PDB-6de2: Crystal structure of the double mutant (D52N/L375F) of the full-l...

Basic information

• Entry: Database: PDB / ID: 6de2
• Title: Crystal structure of the double mutant (D52N/L375F) of the full-length NT5C2 in the active state
• Components: Cytosolic purine 5'-nucleotidase
• Keywords: HYDROLASE

Function / homology

Function:

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

Domain/homology:

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

Component:

ADENOSINE-5'-TRIPHOSPHATE / PHOSPHATE ION / Cytosolic purine 5'-nucleotidase

• Biological species: Homo sapiens (human)
• Method: X-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.1 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 / Derived calculations / Refinement description Category: chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model / pdbx_struct_conn_angle / struct_conn Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession / _pdbx_struct_conn_angle.ptnr1_auth_comp_id / _pdbx_struct_conn_angle.ptnr1_auth_seq_id / _pdbx_struct_conn_angle.ptnr1_label_asym_id / _pdbx_struct_conn_angle.ptnr1_label_atom_id / _pdbx_struct_conn_angle.ptnr1_label_comp_id / _pdbx_struct_conn_angle.ptnr3_auth_comp_id / _pdbx_struct_conn_angle.ptnr3_auth_seq_id / _pdbx_struct_conn_angle.ptnr3_label_asym_id / _pdbx_struct_conn_angle.ptnr3_label_atom_id / _pdbx_struct_conn_angle.ptnr3_label_comp_id / _pdbx_struct_conn_angle.value / _struct_conn.pdbx_dist_value / _struct_conn.ptnr1_auth_comp_id / _struct_conn.ptnr1_auth_seq_id / _struct_conn.ptnr1_label_asym_id / _struct_conn.ptnr1_label_atom_id / _struct_conn.ptnr1_label_comp_id / _struct_conn.ptnr2_auth_comp_id / _struct_conn.ptnr2_auth_seq_id / _struct_conn.ptnr2_label_asym_id / _struct_conn.ptnr2_label_atom_id / _struct_conn.ptnr2_label_comp_id / _struct_conn.ptnr2_symmetry

Assembly

Deposited unit

A: Cytosolic purine 5'-nucleotidase

hetero molecules

Summary:

• 67.8 kDa, 1 polymers

Component details:

	Theoretical mass	Number of molelcules
Total (without water)	67,802	7
Polymers	66,990	1
Non-polymers	813	6
Water	7,909	439

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.
• 271 kDa, 4 polymers

Component details:

	Theoretical mass	Number of molelcules
Total (without water)	271,210	28
Polymers	267,959	4
Non-polymers	3,251	24
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	15480 Å2
ΔGint	-13 kcal/mol
Surface area	75210 Å2
Method	PISA

Unit cell

Length a, b, c (Å)	91.530, 126.717, 130.198
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-602- MG

Components

Protein , 1 types, 1 molecules A

#1: Protein

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

Details:

Mass: 66989.750 Da / Num. of mol.: 1 / Mutation: D52N, L375F
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

Non-polymers , 5 types, 445 molecules

#2: Chemical

A-601 ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE

Details:

Mass: 507.181 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C₁₀H₁₆N₅O₁₃P₃ / Comment: ATP, energy-carrying molecule*YM

#3: Chemical

A-602 ChemComp-MG / MAGNESIUM ION

Details:

Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg

#4: Chemical

A-603 ChemComp-PO4 / PHOSPHATE ION

Details:

Mass: 94.971 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: PO₄

#5: Chemical

A-604 A-605 A-606 ChemComp-EDO / 1,2-ETHANEDIOL / ETHYLENE GLYCOL

Details:

Mass: 62.068 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C₂H₆O₂

#6: Water

ChemComp-HOH / water

Details:

Mass: 18.015 Da / Num. of mol.: 439 / 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; Details: 2M ammonium sulfate and 5% (v/v) 2-proponal, 5 mM ATP, 5 mM IMP, and 5 mM MgCl2

Data collection

• Diffraction: Mean temperature: 100 K
• Diffraction source: Source: SYNCHROTRON / Site: ALS / Beamline: 8.2.2 / Wavelength: 0.977 Å
• Detector: Type: ADSC QUANTUM 315r / Detector: CCD / Date: Oct 4, 2015
• Radiation: Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
• Radiation wavelength: Wavelength: 0.977 Å / Relative weight: 1
• Reflection: Resolution: 2.1→48.935 Å / Num. obs: 44471 / % possible obs: 100 % / Observed criterion σ(F): 0 / Observed criterion σ(I): 0 / Redundancy: 14.7 % / B_iso Wilson estimate: 30.64 Ų / CC_1/2: 0.98 / R_merge(I) obs: 0.08 / R_pim(I) all: 0.03 / R_rim(I) all: 0.09 / Χ²: 1.44 / Net I/av σ(I): 35.2 / Net I/σ(I): 35.2
• Reflection shell: Resolution: 2.1→2.15 Å / Redundancy: 14.8 % / R_merge(I) obs: 0.53 / Mean I/σ(I) obs: 4.9 / CC_1/2: 0.95 / R_pim(I) all: 0.14 / R_rim(I) all: 0.55 / Χ²: 0.78 / % 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: 6DDK

