+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-22518 | |||||||||
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Title | SpCas9 delta4CE Ternary Complex | |||||||||
Map data | SpCas9 delta4CE Ternary Complex | |||||||||
Sample |
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Keywords | CRISPR-Cas / DNA-binding / engineered / RNA BINDING PROTEIN | |||||||||
Biological species | Streptococcus pyogenes (bacteria) / Escherichia coli (E. coli) | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 6.2 Å | |||||||||
Authors | Sham A / Laughlin TG / Savage DF | |||||||||
Funding support | United States, 1 items
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Citation | Journal: Nat Commun / Year: 2021 Title: Comprehensive deletion landscape of CRISPR-Cas9 identifies minimal RNA-guided DNA-binding modules. Authors: Arik Shams / Sean A Higgins / Christof Fellmann / Thomas G Laughlin / Benjamin L Oakes / Rachel Lew / Shin Kim / Maria Lukarska / Madeline Arnold / Brett T Staahl / Jennifer A Doudna / David F Savage / Abstract: Proteins evolve through the modular rearrangement of elements known as domains. Extant, multidomain proteins are hypothesized to be the result of domain accretion, but there has been limited ...Proteins evolve through the modular rearrangement of elements known as domains. Extant, multidomain proteins are hypothesized to be the result of domain accretion, but there has been limited experimental validation of this idea. Here, we introduce a technique for genetic minimization by iterative size-exclusion and recombination (MISER) for comprehensively making all possible deletions of a protein. Using MISER, we generate a deletion landscape for the CRISPR protein Cas9. We find that the catalytically-dead Streptococcus pyogenes Cas9 can tolerate large single deletions in the REC2, REC3, HNH, and RuvC domains, while still functioning in vitro and in vivo, and that these deletions can be stacked together to engineer minimal, DNA-binding effector proteins. In total, our results demonstrate that extant proteins retain significant modularity from the accretion process and, as genetic size is a major limitation for viral delivery systems, establish a general technique to improve genome editing and gene therapy-based therapeutics. | |||||||||
History |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | EM map: SurfViewMolmilJmol/JSmol |
Supplemental images |
-Downloads & links
-EMDB archive
Map data | emd_22518.map.gz | 1.5 MB | EMDB map data format | |
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Header (meta data) | emd-22518-v30.xml emd-22518.xml | 26.5 KB 26.5 KB | Display Display | EMDB header |
FSC (resolution estimation) | emd_22518_fsc.xml | 4.7 KB | Display | FSC data file |
Images | emd_22518.png | 93.9 KB | ||
Masks | emd_22518_msk_1.map | 8 MB | Mask map | |
Others | emd_22518_additional_1.map.gz emd_22518_additional_2.map.gz emd_22518_additional_3.map.gz emd_22518_half_map_1.map.gz emd_22518_half_map_2.map.gz | 4.3 MB 6 MB 5.3 MB 6 MB 6 MB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-22518 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-22518 | HTTPS FTP |
-Related structure data
Similar structure data | |
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EM raw data | EMPIAR-10508 (Title: Cryo-EM structure of the SpCas9 delta4CE Ternary Complex Data size: 1.5 TB Data #1: Unaligned multi-frame, non-gain-corrected frames in LZW-TIFF of SpCas9 delta4CE Ternary Complex [micrographs - multiframe]) |
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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-Map
File | Download / File: emd_22518.map.gz / Format: CCP4 / Size: 8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | SpCas9 delta4CE Ternary Complex | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 1.8 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Mask #1
File | emd_22518_msk_1.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Additional map: #3
File | emd_22518_additional_1.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Additional map: #1
File | emd_22518_additional_2.map | ||||||||||||
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Density Histograms |
-Additional map: #2
File | emd_22518_additional_3.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Half map: #2
File | emd_22518_half_map_1.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Half map: #1
File | emd_22518_half_map_2.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Sample components
-Entire : SpCas9 delta4CE Ternary Complex
Entire | Name: SpCas9 delta4CE Ternary Complex |
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Components |
