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- PDB-2px8: Crystal structure of the Murray Valley Encephalitis Virus NS5 2'-... -

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Basic information

Entry
Database: PDB / ID: 2px8
TitleCrystal structure of the Murray Valley Encephalitis Virus NS5 2'-O Methyltransferase domain in complex with SAH and 7M-GTP
ComponentsGenome polyprotein [Contains: Capsid protein C (Core protein); Envelope protein M (Matrix protein); Major envelope protein E; Non-structural protein 1 (NS1); Non-structural protein 2A (NS2A); Flavivirin protease NS2B regulatory subunit; Flavivirin protease NS3 catalytic subunit; Non-structural protein 4A (NS4A); Non-structural protein 4B (NS4B); RNA-directed RNA polymerase (EC 2.7.7.48) (NS5)]
KeywordsTRANSFERASE / Murray Valley Encephalitis Virus / Methyltransferase / SAH / 7M-GTP / 3PO / Structural Genomics / Oxford Protein Production Facility / OPPF
Function / homology
Function and homology information


flavivirin / symbiont-mediated suppression of host JAK-STAT cascade via inhibition of STAT2 activity / symbiont-mediated suppression of host JAK-STAT cascade via inhibition of STAT1 activity / viral capsid / double-stranded RNA binding / nucleoside-triphosphate phosphatase / clathrin-dependent endocytosis of virus by host cell / mRNA (guanine-N7)-methyltransferase / methyltransferase cap1 / methyltransferase cap1 activity ...flavivirin / symbiont-mediated suppression of host JAK-STAT cascade via inhibition of STAT2 activity / symbiont-mediated suppression of host JAK-STAT cascade via inhibition of STAT1 activity / viral capsid / double-stranded RNA binding / nucleoside-triphosphate phosphatase / 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 / symbiont-mediated suppression of host innate immune response / 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 / Vaccinia Virus protein VP39 / 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 / DNA/RNA polymerase superfamily / Rossmann fold / P-loop containing nucleoside triphosphate hydrolase / 3-Layer(aba) Sandwich / Alpha Beta
Similarity search - Domain/homology
TRIPHOSPHATE / 7N-METHYL-8-HYDROGUANOSINE-5'-TRIPHOSPHATE / S-ADENOSYL-L-HOMOCYSTEINE / Genome polyprotein
Similarity search - Component
Biological speciesMurray valley encephalitis virus
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.2 Å
AuthorsAssenberg, R. / Ren, J. / Verma, A. / Walter, T.S. / Alderton, D. / Hurrelbrink, R.J. / Fuller, S.D. / Owens, R.J. / Stuart, D.I. / Grimes, J.M. / Oxford Protein Production Facility (OPPF)
CitationJournal: J.Gen.Virol. / Year: 2007
Title: Crystal structure of the Murray Valley encephalitis virus NS5 methyltransferase domain in complex with cap analogues.
Authors: Assenberg, R. / Ren, J. / Verma, A. / Walter, T.S. / Alderton, D. / Hurrelbrink, R.J. / Fuller, S.D. / Bressanelli, S. / Owens, R.J. / Stuart, D.I. / Grimes, J.M.
History
DepositionMay 14, 2007Deposition site: RCSB / Processing site: RCSB
Revision 1.0May 29, 2007Provider: repository / Type: Initial release
Revision 1.1May 1, 2008Group: Version format compliance
Revision 1.2Jul 13, 2011Group: Advisory / Version format compliance
Revision 1.3Aug 30, 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 / struct_conn / struct_ncs_dom_lim / 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 / _struct_conn.pdbx_leaving_atom_flag / _struct_ncs_dom_lim.beg_auth_comp_id / _struct_ncs_dom_lim.end_auth_comp_id / _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: Genome polyprotein [Contains: Capsid protein C (Core protein); Envelope protein M (Matrix protein); Major envelope protein E; Non-structural protein 1 (NS1); Non-structural protein 2A (NS2A); Flavivirin protease NS2B regulatory subunit; Flavivirin protease NS3 catalytic subunit; Non-structural protein 4A (NS4A); Non-structural protein 4B (NS4B); RNA-directed RNA polymerase (EC 2.7.7.48) (NS5)]
B: Genome polyprotein [Contains: Capsid protein C (Core protein); Envelope protein M (Matrix protein); Major envelope protein E; Non-structural protein 1 (NS1); Non-structural protein 2A (NS2A); Flavivirin protease NS2B regulatory subunit; Flavivirin protease NS3 catalytic subunit; Non-structural protein 4A (NS4A); Non-structural protein 4B (NS4B); RNA-directed RNA polymerase (EC 2.7.7.48) (NS5)]
hetero molecules


