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- PDB-6ttu: Ubiquitin Ligation to substrate by a cullin-RING E3 ligase at 3.7... -

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Entry
Database: PDB / ID: 6ttu
TitleUbiquitin Ligation to substrate by a cullin-RING E3 ligase at 3.7A resolution: NEDD8-CUL1-RBX1 N98R-SKP1-monomeric b-TRCP1dD-IkBa-UB~UBE2D2
Components
  • CYS-LYS-LYS-ALA-ARG-HIS-ASP-SEP-GLY
  • Cullin-1
  • E3 ubiquitin-protein ligase RBX1
  • F-box/WD repeat-containing protein 1A
  • NEDD8
  • Polyubiquitin-C
  • S-phase kinase-associated protein 1
  • Ubiquitin-conjugating enzyme E2 D2
KeywordsLIGASE / Ubiquitin / E3 ligase / NEDD8 / cullin / CUL1 / RBX1 / SKP1 / TRCP / UBE2D / IkBalpha / Neddylation / Ubiquitylation
Function / homology
Function and homology information


I-kappaB/NF-kappaB complex / protein phosphorylated amino acid binding / : / Parkin-FBXW7-Cul1 ubiquitin ligase complex / negative regulation of myeloid cell differentiation / F-box domain binding / IkBA variant leads to EDA-ID / ubiquitin conjugating enzyme complex / nucleotide-binding oligomerization domain containing 1 signaling pathway / PcG protein complex ...I-kappaB/NF-kappaB complex / protein phosphorylated amino acid binding / : / Parkin-FBXW7-Cul1 ubiquitin ligase complex / negative regulation of myeloid cell differentiation / F-box domain binding / IkBA variant leads to EDA-ID / ubiquitin conjugating enzyme complex / nucleotide-binding oligomerization domain containing 1 signaling pathway / PcG protein complex / (E3-independent) E2 ubiquitin-conjugating enzyme / cullin-RING-type E3 NEDD8 transferase / NEDD8 transferase activity / cullin-RING ubiquitin ligase complex / RIP-mediated NFkB activation via ZBP1 / molecular sequestering activity / cellular response to chemical stress / ubiquitin ligase activator activity / positive regulation of ubiquitin protein ligase activity / Cul7-RING ubiquitin ligase complex / positive regulation of circadian rhythm / ubiquitin-dependent protein catabolic process via the C-end degron rule pathway / SUMOylation of immune response proteins / regulation of proteolysis / maintenance of protein location in nucleus / Loss of Function of FBXW7 in Cancer and NOTCH1 Signaling / nucleotide-binding oligomerization domain containing 2 signaling pathway / positive regulation of protein autoubiquitination / protein neddylation / transcription regulator inhibitor activity / branching involved in mammary gland duct morphogenesis / regulation of canonical NF-kappaB signal transduction / regulation of canonical Wnt signaling pathway / NEDD8 ligase activity / Cul5-RING ubiquitin ligase complex / cellular response to cold / interleukin-1-mediated signaling pathway / negative regulation of response to oxidative stress / non-canonical NF-kappaB signal transduction / mammary gland epithelial cell proliferation / ubiquitin-ubiquitin ligase activity / SCF ubiquitin ligase complex / Cul2-RING ubiquitin ligase complex / Cul4A-RING E3 ubiquitin ligase complex / nuclear localization sequence binding / toll-like receptor 4 signaling pathway / negative regulation of T cell receptor signaling pathway / negative regulation of type I interferon production / Cul4B-RING E3 ubiquitin ligase complex / SCF-dependent proteasomal ubiquitin-dependent protein catabolic process / E2 ubiquitin-conjugating enzyme / Cul3-RING ubiquitin ligase complex / ubiquitin ligase complex scaffold activity / negative regulation of Notch signaling pathway / response to exogenous dsRNA / Prolactin receptor signaling / negative regulation of NF-kappaB transcription factor activity / ligase activity / TGF-beta receptor signaling activates SMADs / ubiquitin conjugating enzyme activity / response to muramyl dipeptide / cullin family protein binding / TRAF6 mediated NF-kB activation / positive regulation of proteolysis / negative regulation of macrophage derived foam cell differentiation / negative regulation of lipid storage / cellular response to organic cyclic compound / protein monoubiquitination / NF-kappaB binding / positive regulation of cholesterol efflux / anatomical structure morphogenesis / ubiquitin-like ligase-substrate adaptor activity / positive regulation of transcription initiation by RNA polymerase II / protein K48-linked ubiquitination / canonical NF-kappaB signal transduction / Nuclear events stimulated by ALK signaling in cancer / regulation of proteasomal protein catabolic process / negative regulation of canonical NF-kappaB signal transduction / ubiquitin ligase complex / negative regulation of smoothened signaling pathway / protein autoubiquitination / lipopolysaccharide-mediated signaling pathway / tumor necrosis factor-mediated signaling pathway / Notch signaling pathway / Maturation of protein E / Maturation of protein E / ER Quality Control Compartment (ERQC) / Myoclonic epilepsy of Lafora / FLT3 signaling by CBL mutants / Prevention of phagosomal-lysosomal fusion / IRAK2 mediated activation of TAK1 complex / Alpha-protein kinase 1 signaling pathway / Glycogen synthesis / IRAK1 recruits IKK complex / IRAK1 recruits IKK complex upon TLR7/8 or 9 stimulation / response to muscle stretch / protein sequestering activity / Regulation of TBK1, IKKε (IKBKE)-mediated activation of IRF3, IRF7 / Regulation of TBK1, IKKε-mediated activation of IRF3, IRF7 upon TLR3 ligation / Membrane binding and targetting of GAG proteins
Similarity search - Function
