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- EMDB-0150: PTC3 holotoxin complex from Photorhabdus luminiscens - Mutant TcC... -

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Basic information

Entry
Database: EMDB / ID: EMD-0150
TitlePTC3 holotoxin complex from Photorhabdus luminiscens - Mutant TcC-D651A
Map dataCryoEM structure of the Photorhadbus luminescens ABC-D651A holotoxin complex
Sample
  • Complex: PTC3 holotoxin complex in prepore state - Mutant TcC-D651A
    • Complex: TcdA1
      • Protein or peptide: TcdA1
    • Complex: TcdB2/TccC3-D651A
      • Protein or peptide: TcdB2,TccC3,TccC3
KeywordsTc / ABC / a-PFT / membrane transport / translocation / pore forming toxin / TOXIN
Function / homology
Function and homology information


extracellular region / identical protein binding / cytoplasm
Similarity search - Function
Insecticide toxin TcdB middle/C-terminal / Insecticide toxin TcdB middle/N-terminal / Insecticide toxin TcdB middle/C-terminal region / Insecticide toxin TcdB middle/N-terminal region / Salmonella virulence plasmid 65kDa B protein / Salmonella virulence plasmid 65kDa B protein / Toxin complex C-like repeat / Tripartite Tc toxins repeat / : / TcdA1, receptor binding domain ...Insecticide toxin TcdB middle/C-terminal / Insecticide toxin TcdB middle/N-terminal / Insecticide toxin TcdB middle/C-terminal region / Insecticide toxin TcdB middle/N-terminal region / Salmonella virulence plasmid 65kDa B protein / Salmonella virulence plasmid 65kDa B protein / Toxin complex C-like repeat / Tripartite Tc toxins repeat / : / TcdA1, receptor binding domain / : / TcdA1, receptor binding domain / ABC toxin, N-terminal domain / ABC toxin N-terminal region / TcA receptor binding domain / TcA receptor binding domain / Insecticidal toxin complex/plasmid virulence protein / Tc toxin complex TcA, C-terminal TcB-binding domain / Neuraminidase-like domain / Salmonella virulence plasmid 28.1kDa A protein / Tc toxin complex TcA C-terminal TcB-binding domain / Neuraminidase-like domain / Rhs repeat-associated core / : / Integrin alpha, N-terminal
Similarity search - Domain/homology
TccC3 / TcdB2 / TcdA1
Similarity search - Component
Biological speciesPhotorhabdus luminescens (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.72 Å
AuthorsGatsogiannis C / Merino F
Funding support Germany, 2 items
OrganizationGrant numberCountry
European Research Council615984 Germany
Max Planck Society Germany
CitationJournal: Nature / Year: 2018
Title: Tc toxin activation requires unfolding and refolding of a β-propeller.
Authors: Christos Gatsogiannis / Felipe Merino / Daniel Roderer / David Balchin / Evelyn Schubert / Anne Kuhlee / Manajit Hayer-Hartl / Stefan Raunser /
Abstract: Tc toxins secrete toxic enzymes into host cells using a unique syringe-like injection mechanism. They are composed of three subunits, TcA, TcB and TcC. TcA forms the translocation channel and the TcB- ...Tc toxins secrete toxic enzymes into host cells using a unique syringe-like injection mechanism. They are composed of three subunits, TcA, TcB and TcC. TcA forms the translocation channel and the TcB-TcC heterodimer functions as a cocoon that shields the toxic enzyme. Binding of the cocoon to the channel triggers opening of the cocoon and translocation of the toxic enzyme into the channel. Here we show in atomic detail how the assembly of the three components activates the toxin. We find that part of the cocoon completely unfolds and refolds into an alternative conformation upon binding. The presence of the toxic enzyme inside the cocoon is essential for its subnanomolar binding affinity for the TcA subunit. The enzyme passes through a narrow negatively charged constriction site inside the cocoon, probably acting as an extruder that releases the unfolded protein with its C terminus first into the translocation channel.
History
DepositionJul 27, 2018-
Header (metadata) releaseAug 15, 2018-
Map releaseOct 3, 2018-
UpdateMay 15, 2024-
Current statusMay 15, 2024Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.03
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.03
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6h6f
  • Surface level: 0.03
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_0150.map.gz / Format: CCP4 / Size: 421.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryoEM structure of the Photorhadbus luminescens ABC-D651A holotoxin complex
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.1 Å/pix.
x 480 pix.
= 528. Å
1.1 Å/pix.
x 480 pix.
= 528. Å
1.1 Å/pix.
x 480 pix.
= 528. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.1 Å
Density
Contour LevelBy AUTHOR: 0.03 / Movie #1: 0.03
Minimum - Maximum-0.11320373 - 0.14066413
Average (Standard dev.)0.0002554831 (±0.0031504855)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions480480480
Spacing480480480
CellA=B=C: 528.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.11.11.1
M x/y/z480480480
origin x/y/z0.0000.0000.000
length x/y/z528.000528.000528.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS480480480
D min/max/mean-0.1130.1410.000

