PDB-7naa: Crystal structure of Mycobacterium tuberculosis H37Rv PknF kinase...

ID or keywords:

Entry

Database: PDB / ID: 7naa

Title

Crystal structure of Mycobacterium tuberculosis H37Rv PknF kinase domain

Components

Non-specific serine/threonine protein kinase

Keywords

TRANSFERASE / pknF / Mtb / Structural Genomics / PSI-Biology / Protein Structure Initiative / Structural Genomics Consortium / SGC / ANTIMICROBIAL PROTEIN

Function / homology

Function and homology information

protein serine/threonine kinase activity => GO:0004674 / membrane => GO:0016020 / non-specific serine/threonine protein kinase / protein serine kinase activity / ATP binding
Similarity search - Function

Biological species

Mycobacterium tuberculosis (bacteria)

Method

X-RAY DIFFRACTION /

SYNCHROTRON /

MOLECULAR REPLACEMENT / Resolution: 2.75 Å

Authors

Oliveira, A.A. / Cabarca, S. / dos Reis, C.V. / Takarada, J.E. / Counago, R.M. / Balan, A. / Structural Genomics Consortium (SGC)

Funding support

Brazil, 2items

Citation

Journal: Curr Res Struct Biol / Year: 2021
Title: Structure of the Mycobacterium tuberculosis c PknF and conformational changes induced in forkhead-associated regulatory domains.
Authors: Cabarca, S. / Frazao de Souza, M. / Albert de Oliveira, A. / Vignoli Muniz, G.S. / Lamy, M.T. / Vinicius Dos Reis, C. / Takarada, J. / Effer, B. / Souza, L.S. / Iriarte de la Torre, L. / ...

History

Deposition	Jun 21, 2021	Deposition site: RCSB / Processing site: RCSB
Revision 1.0	Aug 4, 2021	Provider: repository / Type: Initial release
Revision 1.1	Aug 25, 2021	Group: Database references / Category: citation / citation_author / database_2 Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _database_2.pdbx_DOI / _database_2.pdbx_database_accession
Revision 1.2	Oct 18, 2023	Group: Data collection / Refinement description Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / pdbx_initial_refinement_model / struct_ncs_dom_lim Item: _struct_ncs_dom_lim.beg_auth_comp_id / _struct_ncs_dom_lim.beg_label_asym_id ..._struct_ncs_dom_lim.beg_auth_comp_id / _struct_ncs_dom_lim.beg_label_asym_id / _struct_ncs_dom_lim.beg_label_comp_id / _struct_ncs_dom_lim.beg_label_seq_id / _struct_ncs_dom_lim.end_auth_comp_id / _struct_ncs_dom_lim.end_label_asym_id / _struct_ncs_dom_lim.end_label_comp_id / _struct_ncs_dom_lim.end_label_seq_id

Structure viewer	Molecule: MolmilJmol/JSmol

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PDBx/mmCIF format	7naa.cif.gz	362.4 KB	Display	PDBx/mmCIF format
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Validation report

Summary document	7naa_validation.pdf.gz	1.5 MB	Display	wwPDB validaton report
Full document	7naa_full_validation.pdf.gz	1.5 MB	Display
Data in XML	7naa_validation.xml.gz	34 KB	Display
Data in CIF	7naa_validation.cif.gz	46.5 KB	Display
Arichive directory	https://data.pdbj.org/pub/pdb/validation_reports/na/7naa ftp://data.pdbj.org/pub/pdb/validation_reports/na/7naa	HTTPS FTP

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Related structure data

Related structure data	5m06S S: Starting model for refinement
Similar structure data	1kp5-d 2w45-1 6mda-1 6md7-2 2shp-2 7emn-1 6wu8-2 6atd-1 6md9-1 4ohl-2 Search similar-shape structures by Omokage search (details) Search similar-shape structures by Omokage search (details) Search similar-shape structures by Omokage search (details) Similarity search - Function & homologyF&H Search Similarity search - FunctionF&H Search Similarity search - HomologyF&H Search