: / D domain of beta-TrCP / D domain of beta-TrCP / D domain of beta-TrCP / : / Ankyrin repeats (many copies) / Monooxygenase - #50 / Nedd8-like ubiquitin / A Receptor for Ubiquitination Targets / F-box domain profile. ...: / D domain of beta-TrCP / D domain of beta-TrCP / D domain of beta-TrCP / : / Ankyrin repeats (many copies) / Monooxygenase - #50 / Nedd8-like ubiquitin / A Receptor for Ubiquitination Targets / F-box domain profile. / F-box-like domain superfamily / SKP1 component, dimerisation / S-phase kinase-associated protein 1 / SKP1-like, dimerisation domain superfamily / Skp1 family, dimerisation domain / F-box-like / F-box domain / Zinc finger, RING-H2-type / RING-H2 zinc finger domain / Cullin protein neddylation domain / : / Cullin, conserved site / Cullin family signature. / Cullin repeat-like-containing domain superfamily / Cullin protein, neddylation domain / Cullin / Cullin protein neddylation domain / Potassium Channel Kv1.1; Chain A / Potassium Channel Kv1.1; Chain A / Monooxygenase / Cullin / Cullin, N-terminal / Cullin homology domain / Cullin homology domain superfamily / Cullin family / Cullin family profile. / S-phase kinase-associated protein 1-like / SKP1 component, POZ domain / Skp1 family, tetramerisation domain / Found in Skp1 protein family / Ubiquitin-conjugating enzyme, active site / Ubiquitin-conjugating (UBC) active site signature. / Ubiquitin-conjugating enzyme E2 / Ubiquitin-conjugating enzyme / Ubiquitin-conjugating (UBC) core domain profile. / Ubiquitin-conjugating enzyme E2, catalytic domain homologues / Ubiquitin-conjugating enzyme/RWD-like / YVTN repeat-like/Quinoprotein amine dehydrogenase / 7 Propeller / Methylamine Dehydrogenase; Chain H / SKP1/BTB/POZ domain superfamily / Ankyrin repeats (3 copies) / Ankyrin repeat profile. / Ankyrin repeat region circular profile. / ankyrin repeats / Ankyrin repeat / Ankyrin repeat-containing domain superfamily / Zinc finger RING-type profile. / Zinc finger, RING-type / : / Ubiquitin domain signature. / Ubiquitin conserved site / Ubiquitin domain / Ubiquitin family / Ubiquitin homologues / Ubiquitin domain profile. / Ubiquitin-like domain / G-protein beta WD-40 repeat / Zinc finger, RING/FYVE/PHD-type / Ubiquitin-like domain superfamily / Winged helix DNA-binding domain superfamily / WD40 repeat, conserved site / Trp-Asp (WD) repeats signature. / Trp-Asp (WD) repeats profile. / Trp-Asp (WD) repeats circular profile. / WD domain, G-beta repeat / WD40 repeats / WD40 repeat / WD40-repeat-containing domain superfamily / Winged helix-like DNA-binding domain superfamily / WD40/YVTN repeat-like-containing domain superfamily / Up-down Bundle / 2-Layer Sandwich / Mainly Beta / Mainly Alpha / Alpha Beta
Similarity search - Domain/homology
cullin-RING-type E3 NEDD8 transferase / Polyubiquitin-C / NF-kappa-B inhibitor alpha / Ubiquitin-conjugating enzyme E2 D2 / E3 ubiquitin-protein ligase RBX1 / S-phase kinase-associated protein 1 / Cullin-1 / NEDD8 / F-box/WD repeat-containing protein 1A
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.7 Å
AuthorsBaek, K. / Prabu, J.R. / Schulman, B.A.
Funding support Germany, 1items
OrganizationGrant numberCountry
Max Planck Society Germany
CitationJournal: Nature / Year: 2020
Title: NEDD8 nucleates a multivalent cullin-RING-UBE2D ubiquitin ligation assembly.
Authors: Kheewoong Baek / David T Krist / J Rajan Prabu / Spencer Hill / Maren Klügel / Lisa-Marie Neumaier / Susanne von Gronau / Gary Kleiger / Brenda A Schulman /
Abstract: Eukaryotic cell biology depends on cullin-RING E3 ligase (CRL)-catalysed protein ubiquitylation, which is tightly controlled by the modification of cullin with the ubiquitin-like protein NEDD8. ...Eukaryotic cell biology depends on cullin-RING E3 ligase (CRL)-catalysed protein ubiquitylation, which is tightly controlled by the modification of cullin with the ubiquitin-like protein NEDD8. However, how CRLs catalyse ubiquitylation, and the basis of NEDD8 activation, remain unknown. Here we report the cryo-electron microscopy structure of a chemically trapped complex that represents the ubiquitylation intermediate, in which the neddylated CRL1 promotes the transfer of ubiquitin from the E2 ubiquitin-conjugating enzyme UBE2D to its recruited substrate, phosphorylated IκBα. NEDD8 acts as a nexus that binds disparate cullin elements and the RING-activated ubiquitin-linked UBE2D. Local structural remodelling of NEDD8 and large-scale movements of CRL domains converge to juxtapose the substrate and the ubiquitylation active site. These findings explain how a distinctive ubiquitin-like protein alters the functions of its targets, and show how numerous NEDD8-dependent interprotein interactions and conformational changes synergistically configure a catalytic CRL architecture that is both robust, to enable rapid ubiquitylation of the substrate, and fragile, to enable the subsequent functions of cullin-RING proteins.
History
DepositionDec 30, 2019Deposition site: PDBE / Processing site: PDBE
Revision 1.0Feb 12, 2020Provider: repository / Type: Initial release
Revision 1.1Feb 19, 2020Group: Database references / Source and taxonomy / Structure summary
Category: entity_name_com / entity_src_gen ...entity_name_com / entity_src_gen / struct_ref / struct_ref_seq
Item: _struct_ref.db_code / _struct_ref.pdbx_db_accession / _struct_ref_seq.pdbx_db_accession
Revision 1.2Feb 26, 2020Group: Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.name
Revision 1.3Mar 4, 2020Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.4Oct 23, 2024Group: Data collection / Database references / Structure summary
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_admin.last_update / _pdbx_entry_details.has_protein_modification

