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BMRB: 30133

5KQJ

Solution Structure of Antibiotic-Resistance Factor ANT(2'')-Ia Reveals Substrate-Regulated Conformation Dynamics

Authors

Bacot-Davis, V.R., Berghuis, A.M.

Assembly

2''-aminoglycoside nucleotidyltransferase (E.C.2.7.7.46)

Entity

1. 2''-aminoglycoside nucleotidyltransferase (E.C.2.7.7.46) (polymer, Thiol state: all free), 185 monomers, 20938.28 Da Detail

MDTTQVTLIH KILAAADERN LPLWIGGGWA IDARLGRVTR KHDDIDLTFP GERRGELEAI VEMLGGRVME ELDYGFLAEI GDELLDCEPA WWADEAYEIA EAPQGSCPEA AEGVIAGRPV RCNSWEAIIW DYFYYADEVP PVDWPTKHIE SYRLACTSLG AEKVEVLRAA FRSRYAALEH HHHHH

Formula weight

20938.28 Da

Source organism

Klebsiella pneumoniae

Exptl. method

Refine. method

torsion angle dynamics

Data set

assigned_chemical_shifts

Chem. Shift Complete

Sequence coverage: 97.8 %, Completeness: 86.9 %, Completeness (bb): 93.8 % Detail

Polymer type: polypeptide(L)

	Total	¹H	¹³C	¹⁵N
All	86.9 % (1833 of 2109)	83.8 % (908 of 1084)	90.6 % (759 of 838)	88.8 % (166 of 187)
Backbone	93.8 % (1024 of 1092)	94.4 % (354 of 375)	93.5 % (506 of 541)	93.2 % (164 of 176)
Sidechain	81.0 % (962 of 1188)	77.3 % (548 of 709)	88.0 % (412 of 468)	18.2 % (2 of 11)
Aromatic	43.1 % (93 of 216)	18.5 % (20 of 108)	72.3 % (73 of 101)	0.0 % (0 of 7)
Methyl	94.3 % (200 of 212)	94.3 % (100 of 106)	94.3 % (100 of 106)

1. 2''-aminoglycoside nucleotidyltransferase

MDTTQVTLIH KILAAADERN LPLWIGGGWA IDARLGRVTR KHDDIDLTFP GERRGELEAI VEMLGGRVME ELDYGFLAEI GDELLDCEPA WWADEAYEIA EAPQGSCPEA AEGVIAGRPV RCNSWEAIIW DYFYYADEVP PVDWPTKHIE SYRLACTSLG AEKVEVLRAA FRSRYAALEH HHHHH

Show sample condition

Sample

Solvent system 90% H2O/10% D2O, Pressure 1 atm, Temperature 298 K, pH 7, Details 0.5 mM [U-100% 13C; U-100% 15N] ANT(2'')-Ia, 90% H2O/10% D2O

#	Name	Isotope labeling	Type	Concentration
1	2''-aminoglycoside nucleotidyltransferase	[U-100% 13C; U-100% 15N]		0.5 mM
2	H2O	natural abundance		90 %
3	D2O	natural abundance		10 %

Protein Blocks Logo

Calculated from 15 models in PDB: 5KQJ, Strand ID: A Detail

Release date

2016-07-14

Citation

Effect of solvent and protein dynamics in ligand recognition and inhibition of aminoglycoside adenyltransferase 2″-Ia
Bacot-Davis, V.R., Bassenden, A.V., Sprules, T., Berghuis, A.M.
Protein Sci. (2017), 26, 1852-1863, PubMed 28734024 , DOI 10.1002/pro.3224 , Abstract

Related entities 1. 2''-aminoglycoside nucleotidyltransferase (E.C.2.7.7.46), : 1 : 7 : 1 entities Detail

Experiments performed 15 experiments Detail

1 2D 1H-15N HSQC

Instrument

Varian INOVA - 500 MHz

Sample

State isotropic, Solvent system 90% H2O/10% D2O, Pressure 1 atm, Temperature 298 K, pH 7, Details 0.5 mM [U-100% 13C; U-100% 15N] ANT(2'')-Ia, 90% H2O/10% D2O

#	Name	Isotope labeling	Type	Concentration
1	2''-aminoglycoside nucleotidyltransferase	[U-100% 13C; U-100% 15N]		0.5 mM
2	H2O	natural abundance		90 %
3	D2O	natural abundance		10 %

2 2D 1H-13C HSQC aliphatic

Instrument

Varian INOVA - 800 MHz

Sample

State isotropic, Solvent system 90% H2O/10% D2O, Pressure 1 atm, Temperature 298 K, pH 7, Details 0.5 mM [U-100% 13C; U-100% 15N] ANT(2'')-Ia, 90% H2O/10% D2O

