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- PDB-7aav: Human pre-Bact-2 spliceosome core structure -

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Entry
Database: PDB / ID: 7aav
TitleHuman pre-Bact-2 spliceosome core structure
Components
  • (Pre-mRNA-splicing factor ...) x 2
  • 116 kDa U5 small nuclear ribonucleoprotein component
  • Cell division cycle 5-like protein
  • MINX M3 pre-mRNA
  • Microfibrillar-associated protein 1
  • Pleiotropic regulator 1
  • Pre-mRNA-processing factor 17
  • Pre-mRNA-processing-splicing factor 8
  • Protein BUD31 homolog
  • SNW domain-containing protein 1
  • Spliceosome-associated protein CWC15 homolog
  • U2 snRNAU2 spliceosomal RNA
  • U5 snRNAU5 spliceosomal RNA
  • U6 snRNAU6 spliceosomal RNA
  • Ubiquitin-like protein 5
  • Zinc finger matrin-type protein 2
KeywordsSPLICING / Complex / spliceosome / catalytic activation
Function / homology
Function and homology information


microfibril / regulation of retinoic acid receptor signaling pathway / regulation of vitamin D receptor signaling pathway / embryonic brain development / nuclear retinoic acid receptor binding / U2-type catalytic step 1 spliceosome / Prp19 complex / RNA splicing, via transesterification reactions / positive regulation of androgen receptor activity / mRNA 3'-end processing ...microfibril / regulation of retinoic acid receptor signaling pathway / regulation of vitamin D receptor signaling pathway / embryonic brain development / nuclear retinoic acid receptor binding / U2-type catalytic step 1 spliceosome / Prp19 complex / RNA splicing, via transesterification reactions / positive regulation of androgen receptor activity / mRNA 3'-end processing / pre-mRNA binding / mRNA cis splicing, via spliceosome / U2-type spliceosomal complex / U2-type precatalytic spliceosome / positive regulation of mRNA splicing, via spliceosome / U2-type catalytic step 2 spliceosome / positive regulation by host of viral transcription / positive regulation of vitamin D receptor signaling pathway / Transport of Mature mRNA derived from an Intron-Containing Transcript / Notch binding / nuclear vitamin D receptor binding / Regulation of gene expression in late stage (branching morphogenesis) pancreatic bud precursor cells / RNA Polymerase II Transcription Termination / RUNX3 regulates NOTCH signaling / NOTCH4 Intracellular Domain Regulates Transcription / K63-linked polyubiquitin modification-dependent protein binding / positive regulation of protein targeting to mitochondrion / WD40-repeat domain binding / NOTCH3 Intracellular Domain Regulates Transcription / positive regulation of neurogenesis / precatalytic spliceosome / nuclear androgen receptor binding / Notch-HLH transcription pathway / mRNA Splicing - Minor Pathway / Formation of paraxial mesoderm / positive regulation of transforming growth factor beta receptor signaling pathway / SMAD binding / protein localization to nucleus / spliceosomal tri-snRNP complex assembly / intrinsic apoptotic signaling pathway in response to DNA damage by p53 class mediator / positive regulation of G1/S transition of mitotic cell cycle / U5 snRNA binding / U5 snRNP / retinoic acid receptor signaling pathway / Cajal body / U2 snRNA binding / U6 snRNA binding / pre-mRNA intronic binding / U1 snRNA binding / cellular response to retinoic acid / U4/U6 x U5 tri-snRNP complex / catalytic step 2 spliceosome / mRNA Splicing - Major Pathway / positive regulation of RNA splicing / RNA splicing / positive regulation of protein export from nucleus / response to cocaine / nuclear receptor coactivator activity / DNA damage checkpoint signaling / nuclear receptor binding / Downregulation of SMAD2/3:SMAD4 transcriptional activity / RNA polymerase II transcription regulatory region sequence-specific DNA binding / spliceosomal complex / protein modification process / NOTCH1 Intracellular Domain Regulates Transcription / protein tag activity / mRNA processing / fibrillar center / mRNA splicing, via spliceosome / Constitutive Signaling by NOTCH1 PEST Domain Mutants / Constitutive Signaling by NOTCH1 HD+PEST Domain Mutants / Pre-NOTCH Transcription and Translation / nuclear matrix / positive regulation of protein import into nucleus / calcium-dependent protein binding / transcription corepressor activity / cellular response to xenobiotic stimulus / cellular response to tumor necrosis factor / DNA-binding transcription activator activity, RNA polymerase II-specific / nuclear membrane / cellular response to lipopolysaccharide / transcription coactivator activity / nuclear body / DNA-binding transcription factor activity, RNA polymerase II-specific / nuclear speck / cell cycle / mRNA binding / intracellular membrane-bounded organelle / DNA repair / GTPase activity / negative regulation of DNA-templated transcription / centrosome / chromatin / GTP binding / regulation of transcription by RNA polymerase II / negative regulation of transcription by RNA polymerase II / enzyme binding / positive regulation of transcription by RNA polymerase II / mitochondrion / DNA binding
Similarity search - Function
