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- PDB-6z8k: La Crosse virus polymerase at elongation mimicking stage -

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Basic information

Entry
Database: PDB / ID: 6z8k
TitleLa Crosse virus polymerase at elongation mimicking stage
Components
  • (La Crosse virus 5' vRNA ...) x 2
  • La Crosse virus 3' vRNA (1-16)
  • RNA-directed RNA polymerase L
KeywordsREPLICATION / polymerase / transcription / La Crosse virus
Function / homology
Function and homology information


virion / RNA-directed RNA polymerase / hydrolase activity / viral RNA genome replication / host cell cytoplasm / RNA-directed 5'-3' RNA polymerase activity / Hydrolases; Acting on ester bonds / transcription, DNA-templated / nucleotide binding / metal ion binding
RNA-directed RNA polymerase, negative-strand RNA virus / RNA-dependent RNA polymerase, bunyaviral / RNA-directed RNA polymerase, orthobunyavirus / RNA-directed RNA polymerase L, N-terminal
RNA-directed RNA polymerase L
Biological speciesLa Crosse orthobunyavirus
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.02 Å
AuthorsArragain, B. / Effantin, G. / Schoehn, G. / Cusack, S. / Malet, H.
Funding support France, 1items
OrganizationGrant numberCountry
French National Research AgencyANR-19-CE11-0024-02 France
CitationJournal: Nat Commun / Year: 2020
Title: Pre-initiation and elongation structures of full-length La Crosse virus polymerase reveal functionally important conformational changes.
Authors: Benoît Arragain / Grégory Effantin / Piotr Gerlach / Juan Reguera / Guy Schoehn / Stephen Cusack / Hélène Malet /
Abstract: Bunyavirales is an order of segmented negative-strand RNA viruses comprising several life-threatening pathogens against which no effective treatment is currently available. Replication and ...Bunyavirales is an order of segmented negative-strand RNA viruses comprising several life-threatening pathogens against which no effective treatment is currently available. Replication and transcription of the RNA genome constitute essential processes performed by the virally encoded multi-domain RNA-dependent RNA polymerase. Here, we describe the complete high-resolution cryo-EM structure of La Crosse virus polymerase. It reveals the presence of key protruding C-terminal domains, notably the cap-binding domain, which undergoes large movements related to its role in transcription initiation, and a zinc-binding domain that displays a fold not previously observed. We capture the polymerase structure at pre-initiation and elongation states, uncovering the coordinated movement of the priming loop, mid-thumb ring linker and lid domain required for the establishment of a ten-base-pair template-product RNA duplex before strand separation into respective exit tunnels. These structural details and the observed dynamics of key functional elements will be instrumental for structure-based development of polymerase inhibitors.
Validation Report
SummaryFull reportAbout validation report
History
DepositionJun 2, 2020Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jul 29, 2020Provider: repository / Type: Initial release

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Structure visualization

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Structure viewerMolecule:
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Assembly

Deposited unit
C: La Crosse virus 5' vRNA 1-10
X: La Crosse virus 5' vRNA (9-16)
H: La Crosse virus 3' vRNA (1-16)
T: La Crosse virus 5' vRNA 1-10
P: La Crosse virus 3' vRNA (1-16)
A: RNA-directed RNA polymerase L
hetero molecules


Theoretical massNumber of molelcules
Total (without water)285,2038
Polymers285,1136
Non-polymers902
Water0
1


TypeNameSymmetry operationNumber
identity operation1_5551
Buried area11920 Å2
ΔGint-100 kcal/mol
Surface area90800 Å2
MethodPISA

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Components

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La Crosse virus 5' vRNA ... , 2 types, 3 molecules CTX

#1: RNA chain La Crosse virus 5' vRNA 1-10


Mass: 3217.956 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) La Crosse orthobunyavirus
#2: RNA chain La Crosse virus 5' vRNA (9-16)


Mass: 2509.577 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) La Crosse orthobunyavirus

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RNA chain / Protein , 2 types, 3 molecules HPA

#3: RNA chain La Crosse virus 3' vRNA (1-16)


Mass: 5060.023 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) La Crosse orthobunyavirus
#4: Protein RNA-directed RNA polymerase L / Protein L / Large structural protein / Replicase / Transcriptase


Mass: 266047.469 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) La Crosse orthobunyavirus / Cell line (production host): Hi 5 / Production host: Trichoplusia ni (cabbage looper)
References: UniProt: A5HC98, RNA-directed RNA polymerase, Hydrolases; Acting on ester bonds

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Non-polymers , 2 types, 2 molecules

#5: Chemical ChemComp-ZN / ZINC ION / Zinc


Mass: 65.409 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Zn / Feature type: SUBJECT OF INVESTIGATION
#6: Chemical ChemComp-MG / MAGNESIUM ION / Magnesium


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION

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Details

Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

Component
IDNameTypeEntity IDParent-IDSource
1La Crosse virus polymerase at elongation-mimicking stageCOMPLEX1, 2, 3, 40MULTIPLE SOURCES
2La Crosse virus polymerase at elongation stageCOMPLEX1, 2, 31RECOMBINANT
3RNA-directed RNA polymerase LCOMPLEX41RECOMBINANT
Molecular weightValue: 0.265 MDa / Experimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12La Crosse orthobunyavirus2560547
23La Crosse orthobunyavirus2560547
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
13cabbage looper7111
22synthetic construct (others)32630
Buffer solutionpH: 8 / Details: 20 mM Tris-HCl pH 8, 150 mM NaCl, 2 mM TCEP
Buffer component
IDConc.FormulaBuffer-ID
120 mMTris-HClTris1
2150 mMNaClSodium chloride1
32 mMTCEP1
SpecimenConc.: 0.2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: 30mA / Grid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: UltrAuFoil
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 293 K / Details: blot time: 2 sec, blot force: 1

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 165000 X / Nominal defocus max: -3500 nm / Nominal defocus min: -800 nm / Cs: 2.7 mm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 90 K / Temperature (min): 90 K
Image recordingElectron dose: 1.25 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 2 / Num. of real images: 16498
EM imaging opticsEnergyfilter name: GIF Quantum LS / Energyfilter slit width: 20 eV
Image scansMovie frames/image: 40

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Processing

EM software
IDNameVersionCategory
2SerialEMimage acquisition
4cryoSPARCCTF correction
7Cootmodel fitting
9RELIONinitial Euler assignment
10RELION3.1final Euler assignment
11RELION3.1classification
12RELION3.13D reconstruction
13PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 4065475
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.02 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 59152 / Algorithm: BACK PROJECTION / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL / Space: REAL / Target criteria: Correlation coefficient
Atomic model buildingPDB-ID: 6Z6G
Pdb chain-ID: A

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