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- EMDB-50322: Cas nuclease-CRISPR (cr)RNA ribonucleoprotein (RNP) complex -

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Basic information

Entry
Database: EMDB / ID: EMD-50322
TitleCas nuclease-CRISPR (cr)RNA ribonucleoprotein (RNP) complex
Map data
Sample
  • Complex: Cas13b (cr)RNA complex
    • Protein or peptide: Phage head-tail adaptor
    • RNA: crRNA
KeywordsAcrVIB1 / anti-CRISPR protein / Cas13b / RNA BINDING PROTEIN
Function / homologyPhage head-tail adaptor
Function and homology information
Biological speciesSegatella buccae (bacteria) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.09 Å
AuthorsSchmelz S / Lukat P / Blankenfeldt W
Funding support1 items
OrganizationGrant numberCountry
Not funded
CitationJournal: Acta Crystallogr D Struct Biol / Year: 2019
Title: Macromolecular structure determination using X-rays, neutrons and electrons: recent developments in Phenix.
Authors: Dorothee Liebschner / Pavel V Afonine / Matthew L Baker / Gábor Bunkóczi / Vincent B Chen / Tristan I Croll / Bradley Hintze / Li Wei Hung / Swati Jain / Airlie J McCoy / Nigel W Moriarty ...Authors: Dorothee Liebschner / Pavel V Afonine / Matthew L Baker / Gábor Bunkóczi / Vincent B Chen / Tristan I Croll / Bradley Hintze / Li Wei Hung / Swati Jain / Airlie J McCoy / Nigel W Moriarty / Robert D Oeffner / Billy K Poon / Michael G Prisant / Randy J Read / Jane S Richardson / David C Richardson / Massimo D Sammito / Oleg V Sobolev / Duncan H Stockwell / Thomas C Terwilliger / Alexandre G Urzhumtsev / Lizbeth L Videau / Christopher J Williams / Paul D Adams /
Abstract: Diffraction (X-ray, neutron and electron) and electron cryo-microscopy are powerful methods to determine three-dimensional macromolecular structures, which are required to understand biological ...Diffraction (X-ray, neutron and electron) and electron cryo-microscopy are powerful methods to determine three-dimensional macromolecular structures, which are required to understand biological processes and to develop new therapeutics against diseases. The overall structure-solution workflow is similar for these techniques, but nuances exist because the properties of the reduced experimental data are different. Software tools for structure determination should therefore be tailored for each method. Phenix is a comprehensive software package for macromolecular structure determination that handles data from any of these techniques. Tasks performed with Phenix include data-quality assessment, map improvement, model building, the validation/rebuilding/refinement cycle and deposition. Each tool caters to the type of experimental data. The design of Phenix emphasizes the automation of procedures, where possible, to minimize repetitive and time-consuming manual tasks, while default parameters are chosen to encourage best practice. A graphical user interface provides access to many command-line features of Phenix and streamlines the transition between programs, project tracking and re-running of previous tasks.
History
DepositionMay 16, 2024-
Header (metadata) releaseFeb 12, 2025-
Map releaseFeb 12, 2025-
UpdateApr 2, 2025-
Current statusApr 2, 2025Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_50322.png

Downloads & links

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Map

FileDownload / File: emd_50322.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.91 Å/pix.
x 256 pix.
= 232.96 Å		0.91 Å/pix.
x 256 pix.
= 232.96 Å		0.91 Å/pix.
x 256 pix.
= 232.96 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.91 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.04
Minimum - Maximum-0.6607462 - 1.0835227
Average (Standard dev.)-0.000009755981 (±0.024294013)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 232.96 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_50322_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_50322_half_map_1.map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_50322_half_map_2.map
Projections & Slices
AxesZYX

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Histogram

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Sample components

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Entire : Cas13b (cr)RNA complex

EntireName: Cas13b (cr)RNA complex
Components
  • Complex: Cas13b (cr)RNA complex
    • Protein or peptide: Phage head-tail adaptor
    • RNA: crRNA

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Supramolecule #1: Cas13b (cr)RNA complex

SupramoleculeName: Cas13b (cr)RNA complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Segatella buccae (bacteria)

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Macromolecule #1: Phage head-tail adaptor

