PDB-35yh: Crystal structure of covalent inhibitor 3-(2-chloroacetamido)-N-(...

ID or keywords:

Entry

Database: PDB / ID: 35yh

Title

Crystal structure of covalent inhibitor 3-(2-chloroacetamido)-N-(3-hydroxyphenyl)benzamide bound to Ubiquitin C-terminal Hydrolase-L3

Components

Ubiquitin carboxyl-terminal hydrolase isozyme L3

Keywords

HYDROLASE/HYDROLASE INHIBITOR / UCHL3 / chloroacetamide / covalent inhibitor / HYDROLASE / HYDROLASE-HYDROLASE INHIBITOR complex

Function / homology

Function and homology information

deNEDDylase activity / protein deubiquitination / protein catabolic process / post-translational protein modification / Synthesis of active ubiquitin: roles of E1 and E2 enzymes / ubiquitin binding / peptidase activity / UCH proteinases / Neddylation / ubiquitin-dependent protein catabolic process ...
Similarity search - Function

Biological species

Homo sapiens (human)

Method

X-RAY DIFFRACTION /

MOLECULAR REPLACEMENT / Resolution: 2.6 Å

Authors

Pannala, N. / Patel, R. / Flaherty, D. / Das, C.

Funding support

1items

Citation

Journal: Biorxiv / Year: 2026
Title: Identification, optimization, and structural elucidation of chloroacetamide scaffold as covalent inhibitors for Ubiquitin C-terminal Hydrolase L3
Authors: Beeralingappa, N.C. / Lu, M. / Patel, R. / Pannala, N. / Dhiman, A. / Heil, B.N. / Imhoff, R.D. / Smith, E.G. / Bahler, M.B. / Marsden, H.L. / Allen-Petersen, B.L. / Wendt, M.K. / Das, C. / Flaherty, D.P.

History

Deposition	May 21, 2026	Deposition site: RCSB / Processing site: RCSB
Revision 1.0	Aug 26, 2026	Provider: repository / Type: Initial release

Structure viewer	Molecule: MolmilJmol/JSmol

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Download

PDBx/mmCIF format	35yh.cif.gz	330.1 KB	Display	PDBx/mmCIF format
PDB format	pdb35yh.ent.gz		Display	PDB format
PDBx/mmJSON format	35yh.json.gz		Tree view	PDBx/mmJSON format
Others	Other downloads

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Validation report

Arichive directory	https://data.pdbj.org/pub/pdb/validation_reports/5y/35yh ftp://data.pdbj.org/pub/pdb/validation_reports/5y/35yh	HTTPS FTP

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Related structure data

Related structure data	13cuC 13cvC 13cwC 13dfC C: citing same article (ref.)
Similar structure data	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
Related items in Molecule of the Month	#60 - Dec 2004 Ubiquitin similarity (1) #234 - Jun 2019 MDM2 and Cancer similarity (1)

Deposited unit

B: Ubiquitin carboxyl-terminal hydrolase isozyme L3

A: Ubiquitin carboxyl-terminal hydrolase isozyme L3

C: Ubiquitin carboxyl-terminal hydrolase isozyme L3

D: Ubiquitin carboxyl-terminal hydrolase isozyme L3

hetero molecules

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

1

B: Ubiquitin carboxyl-terminal hydrolase isozyme L3

hetero molecules

defined by author
27.3 kDa, 1 polymers
Search similar-shape structures of this assembly by Omokage search (details)

2

A: Ubiquitin carboxyl-terminal hydrolase isozyme L3

hetero molecules

defined by author
27.3 kDa, 1 polymers
Search similar-shape structures of this assembly by Omokage search (details)

3

C: Ubiquitin carboxyl-terminal hydrolase isozyme L3

hetero molecules

defined by author
27.3 kDa, 1 polymers
Search similar-shape structures of this assembly by Omokage search (details)

4

D: Ubiquitin carboxyl-terminal hydrolase isozyme L3

hetero molecules

defined by author
27.3 kDa, 1 polymers
Search similar-shape structures of this assembly by Omokage search (details)

Unit cell

Length a, b, c (Å)	47.868, 84.991, 123.859
Angle α, β, γ (deg.)	90.00, 97.93, 90.00
Int Tables number	4
Space group name H-M	P12₁1

#1: Protein	Ubiquitin carboxyl-terminal hydrolase isozyme L3 / UCH-L3 / Ubiquitin thioesterase L3 Mass: 26998.436 Da / Num. of mol.: 4 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Homo sapiens (human) / Gene: UCHL3 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P15374, ubiquitinyl hydrolase 1
#2: Chemical	ChemComp-A1DIP / 3-(2-chloroacetamido)-N-(3-hydroxyphenyl)benzamide Mass: 304.728 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C₁₅H₁₃ClN₂O₃ / Feature type: SUBJECT OF INVESTIGATION
#3: Water	ChemComp-HOH / water Mass: 18.015 Da / Num. of mol.: 27 / Source method: isolated from a natural source / Formula: H₂O
Has ligand of interest	Y
Has protein modification	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: 2.31 Å³/Da / Density % sol: 46.77 %
Crystal grow	Temperature: 298 K / Method: vapor diffusion, sitting drop / Details: Bis-tris propane, ammonium sulfate

