PDB-6ddy: Crystal structure of the double mutant (D52N/K359Q) of NT5C2-537X...

Basic information

• Entry: Database: PDB / ID: 6ddy
• Title: Crystal structure of the double mutant (D52N/K359Q) 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 / nucleoside phosphotransferase activity / dGMP metabolic process / GMP metabolic process / Abacavir metabolism / negative regulation of defense response to virus by host / GMP 5'-nucleotidase activity / adenosine metabolic process / IMP-specific 5'-nucleotidase / IMP 5'-nucleotidase activity / IMP metabolic process / Ribavirin ADME / IMP catabolic process / Purine catabolism / allantoin metabolic process / XMP 5'-nucleosidase activity / 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:

PHOSPHATE ION / Cytosolic purine 5'-nucleotidase

• Biological species: Homo sapiens (human)
• Method: X-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.803 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

Assembly

Deposited unit

A: Cytosolic purine 5'-nucleotidase

hetero molecules

Summary:

• 64.1 kDa, 1 polymers

Component details:

	Theoretical mass	Number of molelcules
Total (without water)	64,143	3
Polymers	63,956	1
Non-polymers	187	2
Water	6,233	346

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)	256,571	12
Polymers	255,823	4
Non-polymers	748	8
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	13100 Å2
ΔGint	-75 kcal/mol
Surface area	75810 Å2
Method	PISA

Unit cell

Length a, b, c (Å)	91.480, 126.840, 130.188
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: 63955.703 Da / Num. of mol.: 1 / Fragment: residues 1-536 / Mutation: D52N, K359Q
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-PO4 / PHOSPHATE ION / Phosphate

Details:

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

#3: Chemical

A-602 ChemComp-GOL / GLYCEROL / GLYCERIN / PROPANE-1,2,3-TRIOL / Glycerol

Details:

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

#4: Water

ChemComp-HOH / water / Water

Details:

Mass: 18.015 Da / Num. of mol.: 346 / 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: 7.5 ; Details: 0.1 M HEPES (pH 7.5), 20% (w/v) PEG 1000, 0.1 M ammonium bromide, 5 mM ATP, 5 mM IMP, and 5 mM MgCl2

Data collection

• Diffraction: Mean temperature: 100 K
• Diffraction source: Source: SYNCHROTRON / Site: SSRL / Beamline: BL14-1 / Wavelength: 0.979 Å
• Detector: Type: MARRESEARCH / Detector: CCD / Date: Jun 15, 2015
• Radiation: Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
• Radiation wavelength: Wavelength: 0.979 Å / Relative weight: 1
• Reflection: Resolution: 1.803→45.425 Å / Num. obs: 69294 / % possible obs: 99.4 % / Observed criterion σ(F): 0 / Observed criterion σ(I): 0 / Redundancy: 13.3 % / B_iso Wilson estimate: 35.1 Ų / CC_1/2: 0.98 / R_merge(I) obs: 0.074 / Χ²: 1.1 / Net I/av σ(I): 44.1 / Net I/σ(I): 44.1
• Reflection shell: Resolution: 1.803→1.86 Å / Redundancy: 13.5 % / R_merge(I) obs: 0.51 / Mean I/σ(I) obs: 2.7 / Num. unique obs: 6902 / CC_1/2: 0.92 / Χ²: 0.51 / % 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: 6DDH

6ddh

Resolution: 1.803→45.425 Å / SU ML: 0.17 / Cross valid method: FREE R-VALUE / σ(F): 1.34 / Phase error: 20.9

	Rfactor	Num. reflection	% reflection
Rfree	0.2031	7003	10.11 %
Rwork	0.185	-	-
obs	0.1868	69294	99.02 %

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

Refinement step

Cycle: LAST / Resolution: 1.803→45.425 Å

	Protein	Nucleic acid	Ligand	Solvent	Total
Num. atoms	3795	0	11	346	4152

Refine LS restraints

Refine-ID	Type	Dev ideal	Number
X-RAY DIFFRACTION	f_bond_d	0.006	3903
X-RAY DIFFRACTION	f_angle_d	0.799	5269
X-RAY DIFFRACTION	f_dihedral_angle_d	14.136	2304
X-RAY DIFFRACTION	f_chiral_restr	0.052	558
X-RAY DIFFRACTION	f_plane_restr	0.005	668

