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- EMDB-35191: Lb2Cas12a RNA DNA complex -

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Basic information

Entry
Database: EMDB / ID: EMD-35191
TitleLb2Cas12a RNA DNA complex
Map data
Sample
  • Complex: LbCas12a-crRNA-DNA ternary complex
    • Protein or peptide: Lb2Cas12a
    • RNA: RNA (33-MER)
    • DNA: DNA (25-MER)
    • DNA: DNA (5'-D(*AP*GP*TP*GP*CP*TP*TP*TP*A)-3')
Biological speciesLachnospiraceae bacterium MA2020 (bacteria) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.95 Å
AuthorsLi J / Sivaraman J / Satoru M
Funding support Singapore, 1 items
OrganizationGrant numberCountry
Ministry of Education (MoE, Singapore)R154-000-A39-112 Singapore
CitationJournal: PLoS Biol / Year: 2023
Title: Structures of apo Cas12a and its complex with crRNA and DNA reveal the dynamics of ternary complex formation and target DNA cleavage.
Authors: Li Jianwei / Chacko Jobichen / Satoru Machida / Sun Meng / Randy J Read / Chen Hongying / Shi Jian / Yuren Adam Yuan / J Sivaraman /
Abstract: Cas12a is a programmable nuclease for adaptive immunity against invading nucleic acids in CRISPR-Cas systems. Here, we report the crystal structures of apo Cas12a from Lachnospiraceae bacterium ...Cas12a is a programmable nuclease for adaptive immunity against invading nucleic acids in CRISPR-Cas systems. Here, we report the crystal structures of apo Cas12a from Lachnospiraceae bacterium MA2020 (Lb2) and the Lb2Cas12a+crRNA complex, as well as the cryo-EM structure and functional studies of the Lb2Cas12a+crRNA+DNA complex. We demonstrate that apo Lb2Cas12a assumes a unique, elongated conformation, whereas the Lb2Cas12a+crRNA binary complex exhibits a compact conformation that subsequently rearranges to a semi-open conformation in the Lb2Cas12a+crRNA+DNA ternary complex. Notably, in solution, apo Lb2Cas12a is dynamic and can exist in both elongated and compact forms. Residues from Met493 to Leu523 of the WED domain undergo major conformational changes to facilitate the required structural rearrangements. The REC lobe of Lb2Cas12a rotates 103° concomitant with rearrangement of the hinge region close to the WED and RuvC II domains to position the RNA-DNA duplex near the catalytic site. Our findings provide insight into crRNA recognition and the mechanism of target DNA cleavage.
History
DepositionJan 24, 2023-
Header (metadata) releaseFeb 22, 2023-
Map releaseFeb 22, 2023-
UpdateMay 3, 2023-
Current statusMay 3, 2023Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_35191.png

Downloads & links

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Map

FileDownload / File: emd_35191.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.105 Å
Density
Contour LevelBy AUTHOR: 0.0479
Minimum - Maximum-0.15909009 - 0.45288724
Average (Standard dev.)0.0012037066 (±0.013058879)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 282.88 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: #1

Fileemd_35191_additional_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
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Histogram

Histogram (log scale)

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Half map: #2

Fileemd_35191_half_map_1.map
Projections & Slices
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Histogram

Histogram (log scale)

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Half map: #1

Fileemd_35191_half_map_2.map
Projections & Slices
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Sample components

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Entire : LbCas12a-crRNA-DNA ternary complex

EntireName: LbCas12a-crRNA-DNA ternary complex
Components
  • Complex: LbCas12a-crRNA-DNA ternary complex
    • Protein or peptide: Lb2Cas12a
    • RNA: RNA (33-MER)
    • DNA: DNA (25-MER)
    • DNA: DNA (5'-D(*AP*GP*TP*GP*CP*TP*TP*TP*A)-3')

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Supramolecule #1: LbCas12a-crRNA-DNA ternary complex

SupramoleculeName: LbCas12a-crRNA-DNA ternary complex / type: complex / ID: 1 / Chimera: Yes / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Lachnospiraceae bacterium MA2020 (bacteria)
Molecular weightTheoretical: 140 KDa

