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- PDB-6d6q: Human nuclear exosome-MTR4 RNA complex - overall reconstruction -

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Basic information

Entry
Database: PDB / ID: 6d6q
TitleHuman nuclear exosome-MTR4 RNA complex - overall reconstruction
Components
  • (Exosome complex component ...) x 9
  • DNA/RNA (62-MER)
  • Exosome RNA helicase MTR4
  • Exosome complex exonuclease RRP44
  • Exosome component 10
  • M-phase phosphoprotein 6
  • RNA (5'-R(*AP*GP*CP*AP*CP*CP*GP*UP*AP*AP*AP*GP*AP*CP*GP*C)-3')
KeywordsHYDROLASE / RNA exosome / RNA degradation / ribonuclease / helicase / SF2 / RNA-protein complex / translocase / nuclear
Function / homology
Function and homology information


DNA deamination / nucleolar exosome (RNase complex) / snRNA catabolic process / nuclear polyadenylation-dependent snoRNA catabolic process / nuclear polyadenylation-dependent snRNA catabolic process / TRAMP complex / nuclear polyadenylation-dependent antisense transcript catabolic process / RNA exonuclease activity / nuclear mRNA surveillance of mRNA 3'-end processing / U1 snRNA 3'-end processing ...DNA deamination / nucleolar exosome (RNase complex) / snRNA catabolic process / nuclear polyadenylation-dependent snoRNA catabolic process / nuclear polyadenylation-dependent snRNA catabolic process / TRAMP complex / nuclear polyadenylation-dependent antisense transcript catabolic process / RNA exonuclease activity / nuclear mRNA surveillance of mRNA 3'-end processing / U1 snRNA 3'-end processing / positive regulation of mRNA cis splicing, via spliceosome / regulation of telomerase RNA localization to Cajal body / U5 snRNA 3'-end processing / nuclear polyadenylation-dependent CUT catabolic process / TRAMP-dependent tRNA surveillance pathway / mRNA decay by 3' to 5' exoribonuclease / CUT catabolic process / cytoplasmic exosome (RNase complex) / U4 snRNA 3'-end processing / nuclear polyadenylation-dependent rRNA catabolic process / poly(A)-dependent snoRNA 3'-end processing / exosome (RNase complex) / nuclear exosome (RNase complex) / exonucleolytic trimming to generate mature 3'-end of 5.8S rRNA from tricistronic rRNA transcript (SSU-rRNA, 5.8S rRNA, LSU-rRNA) / Tristetraprolin (TTP, ZFP36) binds and destabilizes mRNA / ATF4 activates genes in response to endoplasmic reticulum stress / histone mRNA catabolic process / Butyrate Response Factor 1 (BRF1) binds and destabilizes mRNA / rRNA catabolic process / nuclear mRNA surveillance / positive regulation of isotype switching / 7S RNA binding / mRNA 3'-UTR AU-rich region binding / isotype switching / telomerase RNA binding / Hydrolases; Acting on ester bonds; Endoribonucleases producing 5'-phosphomonoesters / RNA catabolic process / nuclear-transcribed mRNA catabolic process, nonsense-mediated decay / nuclear-transcribed mRNA catabolic process, deadenylation-dependent decay / KSRP (KHSRP) binds and destabilizes mRNA / maturation of 5.8S rRNA / nuclear chromosome / mRNA catabolic process / negative regulation of telomere maintenance via telomerase / nuclear-transcribed mRNA catabolic process / Major pathway of rRNA processing in the nucleolus and cytosol / RNA processing / catalytic step 2 spliceosome / mRNA Splicing - Major Pathway / Regulation of endogenous retroelements by the Human Silencing Hub (HUSH) complex / guanyl-nucleotide exchange factor activity / small-subunit processome / euchromatin / mRNA splicing, via spliceosome / fibrillar center / rRNA processing / ribosomal small subunit biogenesis / chromosome / Hydrolases; Acting on ester bonds; Exoribonucleases producing 5'-phosphomonoesters / 3'-5'-RNA exonuclease activity / positive regulation of cell growth / endonuclease activity / defense response to virus / RNA polymerase II-specific DNA-binding transcription factor binding / RNA helicase activity / single-stranded RNA binding / RNA helicase / nuclear speck / immune response / intracellular membrane-bounded organelle / DNA repair / nucleotide binding / DNA damage response / nucleolus / positive regulation of transcription by RNA polymerase II / ATP hydrolysis activity / DNA binding / RNA binding / extracellular exosome / nucleoplasm / ATP binding / identical protein binding / membrane / nucleus / metal ion binding / cytosol / cytoplasm
Similarity search - Function
M-phase phosphoprotein 6 / Exosome complex component Rrp43 / M-phase phosphoprotein 6 / : / Mammalian exosome complex component RRP40, N-terminal / Exosome-associated factor Rrp6, N-terminal / Exosome complex exonuclease Rrp6-like / : / PMC2NT (NUC016) domain / Exosome complex exonuclease RRP44, S1 domain ...M-phase phosphoprotein 6 / Exosome complex component Rrp43 / M-phase phosphoprotein 6 / : / Mammalian exosome complex component RRP40, N-terminal / Exosome-associated factor Rrp6, N-terminal / Exosome complex exonuclease Rrp6-like / : / PMC2NT (NUC016) domain / Exosome complex exonuclease RRP44, S1 domain / S1 domain / PIN domain / Exosome complex component RRP45 / Rrp40, S1 domain / : / Exosome complex component RRP40, S1 domain / Exosome complex component CSL4, C-terminal / Exosome complex component, N-terminal domain / Exosome complex component Csl4 / Exosome component EXOSC1/CSL4 / Exosome complex exonuclease RRP4 N-terminal region / RRP4, S1 domain / Rrp44-like cold shock domain / Rrp44-like cold shock domain / : / KH domain / Exosome complex RNA-binding protein 1/RRP40/RRP4 / Dis3-like cold-shock domain 2 / Dis3-like cold-shock domain 2 (CSD2) / GHMP Kinase, N-terminal domain / : / Ribonuclease II/R, conserved site / rRNA-processing arch domain / Ribonuclease II family signature. / Mtr4-like, beta-barrel domain / : / Ribonuclease II/R / RNB domain / RNB / : / ATP-dependent RNA helicase Ski2, C-terminal / ATP-dependent RNA helicase Ski2-like / : / DSHCT (NUC185) domain / Exosome RNA helicase MTR4-like, stalk / DSHCT / : / Helicase and RNase D C-terminal / K Homology domain, type 1 / HRDC domain / HRDC domain / HRDC domain profile. / Exoribonuclease, phosphorolytic domain 2 / 3' exoribonuclease family, domain 2 / Exoribonuclease, phosphorolytic domain 1 / PNPase/RNase PH domain superfamily / Exoribonuclease, PH domain 2 superfamily / 3' exoribonuclease family, domain 1 / HRDC domain superfamily / Large family of predicted nucleotide-binding domains / 3'-5' exonuclease / PIN domain / K Homology domain, type 1 / 3'-5' exonuclease / 3'-5' exonuclease domain / RNA polymerase II/Efflux pump adaptor protein, barrel-sandwich hybrid domain / Ribosomal Protein S8; Chain: A, domain 1 / K Homology domain, type 1 superfamily / PIN-like domain superfamily / Ribosomal Protein S5; domain 2 / HRDC-like superfamily / Ribosomal protein S1-like RNA-binding domain / S1 domain / Nucleic acid-binding proteins / DEAD/DEAH box helicase domain / DEAD/DEAH box helicase / Helicase conserved C-terminal domain / OB fold (Dihydrolipoamide Acetyltransferase, E2P) / helicase superfamily c-terminal domain / Superfamilies 1 and 2 helicase C-terminal domain profile. / Superfamilies 1 and 2 helicase ATP-binding type-1 domain profile. / DEAD-like helicases superfamily / Helicase, C-terminal / Helicase superfamily 1/2, ATP-binding domain / Ribonuclease H superfamily / Ribonuclease H-like superfamily / Ribosomal protein S5 domain 2-type fold / Nucleic acid-binding, OB-fold / Beta Barrel / P-loop containing nucleoside triphosphate hydrolase / 2-Layer Sandwich / Mainly Beta / Alpha Beta