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Supplemental data

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Sample components

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Entire : PTC3 holotoxin complex in prepore state - Mutant TcC-D651A

EntireName: PTC3 holotoxin complex in prepore state - Mutant TcC-D651A
Components
  • Complex: PTC3 holotoxin complex in prepore state - Mutant TcC-D651A
    • Complex: TcdA1
      • Protein or peptide: TcdA1
    • Complex: TcdB2/TccC3-D651A
      • Protein or peptide: TcdB2,TccC3,TccC3

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Supramolecule #1: PTC3 holotoxin complex in prepore state - Mutant TcC-D651A

SupramoleculeName: PTC3 holotoxin complex in prepore state - Mutant TcC-D651A
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: The catalytic protease spartyl protease site is deleted
Molecular weightTheoretical: 1.7 MDa

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Supramolecule #2: TcdA1

SupramoleculeName: TcdA1 / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1 / Details: 5 TcdA1 protomers in the PTC3 holotoxin complex
Source (natural)Organism: Photorhabdus luminescens (bacteria)

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Supramolecule #3: TcdB2/TccC3-D651A

SupramoleculeName: TcdB2/TccC3-D651A / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2
Details: 1 TcdB2/TccC3-D651A monomer in the PTC3 holotoxin complex. The catalytic aspartyl protease site of TccC3 is deleted.
Source (natural)Organism: Photorhabdus luminescens (bacteria)

