PDB-4db5: Crystal structure of Rabbit GITRL

Basic information

• Entry: Database: PDB / ID: 4db5
• Title: Crystal structure of Rabbit GITRL
• Components: Tumor necrosis factor ligand superfamily member 18
• Keywords: IMMUNE SYSTEM / GITRL / GLUCOCORTICOID-INDUCED TNF RECEPTOR LIGAND / TNFRSF18 / Structural genomics / PSI-Biology / New York Structural Genomics Research Consortium / NYSGRC / Atoms-to-Animals: The Immune Function Network / IFN

Function / homology

Function:

tumor necrosis factor receptor superfamily binding / T cell proliferation involved in immune response / positive regulation of leukocyte migration / positive regulation of cell adhesion / positive regulation of tyrosine phosphorylation of STAT protein / tumor necrosis factor-mediated signaling pathway / membrane => GO:0016020 / identical protein binding

Domain/homology:

Tumor necrosis factor ligand superfamily member 18 / Jelly Rolls - #40 / Tumour necrosis factor-like domain superfamily / Jelly Rolls / Sandwich / Mainly Beta

Component:

TNF superfamily member 18

• Biological species: Oryctolagus cuniculus (rabbit)
• Method: X-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.522 Å
• Authors: Kumar, P.R. / Bhosle, R. / Gizzi, A. / Scott Glenn, A. / Chowhury, S. / Hillerich, B. / Seidel, R. / Nathenson, S.G. / Almo, S.C. / New York Structural Genomics Research Consortium (NYSGRC) / Atoms-to-Animals: The Immune Function Network (IFN)
• Citation: Journal: to be published; Title: Crystal structure of GITRL from Oryctolagus cuniculus; Authors: Kumar, P.R. / Bhosle, R. / Gizzi, A. / Scott Glenn, A. / Chowhury, S. / Hillerich, B. / Seidel, R. / Nathenson, S.G. / Almo, S.C.

History

Deposition	Jan 13, 2012	Deposition site: RCSB / Processing site: RCSB
Revision 1.0	Mar 21, 2012	Provider: repository / Type: Initial release
Revision 1.1	Jun 6, 2012	Group: Structure summary
Revision 1.2	Sep 13, 2023	Group: Data collection / Database references / Derived calculations / Refinement description Category: chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model / struct_site Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession / _struct_site.pdbx_auth_asym_id / _struct_site.pdbx_auth_comp_id / _struct_site.pdbx_auth_seq_id

Assembly

Deposited unit

A: Tumor necrosis factor ligand superfamily member 18

hetero molecules

Summary:

• 14.3 kDa, 1 polymers

Component details:

	Theoretical mass	Number of molelcules
Total (without water)	14,337	2
Polymers	14,055	1
Non-polymers	282	1
Water	2,198	122

1

A: Tumor necrosis factor ligand superfamily member 18

hetero molecules

A: Tumor necrosis factor ligand superfamily member 18

hetero molecules

A: Tumor necrosis factor ligand superfamily member 18

hetero molecules

Summary:

• defined by author&software
• 43 kDa, 3 polymers

Component details:

	Theoretical mass	Number of molelcules
Total (without water)	43,012	6
Polymers	42,165	3
Non-polymers	847	3
Water	54	3

Symmetry operations:

Type	Name	Symmetry operation	Number
identity operation	1_555	x,y,z	1
crystal symmetry operation	7_555	-z,-x,y	1
crystal symmetry operation	10_555	-y,z,-x	1

Calculated values:

Buried area	4110 Å2
ΔGint	-23 kcal/mol
Surface area	16900 Å2
Method	PISA

2

A: Tumor necrosis factor ligand superfamily member 18

hetero molecules

x 12

Summary:

• defined by software
• 172 kDa, 12 polymers

Component details:

	Theoretical mass	Number of molelcules
Total (without water)	172,049	24
Polymers	168,661	12
Non-polymers	3,388	12
Water	216	12

Symmetry operations:

Type	Name	Symmetry operation	Number
identity operation	1_555	x,y,z	1
crystal symmetry operation	7_555	-z,-x,y	1
crystal symmetry operation	10_555	-y,z,-x	1
crystal symmetry operation	16_544	x,-y-1/2,-z-1/2	1
crystal symmetry operation	20_544	-z,x-1/2,-y-1/2	1
crystal symmetry operation	24_544	-y,-z-1/2,x-1/2	1
crystal symmetry operation	27_554	-x+1/2,y,-z-1/2	1
crystal symmetry operation	30_554	z+1/2,-x,-y-1/2	1
crystal symmetry operation	33_554	y+1/2,z,x-1/2	1
crystal symmetry operation	38_545	-x+1/2,-y-1/2,z	1
crystal symmetry operation	41_545	z+1/2,x-1/2,y	1
crystal symmetry operation	47_545	y+1/2,-z-1/2,-x	1