#	Name	Isotope labeling	Type	Concentration
1	2''-aminoglycoside nucleotidyltransferase	[U-100% 13C; U-100% 15N]		0.5 mM
2	H2O	natural abundance		90 %
3	D2O	natural abundance		10 %

3 2D 1H-13C HSQC aromatic

Instrument

Varian INOVA - 800 MHz

Sample

State isotropic, Solvent system 90% H2O/10% D2O, Pressure 1 atm, Temperature 298 K, pH 7, Details 0.5 mM [U-100% 13C; U-100% 15N] ANT(2'')-Ia, 90% H2O/10% D2O

#	Name	Isotope labeling	Type	Concentration
1	2''-aminoglycoside nucleotidyltransferase	[U-100% 13C; U-100% 15N]		0.5 mM
2	H2O	natural abundance		90 %
3	D2O	natural abundance		10 %

4 3D HNCO

Instrument

Varian INOVA - 800 MHz

Sample

State isotropic, Solvent system 90% H2O/10% D2O, Pressure 1 atm, Temperature 298 K, pH 7, Details 0.5 mM [U-100% 13C; U-100% 15N] ANT(2'')-Ia, 90% H2O/10% D2O

#	Name	Isotope labeling	Type	Concentration
1	2''-aminoglycoside nucleotidyltransferase	[U-100% 13C; U-100% 15N]		0.5 mM
2	H2O	natural abundance		90 %
3	D2O	natural abundance		10 %

5 3D HNCA

Instrument

Varian INOVA - 800 MHz

Sample

State isotropic, Solvent system 90% H2O/10% D2O, Pressure 1 atm, Temperature 298 K, pH 7, Details 0.5 mM [U-100% 13C; U-100% 15N] ANT(2'')-Ia, 90% H2O/10% D2O

#	Name	Isotope labeling	Type	Concentration
1	2''-aminoglycoside nucleotidyltransferase	[U-100% 13C; U-100% 15N]		0.5 mM
2	H2O	natural abundance		90 %
3	D2O	natural abundance		10 %

6 3D HNCACB

Instrument

Varian INOVA - 800 MHz

Sample

State isotropic, Solvent system 90% H2O/10% D2O, Pressure 1 atm, Temperature 298 K, pH 7, Details 0.5 mM [U-100% 13C; U-100% 15N] ANT(2'')-Ia, 90% H2O/10% D2O

#	Name	Isotope labeling	Type	Concentration
1	2''-aminoglycoside nucleotidyltransferase	[U-100% 13C; U-100% 15N]		0.5 mM
2	H2O	natural abundance		90 %
3	D2O	natural abundance		10 %

7 3D CBCA(CO)NH

Instrument

Varian INOVA - 800 MHz

Sample

State isotropic, Solvent system 90% H2O/10% D2O, Pressure 1 atm, Temperature 298 K, pH 7, Details 0.5 mM [U-100% 13C; U-100% 15N] ANT(2'')-Ia, 90% H2O/10% D2O

#	Name	Isotope labeling	Type	Concentration
1	2''-aminoglycoside nucleotidyltransferase	[U-100% 13C; U-100% 15N]		0.5 mM
2	H2O	natural abundance		90 %
3	D2O	natural abundance		10 %

8 3D HN(CO)CA

Instrument

Varian INOVA - 800 MHz

Sample

State isotropic, Solvent system 90% H2O/10% D2O, Pressure 1 atm, Temperature 298 K, pH 7, Details 0.5 mM [U-100% 13C; U-100% 15N] ANT(2'')-Ia, 90% H2O/10% D2O

#	Name	Isotope labeling	Type	Concentration
1	2''-aminoglycoside nucleotidyltransferase	[U-100% 13C; U-100% 15N]		0.5 mM
2	H2O	natural abundance		90 %
3	D2O	natural abundance		10 %

9 3D HCCH-TOCSY

Instrument

Varian INOVA - 800 MHz

Sample

State isotropic, Solvent system 90% H2O/10% D2O, Pressure 1 atm, Temperature 298 K, pH 7, Details 0.5 mM [U-100% 13C; U-100% 15N] ANT(2'')-Ia, 90% H2O/10% D2O

#	Name	Isotope labeling	Type	Concentration
1	2''-aminoglycoside nucleotidyltransferase	[U-100% 13C; U-100% 15N]		0.5 mM
2	H2O	natural abundance		90 %
3	D2O	natural abundance		10 %

