PDB-2h0e: Crystal Structure of PucM in the absence of substrate

Basic information

• Entry: Database: PDB / ID: 2h0e
• Title: Crystal Structure of PucM in the absence of substrate
• Components: Transthyretin-like protein pucM
• Keywords: HYDROLASE / BETA SANDWITCH / HIU

Function / homology

Function:

hydroxyisourate hydrolase / hydroxyisourate hydrolase activity / urate catabolic process / purine nucleobase metabolic process / identical protein binding

Domain/homology:

Hydroxyisourate hydrolase / Transthyretin/hydroxyisourate hydrolase domain / Transthyretin, conserved site / Transthyretin signature 2. / Transthyretin, thyroxine binding site / Transthyretin signature 1. / Transthyretin/hydroxyisourate hydrolase / Transthyretin/hydroxyisourate hydrolase domain / Transthyretin/hydroxyisourate hydrolase domain superfamily / HIUase/Transthyretin family / Immunoglobulin-like / Sandwich / Mainly Beta

Component:

5-hydroxyisourate hydrolase

• Biological species: Bacillus subtilis (bacteria)
• Method: X-RAY DIFFRACTION / SYNCHROTRON / MAD / Resolution: 2.2 Å
• Authors: Rhee, S.
• Citation: Journal: Proc.Natl.Acad.Sci.Usa / Year: 2006; Title: Structural and functional analysis of PucM, a hydrolase in the ureide pathway and a member of the transthyretin-related protein family.; Authors: Jung, D.-K. / Lee, Y. / Park, S.G. / Park, B.C. / Kim, G.-H. / Rhee, S.

History

Deposition	May 14, 2006	Deposition site: RCSB / Processing site: PDBJ
Revision 1.0	Jun 27, 2006	Provider: repository / Type: Initial release
Revision 1.1	May 1, 2008	Group: Version format compliance
Revision 1.2	Jul 13, 2011	Group: Non-polymer description / Version format compliance
Revision 1.3	Oct 18, 2017	Group: Refinement description / Category: software Item: _software.classification / _software.contact_author / _software.contact_author_email / _software.date / _software.language / _software.location / _software.name / _software.type / _software.version
Revision 1.4	Oct 16, 2024	Group: Data collection / Database references / Derived calculations / Structure summary Category: chem_comp_atom / chem_comp_bond / database_2 / pdbx_entry_details / pdbx_modification_feature / pdbx_struct_special_symmetry / struct_conn / struct_ref_seq_dif / struct_site Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession / _struct_conn.pdbx_leaving_atom_flag / _struct_ref_seq_dif.details / _struct_site.pdbx_auth_asym_id / _struct_site.pdbx_auth_comp_id / _struct_site.pdbx_auth_seq_id

Assembly

Deposited unit

A: Transthyretin-like protein pucM

B: Transthyretin-like protein pucM

hetero molecules

Summary:

• 27.6 kDa, 2 polymers

Component details:

	Theoretical mass	Number of molelcules
Total (without water)	27,592	4
Polymers	27,407	2
Non-polymers	184	2
Water	1,207	67

1

A: Transthyretin-like protein pucM

hetero molecules

A: Transthyretin-like protein pucM

hetero molecules

A: Transthyretin-like protein pucM

hetero molecules

A: Transthyretin-like protein pucM

hetero molecules

Summary:

• defined by author
• 55.2 kDa, 4 polymers

Component details:

	Theoretical mass	Number of molelcules
Total (without water)	55,183	8
Polymers	54,815	4
Non-polymers	368	4
Water	72	4

Symmetry operations:

Type	Name	Symmetry operation	Number
identity operation	1_555	x,y,z	1
crystal symmetry operation	2_575	-x,-y+2,z	1
crystal symmetry operation	7_465	y-1,x+1,-z+1/2	1
crystal symmetry operation	8_665	-y+1,-x+1,-z+1/2	1

2

B: Transthyretin-like protein pucM

hetero molecules

B: Transthyretin-like protein pucM

hetero molecules

B: Transthyretin-like protein pucM

hetero molecules

B: Transthyretin-like protein pucM

hetero molecules

Summary:

• defined by author
• 55.2 kDa, 4 polymers

Component details:

	Theoretical mass	Number of molelcules
Total (without water)	55,183	8
Polymers	54,815	4
Non-polymers	368	4
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	5_556	-x,y,-z+1	1
crystal symmetry operation	6_566	x,-y+1,-z+1	1

Unit cell

Length a, b, c (Å)	73.170, 73.170, 145.020
Angle α, β, γ (deg.)	90.000, 90.000, 90.000
Int Tables number	93
Space group name H-M	P4222

Components on special symmetry positions

ID	Model	Components
1	1	A-200- GOL
2	1	A-203- HOH
3	1	A-228- HOH
4	1	A-251- HOH
5	1	B-204- HOH

• Details: Chain A and B independently forms a homotetrmer.

