PDB-6xj5: Carboxypeptidase G2 modified with a versatile bioconjugate for me...

ID or keywords:

Entry

Database: PDB / ID: 6xj5

Title

Carboxypeptidase G2 modified with a versatile bioconjugate for metalloprotein design

Components

(Carboxypeptidase G2

Glutamate carboxypeptidase) x 2

Keywords

HYDROLASE / metalloprotein design / bivalent metal ion co-ordination / bis-imidazoles /

bioconjugation

Function / homology

Function and homology information

glutamate carboxypeptidase /

carboxypeptidase activity /

metallopeptidase activity /

proteolysis /

metal ion binding
Similarity search - Function

Biological species

Pseudomonas sp. (bacteria)

Method

X-RAY DIFFRACTION /

SYNCHROTRON /

MOLECULAR REPLACEMENT / Resolution: 3.11 Å

Authors

Yachnin, B.J. / Hansen, W.A. / Khare, S.D.

Funding support

United States, 1items

Citation

Journal: J.Inorg.Biochem. / Year: 2023
Title: A Bis(imidazole)-based cysteine labeling tool for metalloprotein assembly.
Authors: Ahmad, R. / Tyryshkin, A.M. / Xie, L. / Hansen, W.A. / Yachnin, B.J. / Emge, T.J. / Mashrai, A. / Khare, S.D. / Knapp, S.

History

Deposition	Jun 22, 2020	Deposition site: RCSB / Processing site: RCSB
Revision 1.0	Jun 30, 2021	Provider: repository / Type: Initial release
Revision 1.1	Apr 19, 2023	Group: Database references / Category: citation / citation_author / database_2 Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _database_2.pdbx_DOI / _database_2.pdbx_database_accession
Revision 1.2	Oct 25, 2023	Group: Data collection / Refinement description Category: chem_comp_atom / chem_comp_bond / pdbx_initial_refinement_model
Revision 1.3	Nov 15, 2023	Group: Data collection / Derived calculations / Category: chem_comp_atom / chem_comp_bond / struct_conn Item: _chem_comp_atom.atom_id / _chem_comp_bond.atom_id_2 / _struct_conn.pdbx_leaving_atom_flag

Structure viewer	Molecule: MolmilJmol/JSmol

-
Download

PDBx/mmCIF format	6xj5.cif.gz	570.1 KB	Display	PDBx/mmCIF format
PDB format	pdb6xj5.ent.gz	440 KB	Display	PDB format
PDBx/mmJSON format	6xj5.json.gz		Tree view	PDBx/mmJSON format
Others	Other downloads

-
Validation report

Arichive directory	https://data.pdbj.org/pub/pdb/validation_reports/xj/6xj5 ftp://data.pdbj.org/pub/pdb/validation_reports/xj/6xj5	HTTPS FTP

-
Related structure data

Related structure data	1cg2S S: Starting model for refinement
Similar structure data	6xj5-3 6xj5-2 6xj5-4 1mxw-3 7kkm-d 1m5b-3 1mxv-3 4m7e-1 Search similar-shape structures by Omokage search (details) Search similar-shape structures by Omokage search (details) 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

Related structure data

1cg2S

S: Starting model for refinement

Similar structure data

-
Links

PDB pages	PDBj / wwPDB / NCBI
Related items in Molecule of the Month	#110 - Feb 2009 Auxin and TIR1 Ubiquitin Ligase similarity (5)

Deposited unit

A: Carboxypeptidase G2

B: Carboxypeptidase G2

C: Carboxypeptidase G2

D: Carboxypeptidase G2

E: Carboxypeptidase G2

F: Carboxypeptidase G2

G: Carboxypeptidase G2

H: Carboxypeptidase G2

hetero molecules

357 kDa, 8 polymers
Search similar-shape structures of this assembly by Omokage search (details)

