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- PDB-6xya: Cap-binding domain of SFTSV L protein -

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Basic information

Entry
Database: PDB / ID: 6xya
TitleCap-binding domain of SFTSV L protein
ComponentsRNA-dependent RNA polymerase
KeywordsVIRAL PROTEIN / bunyavirus / cap binding / cap-snatching / viral polymerase
Function / homology
Function and homology information


host cell endoplasmic reticulum / virion component / host cell endoplasmic reticulum-Golgi intermediate compartment / host cell Golgi apparatus / Hydrolases; Acting on ester bonds / hydrolase activity / RNA-directed RNA polymerase / viral RNA genome replication / RNA-dependent RNA polymerase activity / DNA-templated transcription / metal ion binding
Similarity search - Function
RNA-directed RNA polymerase L, PA-C-like domain / RNA-directed RNA polymerase L, PA-C-like domain / RNA-directed RNA polymerase L, N-terminal / L protein N-terminus / RNA-dependent RNA polymerase, bunyaviral / Bunyavirus RNA dependent RNA polymerase / RNA-directed RNA polymerase, negative-strand RNA virus / RdRp of negative ssRNA viruses with segmented genomes catalytic domain profile.
Similarity search - Domain/homology
7-METHYL-GUANOSINE-5'-TRIPHOSPHATE / RNA-directed RNA polymerase L
Similarity search - Component
Biological speciesSFTS virus AH12
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.35 Å
AuthorsGogrefe, N. / Guenther, S. / Rosenthal, M.
Funding support Germany, European Union, 2items
OrganizationGrant numberCountry
Leibniz AssociationK72/2017 Germany
European Union (EU)653706European Union
Citation
Journal: Nucleic Acids Res / Year: 2020
Title: Structural and functional characterization of the severe fever with thrombocytopenia syndrome virus L protein.
Authors: Dominik Vogel / Sigurdur Rafn Thorkelsson / Emmanuelle R J Quemin / Kristina Meier / Tomas Kouba / Nadja Gogrefe / Carola Busch / Sophia Reindl / Stephan Günther / Stephen Cusack / Kay ...Authors: Dominik Vogel / Sigurdur Rafn Thorkelsson / Emmanuelle R J Quemin / Kristina Meier / Tomas Kouba / Nadja Gogrefe / Carola Busch / Sophia Reindl / Stephan Günther / Stephen Cusack / Kay Grünewald / Maria Rosenthal /
Abstract: The Bunyavirales order contains several emerging viruses with high epidemic potential, including Severe fever with thrombocytopenia syndrome virus (SFTSV). The lack of medical countermeasures, such ...The Bunyavirales order contains several emerging viruses with high epidemic potential, including Severe fever with thrombocytopenia syndrome virus (SFTSV). The lack of medical countermeasures, such as vaccines and antivirals, is a limiting factor for the containment of any virus outbreak. To develop such antivirals a profound understanding of the viral replication process is essential. The L protein of bunyaviruses is a multi-functional and multi-domain protein performing both virus transcription and genome replication and, therefore, is an ideal drug target. We established expression and purification procedures for the full-length L protein of SFTSV. By combining single-particle electron cryo-microscopy and X-ray crystallography, we obtained 3D models covering ∼70% of the SFTSV L protein in the apo-conformation including the polymerase core region, the endonuclease and the cap-binding domain. We compared this first L structure of the Phenuiviridae family to the structures of La Crosse peribunyavirus L protein and influenza orthomyxovirus polymerase. Together with a comprehensive biochemical characterization of the distinct functions of SFTSV L protein, this work provides a solid framework for future structural and functional studies of L protein-RNA interactions and the development of antiviral strategies against this group of emerging human pathogens.
#1: Journal: Biorxiv / Year: 2020
Title: Structural and functional characterization of the Severe fever with thrombocytopenia syndrome virus L protein
Authors: Vogel, D. / Thorkelsson, S.R. / Quemin, E. / Meier, K. / Kouba, T. / Gogrefe, N. / Guenther, S. / Cusack, S. / Grunewald, K. / Rosenthal, M.
History
DepositionJan 30, 2020Deposition site: PDBE / Processing site: PDBE
Revision 1.0Apr 8, 2020Provider: repository / Type: Initial release
Revision 1.1Apr 29, 2020Group: Database references / Category: citation / citation_author
Revision 1.2Jun 10, 2020Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.3Dec 16, 2020Group: Database references / Category: citation / Item: _citation.pdbx_database_id_DOI
Revision 1.4Jan 24, 2024Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / citation / database_2 / pdbx_initial_refinement_model
Item: _citation.journal_id_ISSN / _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
B: RNA-dependent RNA polymerase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)13,4063
Polymers12,8451
Non-polymers5612
Water3,405189
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: isothermal titration calorimetry
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area610 Å2
ΔGint-9 kcal/mol
Surface area6510 Å2
MethodPISA
Unit cell
Length a, b, c (Å)38.974, 44.715, 63.515
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number19
Space group name H-MP212121
Space group name HallP2ac2ab
Symmetry operation#1: x,y,z
#2: x+1/2,-y+1/2,-z
#3: -x,y+1/2,-z+1/2
#4: -x+1/2,-y,z+1/2

