Journal: 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.
Mass: 3217.956 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) La Crosse orthobunyavirus
#2: RNA chain
LaCrossevirus5' 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
LaCrossevirus3' vRNA (1-16)
Mass: 5060.023 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) La Crosse orthobunyavirus
#4: Protein
RNA-directedRNApolymeraseL / 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
Instrument: 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
Microscopy
Model: FEI TITAN KRIOS
Electron gun
Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lens
Mode: BRIGHT FIELD / Nominal magnification: 165000 X / Nominal defocus max: -3500 nm / Nominal defocus min: -800 nm / Cs: 2.7 mm / Alignment procedure: COMA FREE
Specimen holder
Cryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 90 K / Temperature (min): 90 K
Image recording
Electron 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 optics
Energyfilter name: GIF Quantum LS / Energyfilter slit width: 20 eV
Image scans
Movie frames/image: 40
-
Processing
EM software
ID
Name
Version
Category
2
SerialEM
imageacquisition
4
cryoSPARC
CTFcorrection
7
Coot
modelfitting
9
RELION
initialEulerassignment
10
RELION
3.1
finalEulerassignment
11
RELION
3.1
classification
12
RELION
3.1
3Dreconstruction
13
PHENIX
modelrefinement
CTF correction
Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selection
Num. of particles selected: 4065475
Symmetry
Point symmetry: C1 (asymmetric)
3D reconstruction
Resolution: 3.02 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 59152 / Algorithm: BACK PROJECTION / Symmetry type: POINT
Atomic model building
Protocol: AB INITIO MODEL / Space: REAL / Target criteria: Correlation coefficient
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