10 3D H(CCO)NH

Instrument

Varian INOVA - 800 MHz

Sample

State isotropic, Solvent system 90% H2O/10% D2O, Pressure 1 atm, Temperature 298 K, pH 7, Details 0.5 mM [U-100% 13C; U-100% 15N] ANT(2'')-Ia, 90% H2O/10% D2O

#	Name	Isotope labeling	Type	Concentration
1	2''-aminoglycoside nucleotidyltransferase	[U-100% 13C; U-100% 15N]		0.5 mM
2	H2O	natural abundance		90 %
3	D2O	natural abundance		10 %

11 3D C(CO)NH

Instrument

Varian INOVA - 800 MHz

Sample

State isotropic, Solvent system 90% H2O/10% D2O, Pressure 1 atm, Temperature 298 K, pH 7, Details 0.5 mM [U-100% 13C; U-100% 15N] ANT(2'')-Ia, 90% H2O/10% D2O

#	Name	Isotope labeling	Type	Concentration
1	2''-aminoglycoside nucleotidyltransferase	[U-100% 13C; U-100% 15N]		0.5 mM
2	H2O	natural abundance		90 %
3	D2O	natural abundance		10 %

12 3D 1H-15N NOESY

Instrument

Varian INOVA - 800 MHz

Sample

State isotropic, Solvent system 90% H2O/10% D2O, Pressure 1 atm, Temperature 298 K, pH 7, Details 0.5 mM [U-100% 13C; U-100% 15N] ANT(2'')-Ia, 90% H2O/10% D2O

#	Name	Isotope labeling	Type	Concentration
1	2''-aminoglycoside nucleotidyltransferase	[U-100% 13C; U-100% 15N]		0.5 mM
2	H2O	natural abundance		90 %
3	D2O	natural abundance		10 %

13 3D 1H-13C NOESY

Instrument

Varian INOVA - 800 MHz

Sample

State isotropic, Solvent system 90% H2O/10% D2O, Pressure 1 atm, Temperature 298 K, pH 7, Details 0.5 mM [U-100% 13C; U-100% 15N] ANT(2'')-Ia, 90% H2O/10% D2O

#	Name	Isotope labeling	Type	Concentration
1	2''-aminoglycoside nucleotidyltransferase	[U-100% 13C; U-100% 15N]		0.5 mM
2	H2O	natural abundance		90 %
3	D2O	natural abundance		10 %

14 2D 1H-15N HSQC

Instrument

Bruker DRX - 600 MHz

Sample

State isotropic, Solvent system 90% H2O/10% D2O, Pressure 1 atm, Temperature 298 K, pH 7, Details 0.5 mM [U-100% 13C; U-100% 15N] ANT(2'')-Ia, 90% H2O/10% D2O

#	Name	Isotope labeling	Type	Concentration
1	2''-aminoglycoside nucleotidyltransferase	[U-100% 13C; U-100% 15N]		0.5 mM
2	H2O	natural abundance		90 %
3	D2O	natural abundance		10 %

15 2D 1H-1H NOESY

Instrument

Bruker DRX - 600 MHz

Sample

State isotropic, Solvent system 90% H2O/10% D2O, Pressure 1 atm, Temperature 298 K, pH 7, Details 0.5 mM [U-100% 13C; U-100% 15N] ANT(2'')-Ia, 90% H2O/10% D2O

#	Name	Isotope labeling	Type	Concentration
1	2''-aminoglycoside nucleotidyltransferase	[U-100% 13C; U-100% 15N]		0.5 mM
2	H2O	natural abundance		90 %
3	D2O	natural abundance		10 %

NMR combined restraints 6 contents Detail

Properties

Input source #1: NMR data (NEF) - Assigned chemical shifts, Distance restraints	combined_30133_5kqj.nef
Input source #2: Coordindates	5kqj.cif
Diamagnetism of the molecular assembly	True (excluding Oxygen atoms)
Whether the assembly has a disulfide bond	None
Whether the assembly has a other bond	None
Whether the assembly contains a cyclic polymer	None
Overall data processing status	Warning

Disulfide bonds

None

Other bonds (neither disulfide, covalent nor hydrogen bonds, e.g. Zinc–sulphur bond)