1

A: Carboxypeptidase G2

B: Carboxypeptidase G2

hetero molecules

defined by author&software
Evidence: gel filtration, CPG2 has been well established as a dimer in the literature. Gel filtration experiments with this variant confirm that it also behaves as a dimer, as expected.
89 kDa, 2 polymers
Search similar-shape structures of this assembly by Omokage search (details)

2

C: Carboxypeptidase G2

D: Carboxypeptidase G2

hetero molecules

defined by author&software
Evidence: gel filtration, CPG2 has been well established as a dimer in the literature. Gel filtration experiments with this variant confirm that it also behaves as a dimer, as expected.
89.3 kDa, 2 polymers
Search similar-shape structures of this assembly by Omokage search (details)

3

E: Carboxypeptidase G2

F: Carboxypeptidase G2

hetero molecules

defined by author&software
Evidence: gel filtration, CPG2 has been well established as a dimer in the literature. Gel filtration experiments with this variant confirm that it also behaves as a dimer, as expected.
89.3 kDa, 2 polymers
Search similar-shape structures of this assembly by Omokage search (details)

4

G: Carboxypeptidase G2

H: Carboxypeptidase G2

hetero molecules

defined by author&software
Evidence: gel filtration, CPG2 has been well established as a dimer in the literature. Gel filtration experiments with this variant confirm that it also behaves as a dimer, as expected.
89.1 kDa, 2 polymers
Search similar-shape structures of this assembly by Omokage search (details)

Unit cell

Length a, b, c (Å)	72.120, 188.460, 124.500
Angle α, β, γ (deg.)	90.000, 90.170, 90.000
Int Tables number	4
Space group name H-M	P12₁1

Noncrystallographic symmetry (NCS)

NCS domain:
NCS ensembles :

#1: Protein	Carboxypeptidase G2 / Glutamate carboxypeptidase / CPDG2 / Folate hydrolase G2 / Glutamate carboxypeptidase / Pteroylmonoglutamic acid hydrolase G2 Mass: 44006.871 Da / Num. of mol.: 4 / Mutation: S203C Source method: isolated from a genetically manipulated source Source: (gene. exp.) Pseudomonas sp. (strain RS-16) (bacteria) Strain: RS-16 / Gene: cpg2 / Variant: S203C / Plasmid: pSpeedET / Production host: Escherichia coli (E. coli) / References: UniProt: P06621, glutamate carboxypeptidase
#2: Protein	Carboxypeptidase G2 / Glutamate carboxypeptidase / CPDG2 / Folate hydrolase G2 / Glutamate carboxypeptidase / Pteroylmonoglutamic acid hydrolase G2 Mass: 44195.102 Da / Num. of mol.: 4 / Mutation: S203C Source method: isolated from a genetically manipulated source Source: (gene. exp.) Pseudomonas sp. (strain RS-16) (bacteria) Strain: RS-16 / Gene: cpg2 / Variant: S203C / Plasmid: pSpeedET / Production host: Escherichia coli (E. coli) / References: UniProt: P06621, glutamate carboxypeptidase
#3: Chemical	... ChemComp-ZN / ZINC ION Mass: 65.409 Da / Num. of mol.: 56 / Source method: obtained synthetically / Formula: Zn
#4: Chemical	ChemComp-SO4 / SULFATE ION / Sulfate Mass: 96.063 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: SO₄
#5: Water	ChemComp-HOH / water / Water Mass: 18.015 Da / Num. of mol.: 120 / Source method: isolated from a natural source / Formula: H₂O
Has ligand of interest	Y