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Macromolecule #1: TcdA1

MacromoleculeName: TcdA1 / type: protein_or_peptide / ID: 1 / Number of copies: 5 / Enantiomer: LEVO
Source (natural)Organism: Photorhabdus luminescens (bacteria)
Molecular weightTheoretical: 283.229406 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MNESVKEIPD VLKSQCGFNC LTDISHSSFN EFRQQVSEHL SWSETHDLYH DAQQAQKDNR LYEARILKRA NPQLQNAVHL AILAPNAEL IGYNNQFSGR ASQYVAPGTV SSMFSPAAYL TELYREARNL HASDSVYYLD TRRPDLKSMA LSQQNMDIEL S TLSLSNEL ...String:
MNESVKEIPD VLKSQCGFNC LTDISHSSFN EFRQQVSEHL SWSETHDLYH DAQQAQKDNR LYEARILKRA NPQLQNAVHL AILAPNAEL IGYNNQFSGR ASQYVAPGTV SSMFSPAAYL TELYREARNL HASDSVYYLD TRRPDLKSMA LSQQNMDIEL S TLSLSNEL LLESIKTESK LENYTKVMEM LSTFRPSGAT PYHDAYENVR EVIQLQDPGL EQLNASPAIA GLMHQASLLG IN ASISPEL FNILTEEITE GNAEELYKKN FGNIEPASLA MPEYLKRYYN LSDEELSQFI GKASNFGQQE YSNNQLITPV VNS SDGTVK VYRITREYTT NAYQMDVELF PFGGENYRLD YKFKNFYNAS YLSIKLNDKR ELVRTEGAPQ VNIEYSANIT LNTA DISQP FEIGLTRVLP SGSWAYAAAK FTVEEYNQYS FLLKLNKAIR LSRATELSPT ILEGIVRSVN LQLDINTDVL GKVFL TKYY MQRYAIHAET ALILCNAPIS QRSYDNQPSQ FDRLFNTPLL NGQYFSTGDE EIDLNSGSTG DWRKTILKRA FNIDDV SLF RLLKITDHDN KDGKIKNNLK NLSNLYIGKL LADIHQLTID ELDLLLIAVG EGKTNLSAIS DKQLATLIRK LNTITSW LH TQKWSVFQLF IMTSTSYNKT LTPEIKNLLD TVYHGLQGFD KDKADLLHVM APYIAATLQL SSENVAHSVL LWADKLQP G DGAMTAEKFW DWLNTKYTPG SSEAVETQEH IVQYCQALAQ LEMVYHSTGI NENAFRLFVT KPEMFGAATG AAPAHDALS LIMLTRFADW VNALGEKASS VLAAFEANSL TAEQLADAMN LDANLLLQAS IQAQNHQHLP PVTPENAFSC WTSINTILQW VNVAQQLNV APQGVSALVG LDYIQSMKET PTYAQWENAA GVLTAGLNSQ QANTLHAFLD ESRSAALSTY YIRQVAKAAA A IKSRDDLY QYLLIDNQVS AAIKTTRIAE AIASIQLYVN RALENVEENA NSGVISRQFF IDWDKYNKRY STWAGVSQLV YY PENYIDP TMRIGQTKMM DALLQSVSQS QLNADTVEDA FMSYLTSFEQ VANLKVISAY HDNINNDQGL TYFIGLSETD AGE YYWRSV DHSKFNDGKF AANAWSEWHK IDCPINPYKS TIRPVIYKSR LYLLWLEQKE ITKQTGNSKD GYQTETDYRY ELKL AHIRY DGTWNTPITF DVNKKISELK LEKNRAPGLY CAGYQGEDTL LVMFYNQQDT LDSYKNASMQ GLYIFADMAS KDMTP EQSN VYRDNSYQQF DTNNVRRVNN RYAEDYEIPS SVSSRKDYGW GDYYLSMVYN GDIPTINYKA ASSDLKIYIS PKLRII HNG YEGQKRNQCN LMNKYGKLGD KFIVYTSLGV NPNNSSNKLM FYPVYQYSGN TSGLNQGRLL FHRDTTYPSK VEAWIPG AK RSLTNQNAAI GDDYATDSLN KPDDLKQYIF MTDSKGTATD VSGPVEINTA ISPAKVQIIV KAGGKEQTFT ADKDVSIQ P SPSFDEMNYQ FNALEIDGSG LNFINNSASI DVTFTAFAED GRKLGYESFS IPVTLKVSTD NALTLHHNEN GAQYMQWQS YRTRLNTLFA RQLVARATTG IDTILSMETQ NIQEPQLGKG FYATFVIPPY NLSTHGDERW FKLYIKHVVD NNSHIIYSGQ LTDTNINIT LFIPLDDVPL NQDYHAKVYM TFKKSPSDGT WWGPHFVRDD KGIVTINPKS ILTHFESVNV LNNISSEPMD F SGANSLYF WELFYYTPML VAQRLLHEQN FDEANRWLKY VWSPSGYIVH GQIQNYQWNV RPLLEDTSWN SDPLDSVDPD AV AQHDPMH YKVSTFMRTL DLLIARGDHA YRQLERDTLN EAKMWYMQAL HLLGDKPYLP LSTTWSDPRL DRAADITTQN AHD SAIVAL RQNIPTPAPL SLRSANTLTD LFLPQINEVM MNYWQTLAQR VYNLRHNLSI DGQPLYLPIY ATPADPKALL SAAV ATSQG GGKLPESFMS LWRFPHMLEN ARGMVSQLTQ FGSTLQNIIE RQDAEALNAL LQNQAAELIL TNLSIQDKTI EELDA EKTV LEKSKAGAQS RFDSYGKLYD ENINAGENQA MTLRASAAGL TTAVQASRLA GAAADLVPNI FGFAGGGSRW GAIAEA TGY VMEFSANVMN TEADKISQSE TYRRRRQEWE IQRNNAEAEL KQIDAQLKSL AVRREAAVLQ KTSLKTQQEQ TQSQLAF LQ RKFSNQALYN WLRGRLAAIY FQFYDLAVAR CLMAEQAYRW ELNDDSARFI KPGAWQGTYA GLLAGETLML SLAQMEDA H LKRDKRALEV ERTVSLAEVY AGLPKDNGPF SLAQEIDKLV SQGSGSAGSG NNNLAFGAGT DTKTSLQASV SFADLKIRE DYPASLGKIR RIKQISVTLP ALLGPYQDVQ AILSYGDKAG LANGCEALAV SHGMNDSGQF QLDFNDGKFL PFEGIAIDQG TLTLSFPNA SMPEKGKQAT MLKTLNDIIL HIRYTIK