Calculated values:

Buried area	31390 Å2
ΔGint	-152 kcal/mol
Surface area	52660 Å2
Method	PISA

Unit cell

Length a, b, c (Å)	127.047, 127.047, 127.047
Angle α, β, γ (deg.)	90.00, 90.00, 90.00
Int Tables number	196
Space group name H-M	F23

Components on special symmetry positions

ID	Model	Components
1	1	A-303- HOH
2	1	A-404- HOH
3	1	A-405- HOH

• Details: The biological assembly is a trimer generated from the monomer in the asymmetric unit by crystallographic symmetry operations

Components

#1: Protein

A Tumor necrosis factor ligand superfamily member 18

Details:

Mass: 14055.084 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Oryctolagus cuniculus (rabbit) / Gene: GITRL, TNFSF18 / Plasmid: pET28b / Production host: Escherichia coli (E. coli) / Strain (production host): BL21(DE3) / References: UniProt: G1TIM1

#2: Chemical

A-201 ChemComp-B3P / 2-[3-(2-HYDROXY-1,1-DIHYDROXYMETHYL-ETHYLAMINO)-PROPYLAMINO]-2-HYDROXYMETHYL-PROPANE-1,3-DIOL / Bis-tris propane

Details:

Mass: 282.334 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C₁₁H₂₆N₂O₆ / Comment: pH buffer*YM

#3: Water

ChemComp-HOH / water / Water

Details:

Mass: 18.015 Da / Num. of mol.: 122 / Source method: isolated from a natural source / Formula: H₂O

Experimental details

Experiment

• Experiment: Method: X-RAY DIFFRACTION / Number of used crystals: 1

Sample preparation

• Crystal: Density Matthews: 3.04 ų/Da / Density % sol: 59.53 %
• Crystal grow: Temperature: 298 K / Method: vapor diffusion, sitting drop / pH: 7 ; Details: Protein (20 mM Hepes, pH 7.5, 150 mM NaCl, 10% glycerol; Reservoir (0.1M Bis-Tris Propane pH 7.0, 2.4M DL-Malic acid); Cryoprotection (Reservoir + 30% glycerol), Sitting Drop, Vapor Diffusion, temperature 298K, VAPOR DIFFUSION, SITTING DROP

Data collection

• Diffraction: Mean temperature: 100 K
• Diffraction source: Source: SYNCHROTRON / Site: NSLS / Beamline: X29A / Wavelength: 1.075 Å
• Detector: Type: ADSC QUANTUM 315 / Detector: CCD / Date: Sep 27, 2011 / Details: MIRRORS
• Radiation: Monochromator: GRAPHITE / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
• Radiation wavelength: Wavelength: 1.075 Å / Relative weight: 1
• Reflection: Resolution: 1.52→50 Å / Num. obs: 26128 / % possible obs: 100 % / Redundancy: 33.1 % / R_merge(I) obs: 0.051 / Χ²: 1.044 / Net I/σ(I): 12.5

Reflection shell

Resolution (Å)	Redundancy (%)	Rmerge(I) obs	Num. unique all	Χ2	Diffraction-ID	% possible all
1.52-1.55	32.8	0.688	1301	0.986	1	100
1.55-1.57	33	0.612	1278	0.958	1	100
1.57-1.6	32.8	0.49	1329	0.971	1	100
1.6-1.64	33	0.436	1286	0.975	1	100
1.64-1.67	33.1	0.364	1291	0.998	1	100
1.67-1.71	33.2	0.336	1296	1.016	1	100
1.71-1.75	33.3	0.264	1296	1.038	1	100
1.75-1.8	33.2	0.207	1295	1.072	1	100
1.8-1.86	33.4	0.168	1293	1.067	1	100
1.86-1.92	33.3	0.142	1310	1.125	1	100
1.92-1.98	33.3	0.104	1297	1.101	1	100
1.98-2.06	33.4	0.082	1289	1.076	1	100
2.06-2.16	33.4	0.068	1333	1.064	1	100
2.16-2.27	33.3	0.059	1276	1.048	1	100
2.27-2.41	33.5	0.051	1323	1	1	100
2.41-2.6	33.4	0.052	1296	1.159	1	100
2.6-2.86	33	0.051	1328	1.437	1	100
2.86-3.27	32.8	0.042	1307	1.271	1	100
3.27-4.13	31.9	0.027	1337	0.818	1	100
4.13-50	32.1	0.025	1367	0.69	1	99.3