-
Experiment

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

-
Sample preparation

Crystal	Density Matthews: 2.4 Å³/Da / Density % sol: 48.67 %
Crystal grow	Temperature: 277 K / Method: vapor diffusion, hanging drop / pH: 7.5 Details: Reservoir consisted of 750 uL of 0.2 M Tris pH 7.5, 10% PEG 3350, and 5% glycerol. 2 uL of reservoir solution was mixed with 2 uL of protein solution, consisting of 4.7 mg/mL CPG2 in 50 mM ...Details: Reservoir consisted of 750 uL of 0.2 M Tris pH 7.5, 10% PEG 3350, and 5% glycerol. 2 uL of reservoir solution was mixed with 2 uL of protein solution, consisting of 4.7 mg/mL CPG2 in 50 mM Tris 100 mM NaCl pH 7.4 supplemented with 0.1 M ZnSO4

Crystal

Density Matthews: 2.4 Å³/Da / Density % sol: 48.67 %

Crystal grow

Temperature: 277 K / Method: vapor diffusion, hanging drop / pH: 7.5
Details: Reservoir consisted of 750 uL of 0.2 M Tris pH 7.5, 10% PEG 3350, and 5% glycerol. 2 uL of reservoir solution was mixed with 2 uL of protein solution, consisting of 4.7 mg/mL CPG2 in 50 mM ...

-
Data collection

Diffraction	Mean temperature: 93 K / Serial crystal experiment: N
Diffraction source	Source: SYNCHROTRON / Site: APS / Beamline: 23-ID-D / Wavelength: 1.0332 Å
Detector	Type: DECTRIS PILATUS3 6M / Detector: PIXEL / Date: Jun 30, 2019 / Details: Adjustable focus K-B pair Si plus Pt, Rh coatings
Radiation	Monochromator: Double crystal cryo-cooled / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelength	Wavelength: 1.0332 Å / Relative weight: 1
Reflection	Resolution: 3.11→41.5 Å / Num. obs: 59536 / % possible obs: 99.87 % / Redundancy: 6.6 % / CC_1/2: 0.977 / R_merge(I) obs: 0.389 / R_pim(I) all: 0.164 / R_rim(I) all: 0.423 / R_sym value: 0.389 / Net I/σ(I): 4.3
Reflection shell	Resolution: 3.11→3.16 Å / Redundancy: 6.91 % / R_merge(I) obs: 1.949 / Mean I/σ(I) obs: 1.1 / Num. unique obs: 2967 / CC_1/2: 0.358 / R_pim(I) all: 0.793 / R_rim(I) all: 2.106 / R_sym value: 1.949 / % possible all: 99.93

-
Processing

Software

Name	Version	Classification
REFMAC	5.8.0258	refinement
xia2	0.5.898-g5f4e2fb3-dials-1.14	data reduction
PHASER	2.8.3	phasing
xia2	0.5.898-g5f4e2fb3-dials-1.14	data scaling

Refinement

Method to determine structure:

MOLECULAR REPLACEMENT
Starting model: 1CG2

1cg2

Resolution: 3.11→41.5 Å / Cor.coef. F_o:F_c: 0.881 / Cor.coef. F_o:F_c free: 0.845 / WR_factor R_free: 0.305 / WR_factor R_work: 0.269 / SU B: 36.846 / SU ML: 0.611 / Average fsc free: 0.7976 / Average fsc work: 0.8148 / Cross valid method: THROUGHOUT / ESU R Free: 0.603
Details: Hydrogens have been added in their riding positions

	R_factor	Num. reflection	% reflection
R_free	0.3086	2981	5.009 %
R_work	0.2713	56534	-
all	0.273	-	-
obs	-	59515	99.784 %

Solvent computation

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

Displacement parameters

B_iso mean: 62.855 Å²

	B_aniso -1	B_aniso -2	B_aniso -3
1-	-4.783 Å²	0 Å²	1.683 Å²
2-	-	1.256 Å²	0 Å²
3-	-	-	3.517 Å²

