PDB-4nh5: Structure of human Dicer Platform-PAZ-Connector Helix cassette in...

Basic information

• Entry: Database: PDB / ID: 4nh5
• Title: Structure of human Dicer Platform-PAZ-Connector Helix cassette in complex with 14-mer siRNA having 5'-pUU and UU-3' ends (2.55 Angstrom resolution)

Components

• 5'-R(P*UP*UP*GP*CP*GP*AP*AP*UP*UP*CP*GP*CP*UP*U)-3'
• Endoribonuclease Dicer

• Keywords: HYDROLASE/RNA / PAZ domain / platform domain / connector helix / siRNA / RNase III domain / endoribonuclease / pre-miRNA / HYDROLASE-RNA complex

Function / homology

Function:

peripheral nervous system myelin formation / global gene silencing by mRNA cleavage / pre-miRNA binding / negative regulation of Schwann cell proliferation / tRNA-derived small RNA (tsRNA or tRNA-related fragment, tRF) biogenesis / Small interfering RNA (siRNA) biogenesis / apoptotic DNA fragmentation / tRNA decay / ribonuclease III / deoxyribonuclease I activity / positive regulation of Schwann cell differentiation / nerve development / RISC-loading complex / positive regulation of myelination / miRNA metabolic process / RISC complex assembly / ribonuclease III activity / miRNA processing / pre-miRNA processing / siRNA processing / siRNA binding / M-decay: degradation of maternal mRNAs by maternally stored factors / Regulation of MITF-M-dependent genes involved in apoptosis / RISC complex / MicroRNA (miRNA) biogenesis / negative regulation of tumor necrosis factor production / negative regulation of tumor necrosis factor-mediated signaling pathway / neuron projection morphogenesis / RNA endonuclease activity / helicase activity / double-stranded RNA binding / protein domain specific binding / negative regulation of gene expression / perinuclear region of cytoplasm / negative regulation of transcription by RNA polymerase II / RNA binding / extracellular exosome / ATP binding / metal ion binding / nucleus / cytoplasm / cytosol

Domain/homology:

Dicer, double-stranded RNA-binding domain / : / Dicer, partner-binding domain / Dicer, dsRNA-binding domain / paz domain / paz domain / : / Dicer, platform domain / Dicer dimerisation domain / Dicer dimerisation domain superfamily / Dicer dimerisation domain / Dicer double-stranded RNA-binding fold domain profile. / Ribonuclease III family signature. / Ribonuclease III domain / Ribonuclease III family domain profile. / Ribonuclease III family / Ribonuclease III domain / PAZ domain superfamily / PAZ / PAZ domain / PAZ domain profile. / PAZ domain / Double-stranded RNA binding motif / Ribonuclease III, endonuclease domain superfamily / Double stranded RNA-binding domain (dsRBD) profile. / Double-stranded RNA-binding domain / DEAD/DEAH box helicase domain / DEAD/DEAH box helicase / Beta Complex / Helicase conserved C-terminal domain / helicase superfamily c-terminal domain / Superfamilies 1 and 2 helicase C-terminal domain profile. / Superfamilies 1 and 2 helicase ATP-binding type-1 domain profile. / DEAD-like helicases superfamily / Helicase, C-terminal / Helicase superfamily 1/2, ATP-binding domain / P-loop containing nucleoside triphosphate hydrolase / Mainly Beta

Component:

RNA / RNA (> 10) / Endoribonuclease Dicer

• Biological species: Homo sapiens (human)
• Method: X-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / molecular replacement / Resolution: 2.55 Å
• Authors: Tian, Y. / Simanshu, D.K. / Patel, D.J.
• Citation: Journal: Mol.Cell / Year: 2014; Title: A Phosphate-Binding Pocket within the Platform-PAZ-Connector Helix Cassette of Human Dicer.; Authors: Tian, Y. / Simanshu, D.K. / Ma, J.B. / Park, J.E. / Heo, I. / Kim, V.N. / Patel, D.J.