LS refinement shell

Resolution (Å)	Rfactor Rfree	Num. reflection Rfree	Rfactor Rwork	Num. reflection Rwork	Refine-ID	% reflection obs (%)
1.8027-1.8231	0.3072	230	0.2385	1817	X-RAY DIFFRACTION	88
1.8231-1.8446	0.2714	213	0.2353	2100	X-RAY DIFFRACTION	100
1.8446-1.8671	0.2725	213	0.2201	2081	X-RAY DIFFRACTION	100
1.8671-1.8907	0.2054	239	0.2034	2087	X-RAY DIFFRACTION	100
1.8907-1.9156	0.2343	233	0.1994	2069	X-RAY DIFFRACTION	100
1.9156-1.9418	0.2201	234	0.2006	2051	X-RAY DIFFRACTION	100
1.9418-1.9696	0.2472	234	0.2123	2081	X-RAY DIFFRACTION	100
1.9696-1.999	0.2431	215	0.2011	2110	X-RAY DIFFRACTION	100
1.999-2.0302	0.2334	249	0.1951	2067	X-RAY DIFFRACTION	100
2.0302-2.0635	0.2155	234	0.1937	2063	X-RAY DIFFRACTION	100
2.0635-2.0991	0.2155	228	0.1874	2068	X-RAY DIFFRACTION	100
2.0991-2.1373	0.2246	253	0.184	2071	X-RAY DIFFRACTION	100
2.1373-2.1784	0.2215	230	0.1936	2085	X-RAY DIFFRACTION	100
2.1784-2.2228	0.23	242	0.1868	2089	X-RAY DIFFRACTION	100
2.2228-2.2712	0.2179	250	0.191	2052	X-RAY DIFFRACTION	100
2.2712-2.324	0.2454	231	0.1877	2085	X-RAY DIFFRACTION	100
2.324-2.3821	0.2123	222	0.1839	2081	X-RAY DIFFRACTION	100
2.3821-2.4465	0.2305	201	0.1926	2137	X-RAY DIFFRACTION	100
2.4465-2.5185	0.2184	252	0.1962	2072	X-RAY DIFFRACTION	100
2.5185-2.5998	0.2327	211	0.1938	2125	X-RAY DIFFRACTION	100
2.5998-2.6927	0.2055	246	0.1948	2082	X-RAY DIFFRACTION	100
2.6927-2.8005	0.229	224	0.1987	2104	X-RAY DIFFRACTION	100
2.8005-2.9279	0.219	252	0.1963	2076	X-RAY DIFFRACTION	100
2.9279-3.0822	0.2173	236	0.1989	2122	X-RAY DIFFRACTION	100
3.0822-3.2753	0.2464	221	0.1994	2111	X-RAY DIFFRACTION	100
3.2753-3.5281	0.2015	237	0.1878	2086	X-RAY DIFFRACTION	98
3.5281-3.883	0.1781	231	0.1715	2042	X-RAY DIFFRACTION	97
3.883-4.4444	0.1616	248	0.154	2047	X-RAY DIFFRACTION	96
4.4444-5.5979	0.163	238	0.1604	2119	X-RAY DIFFRACTION	98
5.5979-45.4394	0.192	256	0.1955	2111	X-RAY DIFFRACTION	94

Refinement TLS params.

Method: refined / Origin x: 0.1863 Å / Origin y: 39.0901 Å / Origin z: 19.6234 Å

	11	12	13	21	22	23	31	32	33
T	0.2982 Å2	0.003 Å2	-0.0023 Å2	-	0.2597 Å2	0.0156 Å2	-	-	0.267 Å2
L	0.836 °2	-0.056 °2	0.1338 °2	-	0.2885 °2	0.0942 °2	-	-	0.4646 °2
S	0.0067 Å °	0.0258 Å °	-0.0411 Å °	0.0712 Å °	0.0066 Å °	0.0134 Å °	0.1074 Å °	0.0083 Å °	0 Å °

• Refinement TLS group: Selection details: all