Similarity search - Domain/homology
PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER / DNA/RNA hybrid / DNA/RNA hybrid (> 10) / RNA / RNA (> 10) / Exosome RNA helicase MTR4 / Exosome complex component 10 / Exosome complex component RRP45 / Exosome complex component RRP4 / Exosome complex component RRP42 ...PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER / DNA/RNA hybrid / DNA/RNA hybrid (> 10) / RNA / RNA (> 10) / Exosome RNA helicase MTR4 / Exosome complex component 10 / Exosome complex component RRP45 / Exosome complex component RRP4 / Exosome complex component RRP42 / Exosome complex component MTR3 / Exosome complex component RRP43 / M-phase phosphoprotein 6 / Exosome complex component RRP41 / Exosome complex component RRP46 / Exosome complex component RRP40 / Exosome complex exonuclease RRP44 / Exosome complex component CSL4
Similarity search - Component
Biological speciesHomo sapiens (human)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.45 Å
AuthorsWeick, E.-M. / Lima, C.D.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM118080 United States
Howard Hughes Medical Institute (HHMI) United States
CitationJournal: Cell / Year: 2018
Title: Helicase-Dependent RNA Decay Illuminated by a Cryo-EM Structure of a Human Nuclear RNA Exosome-MTR4 Complex.
Authors: Eva-Maria Weick / M Rhyan Puno / Kurt Januszyk / John C Zinder / Michael A DiMattia / Christopher D Lima /
Abstract: The ribonucleolytic RNA exosome interacts with RNA helicases to degrade RNA. To understand how the 3' to 5' Mtr4 helicase engages RNA and the nuclear exosome, we reconstituted 14-subunit Mtr4- ...The ribonucleolytic RNA exosome interacts with RNA helicases to degrade RNA. To understand how the 3' to 5' Mtr4 helicase engages RNA and the nuclear exosome, we reconstituted 14-subunit Mtr4-containing RNA exosomes from Saccharomyces cerevisiae, Schizosaccharomyces pombe, and human and show that they unwind structured substrates to promote degradation. We loaded a human exosome with an optimized DNA-RNA chimera that stalls MTR4 during unwinding and determined its structure to an overall resolution of 3.45 Å by cryoelectron microscopy (cryo-EM). The structure reveals an RNA-engaged helicase atop the non-catalytic core, with RNA captured within the central channel and DIS3 exoribonuclease active site. MPP6 tethers MTR4 to the exosome through contacts to the RecA domains of MTR4. EXOSC10 remains bound to the core, but its catalytic module and cofactor C1D are displaced by RNA-engaged MTR4. Competition for the exosome core may ensure that RNA is committed to degradation by DIS3 when engaged by MTR4.
History
DepositionApr 22, 2018Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jun 20, 2018Provider: repository / Type: Initial release
Revision 1.1Jun 27, 2018Group: Data collection / Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.name
Revision 1.2Nov 20, 2019Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.3Mar 13, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