Components

#1: Protein

A B Transthyretin-like protein pucM

Details:

Mass: 13703.716 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Bacillus subtilis (bacteria) / Plasmid: pET15b / Species (production host): Escherichia coli / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / References: UniProt: O32142

#2: Chemical

A-200 B-201 ChemComp-GOL / GLYCEROL / GLYCERIN / PROPANE-1,2,3-TRIOL

Details:

Mass: 92.094 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C₃H₈O₃

#3: Water

ChemComp-HOH / water

Details:

Mass: 18.015 Da / Num. of mol.: 67 / Source method: isolated from a natural source / Formula: H₂O

• Has protein modification: Y

Experimental details

Experiment

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

Sample preparation

• Crystal: Density Matthews: 3.54 ų/Da / Density % sol: 65.25 %
• Crystal grow: Temperature: 295 K / Method: vapor diffusion / pH: 4.2 / Details: pH 4.2, VAPOR DIFFUSION, temperature 295K

Data collection

Diffraction

ID	Mean temperature (K)	Crystal-ID
1	100	1
2		1
3		1

• Diffraction source: Source: SYNCHROTRON / Site: PAL/PLS / Beamline: 6B / Wavelength: 0.97948, 0.97964, 0.97180
• Detector: Type: BRUKER PROTEUM 300 / Detector: CCD / Date: May 12, 2005
• Radiation: Protocol: MAD / Monochromatic (M) / Laue (L): M / Scattering type: x-ray

Radiation wavelength

ID	Wavelength (Å)	Relative weight
1	0.97948	1
2	0.97964	1
3	0.9718	1

Reflection

D res high: 2.2 Å / D res low: 50 Å

Redundancy (%)	ID	Av σ(I) over netI	Number	Rmerge(I) obs	Χ2	Num. obs	% possible obs
13	1	11.8	267338	0.1	1.08	20584	99
12.9	2	13.3	263156	0.082	1.04	20427	98.2
12.8	3	12.8	261383	0.084	1.03	20441	98.2

Diffraction reflection shell

Highest resolution (Å)	Lowest resolution (Å)	% possible obs (%)	ID	Rmerge(I) obs	Chi squared	Redundancy
4.74	50	99.7	1	0.078	2.898	12.7
3.76	4.74	100	1	0.066	1.335	13.5
3.29	3.76	100	1	0.078	1.092	14
2.99	3.29	100	1	0.106	0.926	14.3
2.77	2.99	100	1	0.171	0.822	14.4
2.61	2.77	100	1	0.251	0.725	14.5
2.48	2.61	100	1	0.322	0.688	14.2
2.37	2.48	100	1	0.392	0.677	13
2.28	2.37	98.3	1	0.441	0.645	10.7
2.2	2.28	91.7	1	0.443	0.663	7.8
4.74	50	99.6	2	0.052	1.773	12.7
3.76	4.74	99.8	2	0.051	1.189	13.5
3.29	3.76	100	2	0.066	1.112	14
2.99	3.29	100	2	0.095	0.991	14.3
2.77	2.99	100	2	0.165	0.908	14.4
2.61	2.77	100	2	0.252	0.86	14.4
2.48	2.61	100	2	0.324	0.846	14
2.37	2.48	98.9	2	0.392	0.836	12.5
2.28	2.37	95.5	2	0.441	0.819	10.5
2.2	2.28	88.1	2	0.444	0.859	7.7
4.74	50	99.7	3	0.053	1.828	12.7
3.76	4.74	100	3	0.051	1.156	13.5
3.29	3.76	99.9	3	0.067	1.089	14
2.99	3.29	100	3	0.102	0.997	14.3
2.77	2.99	100	3	0.182	0.893	14.4
2.61	2.77	100	3	0.281	0.853	14.4
2.48	2.61	100	3	0.352	0.821	13.8
2.37	2.48	99.5	3	0.43	0.82	12
2.28	2.37	95.8	3	0.471	0.794	10.1
2.2	2.28	86.5	3	0.506	0.922	7.7