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Links

PDB pages	PDBj / wwPDB / NCBI

Deposited unit

A: Non-specific serine/threonine protein kinase

B: Non-specific serine/threonine protein kinase

C: Non-specific serine/threonine protein kinase

D: Non-specific serine/threonine protein kinase

hetero molecules

123 kDa, 4 polymers
Search similar-shape structures of this assembly by Omokage search (details)

1

A: Non-specific serine/threonine protein kinase

B: Non-specific serine/threonine protein kinase

hetero molecules

defined by author
Evidence: gel filtration
61.7 kDa, 2 polymers
Search similar-shape structures of this assembly by Omokage search (details)

2

C: Non-specific serine/threonine protein kinase

D: Non-specific serine/threonine protein kinase

hetero molecules

defined by author
61.7 kDa, 2 polymers
Search similar-shape structures of this assembly by Omokage search (details)

Unit cell

Length a, b, c (Å)	100.127, 239.330, 53.982
Angle α, β, γ (deg.)	90.00, 90.00, 90.00
Int Tables number	18
Space group name H-M	P2₁2₁2

Noncrystallographic symmetry (NCS)

NCS domain:

ID	Ens-ID	Details
1	1	A
2	1	B
1	2	A
2	2	C
1	3	A
2	3	D
1	4	B
2	4	C
1	5	B
2	5	D
1	6	C
2	6	D

NCS domain segments:

Component-ID: _ / Refine code: _

Dom-ID	Ens-ID	Beg auth comp-ID	Beg label comp-ID	End auth comp-ID	End label comp-ID	Auth asym-ID	Label asym-ID	Auth seq-ID	Label seq-ID
1	1	LEU	LEU	LEU	LEU	A	A	3 - 278	3 - 278
2	1	LEU	LEU	LEU	LEU	B	B	3 - 278	3 - 278
1	2	GLY	GLY	ARG	ARG	A	A	24 - 277	24 - 277
2	2	GLY	GLY	ARG	ARG	C	C	24 - 277	24 - 277
1	3	ALA	ALA	ARG	ARG	A	A	4 - 277	4 - 277
2	3	ALA	ALA	ARG	ARG	D	D	4 - 277	4 - 277
1	4	GLY	GLY	ARG	ARG	B	B	24 - 277	24 - 277
2	4	GLY	GLY	ARG	ARG	C	C	24 - 277	24 - 277
1	5	ALA	ALA	ARG	ARG	B	B	4 - 277	4 - 277
2	5	ALA	ALA	ARG	ARG	D	D	4 - 277	4 - 277
1	6	GLY	GLY	HIS	HIS	C	C	24 - 276	24 - 276
2	6	LEU	LEU	HIS	HIS	D	D	18 - 276	18 - 276

NCS ensembles :

ID
1
2
3
4
5
6

#1: Protein	Non-specific serine/threonine protein kinase Mass: 30371.281 Da / Num. of mol.: 4 / Fragment: Protein kinase domain, residues 1-278 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Mycobacterium tuberculosis (bacteria) Gene: pknF_1, pknF, pknF_2, C0094_09540, DSI38_08510, E5M05_19500, E5M52_18375, E5M78_18220, ERS007741_01296, ERS013471_03424, ERS027661_00065, ERS094182_03016, F6W99_00312, FRD82_04005, GCL30_17545, SAMEA2683035_01332 Plasmid: pOP5GT / Details (production host): gst tag / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) References: UniProt: A0A045H1A5, non-specific serine/threonine protein kinase
#2: Chemical	ChemComp-V17 / (4-{[4-(1-benzothiophen-2-yl)pyrimidin-2-yl]amino}phenyl)[4-(pyrrolidin-1-yl)piperidin-1-yl]methanone Mass: 483.628 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C₂₈H₂₉N₅OS / Feature type: SUBJECT OF INVESTIGATION
#3: Water	ChemComp-HOH / water Mass: 18.015 Da / Num. of mol.: 117 / Source method: isolated from a natural source / Formula: H₂O
Has ligand of interest	Y

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Experiment

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

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Sample preparation

Crystal	Density Matthews: 2.51 Å³/Da / Density % sol: 51.03 % / Description: Orthorhombic
Crystal grow	Temperature: 291.15 K / Method: vapor diffusion, sitting drop / pH: 5.5 Details: PEG 4000, Citrate buffer, Ammonium acetate pH 5.5, 10 mM Ikk16 Temp details: 18