MacromoleculeName: Phage head-tail adaptor / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Segatella buccae (bacteria)
Molecular weightTheoretical: 134.285938 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MQKQDKLFVD RKKNAIFAFP KYITIMENKE KPEPIYYELT DKHFWAAFLN LARHNVYTTI NHINRRLEIA ELKDDGYMMG IKGSWNEQA KKLDKKVRLR DLIMKHFPFL EAAAYEMTNS KSPNNKEQRE KEQSEALSLN NLKNVLFIFL EKLQVLRNYY S HYKYSEES ...String:
MQKQDKLFVD RKKNAIFAFP KYITIMENKE KPEPIYYELT DKHFWAAFLN LARHNVYTTI NHINRRLEIA ELKDDGYMMG IKGSWNEQA KKLDKKVRLR DLIMKHFPFL EAAAYEMTNS KSPNNKEQRE KEQSEALSLN NLKNVLFIFL EKLQVLRNYY S HYKYSEES PKPIFETSLL KNMYKVFDAN VRLVKRDYMH HENIDMQRDF THLNRKKQVG RTKNIIDSPN FHYHFADKEG NM TIAGLLF FVSLFLDKKD AIWMQKKLKG FKDGRNLREQ MTNEVFCRSR ISLPKLKLEN VQTKDWMQLD MLNELVRCPK SLY ERLREK DRESFKVPFD IFSDDYNAEE EPFKNTLVRH QDRFPYFVLR YFDLNEIFEQ LRFQIDLGTY HFSIYNKRIG DEDE VRHLT HHLYGFARIQ DFAPQNQPEE WRKLVKDLDH FETSQEPYIS KTAPHYHLEN EKIGIKFCSA HNNLFPSLQT DKTCN GRSK FNLGTQFTAE AFLSVHELLP MMFYYLLLTK DYSRKESADK VEGIIRKEIS NIYAIYDAFA NNEINSIADL TRRLQN TNI LQGHLPKQMI SILKGRQKDM GKEAERKIGE MIDDTQRRLD LLCKQTNQKI RIGKRNAGLL KSGKIADWLV NDMMRFQ PV QKDQNNIPIN NSKANSTEYR MLQRALALFG SENFRLKAYF NQMNLVGNDN PHPFLAETQW EHQTNILSFY RNYLEARK K YLKGLKPQNW KQYQHFLILK VQKTNRNTLV TGWKNSFNLP RGIFTQPIRE WFEKHNNSKR IYDQILSFDR VGFVAKAIP LYFAEEYKDN VQPFYDYPFN IGNRLKPKKR QFLDKKERVE LWQKNKELFK NYPSEKKKTD LAYLDFLSWK KFERELRLIK NQDIVTWLM FKELFNMATV EGLKIGEIHL RDIDTNTANE ESNNILNRIM PMKLPVKTYE TDNKGNILKE RPLATFYIEE T ETKVLKQG NFKALVKDRR LNGLFSFAET TDLNLEEHPI SKLSVDLELI KYQTTRISIF EMTLGLEKKL IDKYSTLPTD SF RNMLERW LQCKANRPEL KNYVNSLIAV RNAFSHNQYP MYDATLFAEV KKFTLFPSVD TKKIELNIAP QLLEIVGKAI KEI EKSENK N

UniProtKB: Phage head-tail adaptor

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Macromolecule #2: crRNA

MacromoleculeName: crRNA / type: rna / ID: 2 / Details: unprocessed crRNA / Number of copies: 1
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 25.895355 KDa
SequenceString:
CGUCGCCGUC CAGCUCGACC AGGAUGGGAA GUUGCAUCUG CCUUCUUUUU GAAAGGUAAA AACAACAUCG UCCAUUCCGA C

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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

Concentration1 mg/mL
BufferpH: 7 / Details: 20mM HEPES pH7.0 120 mM NaCl 0.5 mM DTT
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 200 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec. / Pretreatment - Atmosphere: AIR / Details: 15 mA
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 283 K / Instrument: FEI VITROBOT MARK IV
Detailsfor complex formation PubCas13b (1mg/ml) was mixed with crRNA in a ratio of 1:0.9 and kept on ice for 10 min prior vitrification.

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Electron microscopy

MicroscopeTFS GLACIOS
Specialist opticsEnergy filter - Name: TFS Selectris / Energy filter - Slit width: 10 eV
SoftwareName: EPU (ver. 3.3.1.5184)
Image recordingFilm or detector model: TFS FALCON 4i (4k x 4k) / Number real images: 3778 / Average electron dose: 40.0 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.6 µm
Sample stageCooling holder cryogen: NITROGEN

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Image processing

Startup modelType of model: INSILICO MODEL / Details: insilco model generated with cryoSPARC in C1
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.09 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. v4.4.0) / Number images used: 356629
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. v4.4.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. v4.4.0)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

6dtd


Chain - Source name: PDB / Chain - Initial model type: experimental model
SoftwareName: UCSF ChimeraX (ver. 1.71)
Output model

PDB-9fcv:
Cas nuclease-CRISPR (cr)RNA ribonucleoprotein (RNP) complex

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