History

Deposition	Nov 4, 2013	Deposition site: RCSB / Processing site: RCSB
Revision 1.0	Mar 5, 2014	Provider: repository / Type: Initial release
Revision 1.1	Mar 12, 2014	Group: Database references
Revision 1.2	Nov 15, 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 1.3	Sep 20, 2023	Group: Data collection / Database references / Refinement description Category: chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model / struct_ref_seq_dif Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession / _struct_ref_seq_dif.details

Assembly

Deposited unit

A: Endoribonuclease Dicer

B: 5'-R(P*UP*UP*GP*CP*GP*AP*AP*UP*UP*CP*GP*CP*UP*U)-3'

Summary:

• 39.3 kDa, 2 polymers

Component details:

	Theoretical mass	Number of molelcules
Total (without water)	39,330	2
Polymers	39,330	2
Non-polymers	0	0
Water	162	9

1

B: 5'-R(P*UP*UP*GP*CP*GP*AP*AP*UP*UP*CP*GP*CP*UP*U)-3'

A: Endoribonuclease Dicer

B: 5'-R(P*UP*UP*GP*CP*GP*AP*AP*UP*UP*CP*GP*CP*UP*U)-3'

Summary:

• defined by author
• 43.7 kDa, 3 polymers

Component details:

	Theoretical mass	Number of molelcules
Total (without water)	43,732	3
Polymers	43,732	3
Non-polymers	0	0
Water	18	1

Symmetry operations:

Type	Name	Symmetry operation	Number
crystal symmetry operation	2_555	-x,y,-z	1
identity operation	1_555	x,y,z	1

2

A: Endoribonuclease Dicer

B: 5'-R(P*UP*UP*GP*CP*GP*AP*AP*UP*UP*CP*GP*CP*UP*U)-3'

A: Endoribonuclease Dicer

B: 5'-R(P*UP*UP*GP*CP*GP*AP*AP*UP*UP*CP*GP*CP*UP*U)-3'

Summary:

• defined by software
• 78.7 kDa, 4 polymers

Component details:

	Theoretical mass	Number of molelcules
Total (without water)	78,660	4
Polymers	78,660	4
Non-polymers	0	0
Water	36	2

Symmetry operations:

Type	Name	Symmetry operation	Number
identity operation	1_555	x,y,z	1
crystal symmetry operation	2_555	-x,y,-z	1

Calculated values:

Buried area	4490 Å2
ΔGint	-39 kcal/mol
Surface area	32920 Å2
Method	PISA

Unit cell

Length a, b, c (Å)	118.286, 84.429, 51.049
Angle α, β, γ (deg.)	90.00, 108.98, 90.00
Int Tables number	5
Space group name H-M	C121

Components

#1: Protein

A Endoribonuclease Dicer / Helicase with RNase motif / Helicase MOI

Details:

Mass: 34928.336 Da / Num. of mol.: 1
Fragment: platform-PAZ-connector helix cassette (UNP residues 765-1065)
Mutation: K822A/K823A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: DICER, DICER1, HERNA, KIAA0928 / Plasmid: pET28b / Production host: Escherichia coli (E. coli) / Strain (production host): BL21(DE3)
References: UniProt: Q9UPY3, Hydrolases; Acting on ester bonds; Endoribonucleases producing 5'-phosphomonoesters

#2: RNA chain

B 5'-R(P*UP*UP*GP*CP*GP*AP*AP*UP*UP*CP*GP*CP*UP*U)-3'

Details:

Mass: 4401.611 Da / Num. of mol.: 1 / Source method: obtained synthetically / Details: siRNA

#3: Water

ChemComp-HOH / water

Details:

Mass: 18.015 Da / Num. of mol.: 9 / Source method: isolated from a natural source / Formula: H₂O

Experimental details

Experiment

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

Sample preparation

• Crystal: Density Matthews: 3.06 ų/Da / Density % sol: 59.86 %
• Crystal grow: Temperature: 293 K / Method: vapor diffusion, hanging drop / pH: 8.5 ; Details: 0.2 M sodium formate, 0.1 M bicine, pH 8.1-8.5, 16-19% PEG5000 MME, VAPOR DIFFUSION, HANGING DROP, temperature 293K

Data collection

• Diffraction: Mean temperature: 100 K
• Diffraction source: Source: SYNCHROTRON / Site: APS / Beamline: 24-ID-E / Wavelength: 0.97918 Å
• Detector: Type: ADSC QUANTUM 315 / Detector: CCD / Date: Apr 12, 2010
• Radiation: Monochromator: Si(220) / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
• Radiation wavelength: Wavelength: 0.97918 Å / Relative weight: 1
• Reflection: Resolution: 2.55→30 Å / Num. all: 15564 / Num. obs: 15564 / % possible obs: 94.9 % / Redundancy: 3 % / B_iso Wilson estimate: 66.25 Ų / R_merge(I) obs: 0.071 / Χ²: 2.362 / Net I/σ(I): 12.2

