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- EMDB-9731: Structure of a substrate engaged SecA-SecY protein translocation ... -

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Basic information

Entry
Database: EMDB / ID: EMD-9731
TitleStructure of a substrate engaged SecA-SecY protein translocation machine
Map data
Sample
  • Complex: SecA-SecY complex
    • Protein or peptide: x 7 types
  • Ligand: x 4 types
KeywordsSecA / SecY / Translocation / Cryo-EM / PROTEIN TRANSPORT
Function / homology
Function and homology information


outer membrane protein complex / cell envelope Sec protein transport complex / protein-exporting ATPase activity / monoatomic ion transmembrane transporter activity / protein-secreting ATPase / protein transport by the Sec complex / intracellular protein transmembrane transport / detection of virus / SRP-dependent cotranslational protein targeting to membrane, translocation / outer membrane ...outer membrane protein complex / cell envelope Sec protein transport complex / protein-exporting ATPase activity / monoatomic ion transmembrane transporter activity / protein-secreting ATPase / protein transport by the Sec complex / intracellular protein transmembrane transport / detection of virus / SRP-dependent cotranslational protein targeting to membrane, translocation / outer membrane / protein import / signal sequence binding / porin activity / pore complex / protein transmembrane transporter activity / protein secretion / protein targeting / monoatomic ion transport / bioluminescence / generation of precursor metabolites and energy / cell outer membrane / outer membrane-bounded periplasmic space / symbiont entry into host cell / membrane raft / DNA damage response / ATP binding / identical protein binding / membrane / metal ion binding / plasma membrane / cytoplasm
Similarity search - Function
Outer membrane protein OmpA-like, transmembrane domain / Outer membrane protein, OmpA / OmpA-like transmembrane domain / SecE subunit of protein translocation complex, bacterial-like / SecE superfamily / SEC-C motif / SEC-C motif / Protein translocase subunit SecA / SecA DEAD-like, N-terminal / SecA Wing/Scaffold ...Outer membrane protein OmpA-like, transmembrane domain / Outer membrane protein, OmpA / OmpA-like transmembrane domain / SecE subunit of protein translocation complex, bacterial-like / SecE superfamily / SEC-C motif / SEC-C motif / Protein translocase subunit SecA / SecA DEAD-like, N-terminal / SecA Wing/Scaffold / SecA, preprotein cross-linking domain / SecA motor DEAD / SecA conserved site / SecA, Wing/Scaffold superfamily / SecA, preprotein cross-linking domain superfamily / SecA, C-terminal helicase domain / SecA preprotein cross-linking domain / SecA Wing and Scaffold domain / SecA DEAD-like domain / SecA P-loop domain / SecA family signature. / SecA family profile. / SecA DEAD-like domain / SecA preprotein cross-linking domain / Outer membrane protein, OmpA-like, conserved site / Protein translocase subunit SecY / OmpA-like domain. / Outer membrane protein, bacterial / : / OmpA-like domain superfamily / OmpA family / OmpA-like domain / OmpA-like domain profile. / Protein secE/sec61-gamma signature. / Protein secY signature 1. / Protein secY signature 2. / SecE/Sec61-gamma subunits of protein translocation complex / Protein translocase complex, SecE/Sec61-gamma subunit / SecY/SEC61-alpha family / SecY domain superfamily / SecY conserved site / SecY / Outer membrane protein/outer membrane enzyme PagP, beta-barrel / Green fluorescent protein, GFP / Green fluorescent protein-related / Green fluorescent protein / Green fluorescent protein / Helicase, C-terminal / Helicase superfamily 1/2, ATP-binding domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Protein translocase subunit SecE / Protein translocase subunit SecY / Outer membrane protein A / Protein translocase subunit SecA / Green fluorescent protein
Similarity search - Component
Biological speciesBacillus subtilis (bacteria) / Bacillus subtilis (strain 168) (bacteria) / Geobacillus thermodenitrificans (strain NG80-2) (bacteria) / Lama glama (llama) / Escherichia coli (E. coli) / Aequorea victoria (jellyfish)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.45 Å
AuthorsMa CY / Wu XF
Funding support China, 1 items
OrganizationGrant numberCountry
Ministry of Science and Technology (China)2016YFA0500700 China
CitationJournal: Nat Commun / Year: 2019
Title: Structure of the substrate-engaged SecA-SecY protein translocation machine.
Authors: Chengying Ma / Xiaofei Wu / Dongjie Sun / Eunyong Park / Marco A Catipovic / Tom A Rapoport / Ning Gao / Long Li /
Abstract: The Sec61/SecY channel allows the translocation of many proteins across the eukaryotic endoplasmic reticulum membrane or the prokaryotic plasma membrane. In bacteria, most secretory proteins are ...The Sec61/SecY channel allows the translocation of many proteins across the eukaryotic endoplasmic reticulum membrane or the prokaryotic plasma membrane. In bacteria, most secretory proteins are transported post-translationally through the SecY channel by the SecA ATPase. How a polypeptide is moved through the SecA-SecY complex is poorly understood, as structural information is lacking. Here, we report an electron cryo-microscopy (cryo-EM) structure of a translocating SecA-SecY complex in a lipid environment. The translocating polypeptide chain can be traced through both SecA and SecY. In the captured transition state of ATP hydrolysis, SecA's two-helix finger is close to the polypeptide, while SecA's clamp interacts with the polypeptide in a sequence-independent manner by inducing a short β-strand. Taking into account previous biochemical and biophysical data, our structure is consistent with a model in which the two-helix finger and clamp cooperate during the ATPase cycle to move a polypeptide through the channel.
History
DepositionNov 21, 2018-
Header (metadata) releaseJun 12, 2019-
Map releaseJun 12, 2019-
UpdateNov 15, 2023-
Current statusNov 15, 2023Processing site: PDBj / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0242
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 0.0242
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6itc
  • Surface level: 0.0242
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_9731.png

