PDB-6ddh: Crystal structure of the double mutant (D52N/R367Q) of NT5C2-537X...

Basic information

• Entry: Database: PDB / ID: 6ddh
• Title: Crystal structure of the double mutant (D52N/R367Q) of NT5C2-537X in the active state, Northeast Structural Genomics Target
• Components: Cytosolic purine 5'-nucleotidase
• Keywords: HYDROLASE / Structural Genomics / PSI-Biology / Northeast Structural Genomics Consortium / NESG

Function / homology

Function:

nucleoside phosphotransferase / GMP catabolic process to guanine / nucleoside phosphotransferase activity / GMP metabolic process / : / Abacavir metabolism / dGMP metabolic process / negative regulation of defense response to virus by host / adenosine metabolic process / amide catabolic process / : / dGMP catabolic process / IMP-specific 5'-nucleotidase / : / IMP catabolic process / Ribavirin ADME / IMP metabolic process / Purine catabolism / 5'-nucleotidase / allantoin metabolic process / 5'-nucleotidase activity / protein K48-linked ubiquitination / ubiquitin protein ligase activity / ATP binding / metal ion binding / identical protein binding / cytoplasm / cytosol

Domain/homology:

HAD-superfamily hydrolase, subfamily IG, 5'-nucleotidase / Purine 5'-nucleotidase / 5' nucleotidase family / HAD superfamily / HAD-like superfamily

Component:

INOSINIC ACID / Cytosolic purine 5'-nucleotidase

• Biological species: Homo sapiens (human)
• Method: X-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.35 Å
• Authors: Forouhar, F. / Dieck, C.L. / Tzoneva, G. / Carpenter, Z. / Ambesi-Impiombato, A. / Sanchez-Martin, M. / Kirschner-Schwabe, R. / Lew, S. / Seetharaman, J. / Ferrando, A.A. / Tong, L. / Northeast Structural Genomics Consortium (NESG)

Funding support

United States, 4items

Organization	Grant number	Country
National Institutes of Health/National Cancer Institute (NIH/NCI)	CA206501	United States
National Institutes of Health/National Cancer Institute (NIH/NCI)	P30CA013696	United States
National Institutes of Health/National Center for Research Resources (NIH/NCRR)	S10OD012018	United States
National Institutes of Health/National Cancer Institute (NIH/NCI)	T32-CA09503	United States

• Citation: Journal: Cancer Cell / Year: 2018; Title: Structure and Mechanisms of NT5C2 Mutations Driving Thiopurine Resistance in Relapsed Lymphoblastic Leukemia.; Authors: Dieck, C.L. / Tzoneva, G. / Forouhar, F. / Carpenter, Z. / Ambesi-Impiombato, A. / Sanchez-Martin, M. / Kirschner-Schwabe, R. / Lew, S. / Seetharaman, J. / Tong, L. / Ferrando, A.A.

History

Deposition	May 10, 2018	Deposition site: RCSB / Processing site: RCSB
Revision 1.0	Jul 4, 2018	Provider: repository / Type: Initial release
Revision 1.1	Jul 25, 2018	Group: Data collection / Database references / Category: citation Item: _citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year
Revision 1.2	Dec 4, 2019	Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.3	Oct 11, 2023	Group: Data collection / Database references / Refinement description Category: chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession
Revision 1.4	Dec 25, 2024	Group: Advisory / Derived calculations / Structure summary Category: pdbx_entry_details / pdbx_validate_close_contact / struct_conn

Assembly

Deposited unit

A: Cytosolic purine 5'-nucleotidase

hetero molecules

Summary:

• 64.3 kDa, 1 polymers

Component details:

	Theoretical mass	Number of molelcules
Total (without water)	64,276	2
Polymers	63,928	1
Non-polymers	348	1
Water	2,288	127

1

A: Cytosolic purine 5'-nucleotidase

hetero molecules

A: Cytosolic purine 5'-nucleotidase

hetero molecules

A: Cytosolic purine 5'-nucleotidase

hetero molecules

A: Cytosolic purine 5'-nucleotidase

hetero molecules

Summary:

• defined by author&software
• Evidence: gel filtration, crystal structure also suggests tetrameric assembly.
• 257 kDa, 4 polymers

Component details:

	Theoretical mass	Number of molelcules
Total (without water)	257,104	8
Polymers	255,711	4
Non-polymers	1,393	4
Water	72	4

Symmetry operations:

Type	Name	Symmetry operation	Number
identity operation	1_555	x,y,z	1
crystal symmetry operation	2_555	-x,-y,z	1
crystal symmetry operation	3_556	-x,y,-z+1	1
crystal symmetry operation	4_556	x,-y,-z+1	1

Calculated values:

Buried area	14420 Å2
ΔGint	-58 kcal/mol
Surface area	75750 Å2
Method	PISA

Unit cell

Length a, b, c (Å)	91.916, 127.762, 130.305
Angle α, β, γ (deg.)	90.00, 90.00, 90.00
Int Tables number	23
Space group name H-M	I222

Components

#1: Protein

A Cytosolic purine 5'-nucleotidase / Cytosolic 5'-nucleotidase II

Details:

Mass: 63927.688 Da / Num. of mol.: 1 / Fragment: residues 1-536 / Mutation: D52N, R367Q
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: NT5C2, NT5B, NT5CP, PNT5 / Plasmid: pLOC_NT5C2 / Cell (production host): Rosetta 2(DE3) / Production host: Escherichia coli (E. coli) / References: UniProt: P49902, 5'-nucleotidase

#2: Chemical

A-601 ChemComp-IMP / INOSINIC ACID

Details:

Mass: 348.206 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C₁₀H₁₃N₄O₈P

#3: Water

ChemComp-HOH / water

Details:

Mass: 18.015 Da / Num. of mol.: 127 / Source method: isolated from a natural source / Formula: H₂O

• Has protein modification: N

Experimental details

Experiment

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

Sample preparation

• Crystal: Density Matthews: 3 ų/Da / Density % sol: 58 %
• Crystal grow: Temperature: 293 K / Method: microbatch / pH: 7.5 ; Details: 0.1 M HEPES (pH 7.5), 20% (w/v) PEG 1000, and 0.1 M ammonium nitrate, 5 mM ATP, 5 mM IMP, 5 mM MgCl2

Data collection

• Diffraction: Mean temperature: 100 K
• Diffraction source: Source: SYNCHROTRON / Site: NSLS / Beamline: X4C / Wavelength: 0.979 Å
• Detector: Type: MARRESEARCH / Detector: CCD / Date: Aug 14, 2014
• Radiation: Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
• Radiation wavelength: Wavelength: 0.979 Å / Relative weight: 1
• Reflection: Resolution: 2.35→48.661 Å / Num. obs: 32593 / % possible obs: 100 % / Observed criterion σ(F): 0 / Observed criterion σ(I): 0 / Redundancy: 7.4 % / B_iso Wilson estimate: 37.7 Ų / CC_1/2: 0.98 / R_merge(I) obs: 0.09 / R_pim(I) all: 0.04 / R_rim(I) all: 0.1 / Χ²: 1.01 / Net I/av σ(I): 28 / Net I/σ(I): 28
• Reflection shell: Resolution: 2.35→2.39 Å / Redundancy: 6.8 % / R_merge(I) obs: 0.48 / Mean I/σ(I) obs: 4 / Num. unique obs: 1622 / CC_1/2: 0.98 / R_pim(I) all: 0.2 / R_rim(I) all: 0.52 / Χ²: 0.83 / % possible all: 99.9

Processing

Software

Name	Version	Classification
PHENIX	(1.12_2829)	refinement
HKL-2000		data reduction
HKL-2000		data scaling
COMO		phasing

Refinement

Method to determine structure: MOLECULAR REPLACEMENT
Starting model: 2XCW

2xcw

Resolution: 2.35→48.661 Å / SU ML: 0.27 / Cross valid method: THROUGHOUT / σ(F): 1.34 / Phase error: 23.49