Reflection shell

Resolution (Å)	Redundancy (%)	Rmerge(I) obs	Num. unique all	Χ2	Diffraction-ID	% possible all
2.55-2.64	3.3	0.506	1515	1.379	1	99.9
2.64-2.75	3.3	0.381	1504	1.492	1	99.5
2.75-2.87	3.3	0.282	1518	1.659	1	99.7
2.87-3.02	3.3	0.183	1524	1.945	1	99.5
3.02-3.21	3.1	0.126	1509	2.167	1	99.3
3.21-3.46	3.1	0.095	1483	2.538	1	97.6
3.46-3.81	2.9	0.069	1477	2.957	1	95.7
3.81-4.36	2.7	0.056	1375	3.317	1	90.3
4.36-5.48	2.6	0.053	1306	3.697	1	83.9
5.48-30	2.7	0.049	1324	4.137	1	84.5

Phasing

• Phasing: Method: molecular replacement

Processing

Software

Name	Version	Classification	NB
DENZO		data reduction
SCALEPACK		data scaling
PHASER		phasing
PHENIX	1.8.2_1309	refinement
PDB_EXTRACT	3.11	data extraction
HKL-2000		data reduction

Refinement

Method to determine structure: MOLECULAR REPLACEMENT
Starting model: PDB ENTRY 4NGD

4ngd

Resolution: 2.55→27.964 Å / Occupancy max: 1 / Occupancy min: 1 / FOM work R set: 0.7303 / SU ML: 0.43 / Cross valid method: THROUGHOUT / σ(F): 1.34 / Phase error: 32.85 / Stereochemistry target values: ML

	Rfactor	Num. reflection	% reflection	Selection details
Rfree	0.2736	1484	9.99 %	RANDOM
Rwork	0.2113	-	-	-
obs	0.2177	14848	95.49 %	-

• Solvent computation: Shrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
• Displacement parameters: B_iso max: 236.92 Ų / B_iso mean: 83.3458 Ų / B_iso min: 41.28 Ų

Refinement step

Cycle: LAST / Resolution: 2.55→27.964 Å

	Protein	Nucleic acid	Ligand	Solvent	Total
Num. atoms	2176	285	0	9	2470

Refine LS restraints

Refine-ID	Type	Dev ideal	Number
X-RAY DIFFRACTION	f_bond_d	0.006	2551
X-RAY DIFFRACTION	f_angle_d	0.978	3542
X-RAY DIFFRACTION	f_chiral_restr	0.065	420
X-RAY DIFFRACTION	f_plane_restr	0.005	399
X-RAY DIFFRACTION	f_dihedral_angle_d	15.024	985

LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Total num. of bins used: 11

Resolution (Å)	Rfactor Rfree	Num. reflection Rfree	Rfactor Rwork	Num. reflection Rwork	Num. reflection all	% reflection obs (%)
2.55-2.6323	0.4667	137	0.3277	1239	1376	99
2.6323-2.7264	0.3594	139	0.2968	1256	1395	99
2.7264-2.8354	0.3622	140	0.2685	1267	1407	99
2.8354-2.9643	0.3564	139	0.2379	1251	1390	100
2.9643-3.1204	0.3124	141	0.2208	1268	1409	99
3.1204-3.3156	0.2875	139	0.2059	1251	1390	99
3.3156-3.5712	0.2496	138	0.2122	1248	1386	99
3.5712-3.9297	0.2744	136	0.2087	1219	1355	95
3.9297-4.4963	0.27	130	0.194	1164	1294	91
4.4963-5.6571	0.2568	122	0.2064	1096	1218	86
5.6571-27.9659	0.2253	123	0.1906	1105	1228	84

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	0.9893	0.2201	-0.1889	3.2361	-0.0382	3.0703	-0.0522	-0.0858	0.0267	0.0023	-0.0665	0.0228	0.2684	0.0802	0.1435	0.4784	0.0742	0.0326	0.5285	-0.0027	0.5236	-22.9533	14.7066	7.0797
2	6.5891	3.4102	-2.7169	1.7708	-0.913	3.4047	-0.9301	0.625	-0.3903	0.3315	0.9298	-1.2054	-0.0715	0.0552	-0.0059	0.8522	0.0397	0.1223	1.0409	-0.0527	1.4308	-1.7753	-6.4338	2.128
3	0.5135	0.2012	0.8899	2.7061	-0.4103	5.3593	0.0505	0.253	-0.5476	-0.8449	0.256	-0.1542	-0.4195	0.2421	-0.2891	0.5206	-0.0273	0.0023	0.3481	-0.1013	0.4931	-27.7247	12.4354	8.9677

Refinement TLS group

ID	Refine-ID	Refine TLS-ID	Selection details	Auth asym-ID	Auth seq-ID
1	X-RAY DIFFRACTION	1	(chain A and resid 756:1053)	A	756 - 1053
2	X-RAY DIFFRACTION	2	chain B	B	1 - 14
3	X-RAY DIFFRACTION	3	(chain A and resid 1101:1109)	A	1101 - 1109