Downloads & links

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Map

FileDownload / File: emd_9731.map.gz / Format: CCP4 / Size: 52.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.05 Å/pix.
x 240 pix.
= 252. Å		1.05 Å/pix.
x 240 pix.
= 252. Å		1.05 Å/pix.
x 240 pix.
= 252. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.05 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0242 / Movie #1: 0.0242
Minimum - Maximum-0.09917147 - 0.14502499
Average (Standard dev.)0.0003700985 (±0.0036889222)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions240240240
Spacing240240240
CellA=B=C: 251.99998 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.051.051.05
M x/y/z240240240
origin x/y/z0.0000.0000.000
length x/y/z252.000252.000252.000
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ480480480
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS240240240
D min/max/mean-0.0990.1450.000

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Supplemental data

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Sample components

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Entire : SecA-SecY complex

EntireName: SecA-SecY complex
Components
  • Complex: SecA-SecY complex
    • Protein or peptide: Protein translocase subunit SecA
    • Protein or peptide: Protein translocase subunit SecY
    • Protein or peptide: Protein translocase subunit SecE
    • Protein or peptide: Nanobody
    • Protein or peptide: Translocating peptide
    • Protein or peptide: Green fluorescent protein
    • Protein or peptide: Nanobody
  • Ligand: MAGNESIUM ION
  • Ligand: BERYLLIUM TRIFLUORIDE ION
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: (1R)-2-{[{[(2S)-2,3-DIHYDROXYPROPYL]OXY}(HYDROXY)PHOSPHORYL]OXY}-1-[(PALMITOYLOXY)METHYL]ETHYL (11E)-OCTADEC-11-ENOATE

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Supramolecule #1: SecA-SecY complex

SupramoleculeName: SecA-SecY complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#7
Source (natural)Organism: Bacillus subtilis (bacteria)

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Macromolecule #1: Protein translocase subunit SecA

