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- PDB-4m9t: NS2B-NS3 protease from dengue virus in the presence of DTNB, a co... -

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Basic information

Entry
Database: PDB / ID: 4m9t
TitleNS2B-NS3 protease from dengue virus in the presence of DTNB, a covalent allosteric inhibitor
ComponentsNS2B-NS3 protease
KeywordsVIRAL PROTEIN / serine protease / allosteric inhibition / dengue virus protease / trypsin-like protease / conformational flexibility
Function / homology
Function and homology information


flavivirin / symbiont-mediated suppression of host JAK-STAT cascade via inhibition of host TYK2 activity / host cell mitochondrion / symbiont-mediated suppression of host JAK-STAT cascade via inhibition of STAT2 activity / symbiont-mediated suppression of host cytoplasmic pattern recognition receptor signaling pathway via inhibition of MAVS activity / ribonucleoside triphosphate phosphatase activity / viral capsid / double-stranded RNA binding / nucleoside-triphosphate phosphatase / channel activity ...flavivirin / symbiont-mediated suppression of host JAK-STAT cascade via inhibition of host TYK2 activity / host cell mitochondrion / symbiont-mediated suppression of host JAK-STAT cascade via inhibition of STAT2 activity / symbiont-mediated suppression of host cytoplasmic pattern recognition receptor signaling pathway via inhibition of MAVS activity / ribonucleoside triphosphate phosphatase activity / viral capsid / double-stranded RNA binding / nucleoside-triphosphate phosphatase / channel activity / monoatomic ion transmembrane transport / clathrin-dependent endocytosis of virus by host cell / mRNA (guanine-N7)-methyltransferase / methyltransferase cap1 / methyltransferase cap1 activity / mRNA 5'-cap (guanine-N7-)-methyltransferase activity / RNA helicase activity / protein dimerization activity / host cell perinuclear region of cytoplasm / host cell endoplasmic reticulum membrane / RNA helicase / symbiont-mediated suppression of host type I interferon-mediated signaling pathway / symbiont-mediated activation of host autophagy / RNA-directed RNA polymerase / serine-type endopeptidase activity / viral RNA genome replication / RNA-directed RNA polymerase activity / fusion of virus membrane with host endosome membrane / viral envelope / virion attachment to host cell / host cell nucleus / virion membrane / structural molecule activity / ATP hydrolysis activity / proteolysis / extracellular region / ATP binding / metal ion binding / membrane
Similarity search - Function
Flavivirus capsid protein C superfamily / Flavivirus non-structural protein NS2B / Genome polyprotein, Flavivirus / : / Flavivirus non-structural protein NS4A / Flavivirus non-structural protein NS2B / Flavivirus non-structural protein NS4B / mRNA cap 0/1 methyltransferase / Flavivirus non-structural protein NS4B / Flavivirus non-structural protein NS4A ...Flavivirus capsid protein C superfamily / Flavivirus non-structural protein NS2B / Genome polyprotein, Flavivirus / : / Flavivirus non-structural protein NS4A / Flavivirus non-structural protein NS2B / Flavivirus non-structural protein NS4B / mRNA cap 0/1 methyltransferase / Flavivirus non-structural protein NS4B / Flavivirus non-structural protein NS4A / Flavivirus NS2B domain profile. / mRNA cap 0 and cap 1 methyltransferase (EC 2.1.1.56 and EC 2.1.1.57) domain profile. / Flavivirus non-structural protein NS2A / Flavivirus non-structural protein NS2A / Flavivirus NS3, petidase S7 / Peptidase S7, Flavivirus NS3 serine protease / Flavivirus NS3 protease (NS3pro) domain profile. / RNA-directed RNA polymerase, thumb domain, Flavivirus / Flavivirus RNA-directed RNA polymerase, thumb domain / RNA-directed RNA polymerase, flavivirus / Flavivirus RNA-directed RNA polymerase, fingers and palm domains / Flavivirus capsid protein C / Flavivirus capsid protein C / Flavivirus non-structural Protein NS1 / Flavivirus non-structural protein NS1 / Envelope glycoprotein M, flavivirus / Envelope glycoprotein M superfamily, flavivirus / Flavivirus envelope glycoprotein M / Flavivirus polyprotein propeptide / Flavivirus polyprotein propeptide superfamily / Flavivirus polyprotein propeptide / Flavivirus envelope glycoprotein E, stem/anchor domain / : / Flavivirus NS3 helicase, C-terminal helical domain / Flavivirus envelope glycoprotein E, Stem/Anchor domain / Flavivirus envelope glycoprotein E, Stem/Anchor domain superfamily / Flavivirus glycoprotein E, immunoglobulin-like domain / Flavivirus glycoprotein, immunoglobulin-like domain / Flavivirus glycoprotein central and dimerisation domain / Flavivirus glycoprotein, central and dimerisation domains / Flaviviral glycoprotein E, central domain, subdomain 1 / Flaviviral glycoprotein E, central domain, subdomain 2 / Ribosomal RNA methyltransferase, FtsJ domain / FtsJ-like methyltransferase / Flavivirus/Alphavirus glycoprotein, immunoglobulin-like domain superfamily / Flavivirus glycoprotein, central and dimerisation domain superfamily / Flaviviral glycoprotein E, dimerisation domain / DEAD box, Flavivirus / Flavivirus DEAD domain / helicase superfamily c-terminal domain / Immunoglobulin E-set / 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 / RNA-directed RNA polymerase, catalytic domain / RdRp of positive ssRNA viruses catalytic domain profile. / S-adenosyl-L-methionine-dependent methyltransferase superfamily / Peptidase S1, PA clan, chymotrypsin-like fold / Peptidase S1, PA clan / DNA/RNA polymerase superfamily / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Genome polyprotein / Genome polyprotein
Similarity search - Component
Biological speciesDengue virus 2
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.74 Å
AuthorsBell, J.A. / Yildiz, M. / Hardy, J.A.
CitationJournal: Acs Chem.Biol. / Year: 2013
Title: Allosteric Inhibition of the NS2B-NS3 Protease from Dengue Virus.
Authors: Yildiz, M. / Ghosh, S. / Bell, J.A. / Sherman, W. / Hardy, J.A.
History
DepositionAug 15, 2013Deposition site: RCSB / Processing site: RCSB
Revision 1.0Nov 27, 2013Provider: repository / Type: Initial release
Revision 1.1Dec 4, 2013Group: Data collection
Revision 1.2Feb 5, 2014Group: Database references
Revision 1.3Aug 2, 2017Group: Refinement description / Source and taxonomy / Category: entity_src_gen / software
Revision 1.4Sep 20, 2023Group: Data collection / Database references ...Data collection / Database references / Derived calculations / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model / software / struct_ref_seq_dif / struct_site
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _software.name / _struct_ref_seq_dif.details / _struct_site.pdbx_auth_asym_id / _struct_site.pdbx_auth_comp_id / _struct_site.pdbx_auth_seq_id