UniProtKB: TcdA1

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Macromolecule #2: TcdB2,TccC3,TccC3

MacromoleculeName: TcdB2,TccC3,TccC3 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Photorhabdus luminescens (bacteria)
Molecular weightTheoretical: 273.634656 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MQNSQDFSIT ELSLPKGGGA ITGMGEALTP TGPDGMAALS LPLPISAGRG YAPAFTLNYN SGAGNSPFGL GWDCNVMTIR RRTHFGVPH YDETDTFLGP EGEVLVVADQ PRDESTLQGI NLGATFTVTG YRSRLESHFS RLEYWQPKTT GKTDFWLIYS P DGQVHLLG ...String:
MQNSQDFSIT ELSLPKGGGA ITGMGEALTP TGPDGMAALS LPLPISAGRG YAPAFTLNYN SGAGNSPFGL GWDCNVMTIR RRTHFGVPH YDETDTFLGP EGEVLVVADQ PRDESTLQGI NLGATFTVTG YRSRLESHFS RLEYWQPKTT GKTDFWLIYS P DGQVHLLG KSPQARISNP SQTTQTAQWL LEASVSSRGE QIYYQYRAED DTGCEADEIT HHLQATAQRY LHIVYYGNRT AS ETLPGLD GSAPSQADWL FYLVFDYGER SNNLKTPPAF STTGSWLCRQ DRFSRYEYGF EIRTRRLCRQ VLMYHHLQAL DSK ITEHNG PTLVSRLILN YDESAIASTL VFVRRVGHEQ DGNVVTLPPL ELAYQDFSPR HHAHWQPMDV LANFNAIQRW QLVD LKGEG LPGLLYQDKG AWWYRSAQRL GEIGSDAVTW EKMQPLSVIP SLQSNASLVD INGDGQLDWV ITGPGLRGYH SQRPD GSWT RFTPLNALPV EYTHPRAQLA DLMGAGLSDL VLIGPKSVRL YANTRDGFAK GKDVVQSGDI TLPVPGADPR KLVAFS DVL GSGQAHLVEV SATKVTCWPN LGRGRFGQPI TLPGFSQPAT EFNPAQVYLA DLDGSGPTDL IYVHTNRLDI FLNKSGN GF AEPVTLRFPE GLRFDHTCQL QMADVQGLGV ASLILSVPHM SPHHWRCDLT NMKPWLLNEM NNNMGVHHTL RYRSSSQF W LDEKAAALTT GQTPVCYLPF PIHTLWQTET EDEISGNKLV TTLRYARGAW DGREREFRGF GYVEQTDSHQ LAQGNAPER TPPALTKNWY ATGLPVIDNA LSTEYWRDDQ AFAGFSPRFT TWQDNKDVPL TPEDDNSRYW FNRALKGQLL RSELYGLDDS TNKHVPYTV TEFRSQVRRL QHTDSRYPVL WSSVVESRNY HYERIASDPQ CSQNITLSSD RFGQPLKQLS VQYPRRQQPA I NLYPDTLP DKLLANSYDD QQRQLRLTYQ QSSWHHLTNN TVRVLGLPDS TRSDIFTYGA ENVPAGGLNL ELLSDKNSLI AD DKPREYL GQQKTAYTDG QNTTPLQTPT RQALIAFTET TVFNQSTLSA FNGSIPSDKL STTLEQAGYQ QTNYLFPRTG EDK VWVAHH GYTDYGTAAQ FWRPQKQSNT QLTGKITLIW DANYCVVVQT RDAAGLTTSA KYDWRFLTPV QLTDINDNQH LITL DALGR PITLRFWGTE NGKMTGYSSP EKASFSPPSD VNAAIELKKP LPVAQCQVYA PESWMPVLSQ KTFNRLAEQD WQKLY NARI ITEDGRICTL AYRRWVQSQK AIPQLISLLN NGPRLPPHSL TLTTDRYDHD PEQQIRQQVV FSDGFGRLLQ AAARHE AGM ARQRNEDGSL IINVQHTENR WAVTGRTEYD NKGQPIRTYQ PYFLNDWRYV SNDSARQEKE AYADTHVYDP IGREIKV IT AKGWFRRTLF TPWFTVNEDE NDTAAEVKKV KMMKNIDPKL YQKTPTVSVY DNRGLIIRNI DFHRTTANGD PDTRITRH Q YDIHGHLNQS IDPRLYEAKQ TNNTIKPNFL