[English] 日本語
Yorodumi
- EMDB-48340: FnoCas12a bridge helix variant state 4a -

+
Open data


ID or keywords:

Loading...

-
Basic information

Entry
Database: EMDB / ID: EMD-48340
TitleFnoCas12a bridge helix variant state 4a
Map data
Sample
  • Complex: Ternary complex of FnoCRISPR-Cas12a
    • Protein or peptide: CRISPR-associated endonuclease Cas12a
    • RNA: RNA (36-MER)
    • DNA: TS DNA
    • DNA: NTS DNA
KeywordsCas12a / bridge helix / loop-to-helical transition / conformational cascade / allostery / off-target DNA cleavage / DNA BINDING PROTEIN
Function / homology
Function and homology information


Bacillus subtilis ribonuclease / deoxyribonuclease I / deoxyribonuclease I activity / defense response to virus / lyase activity / DNA binding / RNA binding
Similarity search - Function
: / CRISPR-associated endonuclease Cpf1 PI domain / : / CRISPR-associated endonuclease Cpf1 REC2 domain / CRISPR-associated endonuclease Cas12a / Cas12a, REC1 domain / Cas12a, RuvC nuclease domain / Cas12a, nuclease domain / Alpha helical recognition lobe domain / Nuclease domain / RuvC nuclease domain
Similarity search - Domain/homology
CRISPR-associated endonuclease Cas12a
Similarity search - Component
Biological speciesFrancisella tularensis subsp. novicida U112 (bacteria) / Francisella tularensis subsp. novicida (bacteria) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.63 Å
AuthorsGanguly C / Thomas LM / Aribam SD / Martin L / Rajan R
Funding support United States, 2 items
OrganizationGrant numberCountry
National Science Foundation (NSF, United States)MCB-1716423 United States
National Science Foundation (NSF, United States) United States
CitationJournal: Nat Commun / Year: 2026
Title: Bridge helix of Cas12a is an allosteric regulator of R-loop formation and RuvC activation.
Authors: Chhandosee Ganguly / Swarmistha Devi Aribam / Alberto Monteiro Dos Santos / Lindsie Martin / Leonard M Thomas / Yihan Shao / Rakhi Rajan /
Abstract: CRISPR-Cas12a, an RNA-based DNA targeting system, is widely used for genome editing and biomarker detection. To mitigate the off-target DNA cleavage of Cas12a, we previously developed a Francisella ...CRISPR-Cas12a, an RNA-based DNA targeting system, is widely used for genome editing and biomarker detection. To mitigate the off-target DNA cleavage of Cas12a, we previously developed a Francisella novicida Cas12a variant (FnoCas12a) by introducing double proline substitutions (K969P/D970P) in a conserved arginine-rich helix called the bridge helix (BH). In this work, we use a combinatorial approach to understand the molecular mechanisms of BH-mediated activation of Cas12a for DNA cleavage. We report five structures of FnoCas12a that are at different states of conformational activation. Comparison of the variant and wild-type (FnoCas12a) structures, along with activity assays and computational simulations, establishes the loop-to-helical transition and bending of the BH as an allosteric trigger for RNA-DNA hybrid propagation. These changes track with the previously reported coupled remodeling of BH and helix 1 of RuvC motif-II as well as the REC lobe movements needed to accommodate the growing hybrid. The transition of the BH is essential for the loop-to-helical transition of the "lid", which in turn opens the RuvC active site pocket for DNA entry and cleavage. Pairwise 3D structural comparison of the BH and RuvC of Cas12 and Cas9 families provides insight into the diversity of BH's structural organization in these mechanistically similar enzymes.
History
DepositionDec 18, 2024-
Header (metadata) releaseFeb 11, 2026-
Map releaseFeb 11, 2026-
UpdateFeb 11, 2026-
Current statusFeb 11, 2026Processing site: RCSB / Status: Released

-
Structure visualization

Supplemental images

emd_48340.png

Downloads & links

-
Map

FileDownload / File: emd_48340.map.gz / Format: CCP4 / Size: 83.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.86 Å/pix.
x 280 pix.
= 240.8 Å		0.86 Å/pix.
x 280 pix.
= 240.8 Å		0.86 Å/pix.
x 280 pix.
= 240.8 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.86 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.033
Minimum - Maximum-0.17752452 - 0.3305755
Average (Standard dev.)-0.0006636927 (±0.011342453)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions280280280
Spacing280280280
CellA=B=C: 240.8 Å
α=β=γ: 90.0 °

-
Supplemental data

-
Half map: #2

Fileemd_48340_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

-
Half map: #1

Fileemd_48340_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

-
Sample components

-
Entire : Ternary complex of FnoCRISPR-Cas12a

EntireName: Ternary complex of FnoCRISPR-Cas12a
Components
  • Complex: Ternary complex of FnoCRISPR-Cas12a
    • Protein or peptide: CRISPR-associated endonuclease Cas12a
    • RNA: RNA (36-MER)
    • DNA: TS DNA
    • DNA: NTS DNA

-
Supramolecule #1: Ternary complex of FnoCRISPR-Cas12a

SupramoleculeName: Ternary complex of FnoCRISPR-Cas12a / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Francisella tularensis subsp. novicida U112 (bacteria)
Molecular weightTheoretical: 1.52 MDa

