PDB-29da: Crystal structure of enterovirus D68-3Cpro in complex with RK-496

Basic information

• Entry: Database: PDB / ID: 29da
• Title: Crystal structure of enterovirus D68-3Cpro in complex with RK-496
• Components: Genome polyprotein
• Keywords: VIRAL PROTEIN / 3Cpro / enterovirus / inhibitor

Function / homology

Function:

picornain 2A / symbiont-mediated suppression of host mRNA export from nucleus / symbiont genome entry into host cell via pore formation in plasma membrane / picornain 3C / T=pseudo3 icosahedral viral capsid / host cell cytoplasmic vesicle membrane / ribonucleoside triphosphate phosphatase activity / nucleoside-triphosphate phosphatase / channel activity / monoatomic ion transmembrane transport / RNA helicase activity / symbiont-mediated suppression of host innate immune response / endocytosis involved in viral entry into host cell / symbiont-mediated activation of host autophagy / RNA-directed RNA polymerase / cysteine-type endopeptidase activity / viral RNA genome replication / RNA-directed RNA polymerase activity / virion attachment to host cell / host cell nucleus / structural molecule activity / proteolysis / DNA-templated transcription / RNA binding / zinc ion binding / ATP binding

Domain/homology:

: / Picornavirus coat protein / Poliovirus 3A protein-like / Poliovirus 3A protein like / Picornavirus 2B protein / Poliovirus core protein 3a, soluble domain / Picornavirus 2B protein / Peptidase C3, picornavirus core protein 2A / Picornavirus core protein 2A / Picornavirus coat protein VP4 / Picornavirus coat protein (VP4) / Peptidase C3A/C3B, picornaviral / 3C cysteine protease (picornain 3C) / Picornavirales 3C/3C-like protease domain / Picornavirales 3C/3C-like protease domain profile. / Picornavirus capsid / picornavirus capsid protein / Helicase, superfamily 3, single-stranded RNA virus / Superfamily 3 helicase of positive ssRNA viruses domain profile. / Helicase, superfamily 3, single-stranded DNA/RNA virus / RNA helicase / Picornavirus/Calicivirus coat protein / Viral coat protein subunit / Reverse transcriptase/Diguanylate cyclase domain / RNA-directed RNA polymerase, C-terminal domain / Viral RNA-dependent RNA polymerase / RNA-directed RNA polymerase, catalytic domain / RdRp of positive ssRNA viruses catalytic domain profile. / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / Peptidase S1, PA clan, chymotrypsin-like fold / Peptidase S1, PA clan / DNA/RNA polymerase superfamily / P-loop containing nucleoside triphosphate hydrolase

Component:

: / Genome polyprotein

• Biological species: Enterovirus
• Method: X-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.08 Å
• Authors: El Kilani, H.

Funding support

European Union, 1items

Organization	Grant number	Country
European Commission		European Union

• Citation: Journal: Commun Chem / Year: 2026; Title: Structure-based macrocyclization of alpha-ketoamides leads to potent inhibitors of coronaviral and enteroviral proteases.; Authors: Akula, R.K. / El Kilani, H. / Metzen, A. / Joshi, S. / Durmaz, H. / Veenstra, R. / Roske, J. / Hurdiss, D.L. / Van Kuppeveld, F.J.M. / Rox, K. / Hilgenfeld, R. / Bronstrup, M.

History

Deposition	Mar 6, 2026	Deposition site: PDBE / Processing site: PDBE
Revision 1.0	Aug 19, 2026	Provider: repository / Type: Initial release

Assembly

Deposited unit

A: Genome polyprotein

hetero molecules

Summary:

• 21.6 kDa, 1 polymers

Component details:

	Theoretical mass	Number of molelcules
Total (without water)	21,580	2
Polymers	21,025	1
Non-polymers	556	1
Water	1,045	58

1

Summary:

• Idetical with deposited unit
• defined by author&software
• Evidence: native gel electrophoresis

Symmetry operations:

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

Calculated values:

Buried area	0 Å2
ΔGint	0 kcal/mol
Surface area	9150 Å2
Method	PISA

Unit cell

Length a, b, c (Å)	56.210, 56.210, 170.660
Angle α, β, γ (deg.)	90.000, 90.000, 120.000
Int Tables number	152
Space group name H-M	P3121

Components

#1: Protein

A Genome polyprotein

Details:

Mass: 21024.873 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Enterovirus / Production host: Escherichia coli (E. coli)
References: UniProt: A1E4A3, picornain 2A, nucleoside-triphosphate phosphatase, picornain 3C, RNA-directed RNA polymerase

#2: Chemical

A-201 ChemComp-A1JQY / 1-[(3~{S},6~{S},7~{R})-7-oxidanyl-4,8-bis(oxidanylidene)-6-[[(3~{S})-2-oxidanylidenepyrrolidin-3-yl]methyl]-5,9-diazabicyclo[14.3.1]icosa-1(19),16(20),17-trien-3-yl]-3-[2,2,2-tris(fluoranyl)ethyl]urea

Details:

Mass: 555.590 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C₂₆H₃₆F₃N₅O₅ / Feature type: SUBJECT OF INVESTIGATION

#3: Water

ChemComp-HOH / water

Details:

Mass: 18.015 Da / Num. of mol.: 58 / Source method: isolated from a natural source / Formula: H₂O

• Has ligand of interest: Y
• Has protein modification: Y

Experimental details

Experiment

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

Sample preparation

• Crystal: Density Matthews: 3.7 ų/Da / Density % sol: 66.77 %
• Crystal grow: Temperature: 277 K / Method: vapor diffusion, sitting drop; Details: 0.1 M Tris-HCl pH 8, 0.2 M Ammonium acetate, 25% PEG 3350

Data collection

• Diffraction: Mean temperature: 100 K / Serial crystal experiment: N
• Diffraction source: Source: SYNCHROTRON / Site: PETRA III, DESY / Beamline: P11 / Wavelength: 1.0332 Å
• Detector: Type: DECTRIS EIGER X 16M / Detector: PIXEL / Date: Jan 9, 2026
• Radiation: Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
• Radiation wavelength: Wavelength: 1.0332 Å / Relative weight: 1
• Reflection: Resolution: 2.08→48.68 Å / Num. obs: 19582 / % possible obs: 99.87 % / Redundancy: 9.8 % / CC_1/2: 0.999 / Net I/σ(I): 15.07
• Reflection shell: Resolution: 2.08→2.154 Å / Num. unique obs: 1930 / CC_1/2: 0.674

Processing

Software

Name	Version	Classification
REFMAC	5.8.0425	refinement
XDS		data reduction
XDS		data scaling
MOLREP		phasing

Refinement

Method to determine structure: MOLECULAR REPLACEMENT / Resolution: 2.08→48.679 Å / Cor.coef. F_o:F_c: 0.968 / Cor.coef. F_o:F_c free: 0.957 / SU B: 6.094 / SU ML: 0.147 / Cross valid method: FREE R-VALUE / ESU R: 0.151 / ESU R Free: 0.144
Details: Hydrogens have been added in their riding positions

	Rfactor	Num. reflection	% reflection
Rfree	0.2338	980	5.005 %
Rwork	0.2002	18602	-
all	0.202	-	-
obs	-	19582	99.883 %

• 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: 60.368 Ų

	Baniso -1	Baniso -2	Baniso -3
1-	1.809 Å2	0.904 Å2	0 Å2
2-	-	1.809 Å2	-0 Å2
3-	-	-	-5.868 Å2

