PDB-2x0k: Crystal structure of modular FAD synthetase from Corynebacterium ...

Basic information

• Entry: Database: PDB / ID: 2x0k
• Title: Crystal structure of modular FAD synthetase from Corynebacterium ammoniagenes
• Components: RIBOFLAVIN BIOSYNTHESIS PROTEIN RIBF
• Keywords: TRANSFERASE / RIBOFLAVIN KINASE / NUCLEOTIDE-BINDING / ATP-BINDING / MULTIFUNCTIONAL ENZYME / NUCLEOTIDYLTRANSFERASE

Function / homology

Function:

riboflavin kinase / FAD synthase / FMN adenylyltransferase activity / FAD biosynthetic process / riboflavin kinase activity / FMN biosynthetic process / riboflavin biosynthetic process / phosphorylation / ATP binding

Domain/homology:

Riboflavin kinase, bacterial / FAD synthetase / FAD synthetase / Riboflavin kinase domain, bacterial/eukaryotic / Riboflavin kinase / Riboflavin kinase / Riboflavin kinase / Riboflavin kinase-like / Riboflavin kinase domain superfamily / HUPs / Elongation Factor Tu (Ef-tu); domain 3 / Rossmann-like alpha/beta/alpha sandwich fold / Beta Barrel / Rossmann fold / 3-Layer(aba) Sandwich / Mainly Beta / Alpha Beta

Component:

PYROPHOSPHATE / Bifunctional riboflavin kinase/FMN adenylyltransferase

• Biological species: CORYNEBACTERIUM AMMONIAGENES (bacteria)
• Method: X-RAY DIFFRACTION / SYNCHROTRON / MAD / Resolution: 1.95 Å
• Authors: Herguedas, B. / Hermoso, J.A. / Martinez-Julvez, M. / Medina, M. / Frago, S.
• Citation: Journal: J.Mol.Biol. / Year: 2010; Title: Oligomeric State in the Crystal Structure of Modular Fad Synthetase Provides Insights Into its Sequential Catalysis in Prokaryotes; Authors: Herguedas, B. / Martinez-Julvez, M. / Frago, S. / Medina, M. / Hermoso, J.A.

History

Deposition	Dec 15, 2009	Deposition site: PDBE / Processing site: PDBE
Revision 1.0	May 26, 2010	Provider: repository / Type: Initial release
Revision 1.1	Jul 13, 2011	Group: Advisory / Version format compliance
Revision 1.2	May 8, 2024	Group: Data collection / Database references / Derived calculations / Other Category: chem_comp_atom / chem_comp_bond / database_2 / pdbx_database_status / struct_site Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession / _pdbx_database_status.status_code_sf / _struct_site.pdbx_auth_asym_id / _struct_site.pdbx_auth_comp_id / _struct_site.pdbx_auth_seq_id

Assembly

Deposited unit

A: RIBOFLAVIN BIOSYNTHESIS PROTEIN RIBF

B: RIBOFLAVIN BIOSYNTHESIS PROTEIN RIBF

hetero molecules

Summary:

• 74.3 kDa, 2 polymers

Component details:

	Theoretical mass	Number of molelcules
Total (without water)	74,313	6
Polymers	73,765	2
Non-polymers	548	4
Water	9,422	523

1

A: RIBOFLAVIN BIOSYNTHESIS PROTEIN RIBF

B: RIBOFLAVIN BIOSYNTHESIS PROTEIN RIBF

hetero molecules

A: RIBOFLAVIN BIOSYNTHESIS PROTEIN RIBF

B: RIBOFLAVIN BIOSYNTHESIS PROTEIN RIBF

hetero molecules

A: RIBOFLAVIN BIOSYNTHESIS PROTEIN RIBF

B: RIBOFLAVIN BIOSYNTHESIS PROTEIN RIBF

hetero molecules

Summary:

• defined by author&software
• 223 kDa, 6 polymers

Component details:

	Theoretical mass	Number of molelcules
Total (without water)	222,938	18
Polymers	221,294	6
Non-polymers	1,644	12
Water	108	6

Symmetry operations:

Type	Name	Symmetry operation	Number
identity operation	1_555	x,y,z	1
crystal symmetry operation	12_664	-y+3/2,-z+1,x-1/2	1
crystal symmetry operation	6_566	z+1/2,-x+3/2,-y+1	1

Calculated values:

Buried area	19770 Å2
ΔGint	-177.2 kcal/mol
Surface area	82800 Å2
Method	PISA

2

A: RIBOFLAVIN BIOSYNTHESIS PROTEIN RIBF

hetero molecules

Summary:

• defined by author&software
• 37.2 kDa, 1 polymers

Component details:

	Theoretical mass	Number of molelcules
Total (without water)	37,156	3
Polymers	36,882	1
Non-polymers	274	2
Water	18	1

Symmetry operations:

Type	Name	Symmetry operation	Number
identity operation	1_555	x,y,z	1

Calculated values:

Method	PISA

3

B: RIBOFLAVIN BIOSYNTHESIS PROTEIN RIBF

hetero molecules

Summary:

• defined by author&software
• 37.2 kDa, 1 polymers

Component details:

	Theoretical mass	Number of molelcules
Total (without water)	37,156	3
Polymers	36,882	1
Non-polymers	274	2
Water	18	1

Symmetry operations:

Type	Name	Symmetry operation	Number
identity operation	1_555	x,y,z	1

Calculated values:

Method	PISA

Unit cell

Length a, b, c (Å)	133.472, 133.472, 133.472
Angle α, β, γ (deg.)	90.00, 90.00, 90.00
Int Tables number	198
Space group name H-M	P213

Components

#1: Protein

A B RIBOFLAVIN BIOSYNTHESIS PROTEIN RIBF

Details:

Mass: 36882.359 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) CORYNEBACTERIUM AMMONIAGENES (bacteria)
Plasmid: PET28 / Production host: ESCHERICHIA COLI (E. coli) / Strain (production host): BL21(DE3) / References: UniProt: Q59263

#2: Chemical

A-1339 B-2339 ChemComp-SO4 / SULFATE ION

Details:

Mass: 96.063 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: SO₄

#3: Chemical

A-1340 B-2340 ChemComp-PPV / PYROPHOSPHATE

Details:

Mass: 177.975 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: H₄O₇P₂

#4: Water

ChemComp-HOH / water

Details:

Mass: 18.015 Da / Num. of mol.: 523 / 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: 2.68 ų/Da / Density % sol: 54.2 % / Description: NONE
• Crystal grow: pH: 7.5 / Details: 1.5M LI2SO4, 0.1 M HEPES-NAOH, PH 7.5

Data collection

Diffraction

ID	Mean temperature (K)	Crystal-ID
1	100	1
2		1

Diffraction source

Source	Site	Beamline	ID	Wavelength	Wavelength (Å)
SYNCHROTRON	ESRF	ID14-2	1	0.933
SYNCHROTRON	ESRF	BM16	2		0.97919, 0.97942, 0.90752

Detector

Type	ID	Detector
ADSC CCD	1	CCD
ADSC CCD	2	CCD

Radiation

ID	Protocol	Monochromatic (M) / Laue (L)	Scattering type	Wavelength-ID
1	MAD	M	x-ray	1
2		M	x-ray	1

Radiation wavelength

ID	Wavelength (Å)	Relative weight
1	0.933	1
2	0.97919	1
3	0.97942	1
4	0.90752	1

• Reflection: Resolution: 1.95→54.23 Å / Num. obs: 57834 / % possible obs: 100 % / Observed criterion σ(I): 3 / Redundancy: 11.4 % / R_merge(I) obs: 0.09 / Net I/σ(I): 19.9
• Reflection shell: Resolution: 1.95→2.06 Å / Redundancy: 8.7 % / R_merge(I) obs: 0.46 / Mean I/σ(I) obs: 2.7 / % possible all: 100

Processing

Software

Name	Version	Classification
REFMAC	5.2.0019	refinement
MOSFLM		data reduction
SCALA		data scaling
SHELX		phasing

Refinement

Method to determine structure: MAD
Starting model: NONE

Resolution: 1.95→94.49 Å / Cor.coef. F_o:F_c: 0.943 / Cor.coef. F_o:F_c free: 0.93 / SU B: 6.813 / SU ML: 0.106 / TLS residual ADP flag: LIKELY RESIDUAL / Cross valid method: THROUGHOUT / ESU R: 0.182 / ESU R Free: 0.158 / Stereochemistry target values: MAXIMUM LIKELIHOOD
Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS. ATOM RECORD CONTAINS RESIDUAL B FACTORS ONLY. ALTERNATIVE CONFORMATIONS WITH 0.5 OCCUPANCY HAVE BEEN MODELED FOR RESIDUES A195, B1195, A323-A331 AND B1321-B1331.