WQYDLTGNPL CTESIDAGRT VTLNDIEGRP LLTVTATGVI QTRQYETSS LPGRLLSVAE QTPEEKTSRI TERLIWAGNT EAEKDHNLAG QCVRHYDTAG VTRLESLSLT GTVLSQSSQL LIDTQEANWT GDNETVWQN MLADDIYTTL STFDATGALL TQTDAKGNIQ RLAYDVAGQL NGSWLTLKGQ TEQVIIKSLT YSAAGQKLRE E HGNDVITE YSYEPETQRL IGIKTRRPSD TKVLQDLRYE YDPVGNVISI RNDAEATRFW HNQKVMPENT YTYDSLYQLI SA TGREMAN IGQQSHQFPS PALPSDNNTY TNYTRTYTYD RGGNLTKIQH SSPATQNNYT TNITVSNRSN RAVLSTLTED PAQ VDALFD AGGHQNTLIS GQNLNWNTRG ELQQVTLVKR DKGANDDREW YRYSGDGRRM LKINEQQASN NAQTQRVTYL PNLE LRLTQ NSTATTEDLQ VITVGEAGRA QVRVLHWESG KPEDIDNNQL RYSYDNLIGS SQLELDSEGQ IISEEEYYPY GGTAL WAAR NQTEASYKTI RYSGKERDAT GLYYYGYRYY QPWIGRWLSS APAGTIDGLN LYRMVRNNPV TLLDPDGLMP TIAERI AAL KKNKVTDSAP SPANATNVAI NIRPPVAPKP SLPKASTSSQ PTTHPIGAAN IKPTTSGSSI VAPLSPVGNK STSEISL PE SAQSSSSSTT STNLQKKSFT LYRADNRSFE EMQSKFPEGF KAWTPLDTKM ARQFASIFIG QKDTSNLPKE TVKNISTW G AKPKLKDLSN YIKYTKDKST VWVSTAINTE AGGQSSGAPL HKIDMDLYEF AIDGQKLNPL PEGRTKNMVP SLLLDTPQI ETSSIIALNH GPVNDAEISF LTTIPLKNVK PHKR

UniProtKB: TcdB2, TccC3, TccC3

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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

BufferpH: 8
Component:
ConcentrationFormula
20.0 mMTRIS
150.0 mMNaCl
0.02 %Tween20
GridModel: C-flat-2/1 / Material: COPPER / Mesh: 400 / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Instrument: GATAN CRYOPLUNGE 3

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: FEI FALCON II (4k x 4k) / Number real images: 4958 / Average electron dose: 2.2 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: OTHER / Imaging mode: BRIGHT FIELD / Cs: 0.0 mm / Nominal magnification: 59000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 137733
Startup modelType of model: EMDB MAP
EMDB ID:
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.72 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: SPHIRE / Number images used: 132000
Initial angle assignmentType: OTHER / Software - Name: SPHIRE
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: SPHIRE

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Overall B value: 49.43 / Target criteria: Model to Map FSC
Output model

PDB-6h6f:
PTC3 holotoxin complex from Photorhabdus luminiscens - Mutant TcC-D651A

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