PDB-3ebr: Crystal structure of an rmlc-like cupin protein (reut_a0381) from...

Basic information

• Entry: Database: PDB / ID: 3ebr
• Title: Crystal structure of an rmlc-like cupin protein (reut_a0381) from ralstonia eutropha jmp134 at 2.60 A resolution
• Components: uncharacterized RmlC-like cupin
• Keywords: UNKNOWN FUNCTION / Structural genomics / Joint Center for Structural Genomics / JCSG / Protein Structure Initiative / PSI-2

Function / homology

Domain/homology:

ChrR-like cupin domain / ChrR Cupin-like domain / RmlC-like cupin domain superfamily / Jelly Rolls / RmlC-like jelly roll fold / Jelly Rolls / Sandwich / Mainly Beta

Component:

DI(HYDROXYETHYL)ETHER / ChrR-like cupin domain-containing protein

• Biological species: Ralstonia eutropha JMP134 (bacteria)
• Method: X-RAY DIFFRACTION / SYNCHROTRON / MAD / Resolution: 2.6 Å
• Authors: Joint Center for Structural Genomics (JCSG)
• Citation: Journal: To be published; Title: Crystal structure of RmlC-like cupin with unknown function (YP_294607.1) from RALSTONIA EUTROPHA JMP134 at 2.60 A resolution; Authors: Joint Center for Structural Genomics (JCSG)

History

Deposition	Aug 28, 2008	Deposition site: RCSB / Processing site: RCSB
Revision 1.0	Sep 9, 2008	Provider: repository / Type: Initial release
Revision 1.1	Jul 13, 2011	Group: Advisory / Version format compliance
Revision 1.2	Oct 11, 2017	Group: Data collection / Refinement description / Category: reflns_shell / software Item: _reflns_shell.percent_possible_all / _software.classification / _software.name
Revision 1.3	Jul 24, 2019	Group: Data collection / Derived calculations / Refinement description Category: pdbx_struct_special_symmetry / software / struct_conn Item: _software.classification / _software.contact_author / _software.contact_author_email / _software.language / _software.location / _software.name / _software.type / _software.version / _struct_conn.pdbx_leaving_atom_flag
Revision 1.4	Feb 1, 2023	Group: Database references / Derived calculations / Category: database_2 / struct_ref_seq_dif / struct_site Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession / _struct_ref_seq_dif.details / _struct_site.pdbx_auth_asym_id / _struct_site.pdbx_auth_comp_id / _struct_site.pdbx_auth_seq_id
Revision 1.5	Oct 9, 2024	Group: Data collection / Structure summary Category: chem_comp_atom / chem_comp_bond / pdbx_entry_details / pdbx_modification_feature Item: _pdbx_entry_details.has_protein_modification

Assembly

Deposited unit

A: uncharacterized RmlC-like cupin

hetero molecules

Summary:

• 18.7 kDa, 1 polymers

Component details:

	Theoretical mass	Number of molelcules
Total (without water)	18,685	5
Polymers	18,261	1
Non-polymers	424	4
Water	450	25

1

A: uncharacterized RmlC-like cupin

hetero molecules

A: uncharacterized RmlC-like cupin

hetero molecules

A: uncharacterized RmlC-like cupin

hetero molecules

A: uncharacterized RmlC-like cupin

hetero molecules

Summary:

• defined by author&software
• 74.7 kDa, 4 polymers

Component details:

	Theoretical mass	Number of molelcules
Total (without water)	74,740	20
Polymers	73,043	4
Non-polymers	1,698	16
Water	72	4

Symmetry operations:

Type	Name	Symmetry operation	Number
identity operation	1_555	x,y,z	1
crystal symmetry operation	2_655	-x+1,-y,z	1
crystal symmetry operation	5_656	-x+1,y,-z+1	1
crystal symmetry operation	6_556	x,-y,-z+1	1

Calculated values:

Buried area	16580 Å2
ΔGint	-42 kcal/mol
Surface area	23020 Å2
Method	PISA

Unit cell

Length a, b, c (Å)	91.157, 91.157, 48.753
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-162- PEG

Components

#1: Protein

A uncharacterized RmlC-like cupin

Details:

Mass: 18260.641 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Ralstonia eutropha JMP134 (bacteria) / Gene: YP_294607.1, Reut_A0381 / Plasmid: SpeedET / Production host: Escherichia Coli (E. coli) / Strain (production host): HK100 / References: UniProt: Q476C0

#2: Chemical

A-159 A-160 A-161 A-162
ChemComp-PEG / DI(HYDROXYETHYL)ETHER

Details:

Mass: 106.120 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C₄H₁₀O₃

#3: Water

ChemComp-HOH / water

Details:

Mass: 18.015 Da / Num. of mol.: 25 / Source method: isolated from a natural source / Formula: H₂O

• Has protein modification: Y
• Sequence details: THE CONSTRUCT WAS EXPRESSED WITH A PURIFICATION TAG MGSDKIHHHHHHENLYFQG. THE TAG WAS REMOVED WITH TEV PROTEASE LEAVING ONLY A GLYCINE (0) FOLLOWED BY THE TARGET SEQUENCE.