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Data collection

Diffraction	Mean temperature: 80 K / Ambient temp details: Liquid Nitrogen / Serial crystal experiment: N
Diffraction source	Source: SYNCHROTRON / Site: APS / Beamline: 24-ID-C / Wavelength: 0.979 Å
Detector	Type: DECTRIS PILATUS3 6M / Detector: PIXEL / Date: Oct 24, 2019
Radiation	Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelength	Wavelength: 0.979 Å / Relative weight: 1
Reflection	Resolution: 2.75→39.9 Å / Num. obs: 34601 / % possible obs: 99.41 % / Redundancy: 5.8 % / CC_1/2: 0.99 / R_merge(I) obs: 0.145 / R_pim(I) all: 0.065 / R_rim(I) all: 0.159 / Net I/σ(I): 11.14
Reflection shell	Resolution: 2.75→2.84 Å / R_merge(I) obs: 1.57 / Mean I/σ(I) obs: 1.38 / Num. unique obs: 3428 / CC_1/2: 0.6 / R_pim(I) all: 0.76 / R_rim(I) all: 1.76 / % possible all: 98.93

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Processing

Software

Name	Version	Classification
REFMAC	5.8.0267	refinement
XDS		data reduction
Aimless		data scaling
PHASER		phasing

Refinement

Method to determine structure:

MOLECULAR REPLACEMENT
Starting model: 5m06

5m06

Resolution: 2.75→39.9 Å / Cor.coef. F_o:F_c: 0.928 / Cor.coef. F_o:F_c free: 0.891 / SU B: 36.199 / SU ML: 0.33 / Cross valid method: FREE R-VALUE / ESU R: 0.827 / ESU R Free: 0.362 / Stereochemistry target values: MAXIMUM LIKELIHOOD
Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS, WITH TLS ADDED

	R_factor	Num. reflection	% reflection	Selection details
R_free	0.27887	1688	4.9 %	RANDOM
R_work	0.2369	-	-	-
obs	0.23888	32914	99.53 %	-

Solvent computation

Ion probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK

Displacement parameters

B_iso mean: 64.4 Å²

	B_aniso -1	B_aniso -2	B_aniso -3
1-	0.87 Å²	-0 Å²	-0 Å²
2-	-	-2.11 Å²	0 Å²
3-	-	-	1.24 Å²