Pre-mRNA-splicing factor 38, C-terminal / Pre-mRNA-splicing factor 38-associated hydrophilic C-term / Micro-fibrillar-associated protein 1, C-terminal / Microfibrillar-associated protein 1 / Ubiquitin-like modifier Hub1/Ubl5 / Microfibril-associated/Pre-mRNA processing / U4/U6.U5 small nuclear ribonucleoprotein component Snu23 / Pre-mRNA-splicing factor 38 / PRP38 family / Pre-mRNA-processing factor 17 ...Pre-mRNA-splicing factor 38, C-terminal / Pre-mRNA-splicing factor 38-associated hydrophilic C-term / Micro-fibrillar-associated protein 1, C-terminal / Microfibrillar-associated protein 1 / Ubiquitin-like modifier Hub1/Ubl5 / Microfibril-associated/Pre-mRNA processing / U4/U6.U5 small nuclear ribonucleoprotein component Snu23 / Pre-mRNA-splicing factor 38 / PRP38 family / Pre-mRNA-processing factor 17 / : / STL11, N-terminal / WD repeat Prp46/PLRG1-like / BUD31/G10-related, conserved site / : / : / G10 protein signature 1. / G10 protein signature 2. / SKI-interacting protein SKIP, SNW domain / SKI-interacting protein, SKIP / SKIP/SNW domain / Myb-like DNA-binding domain / Pre-mRNA-splicing factor Cwf15/Cwc15 / Cwf15/Cwc15 cell cycle control protein / Pre-mRNA-splicing factor Cwc2/Slt11 / G10 protein / Pre-mRNA-splicing factor BUD31 / Pre-mRNA splicing factor component Cdc5p/Cef1, C-terminal / pre-mRNA splicing factor component / Zinc-finger double-stranded RNA-binding / Zinc finger, double-stranded RNA binding / Zinc finger, CCCH-type superfamily / Snu114, GTP-binding domain / 116kDa U5 small nuclear ribonucleoprotein component, N-terminal / 116kDa U5 small nuclear ribonucleoprotein component, C-terminal / 116 kDa U5 small nuclear ribonucleoprotein component N-terminus / zinc finger / Matrin/U1-C-like, C2H2-type zinc finger / U1-like zinc finger / Myb-type HTH DNA-binding domain profile. / Zinc finger, CCCH-type / Zinc finger C3H1-type profile. / Myb domain / Elongation Factor G, domain II / Elongation Factor G, domain III / JAB1/Mov34/MPN/PAD-1 ubiquitin protease / Translation elongation factor EFG/EF2, domain IV / Elongation factor G, domain IV / Elongation factor G, domain IV / Elongation factor G C-terminus / Elongation factor EFG, domain V-like / Elongation factor G C-terminus / EF-G domain III/V-like / SANT SWI3, ADA2, N-CoR and TFIIIB'' DNA-binding domains / SANT/Myb domain / PROCT domain / Prp8 RNase domain IV, fingers region / PROCT (NUC072) domain / PRO8NT domain / PROCN domain / Pre-mRNA-processing-splicing factor 8, U6-snRNA-binding / Pre-mRNA-processing-splicing factor 8, U5-snRNA-binding / RNA recognition motif, spliceosomal PrP8 / PRP8 domain IV core / Pre-mRNA-processing-splicing factor 8, U5-snRNA-binding domain superfamily / Prp8 RNase domain IV, palm region / PRO8NT (NUC069), PrP8 N-terminal domain / PROCN (NUC071) domain / U6-snRNA interacting domain of PrP8 / U5-snRNA binding site 2 of PrP8 / RNA recognition motif of the spliceosomal PrP8 / PRP8 domain IV core / Pre-mRNA-processing-splicing factor 8 / Translation elongation factor EFTu-like, domain 2 / Elongation factor Tu domain 2 / JAB/MPN domain / JAB1/MPN/MOV34 metalloenzyme domain / MPN domain / MPN domain profile. / Zinc finger C2H2 superfamily / Translational (tr)-type GTP-binding domain / Elongation factor Tu GTP binding domain / Translational (tr)-type guanine nucleotide-binding (G) domain profile. / RNA recognition motif / RNA recognition motif / Eukaryotic RNA Recognition Motif (RRM) profile. / RNA recognition motif domain / RNA-binding domain superfamily / Homeobox-like domain superfamily / Ubiquitin family / Ubiquitin-like domain / Small GTP-binding protein domain / Translation protein, beta-barrel domain superfamily / Ribosomal protein S5 domain 2-type fold, subgroup / G-protein beta WD-40 repeat / Ubiquitin-like domain superfamily / WD40 repeat, conserved site / Ribonuclease H-like superfamily / Nucleotide-binding alpha-beta plait domain superfamily / Ribosomal protein S5 domain 2-type fold
Similarity search - Domain/homology
GUANOSINE-5'-TRIPHOSPHATE / D-chiro inositol hexakisphosphate / : / : / RNA / RNA (> 10) / RNA (> 100) / Pleiotropic regulator 1 / Pre-mRNA-processing factor 17 / Protein BUD31 homolog ...GUANOSINE-5'-TRIPHOSPHATE / D-chiro inositol hexakisphosphate / : / : / RNA / RNA (> 10) / RNA (> 100) / Pleiotropic regulator 1 / Pre-mRNA-processing factor 17 / Protein BUD31 homolog / Microfibrillar-associated protein 1 / SNW domain-containing protein 1 / 116 kDa U5 small nuclear ribonucleoprotein component / Pre-mRNA-processing-splicing factor 8 / Pre-mRNA-splicing factor 38A / Zinc finger matrin-type protein 2 / Cell division cycle 5-like protein / Ubiquitin-like protein 5 / Pre-mRNA-splicing factor RBM22 / Spliceosome-associated protein CWC15 homolog
Similarity search - Component
Biological speciesHomo sapiens (human)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.2 Å
AuthorsTownsend, C. / Kastner, B. / Leelaram, M.N. / Bertram, K. / Stark, H. / Luehrmann, R.
Funding support Germany, 1items
OrganizationGrant numberCountry
German Research Foundation (DFG)860 Germany
CitationJournal: Science / Year: 2020
Title: Mechanism of protein-guided folding of the active site U2/U6 RNA during spliceosome activation.
Authors: Cole Townsend / Majety N Leelaram / Dmitry E Agafonov / Olexandr Dybkov / Cindy L Will / Karl Bertram / Henning Urlaub / Berthold Kastner / Holger Stark / Reinhard Lührmann /
Abstract: Spliceosome activation involves extensive protein and RNA rearrangements that lead to formation of a catalytically active U2/U6 RNA structure. At present, little is known about the assembly pathway ...Spliceosome activation involves extensive protein and RNA rearrangements that lead to formation of a catalytically active U2/U6 RNA structure. At present, little is known about the assembly pathway of the latter and the mechanism whereby proteins aid its proper folding. Here, we report the cryo-electron microscopy structures of two human, activated spliceosome precursors (that is, pre-B complexes) at core resolutions of 3.9 and 4.2 angstroms. These structures elucidate the order of the numerous protein exchanges that occur during activation, the mutually exclusive interactions that ensure the correct order of ribonucleoprotein rearrangements needed to form the U2/U6 catalytic RNA, and the stepwise folding pathway of the latter. Structural comparisons with mature B complexes reveal the molecular mechanism whereby a conformational change in the scaffold protein PRP8 facilitates final three-dimensional folding of the U2/U6 catalytic RNA.
History
DepositionSep 4, 2020Deposition site: PDBE / Processing site: PDBE
Revision 1.0Dec 9, 2020Provider: repository / Type: Initial release
Revision 1.1Dec 30, 2020Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation_author.identifier_ORCID

