[English] 日本語
Yorodumi
- PDB-8ea4: V-K CAST Transpososome from Scytonema hofmanni, minor configuration -

+
Open data


ID or keywords:

Loading...

-
Basic information

Entry
Database: PDB / ID: 8ea4
TitleV-K CAST Transpososome from Scytonema hofmanni, minor configuration
Components
  • 30S ribosomal protein S15
  • Cas12k
  • LE_R
  • Non-target_R
  • RE_F
  • RE_R1
  • RE_R2
  • Target-LE
  • TniQ
  • TnsB
  • TnsC
  • sg_RNA
KeywordsDNA BINDING PROTEIN/RNA/DNA / DNA BINDING PROTEIN / DNA BINDING PROTEIN-RNA-DNA complex
Function / homology
Function and homology information


rRNA binding / ribosome / structural constituent of ribosome / ribonucleoprotein complex / translation / cytoplasm
Similarity search - Function
Bacterial TniB / Bacterial TniB protein / : / TniQ / TniQ / Ribosomal protein S15, bacterial-type / Ribosomal protein S15 signature. / Ribosomal protein S15 / Ribosomal_S15 / Ribosomal protein S15 ...Bacterial TniB / Bacterial TniB protein / : / TniQ / TniQ / Ribosomal protein S15, bacterial-type / Ribosomal protein S15 signature. / Ribosomal protein S15 / Ribosomal_S15 / Ribosomal protein S15 / S15/NS1, RNA-binding / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
ADENOSINE-5'-TRIPHOSPHATE / DNA / DNA (> 10) / DNA (> 100) / RNA / RNA (> 10) / RNA (> 100) / TnsC / TniQ (Homology model) / Cas12k / Small ribosomal subunit protein uS15
Similarity search - Component
Biological speciesScytonema hofmannii (bacteria)
Escherichia coli (E. coli)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3 Å
AuthorsRizo, A.R. / Park, J.-U. / Tsai, A.W. / Kellogg, E.K.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)RO1GM144566 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)S10OD030470-01 United States
CitationJournal: Nature / Year: 2023
Title: Structures of the holo CRISPR RNA-guided transposon integration complex.
Authors: Jung-Un Park / Amy Wei-Lun Tsai / Alexandrea N Rizo / Vinh H Truong / Tristan X Wellner / Richard D Schargel / Elizabeth H Kellogg /
Abstract: CRISPR-associated transposons (CAST) are programmable mobile genetic elements that insert large DNA cargos using an RNA-guided mechanism. CAST elements contain multiple conserved proteins: a CRISPR ...CRISPR-associated transposons (CAST) are programmable mobile genetic elements that insert large DNA cargos using an RNA-guided mechanism. CAST elements contain multiple conserved proteins: a CRISPR effector (Cas12k or Cascade), a AAA+ regulator (TnsC), a transposase (TnsA-TnsB) and a target-site-associated factor (TniQ). These components are thought to cooperatively integrate DNA via formation of a multisubunit transposition integration complex (transpososome). Here we reconstituted the approximately 1 MDa type V-K CAST transpososome from Scytonema hofmannii (ShCAST) and determined its structure using single-particle cryo-electon microscopy. The architecture of this transpososome reveals modular association between the components. Cas12k forms a complex with ribosomal subunit S15 and TniQ, stabilizing formation of a full R-loop. TnsC has dedicated interaction interfaces with TniQ and TnsB. Of note, we observe TnsC-TnsB interactions at the C-terminal face of TnsC, which contribute to the stimulation of ATPase activity. Although the TnsC oligomeric assembly deviates slightly from the helical configuration found in isolation, the TnsC-bound target DNA conformation differs markedly in the transpososome. As a consequence, TnsC makes new protein-DNA interactions throughout the transpososome that are important for transposition activity. Finally, we identify two distinct transpososome populations that differ in their DNA contacts near TniQ. This suggests that associations with the CRISPR effector can be flexible. This ShCAST transpososome structure enhances our understanding of CAST transposition systems and suggests ways to improve CAST transposition for precision genome-editing applications.
History
DepositionAug 27, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Nov 30, 2022Provider: repository / Type: Initial release
Revision 1.1Dec 14, 2022Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID / _citation_author.name
Revision 1.2Feb 8, 2023Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.year / _citation_author.identifier_ORCID
Revision 1.3Jun 19, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond

-
Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

-
Assembly

Deposited unit
A: TnsC
B: TnsC
C: TnsC
D: TnsC
E: TnsC
F: TnsC
G: TnsC
H: TnsC
I: TnsC
J: TnsC
K: TnsC
L: TnsC
M: TnsC
O: Cas12k
Q: TniQ
S: 30S ribosomal protein S15
W: TnsB
X: TnsB
Y: TnsB
Z: TnsB
w: TnsB
x: TnsB
y: TnsB
z: TnsB
1: Target-LE
2: LE_R
3: Non-target_R
4: RE_F
5: RE_R1
6: RE_R2
7: sg_RNA
hetero molecules


Theoretical massNumber of molelcules
Total (without water)1,262,32159
Polymers1,255,36331
Non-polymers6,95828
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

-
Components

-
Protein , 5 types, 24 molecules ABCDEFGHIJKLMOQSWXYZwxyz

#1: Protein
TnsC


Mass: 31444.617 Da / Num. of mol.: 13
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Scytonema hofmannii (bacteria) / Production host: Escherichia coli (E. coli) / References: UniProt: A0A8J0PCL3
#2: Protein Cas12k