Refinement step

Cycle: 1 / Resolution: 2.75→39.9 Å

	Protein	Nucleic acid	Ligand	Solvent	Total
Num. atoms	6831	0	140	117	7088

Refine LS restraints

Refine-ID	Type	Dev ideal	Dev ideal target	Number
X-RAY DIFFRACTION	r_bond_refined_d	0.005	0.013	7158
X-RAY DIFFRACTION	r_bond_other_d	0.001	0.017	6510
X-RAY DIFFRACTION	r_angle_refined_deg	1.359	1.643	9813
X-RAY DIFFRACTION	r_angle_other_deg	1.17	1.604	14857
X-RAY DIFFRACTION	r_dihedral_angle_1_deg	5.948	5	921
X-RAY DIFFRACTION	r_dihedral_angle_2_deg	26.238	21.231	333
X-RAY DIFFRACTION	r_dihedral_angle_3_deg	14.057	15	903
X-RAY DIFFRACTION	r_dihedral_angle_4_deg	16.136	15	48
X-RAY DIFFRACTION	r_chiral_restr	0.054	0.2	967
X-RAY DIFFRACTION	r_gen_planes_refined	0.005	0.02	8775
X-RAY DIFFRACTION	r_gen_planes_other	0.001	0.02	1581
X-RAY DIFFRACTION	r_nbd_refined
X-RAY DIFFRACTION	r_nbd_other
X-RAY DIFFRACTION	r_nbtor_refined
X-RAY DIFFRACTION	r_nbtor_other
X-RAY DIFFRACTION	r_xyhbond_nbd_refined
X-RAY DIFFRACTION	r_xyhbond_nbd_other
X-RAY DIFFRACTION	r_metal_ion_refined
X-RAY DIFFRACTION	r_metal_ion_other
X-RAY DIFFRACTION	r_symmetry_vdw_refined
X-RAY DIFFRACTION	r_symmetry_vdw_other
X-RAY DIFFRACTION	r_symmetry_hbond_refined
X-RAY DIFFRACTION	r_symmetry_hbond_other
X-RAY DIFFRACTION	r_symmetry_metal_ion_refined
X-RAY DIFFRACTION	r_symmetry_metal_ion_other
X-RAY DIFFRACTION	r_mcbond_it	1.33	3.746	3732
X-RAY DIFFRACTION	r_mcbond_other	1.33	3.749	3733
X-RAY DIFFRACTION	r_mcangle_it	2.295	5.617	4641
X-RAY DIFFRACTION	r_mcangle_other	2.296	5.614	4641
X-RAY DIFFRACTION	r_scbond_it	1.265	4.007	3426
X-RAY DIFFRACTION	r_scbond_other	1.265	4.01	3427
X-RAY DIFFRACTION	r_scangle_it
X-RAY DIFFRACTION	r_scangle_other	2.181	5.956	5173
X-RAY DIFFRACTION	r_long_range_B_refined	5.13	44.337	7216
X-RAY DIFFRACTION	r_long_range_B_other	5.111	44.288	7206
X-RAY DIFFRACTION	r_rigid_bond_restr
X-RAY DIFFRACTION	r_sphericity_free
X-RAY DIFFRACTION	r_sphericity_bonded

Refine LS restraints NCS

Refine-ID: X-RAY DIFFRACTION / Type: interatomic distance / Weight position: 0.05

Ens-ID	Dom-ID	Auth asym-ID	Number	Rms dev position (Å)
1	1	A	6953	0.08
1	2	B	6953	0.08
2	1	A	6222	0.07
2	2	C	6222	0.07
3	1	A	6440	0.07
3	2	D	6440	0.07
4	1	B	6175	0.09
4	2	C	6175	0.09
5	1	B	6405	0.08
5	2	D	6405	0.08
6	1	C	6014	0.07
6	2	D	6014	0.07

LS refinement shell

Resolution: 2.75→2.821 Å / Total num. of bins used: 20

	R_factor	Num. reflection	% reflection
R_free	0.329	118	-
R_work	0.354	2371	-
obs	-	-	98.73 %

Refinement TLS params.