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Structure visualization

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Assembly

Deposited unit
r: 116 kDa U5 small nuclear ribonucleoprotein component
Q: Protein BUD31 homolog
L: Cell division cycle 5-like protein
R: Spliceosome-associated protein CWC15 homolog
K: Microfibrillar-associated protein 1
G: Pleiotropic regulator 1
Z: MINX M3 pre-mRNA
8: Pre-mRNA-processing factor 17
I: Pre-mRNA-splicing factor 38A
A: Pre-mRNA-processing-splicing factor 8
P: Pre-mRNA-splicing factor RBM22
v: SNW domain-containing protein 1
N: Zinc finger matrin-type protein 2
2: U2 snRNA
5: U5 snRNA
6: U6 snRNA
q: Ubiquitin-like protein 5
hetero molecules


Theoretical massNumber of molelcules
Total (without water)1,079,06520
Polymers1,077,85717
Non-polymers1,2083
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: cross-linking
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Protein , 11 types, 11 molecules rQLRKG8AvNq

#1: Protein 116 kDa U5 small nuclear ribonucleoprotein component / Elongation factor Tu GTP-binding domain-containing protein 2 / SNU114 homolog / hSNU114 / U5 snRNP- ...Elongation factor Tu GTP-binding domain-containing protein 2 / SNU114 homolog / hSNU114 / U5 snRNP-specific protein / 116 kDa / U5-116 kDa