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Structure visualization

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Assembly

Deposited unit
T: F-box/WD repeat-containing protein 1A
S: S-phase kinase-associated protein 1
C: Cullin-1
N: NEDD8
R: E3 ubiquitin-protein ligase RBX1
D: Ubiquitin-conjugating enzyme E2 D2
U: Polyubiquitin-C
I: CYS-LYS-LYS-ALA-ARG-HIS-ASP-SEP-GLY
hetero molecules


Theoretical massNumber of molelcules
Total (without water)227,73611
Polymers227,5408
Non-polymers1963
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area16230 Å2
ΔGint-79 kcal/mol
Surface area74310 Å2
MethodPISA

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Components

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Protein , 7 types, 7 molecules TSCNRDU

#1: Protein F-box/WD repeat-containing protein 1A / E3RSIkappaB / Epididymis tissue protein Li 2a / F-box and WD repeats protein beta-TrCP / ...E3RSIkappaB / Epididymis tissue protein Li 2a / F-box and WD repeats protein beta-TrCP / pIkappaBalpha-E3 receptor subunit


Mass: 68960.797 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: BTRC, BTRCP, FBW1A, FBXW1A / Production host: Escherichia coli (E. coli) / References: UniProt: Q9Y297
#2: Protein S-phase kinase-associated protein 1 / Cyclin-A/CDK2-associated protein p19 / p19A / Organ of Corti protein 2 / OCP-2 / Organ of Corti ...Cyclin-A/CDK2-associated protein p19 / p19A / Organ of Corti protein 2 / OCP-2 / Organ of Corti protein II / OCP-II / RNA polymerase II elongation factor-like protein / SIII / Transcription elongation factor B polypeptide 1-like / p19skp1


Mass: 18679.965 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: SKP1 contains truncations of residues 38-43, 71-82.
Source: (gene. exp.) Homo sapiens (human) / Gene: SKP1, EMC19, OCP2, SKP1A, TCEB1L / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: P63208
#3: Protein Cullin-1 / CUL-1


Mass: 89800.367 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: CUL1 / Production host: Escherichia coli (E. coli) / References: UniProt: Q13616
#4: Protein NEDD8 / Neddylin / Neural precursor cell expressed developmentally down-regulated protein 8 / NEDD-8 / ...Neddylin / Neural precursor cell expressed developmentally down-regulated protein 8 / NEDD-8 / Ubiquitin-like protein Nedd8