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Components

#1: Protein RNA-dependent RNA polymerase /


Mass: 12844.638 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: the first two residues (GP) are remains of an enzymatic cleavage siite and not originally part of the sequence
Source: (gene. exp.) SFTS virus AH12 / Production host: Escherichia coli (E. coli) / References: UniProt: F1BV96
#2: Chemical ChemComp-MGP / 7-METHYL-GUANOSINE-5'-TRIPHOSPHATE


Mass: 538.215 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C11H19N5O14P3 / Feature type: SUBJECT OF INVESTIGATION
#3: Chemical ChemComp-NA / SODIUM ION


Mass: 22.990 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Na
#4: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 189 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestY

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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: 2.15 Å3/Da / Density % sol: 42.91 %
Crystal growTemperature: 293 K / Method: vapor diffusion, sitting drop
Details: Final concentrations of components: 16.6 mM Sodium phosphate (pH 6.5), 50mM NaCl, 6.6% (w/v) Glycerol, 4.7% (v/v) 2-Butanol, 100mM Mes (pH 6.0), 18.3% (w/v) Polyethylene glycol 4000, 1.3 mM ...Details: Final concentrations of components: 16.6 mM Sodium phosphate (pH 6.5), 50mM NaCl, 6.6% (w/v) Glycerol, 4.7% (v/v) 2-Butanol, 100mM Mes (pH 6.0), 18.3% (w/v) Polyethylene glycol 4000, 1.3 mM m7GTP Protein concentration: SFTSV CBD 12.4 mg/ml

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: PETRA III, EMBL c/o DESY / Beamline: P14 (MX2) / Wavelength: 0.9763 Å
DetectorType: DECTRIS EIGER X 16M / Detector: PIXEL / Date: Oct 22, 2019
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9763 Å / Relative weight: 1
ReflectionResolution: 1.35→50 Å / Num. obs: 321482 / % possible obs: 97.59 % / Redundancy: 13.2 % / Biso Wilson estimate: 16.17 Å2 / CC1/2: 1 / Rmerge(I) obs: 0.03094 / Rrim(I) all: 0.03223 / Net I/σ(I): 41.6
Reflection shellResolution: 1.35→1.398 Å / Redundancy: 11.6 % / Rmerge(I) obs: 0.3684 / Mean I/σ(I) obs: 6.32 / Num. unique obs: 25283 / CC1/2: 0.953 / Rrim(I) all: 0.3857 / % possible all: 88.8

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Processing

Software
NameVersionClassification
PHENIX1.17_3644refinement
Cootmodel building
XDSdata reduction
Aimlessdata scaling
PHENIXphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 6qhg

6qhg


Resolution: 1.35→33.22 Å / SU ML: 0.1152 / Cross valid method: FREE R-VALUE / σ(F): 1.36 / Phase error: 20.1928
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflection
Rfree0.1768 1189 4.86 %
Rwork0.1654 23259 -
obs0.166 24448 97.53 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 21.67 Å2
Refinement stepCycle: LAST / Resolution: 1.35→33.22 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms858 0 34 189 1081
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0087976
X-RAY DIFFRACTIONf_angle_d1.18921334
X-RAY DIFFRACTIONf_chiral_restr0.0851140
X-RAY DIFFRACTIONf_plane_restr0.0053170
X-RAY DIFFRACTIONf_dihedral_angle_d21.819147
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
1.35-1.410.24391530.22152630X-RAY DIFFRACTION90.39
1.41-1.490.20951380.19952853X-RAY DIFFRACTION97.17
1.49-1.580.23861590.18412848X-RAY DIFFRACTION97.92
1.58-1.70.19071420.18072918X-RAY DIFFRACTION98.17
1.7-1.870.20351300.17422958X-RAY DIFFRACTION98.88
1.87-2.140.19551570.15952940X-RAY DIFFRACTION99.39
2.14-2.70.16661570.1632986X-RAY DIFFRACTION99.15
2.7-33.220.15391530.15573126X-RAY DIFFRACTION99
Refinement TLS params.Method: refined / Origin x: -12.7327782818 Å / Origin y: -1.59575522964 Å / Origin z: 6.63915482155 Å
111213212223313233
T0.131114570845 Å2-0.00312521636001 Å20.00835286286115 Å2-0.145685374536 Å20.0062997402544 Å2--0.137496080907 Å2
L1.01445262356 °20.575267584135 °20.474901051133 °2-1.47177247372 °20.797603572778 °2--1.12691735711 °2
S0.00903311518126 Å °-0.0180948129555 Å °-0.0440592600533 Å °0.0428044445398 Å °-0.011116269983 Å °-0.00868802250961 Å °0.00595874621952 Å °-0.0189633371023 Å °0.000843006291066 Å °
Refinement TLS groupSelection details: all

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