Mass: 73280.719 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Scytonema hofmannii (bacteria) / Production host: Escherichia coli (E. coli) / References: UniProt: A0A8M0FGU0
#3: Protein TniQ


Mass: 19011.240 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Scytonema hofmannii (bacteria) / Production host: Escherichia coli (E. coli) / References: UniProt: A0A8J0PCL5
#4: Protein 30S ribosomal protein S15


Mass: 10290.816 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: rpsO, HMPREF9346_03742 / Production host: Escherichia coli (E. coli) / References: UniProt: D8EB41
#5: Protein
TnsB


Mass: 66637.070 Da / Num. of mol.: 8
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Scytonema hofmannii (bacteria) / Production host: Escherichia coli (E. coli)

-
DNA chain , 6 types, 6 molecules 123456

#6: DNA chain Target-LE


Mass: 43440.840 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#7: DNA chain LE_R


Mass: 15775.137 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#8: DNA chain Non-target_R


Mass: 21814.982 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#9: DNA chain RE_F


Mass: 23080.830 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#10: DNA chain RE_R1


Mass: 15443.954 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#11: DNA chain RE_R2


Mass: 6086.917 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

-
RNA chain , 1 types, 1 molecules 7

#12: RNA chain sg_RNA


Mass: 85261.250 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

-
Non-polymers , 2 types, 28 molecules

#13: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 15 / Source method: obtained synthetically / Formula: Mg
#14: Chemical
ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE


Mass: 507.181 Da / Num. of mol.: 13 / Source method: obtained synthetically / Formula: C10H16N5O13P3 / Comment: ATP, energy-carrying molecule*YM

-
Details

Has ligand of interestN

-
Experimental details

-
Experiment

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

-
Sample preparation

ComponentName: Sh_CAST Transpososome / Type: COMPLEX / Entity ID: #1-#12 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: [Scytonema hofmanni] UTEX 2349 (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.4
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

-
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 FIELD / Nominal defocus max: 2500 nm / Nominal defocus min: 1000 nm
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

-
Processing

EM software
IDNameCategory
2Leginonimage acquisition
4WarpCTF correction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 67096 / Symmetry type: POINT

+
About Yorodumi

-
News

-
Feb 9, 2022. New format data for meta-information of EMDB entries

New format data for meta-information of EMDB entries

  • Version 3 of the EMDB header file is now the official format.
  • The previous official version 1.9 will be removed from the archive.

Related info.:EMDB header

External links:wwPDB to switch to version 3 of the EMDB data model

-
Aug 12, 2020. Covid-19 info

Covid-19 info

URL: https://pdbjlvh1.pdbj.org/emnavi/covid19.php

New page: Covid-19 featured information page in EM Navigator.

Related info.:Covid-19 info / Mar 5, 2020. Novel coronavirus structure data

+
Mar 5, 2020. Novel coronavirus structure data

Novel coronavirus structure data

Related info.:Yorodumi Speices / Aug 12, 2020. Covid-19 info

External links:COVID-19 featured content - PDBj / Molecule of the Month (242)：Coronavirus Proteases

+
Jan 31, 2019. EMDB accession codes are about to change! (news from PDBe EMDB page)

EMDB accession codes are about to change! (news from PDBe EMDB page)

  • The allocation of 4 digits for EMDB accession codes will soon come to an end. Whilst these codes will remain in use, new EMDB accession codes will include an additional digit and will expand incrementally as the available range of codes is exhausted. The current 4-digit format prefixed with “EMD-” (i.e. EMD-XXXX) will advance to a 5-digit format (i.e. EMD-XXXXX), and so on. It is currently estimated that the 4-digit codes will be depleted around Spring 2019, at which point the 5-digit format will come into force.
  • The EM Navigator/Yorodumi systems omit the EMD- prefix.

Related info.:Q: What is EMD? / ID/Accession-code notation in Yorodumi/EM Navigator

External links:EMDB Accession Codes are Changing Soon! / Contact to PDBj

+
Jul 12, 2017. Major update of PDB

Major update of PDB

  • wwPDB released updated PDB data conforming to the new PDBx/mmCIF dictionary.
  • This is a major update changing the version number from 4 to 5, and with Remediation, in which all the entries are updated.
  • In this update, many items about electron microscopy experimental information are reorganized (e.g. em_software).
  • Now, EM Navigator and Yorodumi are based on the updated data.

External links:wwPDB Remediation / Enriched Model Files Conforming to OneDep Data Standards Now Available in the PDB FTP Archive

-
Yorodumi

Thousand views of thousand structures

  • Yorodumi is a browser for structure data from EMDB, PDB, SASBDB, etc.
  • This page is also the successor to EM Navigator detail page, and also detail information page/front-end page for Omokage search.
  • The word "yorodu" (or yorozu) is an old Japanese word meaning "ten thousand". "mi" (miru) is to see.

Related info.:EMDB / PDB / SASBDB / Comparison of 3 databanks / Yorodumi Search / Aug 31, 2016. New EM Navigator & Yorodumi / Yorodumi Papers / Jmol/JSmol / Function and homology information / Changes in new EM Navigator and Yorodumi

Read more