Refinement step

Cycle: LAST / Resolution: 3.11→41.5 Å

	Protein	Nucleic acid	Ligand	Solvent	Total
Num. atoms	21355	0	66	120	21541

Refine LS restraints

Refine-ID	Type	Dev ideal	Dev ideal target	Number
X-RAY DIFFRACTION	r_bond_refined_d	0.003	0.013	21757
X-RAY DIFFRACTION	r_bond_other_d	0	0.017	19373
X-RAY DIFFRACTION	r_angle_refined_deg	1.144	1.638	29633
X-RAY DIFFRACTION	r_angle_other_deg	1.145	1.578	44294
X-RAY DIFFRACTION	r_dihedral_angle_1_deg	4.069	5	3096
X-RAY DIFFRACTION	r_dihedral_angle_2_deg	26.754	23.672	817
X-RAY DIFFRACTION	r_dihedral_angle_3_deg	12.918	15	2817
X-RAY DIFFRACTION	r_dihedral_angle_other_3_deg	4.714	15	4
X-RAY DIFFRACTION	r_dihedral_angle_4_deg	13.485	15	75
X-RAY DIFFRACTION	r_chiral_restr	0.022	0.2	3091
X-RAY DIFFRACTION	r_gen_planes_refined	0.002	0.02	25923
X-RAY DIFFRACTION	r_gen_planes_other	0	0.02	4298
X-RAY DIFFRACTION	r_nbd_refined	0.219	0.2	5275
X-RAY DIFFRACTION	r_symmetry_nbd_other	0.222	0.2	18596
X-RAY DIFFRACTION	r_nbtor_refined	0.152	0.2	10807
X-RAY DIFFRACTION	r_symmetry_nbtor_other	0.08	0.2	9844
X-RAY DIFFRACTION	r_xyhbond_nbd_refined	0.17	0.2	535
X-RAY DIFFRACTION	r_symmetry_xyhbond_nbd_other	0.09	0.2	7
X-RAY DIFFRACTION	r_metal_ion_refined	0.135	0.2	23
X-RAY DIFFRACTION	r_symmetry_nbd_refined	0.278	0.2	88
X-RAY DIFFRACTION	r_nbd_other	0.297	0.2	221
X-RAY DIFFRACTION	r_symmetry_xyhbond_nbd_refined	0.176	0.2	15
X-RAY DIFFRACTION	r_symmetry_metal_ion_refined	0.095	0.2	3
X-RAY DIFFRACTION	r_mcbond_it	0.762	7.269	12417
X-RAY DIFFRACTION	r_mcbond_other	0.762	7.269	12415
X-RAY DIFFRACTION	r_mcangle_it	1.357	10.903	15502
X-RAY DIFFRACTION	r_mcangle_other	1.357	10.903	15502
X-RAY DIFFRACTION	r_scbond_it	0.654	7.228	9340
X-RAY DIFFRACTION	r_scbond_other	0.653	7.228	9336
X-RAY DIFFRACTION	r_scangle_it	1.002	10.85	14131
X-RAY DIFFRACTION	r_scangle_other	1.002	10.849	14126
X-RAY DIFFRACTION	r_lrange_it	3.973	89.764	23613
X-RAY DIFFRACTION	r_lrange_other	3.955	89.794	23598
X-RAY DIFFRACTION	r_ncsr_local_group_1	0.096	0.05	10566
X-RAY DIFFRACTION	r_ncsr_local_group_2	0.094	0.05	10739
X-RAY DIFFRACTION	r_ncsr_local_group_3	0.085	0.05	10473
X-RAY DIFFRACTION	r_ncsr_local_group_4	0.092	0.05	10576
X-RAY DIFFRACTION	r_ncsr_local_group_5	0.08	0.05	10514
X-RAY DIFFRACTION	r_ncsr_local_group_6	0.078	0.05	10603
X-RAY DIFFRACTION	r_ncsr_local_group_7	0.069	0.05	11006
X-RAY DIFFRACTION	r_ncsr_local_group_8	0.094	0.05	10683
X-RAY DIFFRACTION	r_ncsr_local_group_9	0.092	0.05	10494
X-RAY DIFFRACTION	r_ncsr_local_group_10	0.099	0.05	10478
X-RAY DIFFRACTION	r_ncsr_local_group_11	0.082	0.05	10404
X-RAY DIFFRACTION	r_ncsr_local_group_12	0.08	0.05	10612
X-RAY DIFFRACTION	r_ncsr_local_group_13	0.085	0.05	10691
X-RAY DIFFRACTION	r_ncsr_local_group_14	0.09	0.05	10568
X-RAY DIFFRACTION	r_ncsr_local_group_15	0.091	0.05	10621
X-RAY DIFFRACTION	r_ncsr_local_group_16	0.08	0.05	10606
X-RAY DIFFRACTION	r_ncsr_local_group_17	0.079	0.05	10651
X-RAY DIFFRACTION	r_ncsr_local_group_18	0.085	0.05	10813
X-RAY DIFFRACTION	r_ncsr_local_group_19	0.082	0.05	10653
X-RAY DIFFRACTION	r_ncsr_local_group_20	0.076	0.05	10346
X-RAY DIFFRACTION	r_ncsr_local_group_21	0.07	0.05	10421
X-RAY DIFFRACTION	r_ncsr_local_group_22	0.079	0.05	10576
X-RAY DIFFRACTION	r_ncsr_local_group_23	0.076	0.05	10338
X-RAY DIFFRACTION	r_ncsr_local_group_24	0.081	0.05	10463
X-RAY DIFFRACTION	r_ncsr_local_group_25	0.085	0.05	10563
X-RAY DIFFRACTION	r_ncsr_local_group_26	0.071	0.05	10347
X-RAY DIFFRACTION	r_ncsr_local_group_27	0.072	0.05	10549
X-RAY DIFFRACTION	r_ncsr_local_group_28	0.064	0.05	10699

LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Total num. of bins used: 20

Resolution (Å)	R_factor R_free	Num. reflection R_free	R_factor R_work	Num. reflection R_work	R_factor all	Num. reflection all	Fsc free	Fsc work	% reflection obs (%)	WR_factor R_work
3.11-3.19	0.395	195	0.359	4208	0.361	4405	0.508	0.498	99.9546	0.358
3.19-3.277	0.358	210	0.347	4086	0.347	4300	0.61	0.658	99.907	0.343
3.277-3.372	0.37	153	0.327	3924	0.329	4081	0.67	0.7	99.902	0.323
3.372-3.475	0.36	195	0.307	3853	0.309	4048	0.737	0.77	100	0.298
3.475-3.588	0.329	182	0.289	3737	0.291	3920	0.795	0.815	99.9745	0.28
3.588-3.713	0.295	177	0.281	3538	0.281	3732	0.83	0.833	99.5445	0.27
3.713-3.852	0.352	215	0.278	3427	0.282	3642	0.783	0.838	100	0.267
3.852-4.008	0.301	222	0.26	3356	0.262	3582	0.839	0.869	99.8883	0.25
4.008-4.184	0.32	156	0.239	3171	0.242	3333	0.864	0.897	99.82	0.231
4.184-4.386	0.269	175	0.239	3058	0.241	3235	0.874	0.876	99.9382	0.232
4.386-4.62	0.333	172	0.228	2910	0.233	3083	0.836	0.899	99.9676	0.223
4.62-4.897	0.251	148	0.222	2730	0.223	2882	0.898	0.913	99.8612	0.217
4.897-5.23	0.274	166	0.232	2577	0.234	2744	0.911	0.926	99.9636	0.231
5.23-5.641	0.279	114	0.282	2465	0.282	2581	0.887	0.89	99.9225	0.28
5.641-6.168	0.342	118	0.28	2209	0.283	2329	0.853	0.889	99.9141	0.284
6.168-6.877	0.325	103	0.28	2039	0.282	2142	0.863	0.897	100	0.288
6.877-7.905	0.262	95	0.245	1819	0.246	1914	0.917	0.911	100	0.26
7.905-9.595	0.276	91	0.241	1527	0.243	1619	0.929	0.927	99.9382	0.259
9.595-13.221	0.272	58	0.263	1202	0.263	1264	0.896	0.919	99.6835	0.28
13.221-41.5	0.413	36	0.401	698	0.401	779	0.778	0.871	94.2234	0.431

-
News

-
Feb 9, 2022. New format data for meta-information of EMDB entries

New format data for meta-information of EMDB entries

Version 3 of the EMDB header file is now the official format.
The previous official version 1.9 will be removed from the archive.

