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- EMDB-9191: Structure of double-stranded target DNA engaged Csy complex from ... -

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Basic information

Entry
Database: EMDB / ID: EMD-9191
TitleStructure of double-stranded target DNA engaged Csy complex from Pseudomonas aeruginosa (PA-14)
Map dataComposite sharpened map of the complex generated from multiple focused maps of different sub-regions of the complex. A B-factor of -35 was used for sharpening the map.
Sample
  • Complex: Double-stranded target DNA engaged Csy Complex from Pseudomonas aeruginosa (PA-14)
    • Protein or peptide: CRISPR-associated protein Csy2
    • Protein or peptide: CRISPR-associated protein Csy3
    • Protein or peptide: CRISPR-associated endonuclease Cas6/Csy4
    • RNA: CRISPR RNA (60-MER)
    • DNA: CRISPR target DNA (44-MER)
    • DNA: Non-complementary R-loop DNA strand
    • Protein or peptide: CRISPR-associated protein Csy1
Function / homology
Function and homology information


maintenance of CRISPR repeat elements / defense response to virus / endonuclease activity / Hydrolases; Acting on ester bonds / RNA binding
Similarity search - Function
CRISPR-associated protein Csy1 / CRISPR-associated protein (Cas_Csy1) / CRISPR-associated endoribonuclease Cas6/Csy4, subtype I-F/YPEST / CRISPR-associated endoribonuclease Cas6/Csy4, subtype I-F/YPEST superfamily / CRISPR-associated protein (Cas_Csy4) / CRISPR-associated protein Csy2 / CRISPR-associated protein (Cas_Csy2) / CRISPR-associated protein Csy3 / CRISPR-associated protein (Cas_Csy3)
Similarity search - Domain/homology
CRISPR-associated protein Csy1 / CRISPR-associated protein Csy2 / CRISPR-associated protein Csy3 / CRISPR-associated endonuclease Cas6/Csy4
Similarity search - Component
Biological speciesPseudomonas aeruginosa (strain UCBPP-PA14) (bacteria) / Pseudomonas aeruginosa UCBPP-PA14 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsChowdhury S / Rollins MF / Carter J / Golden SM / Miettinen HM / Santiago-Frangos A / Faith D / Lawrence MC / Wiedenheft B / Lander GC
CitationJournal: Mol Cell / Year: 2019
Title: Structure Reveals a Mechanism of CRISPR-RNA-Guided Nuclease Recruitment and Anti-CRISPR Viral Mimicry.
Authors: MaryClare F Rollins / Saikat Chowdhury / Joshua Carter / Sarah M Golden / Heini M Miettinen / Andrew Santiago-Frangos / Dominick Faith / C Martin Lawrence / Gabriel C Lander / Blake Wiedenheft /
Abstract: Bacteria and archaea have evolved sophisticated adaptive immune systems that rely on CRISPR RNA (crRNA)-guided detection and nuclease-mediated elimination of invading nucleic acids. Here, we present ...Bacteria and archaea have evolved sophisticated adaptive immune systems that rely on CRISPR RNA (crRNA)-guided detection and nuclease-mediated elimination of invading nucleic acids. Here, we present the cryo-electron microscopy (cryo-EM) structure of the type I-F crRNA-guided surveillance complex (Csy complex) from Pseudomonas aeruginosa bound to a double-stranded DNA target. Comparison of this structure to previously determined structures of this complex reveals a ∼180-degree rotation of the C-terminal helical bundle on the "large" Cas8f subunit. We show that the double-stranded DNA (dsDNA)-induced conformational change in Cas8f exposes a Cas2/3 "nuclease recruitment helix" that is structurally homologous to a virally encoded anti-CRISPR protein (AcrIF3). Structural homology between Cas8f and AcrIF3 suggests that AcrIF3 is a mimic of the Cas8f nuclease recruitment helix.
History
DepositionOct 8, 2018-
Header (metadata) releaseOct 24, 2018-
Map releaseOct 24, 2018-
UpdateMar 20, 2019-
Current statusMar 20, 2019Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0595
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.0595
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6ne0
  • Surface level: 0.0595
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_9191.png