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Assembly

Deposited unit
A: Exosome complex component RRP45
B: Exosome complex component RRP41
C: Exosome complex component RRP43
D: Exosome complex component RRP46
E: Exosome complex component RRP42
F: Exosome complex component MTR3
G: Exosome complex component RRP40
H: Exosome complex component RRP4
I: Exosome complex component CSL4
J: Exosome component 10
K: Exosome complex exonuclease RRP44
L: M-phase phosphoprotein 6
M: Exosome RNA helicase MTR4
N: RNA (5'-R(*AP*GP*CP*AP*CP*CP*GP*UP*AP*AP*AP*GP*AP*CP*GP*C)-3')
O: DNA/RNA (62-MER)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)639,17918
Polymers638,58315
Non-polymers5963
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration, monodisperse peak included all fourteen subunits plus the unwound subtrate
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area58270 Å2
ΔGint-249 kcal/mol
Surface area171380 Å2

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Components

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Exosome complex component ... , 9 types, 9 molecules ABCDEFGHI

#1: Protein Exosome complex component RRP45 / EXOSC9 / Autoantigen PM/Scl 1 / Exosome component 9 / P75 polymyositis-scleroderma overlap syndrome- ...EXOSC9 / Autoantigen PM/Scl 1 / Exosome component 9 / P75 polymyositis-scleroderma overlap syndrome-associated autoantigen / Polymyositis/scleroderma autoantigen 1 / Polymyositis/scleroderma autoantigen 75 kDa / PM/Scl-75


Mass: 52828.020 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: EXOSC9, PMSCL1 / Production host: Escherichia coli (E. coli) / References: UniProt: Q06265
#2: Protein Exosome complex component RRP41 / EXOSC4 / Exosome component 4 / Ribosomal RNA-processing protein 41 / p12A


Mass: 26831.473 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: EXOSC4, RRP41, SKI6 / Production host: Escherichia coli (E. coli) / References: UniProt: Q9NPD3
#3: Protein Exosome complex component RRP43 / EXOSC8 / Exosome component 8 / Opa-interacting protein 2 / OIP-2 / Ribosomal RNA-processing protein 43 / p9


Mass: 30285.762 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: EXOSC8, OIP2, RRP43 / Production host: Escherichia coli (E. coli) / References: UniProt: Q96B26
#4: Protein Exosome complex component RRP46 / EXOSC5 / Chronic myelogenous leukemia tumor antigen 28 / Exosome component 5 / Ribosomal RNA- ...EXOSC5 / Chronic myelogenous leukemia tumor antigen 28 / Exosome component 5 / Ribosomal RNA-processing protein 46 / p12B


Mass: 25480.213 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: EXOSC5, CML28, RRP46 / Production host: Escherichia coli (E. coli) / References: UniProt: Q9NQT4
#5: Protein Exosome complex component RRP42 / EXOSC7 / Exosome component 7 / Ribosomal RNA-processing protein 42 / p8


Mass: 32072.633 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: EXOSC7, KIAA0116, RRP42 / Production host: Escherichia coli (E. coli) / References: UniProt: Q15024
#6: Protein Exosome complex component MTR3 / EXOSC6 / Exosome component 6 / mRNA transport regulator 3 homolog / hMtr3 / p11


Mass: 28267.127 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: EXOSC6, MTR3 / Production host: Escherichia coli (E. coli) / References: UniProt: Q5RKV6
#7: Protein Exosome complex component RRP40 / EXOSC3 / Exosome component 3 / Ribosomal RNA-processing protein 40 / p10


Mass: 29796.371 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: EXOSC3, RRP40, CGI-102 / Production host: Escherichia coli (E. coli) / References: UniProt: Q9NQT5
#8: Protein Exosome complex component RRP4 / EXOSC2 / Exosome component 2 / Ribosomal RNA-processing protein 4


Mass: 33184.371 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: EXOSC2, RRP4 / Production host: Escherichia coli (E. coli) / References: UniProt: Q13868
#9: Protein Exosome complex component CSL4 / EXOSC1 / Exosome component 1


Mass: 21690.971 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: EXOSC1, CSL4, CGI-108 / Production host: Escherichia coli (E. coli) / References: UniProt: Q9Y3B2

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Protein , 4 types, 4 molecules JKLM

#10: Protein Exosome component 10 / EXOSC10 / Autoantigen PM/Scl 2 / P100 polymyositis-scleroderma overlap syndrome-associated ...EXOSC10 / Autoantigen PM/Scl 2 / P100 polymyositis-scleroderma overlap syndrome-associated autoantigen / Polymyositis/scleroderma autoantigen 100 kDa / PM/Scl-100 / Polymyositis/scleroderma autoantigen 2