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: NS2B-NS3 protease
hetero molecules


Theoretical massNumber of molelcules
Total (without water)26,3962
Polymers26,3601
Non-polymers351
Water54030
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)61.394, 62.090, 114.347
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number20
Space group name H-MC2221

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Components

#1: Protein NS2B-NS3 protease


Mass: 26360.479 Da / Num. of mol.: 1 / Fragment: UNP RESIDUES 1394-1440, 1476-1660 / Mutation: A125C
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Dengue virus 2 / Gene: NS2B-NS3 / Production host: Escherichia coli (E. coli) / Strain (production host): BL21 (DE3) / References: UniProt: Q91H74, UniProt: P12823*PLUS
#2: Chemical ChemComp-CL / CHLORIDE ION


Mass: 35.453 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Cl
#3: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 30 / Source method: isolated from a natural source / Formula: H2O
Sequence detailsTHIS PROTEIN IS A FUSION PROTEIN CONTAINING RESIDUES 43-95 FROM DENGUE VIRUS NS2B, A GGGGSGGGG ...THIS PROTEIN IS A FUSION PROTEIN CONTAINING RESIDUES 43-95 FROM DENGUE VIRUS NS2B, A GGGGSGGGG LINKER AND RESIDUES IN 1-185 FROM NS3. SIX EXTRA RESIDUES GSHMLE REMAINING AFTER THROMBIN CLEAVAGE ARE ATTACHED TO THE N-TERMINUS. THE OVERALL ARRANGEMENT OF THE FUSION PROTEIN IS GSHMLE-NS2B43THRU95-GGGGSGGGG-NS31THRU185. IN THE PDB COORDINATES, THE NS2B REGION HAS RESIDUE NUMBERS 43-95, RESIDUE 96 IS FROM THE GGGGSGGG LINKER. THE REMAINDER OF THE LINKER AND THE FIRST 17 RESIDUES FROM NS3B ARE DISORDERED IN THIS STRUCTURE. THE NS3 REGION HAS RESIDUE NUMBERS 1018-1171. THOSE NUMBERS CORRESPOND TO RESIDUES IDENTIFIED AS NS3B 18 TO 171 IN THE MANUSCRIPT.