None

Non-standard residues

None

Sequence alignments

Entity_assembly_ID (NMR): A vs Auth_asym_ID (model): A

--------10--------20--------30--------40--------50--------60--------70--------80--------90-------100
MDTTQVTLIHKILAAADERNLPLWIGGGWAIDARLGRVTRKHDDIDLTFPGERRGELEAIVEMLGGRVMEELDYGFLAEIGDELLDCEPAWWADEAYEIA
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
MDTTQVTLIHKILAAADERNLPLWIGGGWAIDARLGRVTRKHDDIDLTFPGERRGELEAIVEMLGGRVMEELDYGFLAEIGDELLDCEPAWWADEAYEIA

-------110-------120-------130-------140-------150-------160-------170-------180-----
EAPQGSCPEAAEGVIAGRPVRCNSWEAIIWDYFYYADEVPPVDWPTKHIESYRLACTSLGAEKVEVLRAAFRSRYAALEHHHHHH
|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
EAPQGSCPEAAEGVIAGRPVRCNSWEAIIWDYFYYADEVPPVDWPTKHIESYRLACTSLGAEKVEVLRAAFRSRYAALEHHHHHH

Chain assignments

Entity_assembly_ID (NMR)	Auth_asym_ID (model)	Length	Unmapped	Conflict	Sequence coverage (%)
A	A	185	0	0	100.0

Content subtype: combined_30133_5kqj.nef

#	Content subtype	Saveframe	Status	# of rows (sets)	Experiment type	Sequence coverage (%)
1	Assigned chemical shifts	assigned_chemical_shifts_1	Warning	2015		97.8 (chain: A, length: 185)
1	Distance restraints	CNS/XPLOR_distance_constraints_10	OK	310 (1)	noe	91.4 (chain: A, length: 185)
2	Distance restraints	CNS/XPLOR_distance_constraints_3	OK	95 (1)	hbond	69.2 (chain: A, length: 185)
3	Distance restraints	DYANA/DIANA_distance_constraints_8	Warning	17 (1)	hbond	17.3 (chain: A, length: 185)
4	Distance restraints	CNS/XPLOR_distance_constraints_9	Warning	34 (1)	hbond	17.3 (chain: A, length: 185)

Assigned chemical shifts

Saveframe: assigned_chemical_shifts_1

Status: Warning

Sequence coverage of experimental data

Entity_assembly_ID: A, Chain length: 185, Sequence coverage: 97.8%

--------10--------20--------30--------40--------50--------60--------70--------80--------90-------100
MDTTQVTLIHKILAAADERNLPLWIGGGWAIDARLGRVTRKHDDIDLTFPGERRGELEAIVEMLGGRVMEELDYGFLAEIGDELLDCEPAWWADEAYEIA
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
MDTTQVTLIHKILAAADERNLPLWIGGGWAIDARLGRVTRKHDDIDLTFPGERRGELEAIVEMLGGRVMEELDYGFLAEIGDELLDCEPAWWADEAYEIA

-------110-------120-------130-------140-------150-------160-------170-------180-----
EAPQGSCPEAAEGVIAGRPVRCNSWEAIIWDYFYYADEVPPVDWPTKHIESYRLACTSLGAEKVEVLRAAFRSRYAALEHHHHHH
|||||| |||||||||||||| ||||||||||||||||| |||| ||||||||||||||||||||||||||||||||||||||||
EAPQGS.PEAAEGVIAGRPVR.NSWEAIIWDYFYYADEV.PVDW.TKHIESYRLACTSLGAEKVEVLRAAFRSRYAALEHHHHHH

Total number of assignments
- ¹H chemical shifts: 1070
- ¹³C chemical shifts: 778
- ¹⁵N chemical shifts: 167

Completeness of assignments

Entity_assemble_ID: A

Excluded Atom_ID in statistics

Comp_index_ID	Comp_ID	Atom_ID	CS value (ppm)
10	HIS	CG	130.096
24	TRP	CD2	126.914
24	TRP	CE2	139.125
29	TRP	CD2	127.637
29	TRP	CE2	136.472
42	HIS	CG	131.072
49	PHE	CG	131.8
74	TYR	CG	124.994
76	PHE	CG	131.999
91	TRP	CD2	128.005
91	TRP	CE2	137.642
92	TRP	CD2	127.333
92	TRP	CE2	136.435
97	TYR	CG	124.759
125	TRP	CD2	127.963
125	TRP	CE2	137.571
130	TRP	CD2	128.569
130	TRP	CE2	138.002
132	TYR	CG	124.435
133	PHE	CG	130.8
134	TYR	CG	124.66
135	TYR	CG	124.988
144	TRP	CD2	128.808
144	TRP	CE2	139.309
148	HIS	CG	130.221
152	TYR	CG	123.287
171	PHE	CG	134.865
175	TYR	CG	124.003
180	HIS	CG	131.384
181	HIS	CG	130.734
182	HIS	CG	129.379
183	HIS	CG	130.186
184	HIS	CG	129.761
185	HIS	CG	130.98