Refinement step

Cycle: LAST / Resolution: 2.08→48.679 Å

	Protein	Nucleic acid	Ligand	Solvent	Total
Num. atoms	1469	0	39	58	1566

Refine LS restraints

Refine-ID	Type	Dev ideal	Dev ideal target	Number
X-RAY DIFFRACTION	r_bond_refined_d	0.007	0.012	1545
X-RAY DIFFRACTION	r_bond_other_d	0.001	0.016	1436
X-RAY DIFFRACTION	r_angle_refined_deg	1.775	1.825	2096
X-RAY DIFFRACTION	r_angle_other_deg	0.677	1.762	3295
X-RAY DIFFRACTION	r_dihedral_angle_1_deg	7.631	5	187
X-RAY DIFFRACTION	r_dihedral_angle_2_deg	7.231	5	13
X-RAY DIFFRACTION	r_dihedral_angle_other_2_deg	5.37	5	4
X-RAY DIFFRACTION	r_dihedral_angle_3_deg	15.699	10	242
X-RAY DIFFRACTION	r_dihedral_angle_other_3_deg	0.43	10	1
X-RAY DIFFRACTION	r_dihedral_angle_6_deg	13.868	10	71
X-RAY DIFFRACTION	r_chiral_restr	0.097	0.2	235
X-RAY DIFFRACTION	r_gen_planes_refined	0.007	0.02	1818
X-RAY DIFFRACTION	r_gen_planes_other	0.001	0.02	366
X-RAY DIFFRACTION	r_nbd_refined	0.202	0.2	270
X-RAY DIFFRACTION	r_symmetry_nbd_other	0.207	0.2	1302
X-RAY DIFFRACTION	r_nbtor_refined	0.183	0.2	761
X-RAY DIFFRACTION	r_symmetry_nbtor_other	0.093	0.2	836
X-RAY DIFFRACTION	r_xyhbond_nbd_refined	0.123	0.2	39
X-RAY DIFFRACTION	r_symmetry_nbd_refined	0.233	0.2	8
X-RAY DIFFRACTION	r_nbd_other	0.195	0.2	76
X-RAY DIFFRACTION	r_symmetry_xyhbond_nbd_refined	0.178	0.2	2
X-RAY DIFFRACTION	r_mcbond_it	5.065	5.869	751
X-RAY DIFFRACTION	r_mcbond_other	5.064	5.874	752
X-RAY DIFFRACTION	r_mcangle_it	6.796	10.531	938
X-RAY DIFFRACTION	r_mcangle_other	6.786	10.529	938
X-RAY DIFFRACTION	r_scbond_it	7.15	6.781	794
X-RAY DIFFRACTION	r_scbond_other	7.146	6.776	792
X-RAY DIFFRACTION	r_scangle_it	10.323	12.048	1158
X-RAY DIFFRACTION	r_scangle_other	10.319	12.048	1159
X-RAY DIFFRACTION	r_lrange_it	12.132	59.248	1614
X-RAY DIFFRACTION	r_lrange_other	12.128	59.27	1614

LS refinement shell

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

Resolution (Å)	Rfactor Rfree	Num. reflection Rfree	Rfactor Rwork	Num. reflection Rwork	Rfactor all	Num. reflection all	Fsc free	Fsc work	% reflection obs (%)	WRfactor Rwork
2.08-2.134	0.383	71	0.386	1347	0.386	1420	0.853	0.863	99.8592	0.381
2.134-2.192	0.442	69	0.384	1298	0.387	1370	0.823	0.853	99.781	0.37
2.192-2.256	0.379	68	0.353	1297	0.354	1368	0.86	0.878	99.7807	0.335
2.256-2.325	0.353	65	0.315	1231	0.317	1297	0.917	0.908	99.9229	0.297
2.325-2.401	0.398	63	0.275	1198	0.28	1262	0.871	0.935	99.9208	0.252
2.401-2.485	0.279	61	0.244	1168	0.246	1230	0.955	0.955	99.9187	0.219
2.485-2.579	0.27	60	0.236	1127	0.238	1187	0.939	0.961	100	0.206
2.579-2.684	0.285	57	0.225	1088	0.228	1147	0.94	0.967	99.8256	0.196
2.684-2.803	0.222	55	0.185	1057	0.187	1112	0.967	0.978	100	0.155
2.803-2.939	0.255	53	0.171	1001	0.175	1054	0.968	0.981	100	0.146
2.939-3.097	0.259	51	0.188	961	0.192	1012	0.947	0.975	100	0.164
3.097-3.284	0.237	48	0.201	915	0.203	963	0.957	0.974	100	0.18
3.284-3.51	0.241	46	0.21	867	0.212	913	0.961	0.974	100	0.195
3.51-3.789	0.236	41	0.213	793	0.214	834	0.972	0.974	100	0.2
3.789-4.149	0.2	39	0.193	742	0.194	781	0.978	0.979	100	0.19
4.149-4.634	0.171	37	0.147	688	0.148	726	0.986	0.987	99.8623	0.154
4.634-5.343	0.162	31	0.132	593	0.134	632	0.985	0.99	98.7342	0.148
5.343-6.524	0.283	28	0.196	532	0.201	560	0.973	0.981	100	0.204
6.524-9.145	0.269	22	0.202	425	0.205	447	0.963	0.978	100	0.216
9.145-48.679	0.135	15	0.175	274	0.173	289	0.982	0.98	100	0.208