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Data collection

Diffraction	Mean temperature: 100 K / Serial crystal experiment: N
Diffraction source	Source: ROTATING ANODE / Type: RIGAKU MICROMAX-007 HF / Wavelength: 1.54184 Å
Detector	Type: DECTRIS EIGER2 R 4M / Detector: PIXEL / Date: Jan 16, 2026
Radiation	Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelength	Wavelength: 1.54184 Å / Relative weight: 1
Reflection	Resolution: 2.6→20.15 Å / Num. obs: 30188 / % possible obs: 99.02 % / Redundancy: 4.9 % / B_iso Wilson estimate: 56.35 Å² / CC_1/2: 0.999 / CC star: 1 / Net I/σ(I): 11.04
Reflection shell	Resolution: 2.6→2.693 Å / Redundancy: 5 % / Mean I/σ(I) obs: 0.9 / Num. unique obs: 3032 / CC_1/2: 0.372 / CC star: 0.736 / % possible all: 99.8

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Processing

Software

Name	Version	Classification
PHENIX	(1.20.1_4487: ???)	refinement
CrysalisPro		data scaling
CrysalisPro		data reduction
PHENIX		phasing
PDB_EXTRACT		data extraction

Refinement

Method to determine structure:

MOLECULAR REPLACEMENT / Resolution: 2.6→20.15 Å / SU ML: 0.45 / Cross valid method: FREE R-VALUE / σ(F): 1.34 / Phase error: 36.91 / Stereochemistry target values: ML

	R_factor	Num. reflection	% reflection
R_free	0.3159	1987	6.59 %
R_work	0.2574	-	-
obs	0.2613	30148	99.27 %

Solvent computation

Shrinkage radii: 0.9 Å / VDW probe radii: 1.1 Å / Solvent model: FLAT BULK SOLVENT MODEL

Refinement step

Cycle: LAST / Resolution: 2.6→20.15 Å

	Protein	Nucleic acid	Ligand	Solvent	Total
Num. atoms	6110	0	80	27	6217

Refine LS restraints

Refine-ID	Type	Dev ideal	Number
X-RAY DIFFRACTION	f_bond_d	0.004	6315
X-RAY DIFFRACTION	f_angle_d	0.655	8548
X-RAY DIFFRACTION	f_dihedral_angle_d	4.082	837
X-RAY DIFFRACTION	f_chiral_restr	0.068	958
X-RAY DIFFRACTION	f_plane_restr	0.005	1113

LS refinement shell

Resolution (Å)	R_factor R_free	Num. reflection R_free	R_factor R_work	Num. reflection R_work	Refine-ID	% reflection obs (%)
2.6-2.66	0.4171	144	0.3914	2039	X-RAY DIFFRACTION	99
2.66-2.74	0.4517	142	0.3662	1974	X-RAY DIFFRACTION	100
2.74-2.82	0.4019	141	0.359	2035	X-RAY DIFFRACTION	100
2.82-2.91	0.418	144	0.3537	1974	X-RAY DIFFRACTION	100
2.91-3.01	0.3896	138	0.3196	2038	X-RAY DIFFRACTION	100
3.01-3.13	0.3227	142	0.3061	1999	X-RAY DIFFRACTION	100
3.13-3.27	0.3326	148	0.2957	2006	X-RAY DIFFRACTION	100
3.27-3.45	0.3425	142	0.2781	2017	X-RAY DIFFRACTION	100
3.45-3.66	0.326	143	0.2585	2025	X-RAY DIFFRACTION	100
3.66-3.94	0.3349	136	0.2419	2000	X-RAY DIFFRACTION	99
3.94-4.33	0.2955	143	0.2194	2014	X-RAY DIFFRACTION	99
4.33-4.95	0.2571	138	0.208	1992	X-RAY DIFFRACTION	99
4.95-6.21	0.3239	141	0.2355	2034	X-RAY DIFFRACTION	99
6.21-20.15	0.2408	145	0.2215	2014	X-RAY DIFFRACTION	97