• Reflection: Resolution: 2.2→50 Å / Num. all: 20441 / Num. obs: 20441 / % possible obs: 98.2 % / Observed criterion σ(F): 0 / Observed criterion σ(I): 0 / Redundancy: 12.8 % / R_merge(I) obs: 0.084 / Χ²: 1.035 / Net I/σ(I): 12.8

Reflection shell

Resolution (Å)	Redundancy (%)	Rmerge(I) obs	Num. unique all	Χ2	Diffraction-ID	% possible all
2.2-2.28	7.7	0.506	1742	0.922	1	86.5
2.28-2.37	10.1	0.471	1950	0.794	1	95.8
2.37-2.48	12	0.43	2018	0.82	1	99.5
2.48-2.61	13.8	0.352	2051	0.821	1	100
2.61-2.77	14.4	0.281	2042	0.853	1	100
2.77-2.99	14.4	0.182	2055	0.893	1	100
2.99-3.29	14.3	0.102	2080	0.997	1	100
3.29-3.76	14	0.067	2087	1.089	1	99.9
3.76-4.74	13.5	0.051	2130	1.156	1	100
4.74-50	12.7	0.053	2286	1.828	1	99.7

Phasing

• Phasing: Method: MAD

Phasing set

ID
1
2
3

• Phasing MAD: D res high: 2.32 Å / D res low: 50 Å / FOM : 0.56 / Reflection: 17215

Phasing MAD set

Clust-ID	Expt-ID	Set-ID	Wavelength (Å)	F double prime refined	F prime refined
1	3 wavelength	1	0.9795	5.21	-6.49
1	3 wavelength	2	0.9796	2.62	-9.3
1	3 wavelength	3	0.9718	2.93	-4.36

Phasing MAD set site

ID	Atom type symbol	B iso	Fract x	Fract y	Fract z	Occupancy
1	Se	32.025	0.1	0.103	0.156	1.597
2	Se	48.219	0.701	0.145	0.006	1.146
3	Se	60	0.17	0.209	0.005	1.548
4	Se	60	0.448	1	0.132	1.098
5	Se	58.055	0.125	0.162	0.088	1.046
6	Se	60	0.063	0.257	0.074	1.329
7	Se	60	0.149	0.255	0.064	0.951
8	Se	60	0.281	0.383	0.047	0.591

Phasing MAD shell

Resolution (Å)	FOM	Reflection
8.39-50	0.82	916
5.28-8.39	0.87	1510
4.12-5.28	0.83	1897
3.49-4.12	0.77	2200
3.08-3.49	0.65	2430
2.79-3.08	0.46	2651
2.57-2.79	0.33	2782
2.39-2.57	0.21	2829

Processing

Software

Name	Version	Classification	NB
DENZO		data reduction
SCALEPACK		data scaling
SOLVE	2.09	phasing
CNS		refinement
PDB_EXTRACT	2	data extraction

Refinement

Method to determine structure: MAD / Resolution: 2.2→50 Å / σ(F): 3393 / Stereochemistry target values: Engh & Huber

	Rfactor	Num. reflection	% reflection	Selection details
Rfree	0.256	1620	7.8 %	RANDOM
Rwork	0.224	-	-	-
obs	0.224	16301	78.5 %	-
all	-	16301	-	-

• Solvent computation: B_sol: 34.267 Ų

Displacement parameters

B_iso mean: 44.352 Ų

	Baniso -1	Baniso -2	Baniso -3
1-	-6.119 Å2	0 Å2	0 Å2
2-	-	-6.119 Å2	0 Å2
3-	-	-	12.238 Å2

Refinement step

Cycle: LAST / Resolution: 2.2→50 Å

	Protein	Nucleic acid	Ligand	Solvent	Total
Num. atoms	1760	0	12	67	1839

Refine LS restraints

Refine-ID	Type	Dev ideal	Dev ideal target
X-RAY DIFFRACTION	c_mcbond_it	1.731	1.5
X-RAY DIFFRACTION	c_scbond_it	2.525	2
X-RAY DIFFRACTION	c_mcangle_it	2.982	2
X-RAY DIFFRACTION	c_scangle_it	3.702	2.5

Xplor file

Refine-ID	Serial no	Param file
X-RAY DIFFRACTION	1	protein_rep.param
X-RAY DIFFRACTION	2	GOL.param
X-RAY DIFFRACTION	3	water_rep.param