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- EMDB-22518: SpCas9 delta4CE Ternary Complex -

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Basic information

Entry
Database: EMDB / ID: EMD-22518
TitleSpCas9 delta4CE Ternary Complex
Map dataSpCas9 delta4CE Ternary Complex
Sample
  • Complex: SpCas9 delta4CE Ternary Complex
    • Protein or peptide: SpCas9 delta4CE
    • RNA: sgRNA
    • DNA: Target-strand
    • DNA: Non-target strand
KeywordsCRISPR-Cas / DNA-binding / engineered / RNA BINDING PROTEIN
Biological speciesStreptococcus pyogenes (bacteria) / Escherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 6.2 Å
AuthorsSham A / Laughlin TG / Savage DF
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS) United States
CitationJournal: 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
DepositionAug 27, 2020-
Header (metadata) releaseSep 22, 2021-
Map releaseSep 22, 2021-
UpdateSep 6, 2023-
Current statusSep 6, 2023Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.031
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.031
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_22518.png

Downloads & links

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Map

FileDownload / File: emd_22518.map.gz / Format: CCP4 / Size: 8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSpCas9 delta4CE Ternary Complex
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.8 Å/pix.
x 128 pix.
= 230.4 Å		1.8 Å/pix.
x 128 pix.
= 230.4 Å		1.8 Å/pix.
x 128 pix.
= 230.4 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.8 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.031 / Movie #1: 0.031
Minimum - Maximum-0.09108114 - 0.17871282
Average (Standard dev.)0.0005681342 (±0.0074739596)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions128128128
Spacing128128128
CellA=B=C: 230.4 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.81.81.8
M x/y/z128128128
origin x/y/z0.0000.0000.000
length x/y/z230.400230.400230.400
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS128128128
D min/max/mean-0.0910.1790.001

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Supplemental data

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Mask #1

Fileemd_22518_msk_1.map
Projections & Slices
AxesZYX

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Additional map: #3

Fileemd_22518_additional_1.map
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Additional map: #1

Fileemd_22518_additional_2.map
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Additional map: #2

Fileemd_22518_additional_3.map
Projections & Slices
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Histogram

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Half map: #2

Fileemd_22518_half_map_1.map
Projections & Slices
AxesZYX

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Histogram

Histogram (log scale)

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Half map: #1

Fileemd_22518_half_map_2.map
Projections & Slices
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Sample components

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Entire : SpCas9 delta4CE Ternary Complex

EntireName: SpCas9 delta4CE Ternary Complex
Components
  • Complex: SpCas9 delta4CE Ternary Complex
    • Protein or peptide: SpCas9 delta4CE
    • RNA: sgRNA
    • DNA: Target-strand
    • DNA: Non-target strand

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Supramolecule #1: SpCas9 delta4CE Ternary Complex

SupramoleculeName: SpCas9 delta4CE Ternary Complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Streptococcus pyogenes (bacteria)
Molecular weightTheoretical: 176.65 KDa

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Macromolecule #1: SpCas9 delta4CE

MacromoleculeName: SpCas9 delta4CE / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Streptococcus pyogenes (bacteria)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: 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

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Macromolecule #2: sgRNA

MacromoleculeName: sgRNA / type: rna / ID: 2
Source (natural)Organism: Escherichia coli (E. coli)
SequenceString:
UCAGCCACAG CUGGGCCUGG GUUUUAGAGC UAGAAAUAGC AAGUUAAAAU AAGGCUAGUC CGUUAUCAAC UUGAAAAAGU GGCACCGAGU CGGUGCUUUU

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Macromolecule #3: Target-strand

MacromoleculeName: Target-strand / type: dna / ID: 3 / Classification: DNA
Source (natural)Organism: Escherichia coli (E. coli)
SequenceString:
CTCACCTTCC TACGGTAGTC GGTGTCGACC CGGACCACCC GACAGTTTTA ACTCG

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Macromolecule #4: Non-target strand

MacromoleculeName: Non-target strand / type: dna / ID: 4 / Classification: DNA
Source (natural)Organism: Escherichia coli (E. coli)
SequenceString:
GAGTGGAAGG ATGCCATCAG CCACAGCTGG GCCTGGTGGG CTGTCAAAAT TGAGC

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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

Concentration0.0053 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
30.0 mMNH2C(CH2OH)3HClTris-HCl
150.0 mMKClPotassium Chloride
5.0 mMMgCl2Magnesium chloride
5.0 mMC4H10O2S2Dithiothreitol
0.1 %C3H8O3Glycerol
GridModel: 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
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 289 K / Instrument: FEI VITROBOT MARK IV / Details: 10 s incubation, blot 5 seconds.

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
Image recordingFilm 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
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Calibrated defocus max: 3.8000000000000003 µm / Calibrated defocus min: 1.5 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal magnification: 45000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 288416
Startup modelType of model: OTHER
Details: ab initio via stochastic gradient descent as implemented in RELION
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 6.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1b) / Number images used: 167245
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1b)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1b)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

5y36

RefinementSpace: REAL / Protocol: RIGID BODY FIT / Overall B value: 395 / Target criteria: CC

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