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	5.0838	-4.9871	-4.9166	9.2751	4.5477	4.7356	-0.6862	0.5058	0.2356	0.6796	0.4097	0.5034	0.7745	-0.023	0.3049	0.4678	-0.1177	-0.0402	0.6821	0.1501	0.6018	23.1089	-15.2254	62.2893
2	2.5731	2.0539	-1.2928	3.1331	-2.2095	1.8309	-0.1556	-0.2737	-0.5851	0.0017	-0.3124	-0.4812	0.3104	-0.2879	0.3456	0.3639	0.0542	0.0099	0.5732	0.1601	0.5061	12.4862	-18.4392	59.7334
3	2.0479	1.8173	0.5622	3.9938	0.7918	3.9136	0.0177	0.0773	0.1414	-0.0572	0.1182	0.2953	0.234	-0.2603	-0.0541	0.2449	-0.0628	-0	0.3059	0.0329	0.3592	11.0688	0.2372	47.9667
4	1.4603	0.3602	0.4664	3.4073	-0.1928	1.9506	0.1183	-0.4666	-0.1615	0.2367	-0.0483	-0.2117	0.0544	0.0096	-0.0358	0.2275	-0.0465	-0.0127	0.4524	0.0834	0.2964	13.6625	-6.1967	57.9068
5	1.8646	-1.4424	0.6741	8.0828	-0.6903	0.2444	-0.1129	0.4099	1.1541	0.6031	-0.4736	-0.0632	-0.1395	-0.404	0.487	0.5167	-0.0245	-0.2565	0.4463	-0.0336	0.6393	24.779	10.6807	20.4177
6	5.4137	4.5444	-0.4233	4.4658	-2.1862	5.4562	-1.0013	0.1546	-0.1133	0.0157	0.2657	1.2302	-0.2497	0.0544	0.6112	0.9951	0.0208	0.0452	0.461	0.1499	0.6122	33.4612	15.4181	21.5006
7	2.6272	2.2354	0.7226	3.0946	0.0179	3.5001	-0.2394	0.2702	0.1088	-1.2974	0.0319	-0.6244	-0.4992	0.0044	0.0472	0.6862	0.1035	0.122	0.3021	-0.0368	0.3673	39.1408	8.7388	22.288
8	3.2976	3.0181	1.1119	4.0131	2.1808	1.4652	0.871	-0.2954	-0.682	-0.6152	-0.3208	-0.7437	0.7625	0.3571	-0.2568	0.5599	0.0945	0.0017	0.4107	0.1052	0.4355	38	-7.4044	39.0465
9	2.597	3.4603	0.41	6.2922	0.1732	1.5476	0.3934	-0.1609	0.4657	0.2903	-0.2628	0.4972	-0.2753	0.0488	-0.0424	0.521	0.0423	0.0939	0.3045	-0.0517	0.4246	30.7123	9.3676	37.5506
10	4.635	1.2378	-1.1149	9.2388	-4.4992	6.9169	-0.2525	-0.1698	0.6445	1.995	0.3093	1.2602	-1.0757	-1.3138	-0.1085	0.5882	-0.0065	0.1906	0.5965	-0.241	0.822	23.2975	19.5961	37.5342
11	5.4568	2.5886	-2.8402	4.7105	-2.9209	5.9674	-0.6413	-0.9941	0.0617	0.0395	0.2132	2.2201	0.1564	-0.7672	-0.2341	0.6385	-0.1384	0.3165	0.5532	-0.1674	0.8307	21.0999	8.1561	42.6484
12	1.9806	3.5335	-3.238	7.722	3.8567	4.6164	1.7699	-0.4806	2.5484	-0.0749	-1.7038	1.8042	-1.2956	-2.235	-0.1371	0.7094	0.1047	0.0669	0.8096	0.2375	1.3849	15.5668	9.508	31.8238
13	3.0804	2.6621	-0.2567	6.0698	0.2794	3.4482	0.2585	0.2324	-0.151	-0.0916	0.0145	-0.2197	0.2583	0.1111	-0.2869	0.3446	0.0716	-0.0379	0.3508	0.005	0.3369	34.5936	3.8774	33.9737
14	1.4998	2.6575	-0.1655	8.9467	0.8837	3.7446	0.3514	0.2484	0.0181	-0.5937	-0.2164	-0.8722	0.3164	0.2279	-0.0803	0.4515	0.1353	-0.0014	0.5159	0.12	0.3716	45.1965	7.5513	28.7044
15	1.596	0.7836	0.0951	9.7091	0.9999	4.83	0.2312	0.2637	-0.3876	-0.1858	0.2925	-1.231	1.1317	0.1532	0.1406	0.8652	-0.0295	0.1127	0.4491	0.1162	0.025	37.2825	-0.8018	22.4451
16	4.1084	0.7498	2.8817	3.2522	-3.4272	7.792	-0.1967	-0.053	0.2954	-0.7986	0.7124	-0.3355	-0.7892	0.5147	-0.4434	1.3558	-0.1558	0.4144	0.5016	-0.1952	0.518	51.2524	-12.7572	1.5567
17	1.3582	-0.526	-1.0344	3.4274	0.7936	2.7898	0.1771	0.1716	0.0508	-0.4198	-0.0298	-0.0657	-0.0541	-0.0333	-0.1771	1.1259	0.0057	0.0944	0.5166	-0.0047	0.3803	40.6972	-21.293	0.4753
18	7.126	3.1087	0.341	5.7743	0.5709	7.6749	-0.5793	0.557	-0.6033	-1.3357	-0.1912	2.2543	1.8527	0.7094	1.0862	1.5544	-0.1747	-0.0227	0.6495	0.062	0.7358	18.3775	-41.828	30.758
19	3.0087	0.9371	1.2282	3.0795	-0.4974	0.6759	-0.6946	0.2738	-0.1728	-1.2484	0.0316	-0.873	0.9036	0.127	0.6436	0.8723	0.1156	0.2283	0.3659	-0.0556	0.7119	31.8893	-32.4667	37.6174
20	3.3725	2.3793	1.8563	1.7253	1.2577	1.154	-0.8198	-0.5468	-1.7826	-0.1147	0.4602	-0.9964	0.7554	0.2716	-0.21	1.4282	0.4024	0.6029	0.6164	0.0591	0.9699	41.7365	-40.3555	24.5056
21	3.4074	1.7518	0.3898	4.9805	-0.7786	0.707	-0.0247	-0.1417	-0.6803	0.0343	-0.0995	0.8544	1.8151	0.9189	-0.0107	1.7097	0.136	0.0325	0.6532	0.0128	0.8595	27.499	-39.3684	20.497
22	1.6688	1.1747	-0.3845	4.6535	3.8337	4.4673	-0.8525	0.3257	-1.2022	-1.6098	-0.1131	0.3709	-0.0402	0.2841	0.429	1.4563	0.534	0.3841	0.5787	-0.0376	0.903	32.7432	-35.5521	24.7592
23	6.5623	3.0408	-2.0233	4.5101	-4.9696	5.8804	0.2076	-0.7231	0.1675	-0.5862	-0.5132	-0.09	0.5184	0.7104	0.0993	0.8464	0.1439	0.2879	0.5233	-0.0046	1.0806	35.8762	-34.9793	36.3621
24	4.8496	-3.5393	-0.0911	2.5994	-0.3804	5.4664	0.845	-0.4776	0.1299	-1.0079	-1.8192	-1.3406	-0.2021	0.3045	0.8784	0.8493	-0.1172	0.3322	0.5922	0.1257	0.7289	25.3728	-30.8001	38.89