6ddk

Resolution: 2.1→48.935 Å / SU ML: 0.19 / Cross valid method: FREE R-VALUE / σ(F): 1.34 / Phase error: 17.97

	Rfactor	Num. reflection	% reflection
Rfree	0.1836	4448	10 %
Rwork	0.1532	-	-
obs	0.1563	44471	99.96 %

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

Refinement step

Cycle: LAST / Resolution: 2.1→48.935 Å

	Protein	Nucleic acid	Ligand	Solvent	Total
Num. atoms	3848	0	49	439	4336

Refine LS restraints

Refine-ID	Type	Dev ideal	Number
X-RAY DIFFRACTION	f_bond_d	0.006	3994
X-RAY DIFFRACTION	f_angle_d	0.836	5394
X-RAY DIFFRACTION	f_dihedral_angle_d	14.07	2351
X-RAY DIFFRACTION	f_chiral_restr	0.052	571
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 (%)
2.1-2.1239	0.2369	167	0.1811	1304	X-RAY DIFFRACTION	100
2.1239-2.1489	0.2102	142	0.1631	1318	X-RAY DIFFRACTION	100
2.1489-2.1751	0.2227	160	0.161	1313	X-RAY DIFFRACTION	100
2.1751-2.2026	0.1976	175	0.158	1262	X-RAY DIFFRACTION	100
2.2026-2.2316	0.2181	146	0.161	1334	X-RAY DIFFRACTION	100
2.2316-2.2621	0.2089	148	0.165	1335	X-RAY DIFFRACTION	100
2.2621-2.2945	0.1936	147	0.1558	1297	X-RAY DIFFRACTION	100
2.2945-2.3287	0.1716	159	0.1509	1304	X-RAY DIFFRACTION	100
2.3287-2.3651	0.2151	135	0.1452	1355	X-RAY DIFFRACTION	100
2.3651-2.4039	0.2304	136	0.1488	1306	X-RAY DIFFRACTION	100
2.4039-2.4453	0.1923	146	0.1438	1334	X-RAY DIFFRACTION	100
2.4453-2.4898	0.1925	147	0.1456	1334	X-RAY DIFFRACTION	100
2.4898-2.5377	0.2285	141	0.1504	1341	X-RAY DIFFRACTION	100
2.5377-2.5895	0.1975	141	0.1494	1297	X-RAY DIFFRACTION	100
2.5895-2.6458	0.1873	136	0.1433	1340	X-RAY DIFFRACTION	100
2.6458-2.7073	0.1939	128	0.1466	1344	X-RAY DIFFRACTION	100
2.7073-2.775	0.1943	125	0.1555	1364	X-RAY DIFFRACTION	100
2.775-2.85	0.1978	149	0.1567	1319	X-RAY DIFFRACTION	100
2.85-2.9339	0.1971	134	0.1564	1348	X-RAY DIFFRACTION	100
2.9339-3.0286	0.185	156	0.1606	1318	X-RAY DIFFRACTION	100
3.0286-3.1368	0.1966	141	0.169	1339	X-RAY DIFFRACTION	100
3.1368-3.2624	0.1619	139	0.1641	1352	X-RAY DIFFRACTION	100
3.2624-3.4108	0.1858	166	0.1592	1311	X-RAY DIFFRACTION	100
3.4108-3.5906	0.1717	136	0.1485	1350	X-RAY DIFFRACTION	100
3.5906-3.8155	0.1676	152	0.1486	1347	X-RAY DIFFRACTION	100
3.8155-4.1099	0.1601	176	0.1388	1324	X-RAY DIFFRACTION	100
4.1099-4.5232	0.1519	141	0.1259	1351	X-RAY DIFFRACTION	100
4.5232-5.1771	0.1468	132	0.1279	1389	X-RAY DIFFRACTION	100
5.1771-6.5202	0.1852	152	0.1738	1389	X-RAY DIFFRACTION	100
6.5202-48.9478	0.1987	195	0.1836	1404	X-RAY DIFFRACTION	99

Refinement TLS params.

Method: refined / Origin x: 0.0364 Å / Origin y: 38.942 Å / Origin z: 19.1527 Å

	11	12	13	21	22	23	31	32	33
T	0.2267 Å2	0.0028 Å2	0.0066 Å2	-	0.1999 Å2	0.0115 Å2	-	-	0.2029 Å2
L	0.4433 °2	-0.0853 °2	0.0954 °2	-	0.3222 °2	-0.0016 °2	-	-	0.3444 °2
S	-0.0183 Å °	-0.0502 Å °	-0.0188 Å °	0.0704 Å °	-0.0101 Å °	0.013 Å °	0.1041 Å °	-0.008 Å °	0 Å °

• Refinement TLS group: Selection details: all