PDB-6de0: Crystal structure of the single mutant (D52N) of NT5C2-Q523X in t...

Basic information

• Entry: Database: PDB / ID: 6de0
• Title: Crystal structure of the single mutant (D52N) of NT5C2-Q523X in the active state
• Components: Cytosolic purine 5'-nucleotidase
• Keywords: HYDROLASE

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 / dGMP catabolic process / adenosine metabolic process / : / amide 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:

PHOSPHATE ION / Cytosolic purine 5'-nucleotidase

• Biological species: Homo sapiens (human)
• Method: X-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.05 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.

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

Assembly

Deposited unit

A: Cytosolic purine 5'-nucleotidase

hetero molecules

Summary:

• 63 kDa, 1 polymers

Component details:

	Theoretical mass	Number of molelcules
Total (without water)	62,977	6
Polymers	62,505	1
Non-polymers	472	5
Water	6,413	356

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.
• 252 kDa, 4 polymers

Component details:

	Theoretical mass	Number of molelcules
Total (without water)	251,908	24
Polymers	250,020	4
Non-polymers	1,888	20
Water	72	4

Symmetry operations:

Type	Name	Symmetry operation	Number
identity operation	1_555	x,y,z	1
crystal symmetry operation	2_565	-x,-y+1,z	1
crystal symmetry operation	3_555	-x,y,-z	1
crystal symmetry operation	4_565	x,-y+1,-z	1

Calculated values:

Buried area	16160 Å2
ΔGint	-147 kcal/mol
Surface area	74420 Å2
Method	PISA

Unit cell

Length a, b, c (Å)	91.063, 126.649, 130.576
Angle α, β, γ (deg.)	90.00, 90.00, 90.00
Int Tables number	23
Space group name H-M	I222

Components on special symmetry positions

ID	Model	Components
1	1	A-735- HOH

Components

#1: Protein

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

Details:

Mass: 62505.035 Da / Num. of mol.: 1 / Fragment: residues 1-523 / Mutation: D52N
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 A-602 A-603 A-604
ChemComp-PO4 / PHOSPHATE ION

Details:

Mass: 94.971 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: PO₄

#3: Chemical

A-605 ChemComp-GOL / GLYCEROL / GLYCERIN / PROPANE-1,2,3-TRIOL

Details:

Mass: 92.094 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C₃H₈O₃

#4: Water

ChemComp-HOH / water

Details:

Mass: 18.015 Da / Num. of mol.: 356 / 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 ų/Da / Density % sol: 57 %
• Crystal grow: Temperature: 293 K / Method: microbatch / pH: 7 / Details: 2M ammonium sulfate and 5% (v/v) 2-proponal

Data collection

• Diffraction: Mean temperature: 100 K
• Diffraction source: Source: SYNCHROTRON / Site: SSRL / Beamline: BL14-1 / Wavelength: 0.979 Å
• Detector: Type: RAYONIX MX325HE / Detector: CCD / Date: May 27, 2016
• Radiation: Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
• Radiation wavelength: Wavelength: 0.979 Å / Relative weight: 1
• Reflection: Resolution: 2.05→48.939 Å / Num. obs: 47644 / % possible obs: 100 % / Observed criterion σ(F): 0 / Observed criterion σ(I): 0 / Redundancy: 7.4 % / B_iso Wilson estimate: 33.34 Ų / CC_1/2: 0.98 / R_merge(I) obs: 0.057 / R_pim(I) all: 0.027 / R_rim(I) all: 0.072 / Χ²: 1.297 / Net I/av σ(I): 36.6 / Net I/σ(I): 36.6
• Reflection shell: Resolution: 2.05→2.09 Å / Redundancy: 7.4 % / R_merge(I) obs: 0.43 / Mean I/σ(I) obs: 4.3 / Num. unique obs: 2371 / CC_1/2: 0.94 / R_pim(I) all: 0.17 / R_rim(I) all: 0.46 / Χ²: 0.77 / % possible all: 100

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: 6DDY

6ddy

Resolution: 2.05→48.939 Å / SU ML: 0.19 / Cross valid method: FREE R-VALUE / σ(F): 1.36 / Phase error: 19.45

	Rfactor	Num. reflection	% reflection
Rfree	0.199	4782	10.04 %
Rwork	0.1663	-	-
obs	0.1697	47644	99.99 %

