DIFFRACTIONr_bond_other_d0.0010.01712207
X-RAY DIFFRACTIONr_angle_refined_deg1.8691.65318269
X-RAY DIFFRACTIONr_angle_other_deg1.3781.57628255
X-RAY DIFFRACTIONr_dihedral_angle_1_deg8.16551741
X-RAY DIFFRACTIONr_dihedral_angle_2_deg35.5523.528564
X-RAY DIFFRACTIONr_dihedral_angle_3_deg15.497152052
X-RAY DIFFRACTIONr_dihedral_angle_4_deg23.4221544
X-RAY DIFFRACTIONr_chiral_restr0.0830.21790
X-RAY DIFFRACTIONr_gen_planes_refined0.0030.0215334
X-RAY DIFFRACTIONr_gen_planes_other00.022968
LS refinement shellResolution: 2.004→2.056 Å / Rfactor Rfree error: 0 / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.278 671 -
Rwork0.25 12034 -
all-12705 -
obs--95.43 %
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)
10.6563-0.01270.23321.5650.5581.89120.009-0.03-0.01480.04340.03040.10560.1099-0.1024-0.03930.01530.010.02240.09470.02310.086-50.0335.81612.57
21.308-0.45230.38690.9467-0.45492.2780.0233-0.06910.07250.0190.0605-0.0541-0.1623-0.0803-0.08380.0202-0.00720.030.0956-0.00140.0941-28.80641.49212.888
31.07850.3302-0.41560.7889-0.12881.93590.0445-0.0463-0.04260.03040.0088-0.0680.02810.1349-0.05330.0078-0.00230.01130.0687-0.0050.0713-34.61420.22512.53
40.8784-0.32550.57552.6444-0.35921.6914-0.1838-0.11120.00460.3610.1534-0.2228-0.303-0.00330.03040.14770.0622-0.01080.128-0.01330.0282-50.23261.52631.959
53.3744-2.51781.79054.5462-2.47671.6396-0.2047-0.24340.15830.91270.2678-0.03-0.1878-0.1167-0.06310.60180.0475-0.09650.1265-0.05720.0742-55.6596.04437.987
61.1322-1.15830.04882.4005-0.14830.801-0.0633-0.0452-0.03680.16480.05620.0656-0.08660.0050.00710.04250.01370.02370.0934-0.00040.061-59.55468.32112.687
71.77410.88470.92453.79890.51582.0855-0.06970.03960.10170.59590.0497-0.5399-0.10430.1590.020.2030.0207-0.14040.0219-0.00940.1397-49.718100.64322.463
83.0321-1.0756-0.44371.0863-0.50661.6629-0.0035-0.4684-0.13150.0780.01480.16320.04430.1908-0.01130.0254-0.01820.01360.27220.02250.0607-6.5129.37231.989
93.39560.3365-0.78861.3531-0.54281.71760.025-0.2844-0.14130.14230.0182-0.0798-0.117-0.1079-0.04320.06960.06840.00630.2550.03520.018326.59117.74137.846
102.82510.1783-0.00710.645-0.01290.60450.0077-0.15320.03010.03020.00730.02090.02120.0412-0.0150.00350.00090.00980.1201-0.00020.06973.97333.66312.612
111.2797-0.34830.23282.55-1.43542.4273-0.0623-0.188-0.1744-0.05890.066-0.020.3249-0.0967-0.00360.07520.02330.04020.22670.04490.090227.3058.48924.356
122.13190.558-0.31450.82410.42661.50930.1031-0.16380.16170.1698-0.09860.07240.0889-0.1204-0.00450.08-0.06030.03290.1119-0.01040.031-56.4666.8531.975
131.31620.60290.65712.93131.79332.27530.1449-0.2326-0.03670.2877-0.13240.1560.1140.0399-0.01250.156-0.1020.07240.292-0.02060.0479-83.326-15.82737.964
141.0090.8759-0.03722.4430.02550.55640.047-0.0436-0.03690.0798-0.0467-0.05670.0434-0.0572-0.00020.0326-0.01970.01290.09870.00140.0534-57.718-4.34312.583
153.1298-0.2706-0.68591.40010.45182.18350.0909-0.23620.22710.0869-0.11280.1966-0.1245-0.24860.02180.0586-0.0520.04750.1454-0.06380.0865-91.348-11.79124.244
Refinement TLS group
IDRefine-IDRefine TLS-IDAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1A119 - 261
2X-RAY DIFFRACTION2B119 - 261
3X-RAY DIFFRACTION3C119 - 261
4X-RAY DIFFRACTION4H1 - 117
5X-RAY DIFFRACTION5H118 - 219
6X-RAY DIFFRACTION6L1 - 112
7X-RAY DIFFRACTION7L113 - 216
8X-RAY DIFFRACTION8K1 - 117
9X-RAY DIFFRACTION9K118 - 218
10X-RAY DIFFRACTION10M1 - 112
11X-RAY DIFFRACTION11M113 - 217
12X-RAY DIFFRACTION12X1 - 117
13X-RAY DIFFRACTION13X118 - 218
14X-RAY DIFFRACTION14Y1 - 112
15X-RAY DIFFRACTION15Y113 - 216

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