Method: refined / Refine-ID: X-RAY DIFFRACTION

ID	L11 (°²)	L12 (°²)	L13 (°²)	L22 (°²)	L23 (°²)	L33 (°²)	S11 (Å °)	S12 (Å °)	S13 (Å °)	S21 (Å °)	S22 (Å °)	S23 (Å °)	S31 (Å °)	S32 (Å °)	S33 (Å °)	T11 (Å²)	T12 (Å²)	T13 (Å²)	T22 (Å²)	T23 (Å²)	T33 (Å²)	Origin x (Å)	Origin y (Å)	Origin z (Å)
1	4.7433	0.737	-2.4198	1.5843	-0.5433	5.1005	-0.0302	-0.3481	-0.0922	0.1936	0.0982	-0.177	0.7087	0.1303	-0.0681	0.4291	0.0317	-0.1027	0.1062	0.063	0.385	26.7923	0.1829	25.7343
2	24.1696	5.2137	0.6363	10.8011	5.1229	2.5864	-0.4086	0.6136	1.0723	1.0883	0.8158	-0.6294	0.5975	0.356	-0.4072	0.4948	0.0839	-0.1649	0.4345	0.0093	0.3269	31.4248	10.6462	11.9198
3	1.1968	-0.0683	-0.4369	0.2166	0.3041	0.6679	-0.0341	0.0932	-0.0863	0.0448	0.0864	-0.0263	-0.0138	-0.0179	-0.0523	0.3316	0.0094	-0.0092	0.1262	0.074	0.6173	21.5402	1.0854	9.5846
4	5.0607	2.0612	-1.004	1.8949	-0.4965	1.6232	0.0524	-0.0712	0.174	0.2204	-0.0167	0.2526	0.1366	0.0511	-0.0357	0.3751	0.037	-0.02	0.1306	0.0832	0.5206	19.5366	0.2114	8.6015
5	1.8462	-0.792	0.0186	1.1579	0.1512	0.2602	-0.13	0.0073	-0.016	0.0456	0.0528	-0.0266	0.2222	-0.0396	0.0772	0.3898	-0.0374	-0.0163	0.1298	0.0084	0.5198	28.6225	-10.3819	-3.8073
6	5.0805	2.7649	-0.8065	7.0101	0.9203	0.4655	0.362	0.5828	0.4358	-0.1482	-0.254	0.1351	-0.1416	-0.2171	-0.108	0.3389	0.0079	-0.0782	0.3051	0.0473	0.3316	24.0066	0.3391	-9.9806
7	17.2056	1.9077	-10.562	1.91	-1.2196	6.4861	-0.3803	-0.1899	1.0626	0.1576	1.0585	0.2867	0.2246	0.0905	-0.6783	0.6267	-0.088	-0.0087	0.8152	0.0162	0.502	30.1918	31.0713	27.4292
8	3.5983	1.3549	0.3956	0.6137	-0.0526	2.3658	0.3882	0.0526	0.276	0.2711	-0.0093	0.0898	-0.0642	0.1717	-0.3789	0.3565	-0.0272	0.1067	0.0889	-0.0484	0.5425	23.2949	21.4144	22.2463
9	1.8697	-0.1211	0.6713	0.032	-0.0365	0.2644	-0.0201	-0.0717	0.4792	-0.0048	-0.0414	-0.0939	0.0354	-0.0282	0.0615	0.366	-0.0062	0.0326	0.0667	0.0256	0.728	11.913	23.3767	14.7237
10	3.4992	-2.733	-3.0924	6.1664	6.8767	7.7946	0.0601	-0.5145	0.2115	-0.1577	0.0024	-0.1671	-0.2782	0.1508	-0.0625	0.358	-0.0312	0.0176	0.2795	0.0869	0.6544	12.4677	22.8795	13.1219
11	0.259	-0.1478	-0.4725	2.2433	0.4839	1.7015	-0.0502	-0.0066	0.2735	-0.0899	-0.0864	0.2065	-0.0466	0.0135	0.1366	0.3068	-0.0015	-0.0805	0.0042	-0.0163	0.8161	-5.3961	29.7466	14.721
12	3.5629	1.5048	0.8347	10.1453	-7.6878	7.0484	-0.4681	-0.428	0.3506	-0.8912	0.1866	-0.1732	0.541	-0.5461	0.2815	0.3536	-0.0886	-0.0263	0.4619	0.0357	0.3874	-4.3147	21.5614	2.7622
13	7.7311	0.0086	0.7249	0.0036	-0.0067	0.0845	-0.0273	-0.4605	-0.5044	0.0247	-0.0124	-0.0378	-0.0346	-0.0269	0.0397	0.5794	-0.1786	-0.0401	0.3252	0.1317	0.5319	59.5747	39.0079	25.3304
14	0.5331	1.2636	0.3128	3.0627	0.8707	0.4368	0.1998	-0.1318	-0.2279	0.2277	-0.1882	-0.5234	-0.2427	0.1051	-0.0116	0.5472	-0.3073	-0.0581	0.1758	0.0254	0.9713	58.0237	44.0432	14.7675
15	5.5665	0.7238	-3.3835	0.1815	-0.4178	2.0633	0.3232	-0.9335	0.0072	0.1119	-0.3117	-0.0744	-0.1771	0.5225	-0.0115	0.3232	-0.1753	-0.0526	0.5276	0.148	0.7403	50.517	33.8981	18.5112
16	0.7966	-0.3152	-1.3758	1.8235	-0.5049	3.1413	0.1598	-0.0059	0.1014	0.1014	-0.1913	-0.0577	-0.2405	0.1069	0.0315	0.3103	-0.0513	0.0532	0.036	-0.0062	0.7379	45.4155	36.0286	7.227
17	1.3944	-1.0061	-1.372	1.2987	0.5838	1.6546	-0.0099	0.039	0.075	0.0344	0.021	-0.0346	0.0021	-0.0399	-0.0111	0.3361	-0.0026	0.0358	0.0748	0.0656	0.6676	50.6882	24.8748	1.1463
18	1.7511	-0.5023	0.2321	1.0738	0.9548	1.3652	0.0066	0.2167	0.2289	-0.2214	0.0057	0.1089	-0.0683	0.0196	-0.0122	0.3237	-0.0278	-0.0413	0.1312	0.1364	0.7229	40.3642	28.0975	-5.9281
19	4.5181	-1.3361	0.8368	3.4662	-0.6781	1.1333	-0.2875	-0.0198	0.1946	0.6188	-0.0721	-0.9739	-0.2585	0.1229	0.3596	0.3912	-0.2553	-0.1802	0.2205	0.0883	0.7449	65.0681	58.3452	16.966
20	7.1174	1.8382	1.4799	3.5126	0.8598	0.4019	-0.0749	-0.0737	-0.2667	0.2425	0.1034	-0.2676	-0.0675	0.0268	-0.0286	0.5167	-0.1275	-0.1464	0.1205	0.0027	0.5182	56.3548	54.8737	21.0761
21	4.2938	-1.4885	2.9135	4.7052	-1.8809	3.8491	0.1397	-0.4091	0.1765	-0.8781	-0.1109	-0.239	-0.0923	-0.3395	-0.0288	0.5968	-0.0508	0.0762	0.0972	0.0109	0.5237	50.0001	65.8333	5.4608
22	0.3062	-1.0665	0.4441	4.1636	-1.6354	2.492	0.0614	0.0062	0.0538	-0.0201	-0.1997	0.2423	-0.0267	-0.0643	0.1383	0.4315	-0.1188	-0.0285	0.083	-0.144	0.5912	43.1616	59.9194	12.2064
23	0.42	0.4534	-0.7134	3.4473	1.4766	2.9684	-0.0726	0.0195	0.029	-0.1196	-0.281	0.3071	0.0784	-0.2987	0.3536	0.3543	-0.0468	-0.0094	0.0676	-0.175	0.7828	40.3339	73.2128	18.7355
24	1.0065	0.9355	-1.6453	3.0234	1.0358	6.0192	-0.0517	0.0942	0.2835	-0.1653	-0.2586	0.9004	0.004	-0.7475	0.3103	0.1431	-0.0273	-0.1093	0.2037	-0.1865	0.798	29.3715	70.3834	14.1306

