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- EMDB-21607: Internal translation of large subunit transcripts drives synthesi... -

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Basic information

Entry
Database: EMDB / ID: EMD-21607
TitleInternal translation of large subunit transcripts drives synthesis of small subunits in type I CRISPR-Cas Cascade
Map dataType I-D Cascade from Synechocystis with crRNA bound.
Sample
  • Complex: Type I-D Cascade
Biological speciesSynechocystis sp. PCC 6803 (bacteria)
Methodsingle particle reconstruction / negative staining / Resolution: 19.0 Å
AuthorsMcBride TM / Schwartz EA / Kumar A / Taylor DW / Fineran PC / Fagerlund RD
CitationJournal: Mol Cell / Year: 2020
Title: Diverse CRISPR-Cas Complexes Require Independent Translation of Small and Large Subunits from a Single Gene.
Authors: Tess M McBride / Evan A Schwartz / Abhishek Kumar / David W Taylor / Peter C Fineran / Robert D Fagerlund /
Abstract: CRISPR-Cas adaptive immune systems provide prokaryotes with defense against viruses by degradation of specific invading nucleic acids. Despite advances in the biotechnological exploitation of select ...CRISPR-Cas adaptive immune systems provide prokaryotes with defense against viruses by degradation of specific invading nucleic acids. Despite advances in the biotechnological exploitation of select systems, multiple CRISPR-Cas types remain uncharacterized. Here, we investigated the previously uncharacterized type I-D interference complex and revealed that it is a genetic and structural hybrid with similarity to both type I and type III systems. Surprisingly, formation of the functional complex required internal in-frame translation of small subunits from within the large subunit gene. We further show that internal translation to generate small subunits is widespread across diverse type I-D, I-B, and I-C systems, which account for roughly one quarter of CRISPR-Cas systems. Our work reveals the unexpected expansion of protein coding potential from within single cas genes, which has important implications for understanding CRISPR-Cas function and evolution.
History
DepositionMar 30, 2020-
Header (metadata) releaseFeb 10, 2021-
Map releaseFeb 10, 2021-
UpdateFeb 10, 2021-
Current statusFeb 10, 2021Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 1.53
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 1.53
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_21607.png

Downloads & links

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Map

FileDownload / File: emd_21607.map.gz / Format: CCP4 / Size: 11.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationType I-D Cascade from Synechocystis with crRNA bound.
Voxel sizeX=Y=Z: 2.61 Å
Density
Contour LevelBy AUTHOR: 1.53 / Movie #1: 1.53
Minimum - Maximum-0.6001025 - 3.139124
Average (Standard dev.)0.042278785 (±0.30736303)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions144144144
Spacing144144144
CellA=B=C: 375.84 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.612.612.61
M x/y/z144144144
origin x/y/z0.0000.0000.000
length x/y/z375.840375.840375.840
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ320320320
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS144144144
D min/max/mean-0.6003.1390.042

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Supplemental data

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Sample components

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Entire : Type I-D Cascade

EntireName: Type I-D Cascade
Components
  • Complex: Type I-D Cascade

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Supramolecule #1: Type I-D Cascade

SupramoleculeName: Type I-D Cascade / type: complex / ID: 1 / Parent: 0
Source (natural)Organism: Synechocystis sp. PCC 6803 (bacteria)
Recombinant expressionOrganism: Escherichia coli (E. coli)
Molecular weightExperimental: 450 KDa

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Experimental details

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Structure determination

Methodnegative staining
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.045 mg/mL
BufferpH: 7.5
StainingType: NEGATIVE / Material: uranyl acetate
GridModel: C-flat / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Pretreatment - Type: GLOW DISCHARGE

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Electron microscopy

MicroscopeTFS TALOS
Image recordingFilm or detector model: FEI CETA (4k x 4k) / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Number grids imaged: 1 / Number real images: 70 / Average exposure time: 1.0 sec. / Average electron dose: 30.0 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus min: -1.5 µm / Nominal magnification: 57000
Sample stageSpecimen holder model: OTHER

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Image processing

Particle selectionNumber selected: 18696
CTF correctionSoftware - Name: CTFFIND (ver. 4.0)
Startup modelType of model: EMDB MAP
EMDB ID:

2418

Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 19.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 1) / Number images used: 2284
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 1)
Final 3D classificationNumber classes: 4 / Avg.num./class: 1500 / Software - Name: cryoSPARC (ver. 1)

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