Mass: 8661.055 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: Residue 1, Ser, comes from the linker. NEDD8 residue starts from MET.
Source: (gene. exp.) Homo sapiens (human) / Gene: NEDD8 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q15843
#5: Protein E3 ubiquitin-protein ligase RBX1 / E3 ubiquitin-protein transferase RBX1 / Protein ZYP / RING finger protein 75 / RING-box protein 1 / ...E3 ubiquitin-protein transferase RBX1 / Protein ZYP / RING finger protein 75 / RING-box protein 1 / Rbx1 / Regulator of cullins 1 / ROC1


Mass: 12333.067 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: RBX1 contains mutation Asn98Arg (N98R). There are 3 Zinc ions coordinated throughout. This is not meant to align to the C-terminus of the protein.
Source: (gene. exp.) Homo sapiens (human) / Gene: RBX1, RNF75, ROC1 / Production host: Escherichia coli (E. coli)
References: UniProt: P62877, UniProt: H2QLR9*PLUS, RING-type E3 ubiquitin transferase, cullin-RING-type E3 NEDD8 transferase
#6: Protein Ubiquitin-conjugating enzyme E2 D2 / (E3-independent) E2 ubiquitin-conjugating enzyme D2 / E2 ubiquitin-conjugating enzyme D2 / ...(E3-independent) E2 ubiquitin-conjugating enzyme D2 / E2 ubiquitin-conjugating enzyme D2 / Ubiquitin carrier protein D2 / Ubiquitin-conjugating enzyme E2(17)KB 2 / Ubiquitin-conjugating enzyme E2-17 kDa 2 / Ubiquitin-protein ligase D2 / p53-regulated ubiquitin-conjugating enzyme 1


Mass: 16745.121 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: UBE2D2 contains mutations of the following: Cys21Ile, Cys107Ala, Cys111Asp.
Source: (gene. exp.) Homo sapiens (human) / Gene: UBE2D2, PUBC1, UBC4, UBC5B, UBCH4, UBCH5B / Production host: Escherichia coli (E. coli)
References: UniProt: P62837, E2 ubiquitin-conjugating enzyme, (E3-independent) E2 ubiquitin-conjugating enzyme
#7: Protein Polyubiquitin-C


Mass: 8576.831 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: UBC / Production host: Escherichia coli (E. coli) / References: UniProt: P0CG48

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Protein/peptide / Non-polymers , 2 types, 4 molecules I

#8: Protein/peptide CYS-LYS-LYS-ALA-ARG-HIS-ASP-SEP-GLY


Mass: 3782.819 Da / Num. of mol.: 1 / Source method: obtained synthetically / Details: Phosphorylated peptide derived from IkBa. / Source: (synth.) Homo sapiens (human) / References: UniProt: P25963*PLUS
#9: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: Zn

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Details

Has ligand of interestN
Has protein modificationY

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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
1Ubiquitin Ligation to substrate by a cullin-RING E3 ligase at 3.7A resolution: NEDD8-CUL1-RBX1 N98R-SKP1-monomeric b-TRCP1dD-IkBa-UB~UBE2D2COMPLEX#1-#80RECOMBINANT
2S-phase kinase-associated protein 1COMPLEX#51RECOMBINANT
3Cullin-1, E3 ubiquitin-protein ligase RBX1COMPLEX#2, #41RECOMBINANT
4F-box/WD repeat-containing protein 1A, NEDD8, S-phase kinase-associated protein 1, Ubiquitin-conjugating enzyme E2 D2COMPLEX#1, #3, #6-#71RECOMBINANT
5Synthetic peptideCOMPLEX#81RECOMBINANT
Molecular weightValue: 0.210 MDa
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Pan troglodytes (chimpanzee)9598
33Homo sapiens (human)9606
44Homo sapiens (human)9606
55Homo sapiens (human)9606
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
22Escherichia coli (E. coli)562
33Trichoplusia ni (cabbage looper)7111
44Escherichia coli (E. coli)562
55Synthetic construct (others)32630
Buffer solutionpH: 7.8
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE-PROPANE

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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: 70.2 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.15.2_3472: / Classification: refinement
EM software
IDNameCategory
2SerialEMimage acquisition
7Cootmodel fitting
12RELION3D reconstruction
13PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 106257 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00612974
ELECTRON MICROSCOPYf_angle_d0.85417546
ELECTRON MICROSCOPYf_dihedral_angle_d4.0057847
ELECTRON MICROSCOPYf_chiral_restr0.0482004
ELECTRON MICROSCOPYf_plane_restr0.0042222

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