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 2.07 Å3/Da / Density % sol: 40.49 %
Crystal growTemperature: 298 K / Method: vapor diffusion, hanging drop / pH: 8.5
Details: 40% PEG200, 0.1 M Acetic acid, pH 5.5. Crystals were soaked in 40% PEG200, 0.1 M Tris pH 8.5, 5 mM 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB), VAPOR DIFFUSION, HANGING DROP, temperature 298K

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: APS / Beamline: 24-ID-C / Wavelength: 0.979 Å
DetectorType: MARMOSAIC 300 mm CCD / Detector: CCD / Date: Nov 18, 2012
RadiationMonochromator: Si (111) / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.979 Å / Relative weight: 1
ReflectionResolution: 1.74→43.7 Å / Num. all: 19456 / Num. obs: 19455 / % possible obs: 85.3 % / Observed criterion σ(F): 2 / Observed criterion σ(I): 3 / Redundancy: 6.3 % / Biso Wilson estimate: 32.2 Å2 / Rmerge(I) obs: 0.114 / Rsym value: 0.114 / Net I/σ(I): 30.1
Reflection shellHighest resolution: 1.7 Å / Redundancy: 4.6 % / Rmerge(I) obs: 0.114 / Mean I/σ(I) obs: 1.85 / Num. unique all: 19456 / Rsym value: 0.114 / % possible all: 85.3

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Processing

Software
NameClassification
PHASERphasing
PRIME-Xrefinement
HKL-2000data reduction
HKL-2000data scaling
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 2FOM

2fom


Resolution: 1.74→43.66 Å / Rfactor Rfree error: 0.004 / Occupancy max: 1 / Occupancy min: 0.5 / Isotropic thermal model: RESTRAINED / Cross valid method: THROUGHOUT / σ(F): 10000 / σ(I): 10000 / Stereochemistry target values: MAXIMUM LIKELIHOOD
Details: This structure was solved in the presence of the small-molecule NS2B-NS3pro inhibitor DTNB, however density for DTNB could not be observed in the structure.
RfactorNum. reflection% reflectionSelection details
Rfree0.241 996 5.1 %random
Rwork0.215 ---
obs0.217 18265 85 %-
all-19455 --
Solvent computationBsol: 48.0016 Å2 / ksol: 0.42 e/Å3
Displacement parametersBiso max: 91.97 Å2 / Biso mean: 28.8415 Å2 / Biso min: 4.77 Å2
Baniso -1Baniso -2Baniso -3
1--0.141 Å20 Å20 Å2
2--0.337 Å20 Å2
3----0.196 Å2
Refine analyzeLuzzati coordinate error obs: 0.2 Å
Refinement stepCycle: LAST / Resolution: 1.74→43.66 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms1535 0 1 30 1566
Refine LS restraints
Refine-IDTypeDev idealDev ideal target
X-RAY DIFFRACTIONo_bond_d0.013
X-RAY DIFFRACTIONo_angle_deg1.915
X-RAY DIFFRACTIONo_mcbond_it7.021.5
X-RAY DIFFRACTIONo_mcangle_it7.882
X-RAY DIFFRACTIONo_scbond_it11.982
X-RAY DIFFRACTIONo_scangle_it13.442.5
X-RAY DIFFRACTIONo_improper_angle_d0.423
LS refinement shellResolution: 1.74→1.81 Å / Rfactor Rfree error: 0.013 / Total num. of bins used: 10
RfactorNum. reflection% reflection
Rfree0.316 111 5.4 %
Rwork0.332 1945 -
obs-2056 85 %

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