6W9G

Crystal 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

X-RAY DIFFRACTION

Crystallization

Crystalization Experiments
ID	Method	pH	Temperature	Details
1	VAPOR DIFFUSION, SITTING DROP		295	20% PEG MME 2000, 0.1 M Trimethylamine N-oxide, 0.1 M Tris-HCl pH 7.5

Crystal Properties
Matthews coefficient	Solvent content
3.58	65.65

Crystal Data

Unit Cell
Length ( Å )	Angle ( ˚ )
a = 226.677	α = 90
b = 131.2	β = 108.82
c = 97.56	γ = 90

Symmetry
Space Group	C 1 2 1

Diffraction

Diffraction Experiment
ID #	Crystal ID	Scattering Type	Data Collection Temperature	Detector	Detector Type	Details	Collection Date	Monochromator	Protocol
1	1	x-ray	100	PIXEL	DECTRIS PILATUS3 6M		2018-12-05	M	SINGLE WAVELENGTH

Radiation Source
ID #	Source	Type	Wavelength List	Synchrotron Site	Beamline
1	SYNCHROTRON	SSRL BEAMLINE BL9-2	0.97946	SSRL	BL9-2

Data Collection

Overall
ID #	Resolution (High)	Resolution (Low)	Percent Possible (Observed)	R Merge I (Observed)	Rrim I (All)	Rpim I (All)	CC (Half)	Net I Over Average Sigma (I)	Redundancy	Number Reflections (All)	Number Reflections (Observed)	Observed Criterion Sigma (F)	Observed Criterion Sigma (I)	B (Isotropic) From Wilson Plot
1	1.82	92.34	97.2	0.095	0.11	0.054	0.997	9.4	4		233196

Highest Resolution Shell
ID #	Resolution (High)	Resolution (Low)	Percent Possible (All)	Percent Possible (Observed)	R Merge I (Observed)	Rrim I (All)	Rpim I (All)	CC (Half)	Mean I Over Sigma (Observed)	Redundancy	Number Unique Reflections (All)
1	1.82	1.85	98		0.821	0.946	0.462	0.328		4.1

Refinement

Statistics
Diffraction ID	Structure Solution Method	Cross Validation method	Starting model	Resolution (High)	Resolution (Low)	Number Reflections (Observed)	Number Reflections (R-Free)	Percent Reflections (Observed)	R-Factor (Observed)	R-Work	R-Free	R-Free Selection Details	Mean Isotropic B
X-RAY DIFFRACTION	MOLECULAR REPLACEMENT	THROUGHOUT	1i9r	1.82	48.47	221486	11697	97.06	0.1804	0.1789	0.2067	RANDOM	32.814

Temperature Factor Modeling
Anisotropic B[1][1]	Anisotropic B[1][2]	Anisotropic B[1][3]	Anisotropic B[2][2]	Anisotropic B[2][3]	Anisotropic B[3][3]
-0.92		0.35	-0.47		0.93

RMS Deviations
Key	Refinement Restraint Deviation
r_dihedral_angle_2_deg	38.326
r_dihedral_angle_4_deg	20.966
r_dihedral_angle_3_deg	14.512
r_dihedral_angle_1_deg	8.069
r_angle_refined_deg	1.893
r_angle_other_deg	0.989
r_chiral_restr	0.113
r_bond_refined_d	0.015
r_gen_planes_refined	0.004
r_bond_other_d	0.002

RMS Deviations
Key	Refinement Restraint Deviation
r_dihedral_angle_2_deg	38.326
r_dihedral_angle_4_deg	20.966
r_dihedral_angle_3_deg	14.512
r_dihedral_angle_1_deg	8.069
r_angle_refined_deg	1.893
r_angle_other_deg	0.989
r_chiral_restr	0.113
r_bond_refined_d	0.015
r_gen_planes_refined	0.004
r_bond_other_d	0.002
r_gen_planes_other	0.001

Non-Hydrogen Atoms Used in Refinement
Non-Hydrogen Atoms	Number
Protein Atoms	12782
Nucleic Acid Atoms
Solvent Atoms	1238
Heterogen Atoms	488

Software

Software
Software Name	Purpose
Aimless	data scaling
PHASER	phasing
REFMAC	refinement
PDB_EXTRACT	data extraction
MOSFLM	data reduction

RCSB PDB Core Operations are funded by the U.S. National Science Foundation (DBI-2321666), the US Department of Energy (DE-SC0019749), and the National Cancer Institute, National Institute of Allergy and Infectious Diseases, and National Institute of General Medical Sciences of the National Institutes of Health under grant R01GM133198.