Downloads & links

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Map

FileDownload / File: emd_9191.map.gz / Format: CCP4 / Size: 15.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationComposite sharpened map of the complex generated from multiple focused maps of different sub-regions of the complex. A B-factor of -35 was used for sharpening the map.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.15 Å/pix.
x 160 pix.
= 184. Å		1.15 Å/pix.
x 160 pix.
= 184. Å		1.15 Å/pix.
x 160 pix.
= 184. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.15 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0595 / Movie #1: 0.0595
Minimum - Maximum-0.121736825 - 0.27672002
Average (Standard dev.)0.0024644134 (±0.016295806)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions160160160
Spacing160160160
CellA=B=C: 184.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.151.151.15
M x/y/z160160160
origin x/y/z0.0000.0000.000
length x/y/z184.000184.000184.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS160160160
D min/max/mean-0.1220.2770.002

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Supplemental data

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Mask #1

Fileemd_9191_msk_1.map
Projections & Slices
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Additional map: Composite unsharpened map of the complex generated from...

Fileemd_9191_additional_1.map
AnnotationComposite unsharpened map of the complex generated from multiple focused maps of different sub-regions of the complex
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Sharpened non-focused map of the full complex

Fileemd_9191_additional_2.map
AnnotationSharpened non-focused map of the full complex
Projections & Slices
AxesZYX

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Additional map: Unsharpened non-focused map of the full complex

Fileemd_9191_additional_3.map
AnnotationUnsharpened non-focused map of the full complex
Projections & Slices
AxesZYX

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Additional map: Focused map of "Region-1" of the Csy-DNA complex,...

Fileemd_9191_additional_4.map
AnnotationFocused map of "Region-1" of the Csy-DNA complex, comprising the head (Cas6f-crRNA stem loop) - Cas8f C-terminal helix bundle - Cas7.1f and Cas7.2f subunits
Projections & Slices
AxesZYX

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Additional map: Focused map of "Region-2" of the Csy-DNA complex,...

Fileemd_9191_additional_5.map
AnnotationFocused map of "Region-2" of the Csy-DNA complex, comprising all six Cas7f subunits - crRNA - complimentary target DNA strand
Projections & Slices
AxesZYX

Projections

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Additional map: Focused map of "Region-3" of the Csy-DNA complex,...

Fileemd_9191_additional_6.map
AnnotationFocused map of "Region-3" of the Csy-DNA complex, comprising the tail (Cas8f N-terminal half - Cas5f - double-stranded target DNA) - Cas7.6 subunit
Projections & Slices
AxesZYX

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Half map: Unfiltered composite half map #1

Fileemd_9191_half_map_1.map
AnnotationUnfiltered composite half map #1
Projections & Slices
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Half map: Unfiltered composite half map #2

Fileemd_9191_half_map_2.map
AnnotationUnfiltered composite half map #2
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Sample components

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Entire : Double-stranded target DNA engaged Csy Complex from Pseudomonas a...

EntireName: Double-stranded target DNA engaged Csy Complex from Pseudomonas aeruginosa (PA-14)
Components
  • Complex: Double-stranded target DNA engaged Csy Complex from Pseudomonas aeruginosa (PA-14)
    • Protein or peptide: CRISPR-associated protein Csy2
    • Protein or peptide: CRISPR-associated protein Csy3
    • Protein or peptide: CRISPR-associated endonuclease Cas6/Csy4
    • RNA: CRISPR RNA (60-MER)
    • DNA: CRISPR target DNA (44-MER)
    • DNA: Non-complementary R-loop DNA strand
    • Protein or peptide: CRISPR-associated protein Csy1

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Supramolecule #1: Double-stranded target DNA engaged Csy Complex from Pseudomonas a...

