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- EMDB-22855: Partial Cas6-RT-Cas1--Cas2 complex -

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Basic information

Entry
Database: EMDB / ID: EMD-22855
TitlePartial Cas6-RT-Cas1--Cas2 complex
Map data
Sample
  • Complex: Cas6-RT-Cas1--Cas2 complex
    • Protein or peptide: Cas2
    • Protein or peptide: Cas6-RT-Cas1
KeywordsComplex / Hydrolase / CRISPR Cas Protein / Reverse Transcriptase
Biological speciesThiomicrospira sp. (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsHoel CM / Wang JY
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM123496 United States
CitationJournal: Nat Commun / Year: 2021
Title: Structural coordination between active sites of a CRISPR reverse transcriptase-integrase complex.
Authors: Joy Y Wang / Christopher M Hoel / Basem Al-Shayeb / Jillian F Banfield / Stephen G Brohawn / Jennifer A Doudna /
Abstract: CRISPR-Cas systems provide adaptive immunity in bacteria and archaea, beginning with integration of foreign sequences into the host CRISPR genomic locus and followed by transcription and maturation ...CRISPR-Cas systems provide adaptive immunity in bacteria and archaea, beginning with integration of foreign sequences into the host CRISPR genomic locus and followed by transcription and maturation of CRISPR RNAs (crRNAs). In some CRISPR systems, a reverse transcriptase (RT) fusion to the Cas1 integrase and Cas6 maturase creates a single protein that enables concerted sequence integration and crRNA production. To elucidate how the RT-integrase organizes distinct enzymatic activities, we present the cryo-EM structure of a Cas6-RT-Cas1-Cas2 CRISPR integrase complex. The structure reveals a heterohexamer in which the RT directly contacts the integrase and maturase domains, suggesting functional coordination between all three active sites. Together with biochemical experiments, our data support a model of sequential enzymatic activities that enable CRISPR sequence acquisition from RNA and DNA substrates. These findings highlight an expanded capacity of some CRISPR systems to acquire diverse sequences that direct CRISPR-mediated interference.
History
DepositionOct 14, 2020-
Header (metadata) releaseMar 31, 2021-
Map releaseMar 31, 2021-
UpdateMar 6, 2024-
Current statusMar 6, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 9
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 9
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7kft
  • Surface level: 9
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_22855.png

Downloads & links

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Map

FileDownload / File: emd_22855.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesX (Sec.)Y (Row.)Z (Col.)
1.19 Å/pix.
x 400 pix.
= 474.8 Å		1.19 Å/pix.
x 400 pix.
= 474.8 Å		1.19 Å/pix.
x 400 pix.
= 474.8 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.187 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 9.0 / Movie #1: 9
Minimum - Maximum-41.692303000000003 - 62.598267
Average (Standard dev.)-0.000000000005294 (±1.0)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderZYX
Origin000
Dimensions400400400
Spacing400400400
CellA=B=C: 474.80002 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.1871.1871.187
M x/y/z400400400
origin x/y/z0.0000.0000.000
length x/y/z474.800474.800474.800
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ400400400
MAP C/R/S321
start NC/NR/NS000
NC/NR/NS400400400
D min/max/mean-41.69262.598-0.000

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Supplemental data

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Sample components

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Entire : Cas6-RT-Cas1--Cas2 complex

EntireName: Cas6-RT-Cas1--Cas2 complex
Components
  • Complex: Cas6-RT-Cas1--Cas2 complex
    • Protein or peptide: Cas2
    • Protein or peptide: Cas6-RT-Cas1

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Supramolecule #1: Cas6-RT-Cas1--Cas2 complex

SupramoleculeName: Cas6-RT-Cas1--Cas2 complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Thiomicrospira sp. (bacteria)
Molecular weightTheoretical: 470 KDa

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Macromolecule #1: Cas2

MacromoleculeName: Cas2 / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Thiomicrospira sp. (bacteria)
Molecular weightTheoretical: 11.583263 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
SNAMKHYLIC FDVQHDKTRA KLSRLLEKYG PRVQGSVFEV SFKTPDRKRQ LEYKIHQIIK QSNTEENNIR FYNLNKDTIK HSHDINGNP IAQLPAAIVL

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Macromolecule #2: Cas6-RT-Cas1

MacromoleculeName: Cas6-RT-Cas1 / type: protein_or_peptide / ID: 2 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Thiomicrospira sp. (bacteria)
Molecular weightTheoretical: 112.869414 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: SNAMILPSFP DLTGLVVNLK FTARAEFSLN HEMAVDAFLR HSLNLGESYS HHLSIITPEN GRLFYREGDT YRFVVIAMGN QQQTNSIWH TLINHLRKLP DSAPITDKQA PLRNNIKLES LNDLFDGIPV SSKESLDAYT LQRAMEQGLA WHKAANLTEQ P LDIQWYWQ ...String:
SNAMILPSFP DLTGLVVNLK FTARAEFSLN HEMAVDAFLR HSLNLGESYS HHLSIITPEN GRLFYREGDT YRFVVIAMGN QQQTNSIWH TLINHLRKLP DSAPITDKQA PLRNNIKLES LNDLFDGIPV SSKESLDAYT LQRAMEQGLA WHKAANLTEQ P LDIQWYWQ STVRILHADH KQHKGEQRYC RDAVQLTPLL LLKRIYETLN NVATYFGLKT NKNTTENHQA WLKEQAQYIE IQ HPDLYWI DTPYFGKDAE KNTLGGMAGN FTLSLKPGIE PGLLAMLILT QMVGVGQRRT SGLGKYWLKH SLKHAHLILG LKP NRVTRS QTLLDCIIQP HIISQAIAEI EKKTNIDTLN ERTLSQVQSA IGQLRKHQYQ APKLQGFTIP KKDGTERLLA VSPL YDRIL QKAAAIVLTP GLDAIMSQAS YGYRKGLSRQ QVRYEIQNAY RQGYHWVYES DIEDFFDAVY RPQLINRLKS LLGND PLWE QIESWLGQDI HIKDTIIERT PNLGLPQGSP LSPLLANFIL DDFDSDLETH GFKIIRFADD FIILCKSQHE AQQAAH AVE QSLKEVKLSI NVEKTHIIQL NQGFRFLGYL FREDHAIEIA GEKSDGRTTF AAEQTPTNLP PWLANLGTKS PQPLADD DL PKKSYGQIET QGTHLVLAGD AQIITTDNQN LIVKKDDKIT HKISLEQLHA VTLIGLHTMT LPAKHRLLEH KIPVHIAD R TGRYLGAVTS FQPAQNNYKN WFIQLQMCDR EPFAHAIAQQ IVISRIHNQR QTLLKRKAHR KQLQQTLSNL KKLQYKVTA ATKRSSLNGL EGSATREYFQ QFNLFLPEWA HFSKRTRRPP KDPFNVLLSL GYTILYSHTD AILQSAGFIT WKGIYHQQSA AHAALASDI MESYRHLVER YAIYIINHGQ IKQDDFRQEK DHLGQDTIRL SAEARRRYVG GLINRFQKFS KDKTLHQHLY Q QAQQLKNA MHNQQSSQFQ VWKELK

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7.5
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 300
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV / Details: Blot force 1, 3 second blot.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 49.9 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: OTHER / Details: cryoSPARC ab initio
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 129175
Initial angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION (ver. 3.0)
Final angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION (ver. 3.1)

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