Experimental details

Experiment

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

Sample preparation

• Crystal: Density Matthews: 2.77 ų/Da / Density % sol: 55.65 %
• Crystal grow: Temperature: 293 K / Method: vapor diffusion, sitting drop / pH: 6.5 ; Details: 60.0% polyethylene glycol 200, 0.1M HEPES pH 6.5, NANODROP, VAPOR DIFFUSION, SITTING DROP, temperature 293K

Data collection

• Diffraction: Mean temperature: 100 K
• Diffraction source: Source: SYNCHROTRON / Site: SSRL / Beamline: BL11-1 / Wavelength: 0.91837,0.97941,0.97854
• Detector: Type: MARMOSAIC 325 mm CCD / Detector: CCD / Date: Jun 22, 2008 / Details: Flat mirror (vertical focusing)
• Radiation: Monochromator: Single crystal Si(111) bent monochromator (horizontal focusing); Protocol: MAD / Monochromatic (M) / Laue (L): M / Scattering type: x-ray

Radiation wavelength

ID	Wavelength (Å)	Relative weight
1	0.91837	1
2	0.97941	1
3	0.97854	1

• Reflection: Resolution: 2.6→28.831 Å / Num. obs: 6713 / % possible obs: 99.9 % / Redundancy: 4.7 % / B_iso Wilson estimate: 76.248 Ų / R_merge(I) obs: 0.074 / R_sym value: 0.074 / Net I/σ(I): 15.1

Reflection shell

Diffraction-ID: 1

Resolution (Å)	Redundancy (%)	Rmerge(I) obs	Mean I/σ(I) obs	Num. measured all	Num. unique all	Rsym value	% possible all
2.6-2.67	4.8	0.596	2.2	2290	476	0.596	100
2.67-2.74	4.9	0.436	3.1	2283	470	0.436	100
2.74-2.82	4.8	0.375	3.4	2158	451	0.375	100
2.82-2.91	4.8	0.289	4.5	2176	452	0.289	100
2.91-3	4.8	0.203	6.2	2050	430	0.203	100
3-3.11	4.8	0.171	7.7	2026	419	0.171	100
3.11-3.22	4.7	0.118	10.6	1964	414	0.118	100
3.22-3.36	4.8	0.099	13	1844	384	0.099	100
3.36-3.51	4.7	0.079	15.7	1799	383	0.079	100
3.51-3.68	4.8	0.074	19.6	1733	364	0.074	100
3.68-3.88	4.7	0.073	21.7	1575	337	0.073	100
3.88-4.11	4.6	0.067	25.4	1571	339	0.067	100
4.11-4.39	4.6	0.058	29.5	1438	310	0.058	100
4.39-4.75	4.6	0.055	29	1356	294	0.055	100
4.75-5.2	4.5	0.046	30.3	1226	270	0.046	100
5.2-5.81	4.5	0.056	28.4	1122	252	0.056	100
5.81-6.71	4.4	0.069	26.3	989	223	0.069	99.9
6.71-8.22	4.2	0.062	26.5	831	197	0.062	99.8
8.22-11.63	4.1	0.058	27.1	643	157	0.058	99.9
11.63-28.83	3.5	0.054	28.1	318	91	0.054	92.8

Phasing

• Phasing: Method: MAD

Processing

Software

Name	Version	Classification	NB
REFMAC	5.2.0019	refinement
PHENIX		refinement
SHARP		phasing
MolProbity	3beta29	model building
SCALA	3.2.5	data scaling
PDB_EXTRACT	3.006	data extraction
MOSFLM		data reduction

