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- EMDB-40246: Human ER membrane protein complex (EMC) in GDN, Consensus map -

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Basic information

Entry
Database: EMDB / ID: EMD-40246
TitleHuman ER membrane protein complex (EMC) in GDN, Consensus map
Map dataFull map file
Sample
  • Complex: Human ER Membrane Protein Complex
    • Protein or peptide: ER membrane protein complex subunit 1
    • Protein or peptide: ER membrane protein complex subunit 2
  • Protein or peptide: ER membrane protein complex subunit 3
  • Protein or peptide: ER membrane protein complex subunit 4
  • Protein or peptide: ER membrane protein complex subunit 5
  • Protein or peptide: ER membrane protein complex subunit 6
  • Protein or peptide: ER membrane protein complex subunit 7
  • Protein or peptide: ER membrane protein complex subunit 8
  • Protein or peptide: ER membrane protein complex subunit 10
KeywordsInsertase / endoplasmic reticulum / transmembrane chaperone / MEMBRANE PROTEIN
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsTomaleri GP / Nguyen V / Januszyk K / Voorhees RM
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)DP2GM137412 United States
CitationJournal: J Cell Biol / Year: 2023
Title: A selectivity filter in the ER membrane protein complex limits protein misinsertion at the ER.
Authors: Tino Pleiner / Masami Hazu / Giovani Pinton Tomaleri / Vy N Nguyen / Kurt Januszyk / Rebecca M Voorhees /
Abstract: Tail-anchored (TA) proteins play essential roles in mammalian cells, and their accurate localization is critical for proteostasis. Biophysical similarities lead to mistargeting of mitochondrial TA ...Tail-anchored (TA) proteins play essential roles in mammalian cells, and their accurate localization is critical for proteostasis. Biophysical similarities lead to mistargeting of mitochondrial TA proteins to the ER, where they are delivered to the insertase, the ER membrane protein complex (EMC). Leveraging an improved structural model of the human EMC, we used mutagenesis and site-specific crosslinking to map the path of a TA protein from its cytosolic capture by methionine-rich loops to its membrane insertion through a hydrophilic vestibule. Positively charged residues at the entrance to the vestibule function as a selectivity filter that uses charge-repulsion to reject mitochondrial TA proteins. Similarly, this selectivity filter retains the positively charged soluble domains of multipass substrates in the cytosol, thereby ensuring they adopt the correct topology and enforcing the "positive-inside" rule. Substrate discrimination by the EMC provides a biochemical explanation for one role of charge in TA protein sorting and protects compartment integrity by limiting protein misinsertion.
History
DepositionMar 30, 2023-
Header (metadata) releaseMay 17, 2023-
Map releaseMay 17, 2023-
UpdateMay 31, 2023-
Current statusMay 31, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_40246.png

Downloads & links

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Map

FileDownload / File: emd_40246.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationFull map file
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 400 pix.
= 332.8 Å		0.83 Å/pix.
x 400 pix.
= 332.8 Å		0.83 Å/pix.
x 400 pix.
= 332.8 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.832 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.104
Minimum - Maximum-0.16399747 - 0.44918853
Average (Standard dev.)0.0025668677 (±0.014198152)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions400400400
Spacing400400400
CellA=B=C: 332.80002 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: Sharped Full map file

Fileemd_40246_additional_1.map
AnnotationSharped Full map file
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map A

Fileemd_40246_half_map_1.map
AnnotationHalf map A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map B

Fileemd_40246_half_map_2.map
AnnotationHalf map B
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Human ER Membrane Protein Complex

EntireName: Human ER Membrane Protein Complex
Components
  • Complex: Human ER Membrane Protein Complex
    • Protein or peptide: ER membrane protein complex subunit 1
    • Protein or peptide: ER membrane protein complex subunit 2
  • Protein or peptide: ER membrane protein complex subunit 3
  • Protein or peptide: ER membrane protein complex subunit 4
  • Protein or peptide: ER membrane protein complex subunit 5
  • Protein or peptide: ER membrane protein complex subunit 6
  • Protein or peptide: ER membrane protein complex subunit 7
  • Protein or peptide: ER membrane protein complex subunit 8
  • Protein or peptide: ER membrane protein complex subunit 10