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

Refinement step

Cycle: LAST / Resolution: 2.05→48.939 Å

	Protein	Nucleic acid	Ligand	Solvent	Total
Num. atoms	3784	0	26	356	4166

Refine LS restraints

Refine-ID	Type	Dev ideal	Number
X-RAY DIFFRACTION	f_bond_d	0.006	3903
X-RAY DIFFRACTION	f_angle_d	0.805	5270
X-RAY DIFFRACTION	f_dihedral_angle_d	13.748	2300
X-RAY DIFFRACTION	f_chiral_restr	0.05	557
X-RAY DIFFRACTION	f_plane_restr	0.005	665

LS refinement shell

Resolution (Å)	Rfactor Rfree	Num. reflection Rfree	Rfactor Rwork	Num. reflection Rwork	Refine-ID	% reflection obs (%)
2.05-2.0733	0.2246	137	0.1947	1455	X-RAY DIFFRACTION	100
2.0733-2.0977	0.223	154	0.1885	1378	X-RAY DIFFRACTION	100
2.0977-2.1233	0.2653	160	0.1849	1423	X-RAY DIFFRACTION	100
2.1233-2.1502	0.2524	155	0.1798	1416	X-RAY DIFFRACTION	100
2.1502-2.1785	0.2192	146	0.1711	1417	X-RAY DIFFRACTION	100
2.1785-2.2083	0.1883	163	0.1661	1432	X-RAY DIFFRACTION	100
2.2083-2.2398	0.2179	133	0.1697	1418	X-RAY DIFFRACTION	100
2.2398-2.2733	0.2042	161	0.1652	1401	X-RAY DIFFRACTION	100
2.2733-2.3088	0.2117	185	0.1581	1411	X-RAY DIFFRACTION	100
2.3088-2.3466	0.2187	137	0.1658	1419	X-RAY DIFFRACTION	100
2.3466-2.3871	0.2343	156	0.1633	1414	X-RAY DIFFRACTION	100
2.3871-2.4305	0.212	158	0.1628	1420	X-RAY DIFFRACTION	100
2.4305-2.4773	0.1917	158	0.1734	1438	X-RAY DIFFRACTION	100
2.4773-2.5278	0.2496	148	0.1705	1411	X-RAY DIFFRACTION	100
2.5278-2.5828	0.2043	155	0.1709	1414	X-RAY DIFFRACTION	100
2.5828-2.6429	0.2046	165	0.1687	1424	X-RAY DIFFRACTION	100
2.6429-2.709	0.219	172	0.175	1392	X-RAY DIFFRACTION	100
2.709-2.7822	0.2132	173	0.1741	1419	X-RAY DIFFRACTION	100
2.7822-2.864	0.2106	150	0.1754	1426	X-RAY DIFFRACTION	100
2.864-2.9565	0.2261	160	0.184	1424	X-RAY DIFFRACTION	100
2.9565-3.0621	0.2176	161	0.1967	1433	X-RAY DIFFRACTION	100
3.0621-3.1847	0.228	138	0.1935	1462	X-RAY DIFFRACTION	100
3.1847-3.3296	0.2323	171	0.179	1405	X-RAY DIFFRACTION	100
3.3296-3.5051	0.2042	145	0.1654	1466	X-RAY DIFFRACTION	100
3.5051-3.7247	0.1544	172	0.1612	1416	X-RAY DIFFRACTION	100
3.7247-4.0121	0.1562	169	0.145	1425	X-RAY DIFFRACTION	100
4.0121-4.4156	0.1685	169	0.1307	1460	X-RAY DIFFRACTION	100
4.4156-5.054	0.163	149	0.1356	1474	X-RAY DIFFRACTION	100
5.054-6.3653	0.2172	188	0.1817	1447	X-RAY DIFFRACTION	100
6.3653-48.9526	0.1995	194	0.1801	1522	X-RAY DIFFRACTION	100

Refinement TLS params.

Method: refined / Origin x: 0.2176 Å / Origin y: 38.9523 Å / Origin z: 19.4536 Å

	11	12	13	21	22	23	31	32	33
T	0.2534 Å2	0.0048 Å2	0.0001 Å2	-	0.2142 Å2	0.0181 Å2	-	-	0.2205 Å2
L	0.633 °2	-0.119 °2	0.1432 °2	-	0.2962 °2	0.0258 °2	-	-	0.4297 °2
S	-0.0265 Å °	-0.0427 Å °	-0.0309 Å °	0.0937 Å °	0.0088 Å °	0.0232 Å °	0.1257 Å °	0.0079 Å °	0 Å °

• Refinement TLS group: Selection details: all