SupramoleculeName: Double-stranded target DNA engaged Csy Complex from Pseudomonas aeruginosa (PA-14)
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#7
Source (natural)Organism: Pseudomonas aeruginosa (strain UCBPP-PA14) (bacteria)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
Molecular weightTheoretical: 360 KDa

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Macromolecule #1: CRISPR-associated protein Csy2

MacromoleculeName: CRISPR-associated protein Csy2 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Pseudomonas aeruginosa (strain UCBPP-PA14) (bacteria)
Strain: UCBPP-PA14
Molecular weightTheoretical: 36.244074 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MSVTDPEALL LLPRLSIQNA NAISSPLTWG FPSPGAFTGF VHALQRRVGI SLDIELDGVG IVCHRFEAQI SQPAGKRTKV FNLTRNPLN RDGSTAAIVE EGRAHLEVSL LLGVHGDGLD DHPAQEIARQ VQEQAGAMRL AGGSILPWCN ERFPAPNAEL L MLGGSDEQ ...String:
MSVTDPEALL LLPRLSIQNA NAISSPLTWG FPSPGAFTGF VHALQRRVGI SLDIELDGVG IVCHRFEAQI SQPAGKRTKV FNLTRNPLN RDGSTAAIVE EGRAHLEVSL LLGVHGDGLD DHPAQEIARQ VQEQAGAMRL AGGSILPWCN ERFPAPNAEL L MLGGSDEQ RRKNQRRLTR RLLPGFALVS REALLQQHLE TLRTTLPEAT TLDALLDLCR INFEPPATSS EEEASPPDAA WQ VRDKPGW LVPIPAGYNA LSPLYLPGEV RNARDRETPL RFVENLFGLG EWLSPHRVAA LSDLLWYHHA EPDKGLYRWS TPR FVEHAI A

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Macromolecule #2: CRISPR-associated protein Csy3

MacromoleculeName: CRISPR-associated protein Csy3 / type: protein_or_peptide / ID: 2 / Number of copies: 6 / Enantiomer: LEVO
Source (natural)Organism: Pseudomonas aeruginosa (strain UCBPP-PA14) (bacteria)
Strain: UCBPP-PA14
Molecular weightTheoretical: 37.579273 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MSKPILSTAS VLAFERKLDP SDALMSAGAW AQRDASQEWP AVTVREKSVR GTISNRLKTK DRDPAKLDAS IQSPNLQTVD VANLPSDAD TLKVRFTLRV LGGAGTPSAC NDAAYRDKLL QTVATYVNDQ GFAELARRYA HNLANARFLW RNRVGAEAVE V RINHIRQG ...String:
MSKPILSTAS VLAFERKLDP SDALMSAGAW AQRDASQEWP AVTVREKSVR GTISNRLKTK DRDPAKLDAS IQSPNLQTVD VANLPSDAD TLKVRFTLRV LGGAGTPSAC NDAAYRDKLL QTVATYVNDQ GFAELARRYA HNLANARFLW RNRVGAEAVE V RINHIRQG EVARAWRFDA LAIGLRDFKA DAELDALAEL IASGLSGSGH VLLEVVAFAR IGDGQEVFPS QELILDKGDK KG QKSKTLY SVRDAAAIHS QKIGNALRTI DTWYPDEDGL GPIAVEPYGS VTSQGKAYRQ PKQKLDFYTL LDNWVLRDEA PAV EQQHYV IANLIRGGVF GEAEEK

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Macromolecule #3: CRISPR-associated endonuclease Cas6/Csy4