Refinement TLS group

ID	Refine-ID	Refine TLS-ID	Auth asym-ID	Auth seq-ID
1	X-RAY DIFFRACTION	1	A	3 - 60
2	X-RAY DIFFRACTION	2	A	61 - 67
3	X-RAY DIFFRACTION	3	A	68 - 132
4	X-RAY DIFFRACTION	4	A	133 - 159
5	X-RAY DIFFRACTION	5	A	160 - 258
6	X-RAY DIFFRACTION	6	A	259 - 278
7	X-RAY DIFFRACTION	7	B	3 - 9
8	X-RAY DIFFRACTION	8	B	10 - 71
9	X-RAY DIFFRACTION	9	B	72 - 149
10	X-RAY DIFFRACTION	10	B	150 - 181
11	X-RAY DIFFRACTION	11	B	182 - 269
12	X-RAY DIFFRACTION	12	B	270 - 278
13	X-RAY DIFFRACTION	13	C	24 - 33
14	X-RAY DIFFRACTION	14	C	34 - 81
15	X-RAY DIFFRACTION	15	C	82 - 104
16	X-RAY DIFFRACTION	16	C	105 - 153
17	X-RAY DIFFRACTION	17	C	154 - 233
18	X-RAY DIFFRACTION	18	C	234 - 278
19	X-RAY DIFFRACTION	19	D	4 - 65
20	X-RAY DIFFRACTION	20	D	66 - 87
21	X-RAY DIFFRACTION	21	D	88 - 109
22	X-RAY DIFFRACTION	22	D	110 - 155
23	X-RAY DIFFRACTION	23	D	156 - 230
24	X-RAY DIFFRACTION	24	D	231 - 277

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