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Macromolecule #1: Lb2Cas12a

MacromoleculeName: Lb2Cas12a / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Lachnospiraceae bacterium MA2020 (bacteria)
Molecular weightTheoretical: 141.200609 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MYYESLTKQY PVSKTIRNEL IPIGKTLDNI RQNNILESDV KRKQNYEHVK GILDEYHKQL INEALDNCTL PSLKIAAEIY LKNQKEVSD REDFNKTQDL LRKEVVEKLK AHENFTKIGK KDILDLLEKL PSISEDDYNA LESFRNFYTY FTSYNKVREN L YSDKEKSS ...String:
MYYESLTKQY PVSKTIRNEL IPIGKTLDNI RQNNILESDV KRKQNYEHVK GILDEYHKQL INEALDNCTL PSLKIAAEIY LKNQKEVSD REDFNKTQDL LRKEVVEKLK AHENFTKIGK KDILDLLEKL PSISEDDYNA LESFRNFYTY FTSYNKVREN L YSDKEKSS TVAYRLINEN FPKFLDNVKS YRFVKTAGIL ADGLGEEEQD SLFIVETFNK TLTQDGIDTY NSQVGKINSS IN LYNQKNQ KANGFRKIPK MKMLYKQILS DREESFIDEF QSDEVLIDNV ESYGSVLIES LKSSKVSAFF DALRESKGKN VYV KNDLAK TAMSNIVFEN WRTFDDLLNQ EYDLANENKK KDDKYFEKRQ KELKKNKSYS LEHLCNLSED SCNLIENYIH QISD DIENI IINNETFLRI VINEHDRSRK LAKNRKAVKA IKDFLDSIKV LERELKLINS SGQELEKDLI VYSAHEELLV ELKQV DSLY NMTRNYLTKK PFSTEKVKLN FNRSTLLNGW DRNKETDNLG VLLLKDGKYY LGIMNTSANK AFVNPPVAKT EKVFKK VDY KLLPVPNQML PKVFFAKSNI DFYNPSSEIY SNYKKGTHKK GNMFSLEDCH NLIDFFKESI SKHEDWSKFG FKFSDTA SY NDISEFYREV EKQGYKLTYT DIDETYINDL IERNELYLFQ IYNKDFSMYS KGKLNLHTLY FMMLFDQRNI DDVVYKLN G EAEVFYRPAS ISEDELIIHK AGEEIKNKNP NRARTKETST FSYDIVKDKR YSKDKFTLHI PITMNFGVDE VKRFNDAVN SAIRIDENVN VIGIDRGERN LLYVVVIDSK GNILEQISLN SIINKEYDIE TDYHALLDER EGGRDKARKD WNTVENIRDL KAGYLSQVV NVVAKLVLKY NAIICLEDLN FGFKRGRQKV EKQVYQKFEK MLIDKLNYLV IDKSREQTSP KELGGALNAL Q LTSKFKSF KELGKQSGVI YYVPAYLTSK IDPTTGFANL FYMKCENVEK SKRFFDGFDF IRFNALENVF EFGFDYRSFT QR ACGINSK WTVCTNGERI IKYRNPDKNN MFDEKVVVVT DEMKNLFEQY KIPYEDGRNV KDMIISNEEA EFYRRLYRLL QQT LQMRNS TSDGTRDYII SPVKNKREAY FNSELSDGSV PKDADANGAY NIARKGLWVL EQIRQKSEGE KINLAMTNAE WLEY AQTHL L

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Macromolecule #2: RNA (33-MER)

MacromoleculeName: RNA (33-MER) / type: rna / ID: 2 / Number of copies: 1
Source (natural)Organism: Lachnospiraceae bacterium MA2020 (bacteria)
Molecular weightTheoretical: 10.452219 KDa
SequenceString:
AAUUUCUACU AAUUGUAGAU GCCGCUACCC CGA

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Macromolecule #3: DNA (25-MER)

MacromoleculeName: DNA (25-MER) / type: dna / ID: 3 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 7.780007 KDa
SequenceString:
(DT)(DG)(DG)(DT)(DC)(DG)(DG)(DG)(DG)(DT) (DA)(DG)(DC)(DG)(DG)(DC)(DT)(DA)(DA)(DA) (DG)(DC)(DA)(DC)(DT)

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Macromolecule #4: DNA (5'-D(*AP*GP*TP*GP*CP*TP*TP*TP*A)-3')

MacromoleculeName: DNA (5'-D(*AP*GP*TP*GP*CP*TP*TP*TP*A)-3') / type: dna / ID: 4 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 2.745821 KDa
SequenceString:
(DA)(DG)(DT)(DG)(DC)(DT)(DT)(DT)(DA)

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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
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 400 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 50 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 295.15 K

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Electron microscopy

MicroscopeFEI TITAN
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Illumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 81000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
TemperatureMin: 140.0 K / Max: 150.0 K
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 1 / Number real images: 2560 / Average exposure time: 3.49 sec. / Average electron dose: 45.0 e/Å2

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Image processing

Initial angle assignmentType: NOT APPLICABLE
Final angle assignmentType: NOT APPLICABLE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.95 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 173902
FSC plot (resolution estimation)

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