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- EMDB-11095: Masked cryo-EM map of La Crosse virus polymerase cap-binding doma... -

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Basic information

Entry
Database: EMDB / ID: EMD-11095
TitleMasked cryo-EM map of La Crosse virus polymerase cap-binding domain and mid domain
Map data
SampleLa Crosse virus polymerase cap-binding domain and mid domain
Biological speciesLa Crosse orthobunyavirus
Methodsingle particle reconstruction / cryo EM / Resolution: 3.54 Å
AuthorsArragain B / Effantin G / Schoehn G / Cusack S / Malet H
Funding support France, 1 items
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.
History
DepositionMay 28, 2020-
Header (metadata) releaseJul 29, 2020-
Map releaseJul 29, 2020-
UpdateJul 29, 2020-
Current statusJul 29, 2020Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.013
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.013
  • Imaged by UCSF Chimera
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Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_11095.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 300 pix.
= 247.8 Å
0.83 Å/pix.
x 300 pix.
= 247.8 Å
0.83 Å/pix.
x 300 pix.
= 247.8 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.826 Å
Density
Contour LevelBy AUTHOR: 0.013 / Movie #1: 0.013
Minimum - Maximum-0.040666975 - 0.08123269
Average (Standard dev.)0.0000479571 (±0.0011303393)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 247.79999 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.8260.8260.826
M x/y/z300300300
origin x/y/z0.0000.0000.000
length x/y/z247.800247.800247.800
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS300300300
D min/max/mean-0.0410.0810.000

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Supplemental data

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Sample components

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Entire La Crosse virus polymerase cap-binding domain and mid domain

EntireName: La Crosse virus polymerase cap-binding domain and mid domain
Details: Masked cryo-EM map of La Crosse virus full-length polymerase, displaying only the cap-binding domain and the mid domain. Masking, 3D classification and refinement enable to obtain a ...Details: Masked cryo-EM map of La Crosse virus full-length polymerase, displaying only the cap-binding domain and the mid domain. Masking, 3D classification and refinement enable to obtain a structure of the cap-binding domain.
Number of components: 2

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Component #1: protein, La Crosse virus polymerase cap-binding domain and mid domain

ProteinName: La Crosse virus polymerase cap-binding domain and mid domain
Details: Masked cryo-EM map of La Crosse virus full-length polymerase, displaying only the cap-binding domain and the mid domain. Masking, 3D classification and refinement enable to obtain a ...Details: Masked cryo-EM map of La Crosse virus full-length polymerase, displaying only the cap-binding domain and the mid domain. Masking, 3D classification and refinement enable to obtain a structure of the cap-binding domain.
Recombinant expression: No
MassTheoretical: 265 kDa
SourceSpecies: La Crosse orthobunyavirus

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Component #2: protein, La Crosse virus polymerase cap-binding domain and mid domain

ProteinName: La Crosse virus polymerase cap-binding domain and mid domain
Recombinant expression: No
SourceSpecies: La Crosse orthobunyavirus
Source (engineered)Expression System: Trichoplusia ni (cabbage looper)

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Experimental details

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Sample preparation

SpecimenSpecimen state: Particle / Method: cryo EM
Sample solutionSpecimen conc.: 0.2 mg/mL
Buffer solution: 20 mM Tris-HCl pH 8, 150 mM NaCl, 2 mM TCEP
pH: 8
Support film30mA
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Temperature: 90 K / Humidity: 100 % / Details: blot time: 2 sec, blot force: 1.

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
ImagingMicroscope: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 1.25 e/Å2 / Illumination mode: FLOOD BEAM
LensMagnification: 165000 X (nominal) / Cs: 2.7 mm / Imaging mode: BRIGHT FIELD / Defocus: -800.0 - -3500.0 nm / Energy filter: GIF Quantum LS
Specimen HolderModel: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature: (90.0 - 90.0 K)
CameraDetector: GATAN K2 SUMMIT (4k x 4k)

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Image acquisition

Image acquisitionNumber of digital images: 16498

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C1 (asymmetric) / Number of projections: 131058
3D reconstructionAlgorithm: BACK PROJECTION / Software: RELION / Resolution: 3.54 Å / Resolution method: FSC 0.143 CUT-OFF
Details: Signal subtraction of the other polymerase domain and re-centering of the mid and CBD before refinement

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Atomic model buiding

Modeling #1Refinement space: REAL

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