Refinement

Method to determine structure: MAD / Resolution: 2.6→28.831 Å / Cor.coef. F_o:F_c: 0.937 / Cor.coef. F_o:F_c free: 0.925 / Occupancy max: 1 / Occupancy min: 0.5 / SU B: 24.026 / SU ML: 0.245 / TLS residual ADP flag: LIKELY RESIDUAL / Cross valid method: THROUGHOUT / σ(F): 0 / ESU R: 0.669 / ESU R Free: 0.318
Stereochemistry target values: MAXIMUM LIKELIHOOD WITH PHASES
Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS OTHER REFINEMENT REMARKS: 1.HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS. 2.ATOM RECORD CONTAINS RESIDUAL B FACTORS ONLY. 3.A MET-INHIBITION PROTOCOL WAS USED FOR SELENOMETHIONINE INCORPORATION DURING PROTEIN EXPRESSION. THE OCCUPANCY OF THE SE ATOMS IN THE MSE RESIDUES WAS REDUCED TO 0.75 TO ACCOUNT FOR THE REDUCED SCATTERING POWER DUE TO PARTIAL S-MET INCORPORATION. 4.PEG MOLECULES FROM CRYSTALLIZATION ARE MODELED INTO THIS STRUCTURE.

	Rfactor	Num. reflection	% reflection	Selection details
Rfree	0.262	498	7.4 %	RANDOM
Rwork	0.227	-	-	-
obs	0.229	6707	99.79 %	-

• 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 max: 84.51 Ų / B_iso mean: 48.516 Ų / B_iso min: 29.27 Ų

	Baniso -1	Baniso -2	Baniso -3
1-	0.28 Å2	0 Å2	0 Å2
2-	-	0.28 Å2	0 Å2
3-	-	-	-0.56 Å2

Refinement step

Cycle: LAST / Resolution: 2.6→28.831 Å

	Protein	Nucleic acid	Ligand	Solvent	Total
Num. atoms	1225	0	28	25	1278

Refine LS restraints

Refine-ID	Type	Dev ideal	Dev ideal target	Number
X-RAY DIFFRACTION	r_bond_refined_d	0.015	0.022	1298
X-RAY DIFFRACTION	r_bond_other_d	0.004	0.02	848
X-RAY DIFFRACTION	r_angle_refined_deg	1.885	1.933	1764
X-RAY DIFFRACTION	r_angle_other_deg	1.532	3	2063
X-RAY DIFFRACTION	r_dihedral_angle_1_deg	2.404	5	158
X-RAY DIFFRACTION	r_dihedral_angle_2_deg	23.456	23.684	57
X-RAY DIFFRACTION	r_dihedral_angle_3_deg	10.173	15	185
X-RAY DIFFRACTION	r_dihedral_angle_4_deg	5.639	15	5
X-RAY DIFFRACTION	r_chiral_restr	0.07	0.2	190
X-RAY DIFFRACTION	r_gen_planes_refined	0.006	0.02	1439
X-RAY DIFFRACTION	r_gen_planes_other	0.003	0.02	266
X-RAY DIFFRACTION	r_nbd_refined	0.132	0.3	186
X-RAY DIFFRACTION	r_nbd_other	0.12	0.3	790
X-RAY DIFFRACTION	r_nbtor_refined	0.162	0.5	595
X-RAY DIFFRACTION	r_nbtor_other	0.077	0.5	633
X-RAY DIFFRACTION	r_xyhbond_nbd_refined	0.134	0.5	65
X-RAY DIFFRACTION	r_symmetry_vdw_refined	0.09	0.3	18
X-RAY DIFFRACTION	r_symmetry_vdw_other	0.085	0.3	42
X-RAY DIFFRACTION	r_symmetry_hbond_refined	0.184	0.5	8
X-RAY DIFFRACTION	r_mcbond_it	1.077	3	816
X-RAY DIFFRACTION	r_mcbond_other	0.173	3	316
X-RAY DIFFRACTION	r_mcangle_it	1.869	5	1277
X-RAY DIFFRACTION	r_scbond_it	3.098	8	570
X-RAY DIFFRACTION	r_scangle_it	4.343	11	487

LS refinement shell

Resolution: 2.6→2.668 Å / Total num. of bins used: 20

	Rfactor	Num. reflection	% reflection
Rfree	0.512	37	-
Rwork	0.34	439	-
all	-	476	-
obs	-	-	100 %

Refinement TLS params.

Method: refined / Origin x: 34.5814 Å / Origin y: 13.8228 Å / Origin z: 20.91 Å

	11	12	13	21	22	23	31	32	33
T	-0.041 Å2	0.1226 Å2	0.1029 Å2	-	0.0195 Å2	-0.0137 Å2	-	-	0.1081 Å2
L	2.4513 °2	-0.0432 °2	0.1002 °2	-	3.3662 °2	0.3986 °2	-	-	4.187 °2
S	0.4224 Å °	0.2154 Å °	0.394 Å °	-0.2277 Å °	-0.3867 Å °	0.4321 Å °	-0.2683 Å °	-0.4346 Å °	-0.0356 Å °