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-Supramolecule #1: SpCas9 delta4CE Ternary Complex
Supramolecule | Name: SpCas9 delta4CE Ternary Complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all |
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Source (natural) | Organism: Streptococcus pyogenes (bacteria) |
Molecular weight | Theoretical: 176.65 KDa |
-Macromolecule #1: SpCas9 delta4CE
Macromolecule | Name: SpCas9 delta4CE / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO |
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Source (natural) | Organism: Streptococcus pyogenes (bacteria) |
Recombinant expression | Organism: Escherichia coli (E. coli) |
Sequence | String: MKSSHHHHHH ENLYFQSNAT PKKKRKVGGS PKKKRKVGGS PKKKRKVGGS PKKKRKVGIH GVPAATMDKK YSIGLAIGTN SVGWAVITDE YKVPSKKFKV LGNTDRHSIK KNLIGALLFD SGETAEATRL KRTARRRYTR RKNRICYLQE IFSNEMAKVD DSFFHRLEES ...String: MKSSHHHHHH ENLYFQSNAT PKKKRKVGGS PKKKRKVGGS PKKKRKVGGS PKKKRKVGIH GVPAATMDKK YSIGLAIGTN SVGWAVITDE YKVPSKKFKV LGNTDRHSIK KNLIGALLFD SGETAEATRL KRTARRRYTR RKNRICYLQE IFSNEMAKVD DSFFHRLEES FLVEEDKKHE RHPIFGNIVD EVAYHEKYPT IYHLRKKLVD STDKADLRLI YLALAHMIKF RGHFLIEGDL NPDNSTSDAI LLSDILRVNT EITKAPLSAS MIKRYDEHHQ DLTLLKALVR QQLPEKYKEI FFDQSKNGYA GYIDGGASQE EFYKFIKPIL EKMDGTEELL VKLNREDLLR KQRTFDNGSI PHQIHLGELH AILRRQEDFY PFLKDNREKI EKILTFRIPY YVGPLARGNS RFAWMTRKSE ETITPWNFEE VVDKGASAQS FIERMTNFDK NLTSQKAQVS GQGDSLHEHI ANLAGSPAIK KGILQTVKVV DELVKVMGRH KPENIVIEMA RENQTTQKGQ KNSRERMKRI EEGIKELASD NLTKAERGGL SELDKAGFIK RQLVETRQIT KHVAQILDSR MNTKYDENDK LIREVKVITL KSKLVSDFRK DFQFYKVREI NNYHHAHDAY LNAVVGTALI KKYPKLESEF VASTVRKVLS MPQVNIVKKT EVQTGGFSKE SILPKRNSDK LIARKKDWDP KKYGGFDSPT VAYSVLVVAK VEKGKSKKLK SVKELLGITI MERSSFEKNP IDFLEAKGYK EVKKDLIIKL PKYSLFELEN GRKRMLASAG ELQKGNELAL PSKYVNFLYL ASHYEKLKGS PEDNEQKQLF VEQHKHYLDE IIEQISEFSK RVILADANLD KVLSAYNKHR DKPIREQAEN IIHLFTLTNL GAPAAFKYFD TTIDRKRYTS TKEVLDATLI HQSITGLYET RIDLSQLGGD GSPKKKRKVE DPKKKRKVDT QSPKGS |
-Macromolecule #2: sgRNA
Macromolecule | Name: sgRNA / type: rna / ID: 2 |
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Source (natural) | Organism: Escherichia coli (E. coli) |
Sequence | String: UCAGCCACAG CUGGGCCUGG GUUUUAGAGC UAGAAAUAGC AAGUUAAAAU AAGGCUAGUC CGUUAUCAAC UUGAAAAAGU GGCACCGAGU CGGUGCUUUU |
-Macromolecule #3: Target-strand
Macromolecule | Name: Target-strand / type: dna / ID: 3 / Classification: DNA |
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Source (natural) | Organism: Escherichia coli (E. coli) |
Sequence | String: CTCACCTTCC TACGGTAGTC GGTGTCGACC CGGACCACCC GACAGTTTTA ACTCG |
-Macromolecule #4: Non-target strand
Macromolecule | Name: Non-target strand / type: dna / ID: 4 / Classification: DNA |
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Source (natural) | Organism: Escherichia coli (E. coli) |
Sequence | String: GAGTGGAAGG ATGCCATCAG CCACAGCTGG GCCTGGTGGG CTGTCAAAAT TGAGC |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 0.0053 mg/mL | ||||||||||||||||||
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Buffer | pH: 8 Component:
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Grid | Model: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 200 / Support film - #0 - Film type ID: 1 / Support film - #0 - Material: CARBON / Support film - #0 - topology: HOLEY / Support film - #1 - Film type ID: 2 / Support film - #1 - Material: CARBON / Support film - #1 - topology: CONTINUOUS / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 15 sec. / Pretreatment - Atmosphere: OTHER | ||||||||||||||||||
Vitrification | Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 289 K / Instrument: FEI VITROBOT MARK IV / Details: 10 s incubation, blot 5 seconds. |
-Electron microscopy
Microscope | FEI TALOS ARCTICA |
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Electron beam | Acceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN |
Electron optics | C2 aperture diameter: 70.0 µm / Calibrated defocus max: 3.8000000000000003 µm / Calibrated defocus min: 1.5 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal magnification: 45000 |
Sample stage | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN |
Image recording | Film or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 1 / Number real images: 3400 / Average electron dose: 60.0 e/Å2 Details: collected using image-shift for a regular 3x3 array of holes per stage movement |
Experimental equipment | Model: Talos Arctica / Image courtesy: FEI Company |
-Image processing
-Atomic model buiding 1
Initial model | PDB ID: |
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Refinement | Space: REAL / Protocol: RIGID BODY FIT / Overall B value: 395 / Target criteria: CC |