MacromoleculeName: CRISPR-associated endonuclease Cas6/Csy4 / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO / EC number: Hydrolases; Acting on ester bonds
Source (natural)Organism: Pseudomonas aeruginosa (strain UCBPP-PA14) (bacteria)
Strain: UCBPP-PA14
Molecular weightTheoretical: 21.675781 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
FTMDHYLDIR LRPDPEFPPA QLMSVLFGKL HQALVAQGGD RIGVSFPDLD ESRSRLGERL RIHASADDLR ALLARPWLEG LRDHLQFGE PAVVPHPTPY RQVSRVQAKS NPERLRRRLM RRHDLSEEEA RKRIPDTVAR ALDLPFVTLR SQSTGQHFRL F IRHGPLQV TAEEGGFTCY GLSKGGFVPW F

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Macromolecule #7: CRISPR-associated protein Csy1

MacromoleculeName: CRISPR-associated protein Csy1 / type: protein_or_peptide / ID: 7 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Pseudomonas aeruginosa (strain UCBPP-PA14) (bacteria)
Strain: UCBPP-PA14
Molecular weightTheoretical: 49.194168 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MTSPLPTPTW QELRQFIESF IQERLQGKLD KLQPDEDDKR QTLLATHRRE AWLADAARRV GQLQLVTHTL KPIHPDARGS NLHSLPQAP GQPGLAGSHE LGDRLVSDVV GNAAALDVFK FLSLQYQGKN LLNWLTEDSA EALQALSDNA EQAREWRQAF I GITTVKGA ...String:
MTSPLPTPTW QELRQFIESF IQERLQGKLD KLQPDEDDKR QTLLATHRRE AWLADAARRV GQLQLVTHTL KPIHPDARGS NLHSLPQAP GQPGLAGSHE LGDRLVSDVV GNAAALDVFK FLSLQYQGKN LLNWLTEDSA EALQALSDNA EQAREWRQAF I GITTVKGA PASHSLAKQL YFPLPGSGYH LLAPLFPTSL VHHVHALLRE ARFGDAAKAA REARSRQESW PHGFSEYPNL AI QKFGGTK PQNISQLNNE RRGENWLLPS LPPNWQRQNV NAPMRHSSVF EHDFGRTPEV SRLTRTLQRF LAKTVHNNLA IRQ RRAQLV AQICDEALQY AARLRELEPG WSATPGCQLH DAEQLWLDPL RAQTDETFLQ RRLRGDWPAE VGNRFANWLN RAVS SDSQI LGSPEAAQWS QELSKELTMF KEILEDERD

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Macromolecule #4: CRISPR RNA (60-MER)

MacromoleculeName: CRISPR RNA (60-MER) / type: rna / ID: 4 / Number of copies: 1
Source (natural)Organism: Pseudomonas aeruginosa UCBPP-PA14 (bacteria)
Molecular weightTheoretical: 19.265404 KDa
SequenceString:
CUAAGAAAUU CACGGCGGGC UUGAUGUCCG CGUCUACCUG GUUCACUGCC GUGUAGGCAG

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Macromolecule #5: CRISPR target DNA (44-MER)

MacromoleculeName: CRISPR target DNA (44-MER) / type: dna / ID: 5 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Pseudomonas aeruginosa UCBPP-PA14 (bacteria)
Molecular weightTheoretical: 13.584703 KDa
SequenceString:
(DC)(DA)(DG)(DG)(DT)(DA)(DG)(DA)(DC)(DG) (DC)(DG)(DG)(DA)(DC)(DA)(DT)(DC)(DA)(DA) (DG)(DC)(DC)(DC)(DG)(DC)(DC)(DG)(DT) (DG)(DA)(DA)(DG)(DG)(DT)(DG)(DC)(DA)(DG) (DC) (DT)(DT)(DC)(DT)

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Macromolecule #6: Non-complementary R-loop DNA strand