-
Macromolecule #1: CRISPR-associated endonuclease Cas12a

MacromoleculeName: CRISPR-associated endonuclease Cas12a / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: deoxyribonuclease I
Source (natural)Organism: Francisella tularensis subsp. novicida (bacteria)
Molecular weightTheoretical: 152.515516 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: GAASHMSIYQ EFVNKYSLSK TLRFELIPQG KTLENIKARG LILDDEKRAK DYKKAKQIID KYHQFFIEEI LSSVCISEDL LQNYSDVYF KLKKSDDDNL QKDFKSAKDT IKKQISEYIK DSEKFKNLFN QNLIDAKKGQ ESDLILWLKQ SKDNGIELFK A NSDITDID ...String:
GAASHMSIYQ EFVNKYSLSK TLRFELIPQG KTLENIKARG LILDDEKRAK DYKKAKQIID KYHQFFIEEI LSSVCISEDL LQNYSDVYF KLKKSDDDNL QKDFKSAKDT IKKQISEYIK DSEKFKNLFN QNLIDAKKGQ ESDLILWLKQ SKDNGIELFK A NSDITDID EALEIIKSFK GWTTYFKGFH ENRKNVYSSN DIPTSIIYRI VDDNLPKFLE NKAKYESLKD KAPEAINYEQ IK KDLAEEL TFDIDYKTSE VNQRVFSLDE VFEIANFNNY LNQSGITKFN TIIGGKFVNG ENTKRKGINE YINLYSQQIN DKT LKKYKM SVLFKQILSD TESKSFVIDK LEDDSDVVTT MQSFYEQIAA FKTVEEKSIK ETLSLLFDDL KAQKLDLSKI YFKN DKSLT DLSQQVFDDY SVIGTAVLEY ITQQIAPKNL DNPSKKEQEL IAKKTEKAKY LSLETIKLAL EEFNKHRDID KQCRF EEIL ANFAAIPMIF DEIAQNKDNL AQISIKYQNQ GKKDLLQASA EDDVKAIKDL LDQTNNLLHK LKIFHISQSE DKANIL DKD EHFYLVFEEC YFELANIVPL YNKIRNYITQ KPYSDEKFKL NFENSTLANG WDKNKEPDNT AILFIKDDKY YLGVMNK KN NKIFDDKAIK ENKGEGYKKI VYKLLPGANK MLPKVFFSAK SIKFYNPSED ILRIRNHSTH TKNGSPQKGY EKFEFNIE D CRKFIDFYKQ SISKHPEWKD FGFRFSDTQR YNSIDEFYRE VENQGYKLTF ENISESYIDS VVNQGKLYLF QIYNKDFSA YSKGRPNLHT LYWKALFDER NLQDVVYKLN GEAELFYRKQ SIPKKITHPA KEAIANKNKD NPKKESVFEY DLIKDKRFTE DKFFFHCPI TINFKSSGAN KFNDEINLLL KEKANDVHIL SIDRGERHLA YYTLVDGKGN IIKQDTFNII GNDRMKTNYH D KLAAIEKD RDSARPPWKK INNIKEMKEG YLSQVVHEIA KLVIEYNAIV VFEDLNFGFK RGRFKVEKQV YQKLEKMLIE KL NYLVFKD NEFDKTGGVL RAYQLTAPFE TFKKMGKQTG IIYYVPAGFT SKICPVTGFV NQLYPKYESV SKSQEFFSKF DKI CYNLDK GYFEFSFDYK NFGDKAAKGK WTIASFGSRL INFRNSDKNH NWDTREVYPT KELEKLLKDY SIEYGHGECI KAAI CGESD KKFFAKLTSV LNTILQMRNS KTGTELDYLI SPVADVNGNF FDSRQAPKNM PQDADANGAY HIGLKGLMLL GRIKN NQEG KKLNLVIKNE EYFEFVQNRN N

UniProtKB: CRISPR-associated endonuclease Cas12a

-
Macromolecule #2: RNA (36-MER)

MacromoleculeName: RNA (36-MER) / type: rna / ID: 2 / Number of copies: 1
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 13.749146 KDa
SequenceString:
AAUUUCUACU GUUGUAGAUG AGAAGUCAUU UAAUAAGGCC ACU

-
Macromolecule #3: TS DNA

MacromoleculeName: TS DNA / type: dna / ID: 3 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 7.302752 KDa
SequenceString:
(DG)(DC)(DC)(DT)(DT)(DA)(DT)(DT)(DA)(DA) (DA)(DT)(DG)(DA)(DC)(DT)(DT)(DC)(DT)(DC) (DT)(DA)(DA)(DA)

-
Macromolecule #4: NTS DNA

MacromoleculeName: NTS DNA / type: dna / ID: 4 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 7.431839 KDa
SequenceString:
(DT)(DT)(DT)(DA)(DG)(DA)(DG)(DA)(DA)(DG) (DT)(DC)(DA)(DT)(DT)(DT)(DA)(DA)(DT)(DA) (DA)(DG)(DG)(DC)

-
Experimental details

-
Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

-
Sample preparation

BufferpH: 7.5
GridModel: Quantifoil R1.2/1.3 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec. / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 4 K / Instrument: LEICA EM GP

-
Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 1.25 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 1.0 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

+
Image processing

CTF correctionType: NONE
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

6gtg

Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.63 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 65427
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

+
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://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