Mass: 109560.625 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / Cell line: Hela / References: UniProt: Q15029
#2: Protein Protein BUD31 homolog / Protein EDG-2 / Protein G10 homolog


Mass: 17032.850 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / Cell line: Hela / References: UniProt: P41223
#3: Protein Cell division cycle 5-like protein / Cdc5-like protein / Pombe cdc5-related protein


Mass: 92406.883 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / Cell line: HeLa / References: UniProt: Q99459
#4: Protein Spliceosome-associated protein CWC15 homolog


Mass: 26674.447 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / Cell line: Hela / References: UniProt: Q9P013
#5: Protein Microfibrillar-associated protein 1


Mass: 52050.527 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / Cell line: Hela / References: UniProt: P55081
#6: Protein Pleiotropic regulator 1


Mass: 57280.758 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / Cell line: Hela / References: UniProt: O43660
#8: Protein Pre-mRNA-processing factor 17 / Cell division cycle 40 homolog / EH-binding protein 3 / Ehb3 / PRP17 homolog / hPRP17


Mass: 65612.180 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / Cell line: Hela / References: UniProt: O60508
#10: Protein Pre-mRNA-processing-splicing factor 8 / 220 kDa U5 snRNP-specific protein / PRP8 homolog / Splicing factor Prp8 / p220


Mass: 273974.250 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / Cell line: Hela / References: UniProt: Q6P2Q9
#12: Protein SNW domain-containing protein 1 / Nuclear protein SkiP / Nuclear receptor coactivator NCoA-62 / Ski-interacting protein


Mass: 61610.703 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / Cell line: Hela / References: UniProt: Q13573
#13: Protein Zinc finger matrin-type protein 2


Mass: 23664.047 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / Cell line: Hela / References: UniProt: Q96NC0
#17: Protein Ubiquitin-like protein 5


Mass: 8560.945 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / Cell line: Hela / References: UniProt: Q9BZL1

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RNA chain , 4 types, 4 molecules Z256

#7: RNA chain MINX M3 pre-mRNA


Mass: 73712.359 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#14: RNA chain U2 snRNA / U2 spliceosomal RNA


Mass: 60186.445 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / Cell line: Hela / References: GenBank: 36516
#15: RNA chain U5 snRNA / U5 spliceosomal RNA


Mass: 36908.668 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / Cell line: Hela / References: GenBank: 36515
#16: RNA chain U6 snRNA / U6 spliceosomal RNA


Mass: 34098.270 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / Cell line: Hela

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Pre-mRNA-splicing factor ... , 2 types, 2 molecules IP

#9: Protein Pre-mRNA-splicing factor 38A


Mass: 37563.863 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / Cell line: Hela / References: UniProt: Q8NAV1
#11: Protein Pre-mRNA-splicing factor RBM22 / RNA-binding motif protein 22 / Zinc finger CCCH domain-containing protein 16


Mass: 46959.555 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / Cell line: Hela / References: UniProt: Q9NW64

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Non-polymers , 3 types, 3 molecules

#18: Chemical ChemComp-GTP / GUANOSINE-5'-TRIPHOSPHATE / Guanosine triphosphate


Mass: 523.180 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H16N5O14P3 / Comment: GTP, energy-carrying molecule*YM
#19: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg
#20: Chemical ChemComp-KGN / D-chiro inositol hexakisphosphate


Mass: 660.035 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C6H18O24P6

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Details

Has ligand of interestN

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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

Component
IDNameTypeEntity IDParent-IDSource
1pre-Bact-2 spliceosomal core structureCOMPLEX#1-#170MULTIPLE SOURCES
2pre-Bact-2 spliceosomal core structureCOMPLEX#1-#6, #8-#171NATURAL
3MINX M3 pre-mRNACOMPLEX#71RECOMBINANT
Molecular weightUnits: MEGADALTONS / Experimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Homo sapiens (human)9606
23synthetic construct (others)32630
Source (recombinant)Organism: synthetic construct (others)
Buffer solutionpH: 7.9
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid type: Quantifoil R3.5/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE

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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: SPOT SCAN
Electron lensMode: BRIGHT FIELDBright-field microscopy
Image recordingAverage exposure time: 1 sec. / Electron dose: 2.25 e/Å2 / Detector mode: INTEGRATING / Film or detector model: FEI FALCON III (4k x 4k)
Image scansWidth: 4096 / Height: 4096

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Processing

EM software
IDNameVersionCategory
7UCSF Chimeramodel fitting
8Cootmodel fitting
10PHENIXmodel refinement
14RELION33D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 4.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 39336 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL

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