MacromoleculeName: Protein translocase subunit SecA / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Bacillus subtilis (strain 168) (bacteria) / Strain: 168
Molecular weightTheoretical: 88.91618 KDa
Recombinant expressionOrganism: Escherichia coli #1/H766 (bacteria)
SequenceString: MLGILNKMFD PTKRTLNRYE KIANDIDAIR GDYENLSDDA LKHKTIEFKE RLEKGATTDD LLVEAFAVVR EASRRVTGMF PFKVQLMGG VALHDGNIAE MKTGEGKTLT STLPVYLNAL TGKGVHVVTV NEYLASRDAE QMGKIFEFLG LTVGLNLNSM S KDEKREAY ...String:
MLGILNKMFD PTKRTLNRYE KIANDIDAIR GDYENLSDDA LKHKTIEFKE RLEKGATTDD LLVEAFAVVR EASRRVTGMF PFKVQLMGG VALHDGNIAE MKTGEGKTLT STLPVYLNAL TGKGVHVVTV NEYLASRDAE QMGKIFEFLG LTVGLNLNSM S KDEKREAY AADITYSTNN ELGFDYLRDN MVLYKEQMVQ RPLHFAVIDE VDSILIDEAR TPLIISGQAA KSTKLYVQAN AF VRTLKAE KDYTYDIKTK AVQLTEEGMT KAEKAFGIDN LFDVKHVALN HHINQALKAH VAMQKDVDYV VEDGQVVIVD SFT GRLMKG RRYSEGLHQA IEAKEGLEIQ NESMTLATIT FQNYFRMYEK LAGMTGTAKT EEEEFRNIYN MQVVTIPTNR PVVR DDRPD LIYRTMEGKF KAVAEDVAQR YMTGQPVLVG TVAVETSELI SKLLKNKGIP HQVLNAKNHE REAQIIEEAG QKGAV TIAT NMAGRGTDIK LGEGVKELGG LAVVGTERHE SRRIDNQLRG RSGRQGDPGI TQFYLSMEDE LMRRFGAERT MAMLDR FGM DDSTPIQSKM VSRAVESSQK RVEGNNFDSR KQLLQYDDVL RQQREVIYKQ RFEVIDSENL REIVENMIKS SLERAIA AY TPREELPEEW KLDGLVDLIN TTYLDEGALE KSDIFGKEPD EMLELIMDRI ITKYNEKEEQ FGKEQMREFE KVIVLRAV D SKWMDHIDAM DQLRQGIHLR AYAQTNPLRE YQMEGFAMFE HMIESIEDEV AKFVMKAEI

UniProtKB: Protein translocase subunit SecA

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Macromolecule #2: Protein translocase subunit SecY

MacromoleculeName: Protein translocase subunit SecY / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Geobacillus thermodenitrificans (strain NG80-2) (bacteria)
Strain: NG80-2
Molecular weightTheoretical: 46.768301 KDa
Recombinant expressionOrganism: Escherichia coli K-12 (bacteria)
SequenceString: MFRTISNFMR VSDIRNKIIF TLLMLIVFRI GTFIPVPSVN TDVLKLQDQL NAFGVLNIFC GGALQNFSIF AMGVMPYITA SIIVQLLQM DVVPKFAEWS KQGEMGRRKL AQFTRYFTIV LGFIQALGMS YGFNNLAGGM LIQNPGIGTY LLIAVVLTAG T AFLMWLGE ...String:
MFRTISNFMR VSDIRNKIIF TLLMLIVFRI GTFIPVPSVN TDVLKLQDQL NAFGVLNIFC GGALQNFSIF AMGVMPYITA SIIVQLLQM DVVPKFAEWS KQGEMGRRKL AQFTRYFTIV LGFIQALGMS YGFNNLAGGM LIQNPGIGTY LLIAVVLTAG T AFLMWLGE QITAKGVGNG ISIIIFAGIV SGIPTILNQI YAQTFGGLNI VRLLLVALAV VAVIVGVIYI QQAFRKIPIQ YA KRLEGRN PVGGHSTHLP LKVNPAGVIP VIFAVSFLIA PPTIASFFGT NDVTLWIRRT FDYTHPVGMT IYVVLIIAFT YFY AFVQVN PEQMADNLKK QGGYIPGIRP GKNTQEYVTR ILYRLTLVGS LFLAFIAVLP VFFVNFANLP PSAQIGGTSL LIVV GVALE TMKQLESQLV KRHYRGFIK

UniProtKB: Protein translocase subunit SecY

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Macromolecule #3: Protein translocase subunit SecE

MacromoleculeName: Protein translocase subunit SecE / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Geobacillus thermodenitrificans (strain NG80-2) (bacteria)
Strain: NG80-2
Molecular weightTheoretical: 8.2496 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MQRVTNFFKE VVRELKKVSW PNRKELVNYT AVVLATVAFF TVFFAVIDLG ISQLIRLVFE GGHHHHHHHH

UniProtKB: Protein translocase subunit SecE

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Macromolecule #4: Nanobody

MacromoleculeName: Nanobody / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Lama glama (llama)
Molecular weightTheoretical: 12.919544 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
QVQLVETGGG LVQPGGSLRL SCGASGSIFN MYAMGWYRQA PGKRREVVAR IATDDSTMYP DSVKGRFTIS RDNAKNTVYL QMNSLKPED TAVYYCYYQR TVMSQPYWGQ GTQVTVS