Theoretical massNumber of molelcules
Total (without water)63,95212
Polymers60,8302
Non-polymers3,12210
Water7,692427
1
A: Genome polyprotein [Contains: Capsid protein C (Core protein); Envelope protein M (Matrix protein); Major envelope protein E; Non-structural protein 1 (NS1); Non-structural protein 2A (NS2A); Flavivirin protease NS2B regulatory subunit; Flavivirin protease NS3 catalytic subunit; Non-structural protein 4A (NS4A); Non-structural protein 4B (NS4B); RNA-directed RNA polymerase (EC 2.7.7.48) (NS5)]
hetero molecules


Theoretical massNumber of molelcules
Total (without water)31,6895
Polymers30,4151
Non-polymers1,2744
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
2
B: Genome polyprotein [Contains: Capsid protein C (Core protein); Envelope protein M (Matrix protein); Major envelope protein E; Non-structural protein 1 (NS1); Non-structural protein 2A (NS2A); Flavivirin protease NS2B regulatory subunit; Flavivirin protease NS3 catalytic subunit; Non-structural protein 4A (NS4A); Non-structural protein 4B (NS4B); RNA-directed RNA polymerase (EC 2.7.7.48) (NS5)]
hetero molecules


Theoretical massNumber of molelcules
Total (without water)32,2637
Polymers30,4151
Non-polymers1,8486
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)67.579, 87.445, 100.914
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number19
Space group name H-MP212121
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
11A
21B

NCS domain segments:

Component-ID: 1 / Ens-ID: 1 / Beg auth comp-ID: GLY / Beg label comp-ID: GLY / End auth comp-ID: VAL / End label comp-ID: VAL / Refine code: 5 / Auth seq-ID: 5 - 267 / Label seq-ID: 5 - 267

Dom-IDAuth asym-IDLabel asym-ID
1AA
2BB

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Components

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Protein , 1 types, 2 molecules AB

#1: Protein Genome polyprotein [Contains: Capsid protein C (Core protein); Envelope protein M (Matrix protein); Major envelope protein E; Non-structural protein 1 (NS1); Non-structural protein 2A (NS2A); Flavivirin protease NS2B regulatory subunit; Flavivirin protease NS3 catalytic subunit; Non-structural protein 4A (NS4A); Non-structural protein 4B (NS4B); RNA-directed RNA polymerase (EC 2.7.7.48) (NS5)]


Mass: 30415.035 Da / Num. of mol.: 2
Fragment: NS5 2'-O Methyltransferase Domain: Residues 2530-2798
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Murray valley encephalitis virus (strain MVE-1-51)
Genus: Flavivirus / Species: Murray Valley encephalitis virus / Strain: MVE-1-51, MVEV / Gene: NS5 / Plasmid: OPPF2936 / Production host: Escherichia coli (E. coli) / Strain (production host): Rosetta(DE3)pLysS / References: UniProt: P05769, RNA-directed RNA polymerase

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Non-polymers , 6 types, 437 molecules

#2: Chemical ChemComp-MGT / 7N-METHYL-8-HYDROGUANOSINE-5'-TRIPHOSPHATE


Mass: 539.223 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C11H20N5O14P3
#3: Chemical ChemComp-3PO / TRIPHOSPHATE


Mass: 257.955 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: H5O10P3
#4: Chemical ChemComp-SAH / S-ADENOSYL-L-HOMOCYSTEINE


Type: L-peptide linking / Mass: 384.411 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C14H20N6O5S
#5: Chemical ChemComp-GOL / GLYCEROL / GLYCERIN / PROPANE-1,2,3-TRIOL


Mass: 92.094 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C3H8O3
#6: Chemical ChemComp-CL / CHLORIDE ION


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


Mass: 18.015 Da / Num. of mol.: 427 / Source method: isolated from a natural source / Formula: H2O

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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: 2.45 Å3/Da / Density % sol: 49.8 %
Crystal growTemperature: 293 K / Method: vapor diffusion, sitting drop / pH: 6.5
Details: 0.2 M Sodium chloride, 0.1 M Bis-Tris pH 6.5, 25 % w/v PEG 3350, VAPOR DIFFUSION, SITTING DROP, temperature 293K

