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- EMDB-20267: Cryo-EM structure of LbCas12a-crRNA: AcrVA4 (2:2 complex) -

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Basic information

Entry
Database: EMDB / ID: EMD-20267
TitleCryo-EM structure of LbCas12a-crRNA: AcrVA4 (2:2 complex)
Map data3D Reconstruction (State II)
Sample
  • Complex: LbCas12a-crRNA-AcrVA4 Complex (2:2, State I)
    • Complex: LbCas12a
      • Protein or peptide: Cas12a
    • Complex: crRNA
      • RNA: mature crRNA
    • Complex: AcrVA4
      • Protein or peptide: anti-CRISPR VA4
  • Ligand: MAGNESIUM ION
KeywordsCRISPR-Cas / anti-CRISPR / Cas12a / Cpf1 / LbCas12a / AcrVA4 / RNA BINDING PROTEIN-RNA complex
Function / homologyCRISPR-associated endonuclease Cas12a / Cas12a, REC1 domain / Cas12a, RuvC nuclease domain / Cas12a, nuclease domain / Alpha helical recognition lobe domain / Nuclease domain / RuvC nuclease domain / Uncharacterized protein / Cpf1
Function and homology information
Biological speciesLachnospiraceae bacterium ND2006 (bacteria) / Moraxella bovoculi (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.9 Å
AuthorsKnott GJ / Liu JJ
Funding support United States, 1 items
OrganizationGrant numberCountry
Howard Hughes Medical Institute (HHMI) United States
CitationJournal: Acta Crystallogr D Biol Crystallogr / Year: 2010
Title: PHENIX: a comprehensive Python-based system for macromolecular structure solution.
Authors: Paul D Adams / Pavel V Afonine / Gábor Bunkóczi / Vincent B Chen / Ian W Davis / Nathaniel Echols / Jeffrey J Headd / Li-Wei Hung / Gary J Kapral / Ralf W Grosse-Kunstleve / Airlie J McCoy ...Authors: Paul D Adams / Pavel V Afonine / Gábor Bunkóczi / Vincent B Chen / Ian W Davis / Nathaniel Echols / Jeffrey J Headd / Li-Wei Hung / Gary J Kapral / Ralf W Grosse-Kunstleve / Airlie J McCoy / Nigel W Moriarty / Robert Oeffner / Randy J Read / David C Richardson / Jane S Richardson / Thomas C Terwilliger / Peter H Zwart /
Abstract: Macromolecular X-ray crystallography is routinely applied to understand biological processes at a molecular level. However, significant time and effort are still required to solve and complete many ...Macromolecular X-ray crystallography is routinely applied to understand biological processes at a molecular level. However, significant time and effort are still required to solve and complete many of these structures because of the need for manual interpretation of complex numerical data using many software packages and the repeated use of interactive three-dimensional graphics. PHENIX has been developed to provide a comprehensive system for macromolecular crystallographic structure solution with an emphasis on the automation of all procedures. This has relied on the development of algorithms that minimize or eliminate subjective input, the development of algorithms that automate procedures that are traditionally performed by hand and, finally, the development of a framework that allows a tight integration between the algorithms.
History
DepositionJun 6, 2019-
Header (metadata) releaseJul 17, 2019-
Map releaseAug 21, 2019-
UpdateAug 16, 2023-
Current statusAug 16, 2023Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.08
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.08
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6p7n
  • Surface level: 0.08
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-6p7n
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_20267.png

Downloads & links

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Map

FileDownload / File: emd_20267.map.gz / Format: CCP4 / Size: 149.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotation3D Reconstruction (State II)
Voxel sizeX=Y=Z: 0.9 Å
Density
Contour LevelBy EMDB: 0.08 / Movie #1: 0.08
Minimum - Maximum-0.24226066 - 0.6646911
Average (Standard dev.)0.0021240285 (±0.03206585)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions340340340
Spacing340340340
CellA=B=C: 306.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.90.90.9
M x/y/z340340340
origin x/y/z0.0000.0000.000
length x/y/z306.000306.000306.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS340340340
D min/max/mean-0.2420.6650.002