Processing

Software

Name	Version	Classification	NB
SCALEPACK		data scaling
PHENIX	1.7.3_928	refinement
PDB_EXTRACT	3.1	data extraction
CBASS		data collection
HKL-2000		data reduction
HKL-2000		data scaling
PHENIX	1.7.3_928	phasing

Refinement

Method to determine structure: MOLECULAR REPLACEMENT
Starting model: pdb entry 3FC0

3fc0

Resolution: 1.522→29.147 Å / Occupancy max: 1 / Occupancy min: 0.21 / FOM work R set: 0.9095 / SU ML: 0.16 / Cross valid method: THROUGHOUT / σ(F): 1.34 / Phase error: 15.63 / Stereochemistry target values: ML

	Rfactor	Num. reflection	% reflection	Selection details
Rfree	0.1757	1333	5.1 %	RANDOM
Rwork	0.1606	-	-	-
obs	0.1614	26128	99.98 %	-

• Solvent computation: Shrinkage radii: 0.98 Å / VDW probe radii: 1.2 Å / Solvent model: FLAT BULK SOLVENT MODEL / B_sol: 53.362 Ų / k_sol: 0.442 e/ų

Displacement parameters

B_iso max: 57.92 Ų / B_iso mean: 16.9381 Ų / B_iso min: 7.42 Ų

	Baniso -1	Baniso -2	Baniso -3
1-	-0 Å2	0 Å2	0 Å2
2-	-	0 Å2	-0 Å2
3-	-	-	0 Å2

Refinement step

Cycle: LAST / Resolution: 1.522→29.147 Å

	Protein	Nucleic acid	Ligand	Solvent	Total
Num. atoms	961	0	19	122	1102

Refine LS restraints

Refine-ID	Type	Dev ideal	Number
X-RAY DIFFRACTION	f_bond_d	0.007	1101
X-RAY DIFFRACTION	f_angle_d	1.171	1508
X-RAY DIFFRACTION	f_chiral_restr	0.08	160
X-RAY DIFFRACTION	f_plane_restr	0.005	197
X-RAY DIFFRACTION	f_dihedral_angle_d	18.525	424

LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Total num. of bins used: 10 / % reflection obs: 100 %

Resolution (Å)	Rfactor Rfree	Num. reflection Rfree	Rfactor Rwork	Num. reflection Rwork	Num. reflection all
1.5217-1.576	0.2516	144	0.1916	2477	2621
1.576-1.6391	0.1848	124	0.1731	2461	2585
1.6391-1.7137	0.1974	124	0.167	2465	2589
1.7137-1.8041	0.1721	157	0.1621	2424	2581
1.8041-1.9171	0.186	133	0.1584	2482	2615
1.9171-2.0651	0.1691	118	0.1549	2452	2570
2.0651-2.2728	0.1817	129	0.1476	2480	2609
2.2728-2.6015	0.183	139	0.1594	2480	2619
2.6015-3.2769	0.1602	146	0.1691	2489	2635
3.2769-29.1519	0.1657	119	0.1565	2585	2704

Refinement TLS params.

Method: refined / Refine-ID: X-RAY DIFFRACTION

ID	L11 (°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)
1	2.9317	-0.3997	-0.3154	1.7549	0.4299	3.5404	0.0895	-0.0362	0.1924	0.0648	-0.0251	0.4175	-0.2407	-0.2396	-0.0367	0.077	0.0008	0.0572	0.1121	0.007	0.1559	-2.9821	-22.5775	-11.6534
2	0.3186	-0.0502	0.1161	2.1304	0.1728	0.6206	0.0271	0.0215	0.0198	0.0178	0.0263	0.0204	0.0389	-0.0191	-0.0486	0.0738	0.0167	0.0026	0.0741	-0.0071	0.0902	7.2813	-26.2788	-21.6007
3	0.9396	-1.4459	0.0848	4.0802	0.0905	0.6721	0.0594	0.0304	0.0071	-0.1803	-0.0098	0.0859	-0.0468	-0.0444	-0.0546	0.0458	0.0035	0.028	0.0894	-0.0038	0.1076	4.1322	-21.8708	-20.6955

Refinement TLS group

ID	Refine-ID	Refine TLS-ID	Selection details	Auth asym-ID	Auth seq-ID
1	X-RAY DIFFRACTION	1	chain 'A' and (resseq 5:26)	A	5 - 26
2	X-RAY DIFFRACTION	2	chain 'A' and (resseq 27:90)	A	27 - 90
3	X-RAY DIFFRACTION	3	chain 'A' and (resseq 91:125)	A	91 - 125