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Macromolecule #5: Translocating peptide

MacromoleculeName: Translocating peptide / type: protein_or_peptide / ID: 5 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 6.024562 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MAKKTAIAIA VALAGFATVA SYAQYEDGCS GELERQHTFA GGARSISGDG DSPHSYHSG

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Macromolecule #6: Green fluorescent protein

MacromoleculeName: Green fluorescent protein / type: protein_or_peptide / ID: 6 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Aequorea victoria (jellyfish)
Molecular weightTheoretical: 26.813113 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MSKGEELFTG VVPILVELDG DVNGHKFSVS GEGEGDATYG KLTLKFICTT GKLPVPWPTL VTTF(GYS)VQCFS RYPDHM KRH DFFKSAMPEG YVQERTISFK DDGNYKTRAE VKFEGDTLVN RIELKGIDFK EDGNILGHKL EYNYNSHNVY ITADKQK NG IKANFKIRHN ...String:
MSKGEELFTG VVPILVELDG DVNGHKFSVS GEGEGDATYG KLTLKFICTT GKLPVPWPTL VTTF(GYS)VQCFS RYPDHM KRH DFFKSAMPEG YVQERTISFK DDGNYKTRAE VKFEGDTLVN RIELKGIDFK EDGNILGHKL EYNYNSHNVY ITADKQK NG IKANFKIRHN IEDGSVQLAD HYQQNTPIGD GPVLLPDNHY LSTQSALSKD PNEKRDHMVL LEFVTAAGIT HGMDELYK

UniProtKB: Green fluorescent protein

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Macromolecule #7: Nanobody

MacromoleculeName: Nanobody / type: protein_or_peptide / ID: 7 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Lama glama (llama)
Molecular weightTheoretical: 12.368727 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
VALVESGGAL VQPGGSLRLS CAASGFPVNR YSMRWYRQAP GKEREWVAGM SAGDRSSYED SVKGRFTISR DDARNTVYLQ MNSLKPEDT AVYYCNVNVG FEYWGQGTQV TVS

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Macromolecule #8: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 8 / Number of copies: 1 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #9: BERYLLIUM TRIFLUORIDE ION

MacromoleculeName: BERYLLIUM TRIFLUORIDE ION / type: ligand / ID: 9 / Number of copies: 1 / Formula: BEF
Molecular weightTheoretical: 66.007 Da
Chemical component information

ChemComp-BEF:
BERYLLIUM TRIFLUORIDE ION

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Macromolecule #10: ADENOSINE-5'-DIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-DIPHOSPHATE / type: ligand / ID: 10 / Number of copies: 1 / Formula: ADP
Molecular weightTheoretical: 427.201 Da
Chemical component information

ChemComp-ADP:
ADENOSINE-5'-DIPHOSPHATE / ADP, energy-carrying molecule*YM

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Macromolecule #11: (1R)-2-{[{[(2S)-2,3-DIHYDROXYPROPYL]OXY}(HYDROXY)PHOSPHORYL]OXY}-...

MacromoleculeName: (1R)-2-{[{[(2S)-2,3-DIHYDROXYPROPYL]OXY}(HYDROXY)PHOSPHORYL]OXY}-1-[(PALMITOYLOXY)METHYL]ETHYL (11E)-OCTADEC-11-ENOATE
type: ligand / ID: 11 / Number of copies: 2 / Formula: PGV
Molecular weightTheoretical: 749.007 Da
Chemical component information

ChemComp-PGV:
(1R)-2-{[{[(2S)-2,3-DIHYDROXYPROPYL]OXY}(HYDROXY)PHOSPHORYL]OXY}-1-[(PALMITOYLOXY)METHYL]ETHYL (11E)-OCTADEC-11-ENOATE / phospholipid*YM

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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: 7.5
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 QUANTUM (4k x 4k) / Average electron dose: 5.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: OTHER
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.45 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 130153
Initial angle assignmentType: OTHER
Final angle assignmentType: PROJECTION MATCHING

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Atomic model buiding 1

RefinementProtocol: FLEXIBLE FIT
Output model

PDB-6itc:
Structure of a substrate engaged SecA-SecY protein translocation machine

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