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Supplemental data

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Half map: 3D Reconstruction (State II) Half Map A

Fileemd_20267_half_map_1.map
Annotation3D Reconstruction (State II) Half Map A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: 3D Reconstruction (State II) Half Map B

Fileemd_20267_half_map_2.map
Annotation3D Reconstruction (State II) Half Map B
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : LbCas12a-crRNA-AcrVA4 Complex (2:2, State I)

EntireName: LbCas12a-crRNA-AcrVA4 Complex (2:2, State I)
Components
  • Complex: LbCas12a-crRNA-AcrVA4 Complex (2:2, State I)
    • Complex: LbCas12a
      • Protein or peptide: Cas12a
    • Complex: crRNA
      • RNA: mature crRNA
    • Complex: AcrVA4
      • Protein or peptide: anti-CRISPR VA4
  • Ligand: MAGNESIUM ION

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Supramolecule #1: LbCas12a-crRNA-AcrVA4 Complex (2:2, State I)

SupramoleculeName: LbCas12a-crRNA-AcrVA4 Complex (2:2, State I) / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Details: Homodimeric AcrVA4 bound to two copies of the LbCas12a-crRNA complex.

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Supramolecule #2: LbCas12a

SupramoleculeName: LbCas12a / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #2
Source (natural)Organism: Lachnospiraceae bacterium ND2006 (bacteria)

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Supramolecule #3: crRNA

SupramoleculeName: crRNA / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #3
Source (natural)Organism: Lachnospiraceae bacterium ND2006 (bacteria)

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Supramolecule #4: AcrVA4

SupramoleculeName: AcrVA4 / type: complex / ID: 4 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Moraxella bovoculi (bacteria)

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Macromolecule #1: anti-CRISPR VA4

MacromoleculeName: anti-CRISPR VA4 / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Moraxella bovoculi (bacteria)
Molecular weightTheoretical: 27.641422 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: SNAMYEIKLN DTLIHQTDDR VNAFVAYRYL LRRGDLPKCE NIARMYYDGK VIKTDVIDHD SVHSDEQAKV SNNDIIKMAI SELGVNNFK SLIKKQGYPF SNGHINSWFT DDPVKSKTMH NDEMYLVVQA LIRACIIKEI DLYTEQLYNI IKSLPYDKRP N VVYSDQPL ...String:
SNAMYEIKLN DTLIHQTDDR VNAFVAYRYL LRRGDLPKCE NIARMYYDGK VIKTDVIDHD SVHSDEQAKV SNNDIIKMAI SELGVNNFK SLIKKQGYPF SNGHINSWFT DDPVKSKTMH NDEMYLVVQA LIRACIIKEI DLYTEQLYNI IKSLPYDKRP N VVYSDQPL DPNNLDLSEP ELWAEQVGEC MRYAHNDQPC FYIGSTKREL RVNYIVPVIG VRDEIERVMT LEEVRNLHK