Related info.:EMDB header

External links:wwPDB to switch to version 3 of the EMDB data model

-
Aug 12, 2020. Covid-19 info

Covid-19 info

URL: https://pdbj.org/emnavi/covid19.php

New page: Covid-19 featured information page in EM Navigator.

Related info.:Covid-19 info / Mar 5, 2020. Novel coronavirus structure data

+
Mar 5, 2020. Novel coronavirus structure data

+
Jan 31, 2019. EMDB accession codes are about to change! (news from PDBe EMDB page)

+
Jul 12, 2017. Major update of PDB

-
Yorodumi

Thousand views of thousand structures

Yorodumi is a browser for structure data from EMDB, PDB, SASBDB, etc.
This page is also the successor to EM Navigator detail page, and also detail information page/front-end page for Omokage search.
The word "yorodu" (or yorozu) is an old Japanese word meaning "ten thousand". "mi" (miru) is to see.

Related info.:EMDB / PDB / SASBDB / Comparison of 3 databanks / Yorodumi Search / Aug 31, 2016. New EM Navigator & Yorodumi / Yorodumi Papers / Jmol/JSmol / Function and homology information / Changes in new EM Navigator and Yorodumi

-
Movie

-
Structure viewers

+
Viewer log

-
This page

-
This web site

-
Options

+
Open data

-
Basic information

-
Structure visualization

-
Downloads & links

-
Download

-
Validation report

-
Related structure data

-
Links

-
Assembly

-
Components

-
Experimental details

-
Experiment

-
Sample preparation

-
Data collection

-
Processing

+
About Yorodumi

-
News

-
Feb 9, 2022. New format data for meta-information of EMDB entries

-
Aug 12, 2020. Covid-19 info

+
Mar 5, 2020. Novel coronavirus structure data

+
Jan 31, 2019. EMDB accession codes are about to change! (news from PDBe EMDB page)

+
Jul 12, 2017. Major update of PDB

-
Yorodumi

-Movie

-Structure viewers

+Viewer log

-This page

-This web site

-Options

+Open data

-Basic information

-Structure visualization

-Downloads & links

-Download

-Validation report

-Related structure data

-Links

-Assembly

-Components

-Experimental details

-Experiment

-Sample preparation

-Data collection

-Processing

+About Yorodumi

-News

-Feb 9, 2022. New format data for meta-information of EMDB entries

-Aug 12, 2020. Covid-19 info

+Mar 5, 2020. Novel coronavirus structure data

+Jan 31, 2019. EMDB accession codes are about to change! (news from PDBe EMDB page)

+Jul 12, 2017. Major update of PDB

-Yorodumi

-
Movie

-
Structure viewers

+
Viewer log

-
This page

-
This web site

-
Options

+
Open data

-
Basic information

-
Structure visualization

-
Downloads & links

-
Download

-
Validation report

-
Related structure data

-
Links

-
Assembly

-
Components

-
Experimental details

-
Experiment

-
Sample preparation

-
Data collection

-
Processing

+
About Yorodumi

-
News

-
Feb 9, 2022. New format data for meta-information of EMDB entries

-
Aug 12, 2020. Covid-19 info

+
Mar 5, 2020. Novel coronavirus structure data

+
Jan 31, 2019. EMDB accession codes are about to change! (news from PDBe EMDB page)

+
Jul 12, 2017. Major update of PDB

-
Yorodumi