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: ESRF / Beamline: BM14 / Wavelength: 0.9535 Å
DetectorType: MARMOSAIC 225 mm CCD / Detector: CCD / Date: Feb 19, 2006 / Details: mirrors
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9535 Å / Relative weight: 1
ReflectionResolution: 2.2→30 Å / Num. obs: 29231 / % possible obs: 94.2 % / Observed criterion σ(I): -1.5 / Redundancy: 6 % / Rmerge(I) obs: 0.126 / Net I/σ(I): 10.2
Reflection shellResolution: 2.2→2.3 Å / Redundancy: 4.1 % / Rmerge(I) obs: 0.472 / Mean I/σ(I) obs: 2.3 / Num. unique all: 2063 / % possible all: 68.4

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Processing

Software
NameVersionClassification
REFMAC5.2.0019refinement
HKL-2000data collection
HKL-2000data reduction
HKL-2000data scaling
MOLREPphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: PDB entry 2PX2

2px2


Resolution: 2.2→30 Å / Cor.coef. Fo:Fc: 0.955 / Cor.coef. Fo:Fc free: 0.931 / SU B: 10.239 / SU ML: 0.142 / TLS residual ADP flag: LIKELY RESIDUAL / Cross valid method: THROUGHOUT / ESU R: 0.287 / ESU R Free: 0.206 / Stereochemistry target values: MAXIMUM LIKELIHOOD
Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS. CNS program has also been used in refinement
RfactorNum. reflection% reflectionSelection details
Rfree0.2167 1460 5 %RANDOM
Rwork0.16615 ---
obs0.16869 27752 94.17 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: BABINET MODEL WITH MASK
Displacement parametersBiso mean: 35.313 Å2
Baniso -1Baniso -2Baniso -3
1--1.97 Å20 Å20 Å2
2--4.27 Å20 Å2
3----2.3 Å2
Refinement stepCycle: LAST / Resolution: 2.2→30 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms4206 0 190 427 4823
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0070.0214488
X-RAY DIFFRACTIONr_angle_refined_deg1.3792.0226071
X-RAY DIFFRACTIONr_dihedral_angle_1_deg4.4955527
X-RAY DIFFRACTIONr_dihedral_angle_2_deg31.80622.708192
X-RAY DIFFRACTIONr_dihedral_angle_3_deg14.09215764
X-RAY DIFFRACTIONr_dihedral_angle_4_deg10.7191544
X-RAY DIFFRACTIONr_chiral_restr0.0660.2628
X-RAY DIFFRACTIONr_gen_planes_refined0.0030.023289
X-RAY DIFFRACTIONr_nbd_refined0.1820.22101
X-RAY DIFFRACTIONr_nbtor_refined0.2960.22994
X-RAY DIFFRACTIONr_xyhbond_nbd_refined0.1190.2365
X-RAY DIFFRACTIONr_symmetry_vdw_refined0.2080.2107
X-RAY DIFFRACTIONr_symmetry_hbond_refined0.1130.227
X-RAY DIFFRACTIONr_mcbond_it2.93842700
X-RAY DIFFRACTIONr_mcangle_it3.98464198
X-RAY DIFFRACTIONr_scbond_it5.48562129
X-RAY DIFFRACTIONr_scangle_it8.454101873
Refine LS restraints NCS

Dom-ID: 1 / Auth asym-ID: A / Ens-ID: 1 / Refine-ID: X-RAY DIFFRACTION

NumberTypeRms dev position (Å)Weight position
1052medium positional0.180.5
1042loose positional0.515
1052medium thermal2.4820
1042loose thermal3.6630
LS refinement shellResolution: 2.2→2.257 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.33 78 -
Rwork0.206 1424 -
obs--66.73 %
Refinement TLS params.

Method: refined / Refine-ID: X-RAY DIFFRACTION

IDL11 (°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)
11.8322-0.05540.81790.58420.1672.82820.01970.03650.0105-0.0362-0.02930.06050.0444-0.10120.0096-0.1366-0.0048-0.0009-0.18010.0073-0.107410.01-20.419-2.778
21.418-0.1440.9150.3577-0.02812.45050.05490.0227-0.0528-0.0021-0.02390.0090.10060.0328-0.031-0.1462-0.01410.0184-0.168-0.0198-0.1268-18.752-20.228-48.353
Refinement TLS group
IDRefine-IDRefine TLS-IDAuth asym-IDLabel asym-IDAuth seq-IDLabel seq-ID
1X-RAY DIFFRACTION1AA4 - 2674 - 267
2X-RAY DIFFRACTION2BB5 - 2695 - 269

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