MacromoleculeName: Non-complementary R-loop DNA strand / type: dna / ID: 6 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Pseudomonas aeruginosa UCBPP-PA14 (bacteria)
Molecular weightTheoretical: 10.476714 KDa
SequenceString:
(DA)(DG)(DA)(DA)(DG)(DC)(DT)(DG)(DC)(DA) (DC)(DC)(DT)(DT)(DC)(DA)(DC)(DG)(DG)(DC) (DG)(DG)(DG)(DC)(DT)(DT)(DG)(DA)(DT) (DG)(DT)(DC)(DC)(DG)

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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

Concentration2 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
20.0 mMC8H18N2O4SHEPES
100.0 mMKClpotassium chloride
1.0 mMC9H15O6PTCEP
2.0 % (v/v)C3H8O3glycerol
0.05 % (v/v)C47H88O22lauryl maltose neopentyl glycol
GridModel: Quantifoil, UltrAuFoil, R1.2/1.3 / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Atmosphere: OTHER
VitrificationCryogen name: ETHANE / Instrument: HOMEMADE PLUNGER
Details: Freezing was carried out in a cold room at 4 degrees C and relative humidity of 98%. 5 uL sample was applied to plasma cleaned grid and manually blotted with Whatman 1 filter paper for 5-7 ...Details: Freezing was carried out in a cold room at 4 degrees C and relative humidity of 98%. 5 uL sample was applied to plasma cleaned grid and manually blotted with Whatman 1 filter paper for 5-7 sec before plunge freezing..

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
TemperatureMin: 77.0 K / Max: 79.0 K
DetailsObjective astigmatism was corrected at 36000x magnification using Thon rings visualized with a K2 camera.
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Digitization - Dimensions - Width: 7676 pixel / Digitization - Dimensions - Height: 7420 pixel / Digitization - Sampling interval: 2.5 µm / Digitization - Frames/image: 1-56 / Number grids imaged: 4 / Number real images: 3208 / Average exposure time: 14.0 sec. / Average electron dose: 58.0 e/Å2
Details: Data were acquired using Leginon and collected on K2 summit operating in super-resolution mode.
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 1.5 µm / Nominal defocus min: 0.6 µm / Nominal magnification: 36000
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

DetailsSuper-resolution movie frames were Fourier-binned 2 x 2 times to a pixel size of 1.15 Angstrom/pixel prior to dose-weighted frame alignment using MotionCorr2.
Particle selectionNumber selected: 1543677
Details: Particles were initially extracted binned by 2 with a box size of 144 pixels (2.3 Angstrom/pixel). The final processing was carried out with an unbinned particle stack with a box size of 288 ...Details: Particles were initially extracted binned by 2 with a box size of 144 pixels (2.3 Angstrom/pixel). The final processing was carried out with an unbinned particle stack with a box size of 288 pixels (1.15 Angstrom/pixel).
CTF correctionSoftware - Name: CTFFIND (ver. 4)
Startup modelType of model: EMDB MAP
EMDB ID:

8624


Details: This map was low pass-filtered to 60 Angstrom and used as initial model for 3D reconstruction and data processing.
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF
Details: The final map was reconstructed using several focused maps from different subregions of the complex. These were initially aligned to each other and then stitched using the vop maximum ...Details: The final map was reconstructed using several focused maps from different subregions of the complex. These were initially aligned to each other and then stitched using the vop maximum function in UCSF Chimera. The resolution of the final composite map is 3.2 Angstrom.
Number images used: 291227
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 2.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 2.1)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

5uz9

DetailsThe atomic models for Cas5f, Cas8f, Cas6f, and Cas7f from the Csy Acr complex (PDB ID 5UZ9) were used as initial template models for model building. These were individually rigid body-fitted into the reconstructed maps using the fit map function in UCSF Chimera, and residue registers and backbone geometries were fixed in Coot. Models for the crRNA and DNA strands were also manually built into the map using Coot.
RefinementSpace: REAL / Protocol: AB INITIO MODEL
Output model

PDB-6ne0:
Structure of double-stranded target DNA engaged Csy complex from Pseudomonas aeruginosa (PA-14)

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