	Rfactor	Num. reflection	% reflection
Rfree	0.2229	3235	10.02 %
Rwork	0.1817	-	-
obs	0.1859	32292	99.91 %

• Solvent computation: Shrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å

Refinement step

Cycle: LAST / Resolution: 2.35→48.661 Å

	Protein	Nucleic acid	Ligand	Solvent	Total
Num. atoms	3851	0	23	127	4001

Refine LS restraints

Refine-ID	Type	Dev ideal	Number
X-RAY DIFFRACTION	f_bond_d	0.007	3976
X-RAY DIFFRACTION	f_angle_d	0.834	5372
X-RAY DIFFRACTION	f_dihedral_angle_d	13.858	2348
X-RAY DIFFRACTION	f_chiral_restr	0.051	574
X-RAY DIFFRACTION	f_plane_restr	0.005	677

LS refinement shell

Resolution (Å)	Rfactor Rfree	Num. reflection Rfree	Rfactor Rwork	Num. reflection Rwork	Refine-ID	% reflection obs (%)
2.35-2.3851	0.2787	138	0.219	1239	X-RAY DIFFRACTION	100
2.3851-2.4224	0.2921	148	0.2201	1244	X-RAY DIFFRACTION	100
2.4224-2.4621	0.2556	123	0.2003	1256	X-RAY DIFFRACTION	100
2.4621-2.5045	0.2871	134	0.2048	1265	X-RAY DIFFRACTION	100
2.5045-2.5501	0.2849	151	0.206	1242	X-RAY DIFFRACTION	100
2.5501-2.5991	0.2417	142	0.2087	1228	X-RAY DIFFRACTION	100
2.5991-2.6522	0.2527	142	0.1956	1228	X-RAY DIFFRACTION	100
2.6522-2.7098	0.2318	129	0.2006	1290	X-RAY DIFFRACTION	100
2.7098-2.7729	0.2654	115	0.2024	1276	X-RAY DIFFRACTION	100
2.7729-2.8422	0.2405	124	0.2151	1273	X-RAY DIFFRACTION	100
2.8422-2.919	0.2726	139	0.2134	1251	X-RAY DIFFRACTION	100
2.919-3.0049	0.2589	153	0.2186	1240	X-RAY DIFFRACTION	100
3.0049-3.1019	0.3339	136	0.2273	1257	X-RAY DIFFRACTION	100
3.1019-3.2127	0.277	152	0.2208	1236	X-RAY DIFFRACTION	100
3.2127-3.3413	0.2805	121	0.2043	1279	X-RAY DIFFRACTION	100
3.3413-3.4934	0.2195	147	0.2026	1253	X-RAY DIFFRACTION	100
3.4934-3.6775	0.2225	165	0.1932	1244	X-RAY DIFFRACTION	100
3.6775-3.9078	0.1867	141	0.1617	1263	X-RAY DIFFRACTION	100
3.9078-4.2094	0.1808	132	0.154	1273	X-RAY DIFFRACTION	100
4.2094-4.6327	0.1659	154	0.1316	1280	X-RAY DIFFRACTION	100
4.6327-5.3023	0.1823	144	0.1411	1277	X-RAY DIFFRACTION	100
5.3023-6.6776	0.2265	150	0.1803	1299	X-RAY DIFFRACTION	100
6.6776-48.6718	0.1861	155	0.1615	1364	X-RAY DIFFRACTION	100

Refinement TLS params.

Method: refined / Origin x: -0.3093 Å / Origin y: 25.0343 Å / Origin z: 45.859 Å

	11	12	13	21	22	23	31	32	33
T	0.2292 Å2	0.0009 Å2	0.0119 Å2	-	0.2066 Å2	0.0219 Å2	-	-	0.2069 Å2
L	0.6554 °2	0.1871 °2	-0.023 °2	-	0.331 °2	0.0203 °2	-	-	0.299 °2
S	0.0206 Å °	0.0114 Å °	0.1091 Å °	-0.0469 Å °	-0.0139 Å °	0.0278 Å °	-0.1166 Å °	0.0123 Å °	0.0044 Å °

• Refinement TLS group: Selection details: all