Refinement TLS group

ID	Refine-ID	Refine TLS-ID	Selection details
1	X-RAY DIFFRACTION	1	chain 'B' and (resid 6 through 22 )
2	X-RAY DIFFRACTION	2	chain 'B' and (resid 23 through 59 )
3	X-RAY DIFFRACTION	3	chain 'B' and (resid 60 through 94 )
4	X-RAY DIFFRACTION	4	chain 'B' and (resid 95 through 228 )
5	X-RAY DIFFRACTION	5	chain 'A' and (resid 5 through 22 )
6	X-RAY DIFFRACTION	6	chain 'A' and (resid 23 through 38 )
7	X-RAY DIFFRACTION	7	chain 'A' and (resid 39 through 59 )
8	X-RAY DIFFRACTION	8	chain 'A' and (resid 60 through 75 )
9	X-RAY DIFFRACTION	9	chain 'A' and (resid 76 through 110 )
10	X-RAY DIFFRACTION	10	chain 'A' and (resid 111 through 130 )
11	X-RAY DIFFRACTION	11	chain 'A' and (resid 131 through 139 )
12	X-RAY DIFFRACTION	12	chain 'A' and (resid 140 through 161 )
13	X-RAY DIFFRACTION	13	chain 'A' and (resid 162 through 193 )
14	X-RAY DIFFRACTION	14	chain 'A' and (resid 194 through 214 )
15	X-RAY DIFFRACTION	15	chain 'A' and (resid 215 through 228 )
16	X-RAY DIFFRACTION	16	chain 'C' and (resid 6 through 38 )
17	X-RAY DIFFRACTION	17	chain 'C' and (resid 39 through 229 )
18	X-RAY DIFFRACTION	18	chain 'D' and (resid 7 through 33 )
19	X-RAY DIFFRACTION	19	chain 'D' and (resid 34 through 75 )
20	X-RAY DIFFRACTION	20	chain 'D' and (resid 76 through 86 )
21	X-RAY DIFFRACTION	21	chain 'D' and (resid 87 through 126 )
22	X-RAY DIFFRACTION	22	chain 'D' and (resid 127 through 183 )
23	X-RAY DIFFRACTION	23	chain 'D' and (resid 184 through 215 )
24	X-RAY DIFFRACTION	24	chain 'D' and (resid 216 through 230 )

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