All atoms

Atom group	# of target shifts	# of assigned shifts	Completeness (%)
¹H chemical shifts	1084	867	80.0
¹³C chemical shifts	838	744	88.8
¹⁵N chemical shifts	200	167	83.5

Backbone atoms

Atom group	# of target shifts	# of assigned shifts	Completeness (%)
¹H chemical shifts	375	349	93.1
¹³C chemical shifts	370	340	91.9
¹⁵N chemical shifts	176	165	93.8

Side chain atoms

Atom group	# of target shifts	# of assigned shifts	Completeness (%)
¹H chemical shifts	709	518	73.1
¹³C chemical shifts	468	404	86.3
¹⁵N chemical shifts	24	2	8.3

Methyl group atoms

Atom group	# of target shifts	# of assigned shifts	Completeness (%)
¹H chemical shifts	109	101	92.7
¹³C chemical shifts	109	102	93.6

Aromatic group atoms

Atom group	# of target shifts	# of assigned shifts	Completeness (%)
¹H chemical shifts	108	0	0.0
¹³C chemical shifts	101	73	72.3
¹⁵N chemical shifts	7	0	0.0

Redox state of CYS
Peptide bond of PRO
Tautomeric state of HIS
Rotameric state of ILE , LEU, and VAL
Histogram of normalized assigned chemical shifts
Random coil index and derived parameters (S² and NMR RMSD)
- Chain_ID: A

Distance restraints

Saveframe: CNS/XPLOR_distance_constraints_10
- Status: OK
- Experiment type: noe
- Sequence coverage of experimental data
  - Entity_assembly_ID: A, Chain length: 185, Sequence coverage: 91.4%
- Total number of restraints: 309
- Weight of restraints: 1.0 (309)
- Potential type of restraints: square-well-parabolic (309)
- Range of distance target values: 2.72, 4.67 (Å)
- Histogram of distance target values
- Distance restraints per residue
  - Chain_ID: A
- Distance restraints on contact map
- Saveframe: CNS/XPLOR_distance_constraints_3
  - Status: OK
  - Experiment type: hbond
  - Sequence coverage of experimental data
    - Entity_assembly_ID: A, Chain length: 185, Sequence coverage: 69.2%
  - Total number of restraints: 95
  - Weight of restraints: 1.0 (95)
  - Potential type of restraints: square-well-parabolic (95)
  - Range of distance target values: 0.0, 0.0 (Å)
  - Distance restraints per residue
    - Chain_ID: A
  - Distance restraints on contact map
  - Saveframe: DYANA/DIANA_distance_constraints_8
    - Status: Warning
    - Experiment type: hbond
    - Sequence coverage of experimental data
      - Entity_assembly_ID: A, Chain length: 185, Sequence coverage: 17.3%
    - Total number of restraints: 17
    - Weight of restraints: 1.0 (17)
    - Potential type of restraints: upper-bound-parabolic (17)
    - Range of distance target values: 3.3, 3.3 (Å)
    - Distance restraints per residue
      - Chain_ID: A
    - Distance restraints on contact map
    - Saveframe: CNS/XPLOR_distance_constraints_9
      - Status: Warning
      - Experiment type: hbond
      - Sequence coverage of experimental data
        Entity_assembly_ID: A, Chain length: 185, Sequence coverage: 17.3%
      - Total number of restraints: 34
        Total hydrogen bond restraints: 34
        O...H-x: 10
        O...h-N: 10
        Long range: 14
        O...H-x: 7
        O...h-N: 7
      - Weight of restraints: 1.0 (34)
        Total hydrogen bond restraints: 1.0 (34)
        O...H-x: 1.0 (10)
        O...h-N: 1.0 (10)
        Long range: 1.0 (14)
        O...H-x: 1.0 (7)
        O...h-N: 1.0 (7)
      - Potential type of restraints: square-well-parabolic (34)
        Total hydrogen bond restraints: square-well-parabolic (34)
        O...H-x: square-well-parabolic (10)
        O...h-N: square-well-parabolic (10)
        Long range: square-well-parabolic (14)
        O...H-x: square-well-parabolic (7)
        O...h-N: square-well-parabolic (7)
      - Range of distance target values: 2.0, 3.0 (Å)
      - Histogram of distance target values
      - Distance restraints per residue
        Chain_ID: A
      - Distance restraints on contact map

Keywords Antibiotic resistance, aminoglycoside, transferase