UniProtKB: Uncharacterized protein

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Macromolecule #2: Cas12a

MacromoleculeName: Cas12a / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Lachnospiraceae bacterium ND2006 (bacteria)
Molecular weightTheoretical: 144.160609 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: SNAMSKLEKF TNCYSLSKTL RFKAIPVGKT QENIDNKRLL VEDEKRAEDY KGVKKLLDRY YLSFINDVLH SIKLKNLNNY ISLFRKKTR TEKENKELEN LEINLRKEIA KAFKGNEGYK SLFKKDIIET ILPEFLDDKD EIALVNSFNG FTTAFTGFFD N RENMFSEE ...String:
SNAMSKLEKF TNCYSLSKTL RFKAIPVGKT QENIDNKRLL VEDEKRAEDY KGVKKLLDRY YLSFINDVLH SIKLKNLNNY ISLFRKKTR TEKENKELEN LEINLRKEIA KAFKGNEGYK SLFKKDIIET ILPEFLDDKD EIALVNSFNG FTTAFTGFFD N RENMFSEE AKSTSIAFRC INENLTRYIS NMDIFEKVDA IFDKHEVQEI KEKILNSDYD VEDFFEGEFF NFVLTQEGID VY NAIIGGF VTESGEKIKG LNEYINLYNQ KTKQKLPKFK PLYKQVLSDR ESLSFYGEGY TSDEEVLEVF RNTLNKNSEI FSS IKKLEK LFKNFDEYSS AGIFVKNGPA ISTISKDIFG EWNVIRDKWN AEYDDIHLKK KAVVTEKYED DRRKSFKKIG SFSL EQLQE YADADLSVVE KLKEIIIQKV DEIYKVYGSS EKLFDADFVL EKSLKKNDAV VAIMKDLLDS VKSFENYIKA FFGEG KETN RDESFYGDFV LAYDILLKVD HIYDAIRNYV TQKPYSKDKF KLYFQNPQFM GGWDKDKETD YRATILRYGS KYYLAI MDK KYAKCLQKID KDDVNGNYEK INYKLLPGPN KMLPKVFFSK KWMAYYNPSE DIQKIYKNGT FKKGDMFNLN DCHKLID FF KDSISRYPKW SNAYDFNFSE TEKYKDIAGF YREVEEQGYK VSFESASKKE VDKLVEEGKL YMFQIYNKDF SDKSHGTP N LHTMYFKLLF DENNHGQIRL SGGAELFMRR ASLKKEELVV HPANSPIANK NPDNPKKTTT LSYDVYKDKR FSEDQYELH IPIAINKCPK NIFKINTEVR VLLKHDDNPY VIGIDRGERN LLYIVVVDGK GNIVEQYSLN EIINNFNGIR IKTDYHSLLD KKEKERFEA RQNWTSIENI KELKAGYISQ VVHKICELVE KYDAVIALED LNSGFKNSRV KVEKQVYQKF EKMLIDKLNY M VDKKSNPC ATGGALKGYQ ITNKFESFKS MSTQNGFIFY IPAWLTSKID PSTGFVNLLK TKYTSIADSK KFISSFDRIM YV PEEDLFE FALDYKNFSR TDADYIKKWK LYSYGNRIRI FRNPKKNNVF DWEEVCLTSA YKELFNKYGI NYQQGDIRAL LCE QSDKAF YSSFMALMSL MLQMRNSITG RTDVDFLISP VKNSDGIFYD SRNYEAQENA ILPKNADANG AYNIARKVLW AIGQ FKKAE DEKLDKVKIA ISNKEWLEYA QTSVKH

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Macromolecule #3: mature crRNA

MacromoleculeName: mature crRNA / type: rna / ID: 3 / Number of copies: 2
Source (natural)Organism: Lachnospiraceae bacterium ND2006 (bacteria)
Molecular weightTheoretical: 12.815634 KDa
SequenceString:
AAUUUCUACU AAGUGUAGAU AAAGUGCUCA UCAUUGGAAA

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Macromolecule #4: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 4 / Number of copies: 4 / Formula: MG
Molecular weightTheoretical: 24.305 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: 7.5
Component:
ConcentrationFormulaName
20.0 mMC8H18N2O4SHEPES
200.0 mMKClpotassium chloride
1.0 mMC9H15O6Ptris((2-carboxyethyl)phosphine)
0.1 % (v/v)C3H8O3glycerol
GridDetails: unspecified
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Average electron dose: 42.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

5id6

Initial angle assignmentType: NOT APPLICABLE
Final angle assignmentType: NOT APPLICABLE
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 4.9 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 79787

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