	Rfactor	Num. reflection	% reflection	Selection details
Rfree	0.23711	4189	7.3 %	RANDOM
Rwork	0.2044	-	-	-
obs	0.20676	53568	99.93 %	-

• 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: 22.147 Ų

Refinement step

Cycle: LAST / Resolution: 1.95→94.49 Å

	Protein	Nucleic acid	Ligand	Solvent	Total
Num. atoms	5200	0	28	523	5751

Refine LS restraints

Refine-ID	Type	Dev ideal	Dev ideal target	Number
X-RAY DIFFRACTION	r_bond_refined_d	0.006	0.022	5497
X-RAY DIFFRACTION	r_bond_other_d	0.001	0.02	3597
X-RAY DIFFRACTION	r_angle_refined_deg	0.993	1.953	7486
X-RAY DIFFRACTION	r_angle_other_deg	0.793	3	8758
X-RAY DIFFRACTION	r_dihedral_angle_1_deg	5.282	5	695
X-RAY DIFFRACTION	r_dihedral_angle_2_deg	34.912	23.906	256
X-RAY DIFFRACTION	r_dihedral_angle_3_deg	13.397	15	865
X-RAY DIFFRACTION	r_dihedral_angle_4_deg	13	15	38
X-RAY DIFFRACTION	r_chiral_restr	0.059	0.2	848
X-RAY DIFFRACTION	r_gen_planes_refined	0.003	0.02	6217
X-RAY DIFFRACTION	r_gen_planes_other	0.001	0.02	1135
X-RAY DIFFRACTION	r_nbd_refined	0.187	0.2	1061
X-RAY DIFFRACTION	r_nbd_other	0.18	0.2	3688
X-RAY DIFFRACTION	r_nbtor_refined	0.17	0.2	2606
X-RAY DIFFRACTION	r_nbtor_other	0.08	0.2	2852
X-RAY DIFFRACTION	r_xyhbond_nbd_refined	0.116	0.2	399
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	0.144	0.2	14
X-RAY DIFFRACTION	r_symmetry_vdw_other	0.172	0.2	65
X-RAY DIFFRACTION	r_symmetry_hbond_refined	0.101	0.2	11
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	0.697	1.5	4435
X-RAY DIFFRACTION	r_mcbond_other	0.078	1.5	1412
X-RAY DIFFRACTION	r_mcangle_it	0.794	2	5569
X-RAY DIFFRACTION	r_mcangle_other
X-RAY DIFFRACTION	r_scbond_it	1.19	3	2329
X-RAY DIFFRACTION	r_scbond_other
X-RAY DIFFRACTION	r_scangle_it	1.729	4.5	1917
X-RAY DIFFRACTION	r_scangle_other
X-RAY DIFFRACTION	r_long_range_B_refined
X-RAY DIFFRACTION	r_long_range_B_other
X-RAY DIFFRACTION	r_rigid_bond_restr
X-RAY DIFFRACTION	r_sphericity_free
X-RAY DIFFRACTION	r_sphericity_bonded

LS refinement shell

Resolution: 1.95→2.001 Å / Total num. of bins used: 20

	Rfactor	Num. reflection	% reflection
Rfree	0.287	301	-
Rwork	0.227	3913	-
obs	-	-	100 %

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	0.8216	-0.7555	-0.4931	1.3148	0.7039	0.9355	-0.0708	-0.0757	0.0496	0.1044	0.124	-0.0155	0.0674	-0.0024	-0.0533	-0.0206	0.01	0.0143	-0.0262	-0.0004	-0.0428	53.0989	123.8604	37.2396
2	0.7589	-0.0091	0.1655	0.3087	0.2081	0.8433	-0.0039	0.0535	-0.0001	-0.0149	0.027	0.0173	-0.0489	0.0442	-0.0231	0.0021	-0.0116	0.0035	-0.0487	0.0089	-0.0173	57.7015	103.6814	12.1033
3	1.167	-1.5963	0.5314	3.2649	-0.3634	0.8352	0.2888	0.1661	-0.1074	-0.4559	-0.2771	0.1803	0.0981	0.0647	-0.0116	-0.0028	0.0327	-0.0526	-0.0119	-0.0107	-0.0241	39.8266	111.1783	-14.1427
4	0.2484	0.2022	0.5216	1.4034	0.0064	2.0279	-0.0845	-0.0489	0.0583	0.0143	-0.0023	0.0165	-0.0961	0.0619	0.0867	-0.0675	0.0191	-0.054	-0.021	-0.0194	-0.0099	38.728	133.3766	9.6961

Refinement TLS group

ID	Refine-ID	Refine TLS-ID	Auth asym-ID	Auth seq-ID
1	X-RAY DIFFRACTION	1	A	1 - 186
2	X-RAY DIFFRACTION	2	A	187 - 338
3	X-RAY DIFFRACTION	3	B	1001 - 1186
4	X-RAY DIFFRACTION	4	B	1187 - 1338