Mass: 86754.859 Da / Num. of mol.: 1 / Mutation: D313N
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: EXOSC10, PMSCL, PMSCL2, RRP6 / Production host: Escherichia coli (E. coli)
References: UniProt: Q01780, Hydrolases; Acting on ester bonds; Exoribonucleases producing 5'-phosphomonoesters
#11: Protein Exosome complex exonuclease RRP44 / DIS3 / Protein DIS3 homolog / Ribosomal RNA-processing protein 44


Mass: 109267.930 Da / Num. of mol.: 1 / Mutation: D146N, D487N
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: DIS3, KIAA1008, RRP44 / Production host: Escherichia coli (E. coli)
References: UniProt: Q9Y2L1, Hydrolases; Acting on ester bonds; Exoribonucleases producing 5'-phosphomonoesters, Hydrolases; Acting on ester bonds; Endoribonucleases producing 5'-phosphomonoesters
#12: Protein M-phase phosphoprotein 6 / MPP6 / MPHOSPH6


Mass: 19267.961 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: MPHOSPH6, MPP6 / Production host: Escherichia coli (E. coli) / References: UniProt: Q99547
#13: Protein Exosome RNA helicase MTR4 / MTR4 / MTREX / ATP-dependent RNA helicase DOB1 / ATP-dependent RNA helicase SKIV2L2 / Superkiller ...MTR4 / MTREX / ATP-dependent RNA helicase DOB1 / ATP-dependent RNA helicase SKIV2L2 / Superkiller viralicidic activity 2-like 2 / TRAMP-like complex helicase


Mass: 118224.961 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: MTREX, DOB1, KIAA0052, MTR4, SKIV2L2 / Production host: Escherichia coli (E. coli) / References: UniProt: P42285, RNA helicase

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RNA chain / DNA/RNA hybrid , 2 types, 2 molecules NO

#14: RNA chain RNA (5'-R(*AP*GP*CP*AP*CP*CP*GP*UP*AP*AP*AP*GP*AP*CP*GP*C)-3')


Mass: 5143.175 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#15: DNA/RNA hybrid DNA/RNA (62-MER)


Mass: 19487.074 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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Non-polymers , 3 types, 3 molecules

#16: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg
#17: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Zn
#18: Chemical ChemComp-ANP / PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER


Mass: 506.196 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H17N6O12P3 / Comment: AMP-PNP, energy-carrying molecule analogue*YM

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Human nuclear exosome-MTR4 helicase captured after unwinding a DNA/RNA substrate
Type: COMPLEX
Details: Human C1D/Rrp47 also in the sample, but was not observed in density
Entity ID: #1-#15 / Source: RECOMBINANT
Molecular weightValue: 0.69 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMTris-HCl1
2100 mMsodium chlorideNaCl1
30.5 mMmagnesium chlorideMgCl21
42 mMAMPPNP1
51 mMBME1
60.05 %Chapso1
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: Sample was monodisperse upon elution from gel filtration prior to vitrification.
Specimen supportGrid material: GOLD / Grid type: Quantifoil R2/2
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Chamber temperature: 298 K / Details: 30 sec wait time, 2.5 sec blot time

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD
Image recordingElectron dose: 85.23 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of real images: 1439
Image scansMovie frames/image: 50

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Processing

EM software
IDNameVersionCategoryDetails
2Leginon3.2image acquisition
4Gctf1.06CTF correctiondetermine
5RELION2.1.b1CTF correctionapply
8PHENIX1.13-2998model fitting
10PHENIX1.13-2998model refinement
11cryoSPARC0.6.1initial Euler assignmentfrom 0.6.1 to 0.6.5
12RELION2.1b1final Euler assignment
13RELION2.1b1classification
14RELION2.1b13D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 278185
3D reconstructionResolution: 3.45 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 122703
Details: Focused refinements with five strategies required to achieve final model
Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingB value: 73.6 / Protocol: BACKBONE TRACE / Space: REAL / Target criteria: Correlation coefficient
Details: Models were rebuilt manually using Coot, with real space refinement with local scaling followed by Phenix real space refinement with suboptimal global scaling.

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