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Supramolecule #1: Human ER Membrane Protein Complex

SupramoleculeName: Human ER Membrane Protein Complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Homo sapiens (human) / Cell: HEK293

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Macromolecule #1: ER membrane protein complex subunit 1

MacromoleculeName: ER membrane protein complex subunit 1 / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
SequenceString: MAAEWASRFW LWATLLIPAA AVYEDQVGKF DWRQQYVGKV KFASLEFSPG SKKLVVATEK NVIAALNSRT GEILWRHVDK GTAEGAVDA MLLHGQDVIT VSNGGRIMRS WETNIGGLNW EITLDSGSFQ ALGLVGLQES VRYIAVLKKT TLALHHLSSG H LKWVEHLP ...String:
MAAEWASRFW LWATLLIPAA AVYEDQVGKF DWRQQYVGKV KFASLEFSPG SKKLVVATEK NVIAALNSRT GEILWRHVDK GTAEGAVDA MLLHGQDVIT VSNGGRIMRS WETNIGGLNW EITLDSGSFQ ALGLVGLQES VRYIAVLKKT TLALHHLSSG H LKWVEHLP ESDSIHYQMV YSYGSGVVWA LGVVPFSHVN IVKFNVEDGE IVQQVRVSTP WLQHLSGACG VVDEAVLVCP DP SSRSLQT LALETEWELR QIPLQSLDLE FGSGFQPRVL PTQPNPVDAS RAQFFLHLSP SHYALLQYHY GTLSLLKNFP QTA LVSFAT TGEKTVAAVM ACRNEVQKSS SSEDGSMGSF SEKSSSKDSL ACFNQTYTIN LYLVETGRRL LDTTITFSLE QSGT RPERL YIQVFLKKDD SVGYRALVQT EDHLLLFLQQ LAGKVVLWSR EESLAEVVCL EMVDLPLTGA QAELEGEFGK KADGL LGMF LKRLSSQLIL LQAWTSHLWK MFYDARKPRS QIKNEINIDT LARDEFNLQK MMVMVTASGK LFGIESSSGT ILWKQY LPN VKPDSSFKLM VQRTTAHFPH PPQCTLLVKD KESGMSSLYV FNPIFGKWSQ VAPPVLKRPI LQSLLLPVMD QDYAKVL LL IDDEYKVTAF PATRNVLRQL HELAPSIFFY LVDAEQGRLC GYRLRKDLTT ELSWELTIPP EVQRIVKVKG KRSSEHVH S QGRVMGDRSV LYKSLNPNLL AVVTESTDAH HERTFIGIFL IDGVTGRIIH SSVQKKAKGP VHIVHSENWV VYQYWNTKA RRNEFTVLEL YEGTEQYNAT AFSSLDRPQL PQVLQQSYIF PSSISAMEAT ITERGITSRH LLIGLPSGAI LSLPKALLDP RRPEIPTEQ SREENLIPYS PDVQIHAERF INYNQTVSRM RGIYTAPSGL ESTCLVVAYG LDIYQTRVYP SKQFDVLKDD Y DYVLISSV LFGLVFATMI TKRLAQVKLL NRAWR

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Macromolecule #2: ER membrane protein complex subunit 2

MacromoleculeName: ER membrane protein complex subunit 2 / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
SequenceString: MAKVSELYDV TWEEMRDKMR KWREENSRNS EQIVEVGEEL INEYASKLGD DIWIIYEQVM IAALDYGRDD LALFCLQELR RQFPGSHRV KRLTGMRFEA MERYDDAIQL YDRILQEDPT NTAARKRKIA IRKAQGKNVE AIRELNEYLE QFVGDQEAWH E LAELYINE ...String:
MAKVSELYDV TWEEMRDKMR KWREENSRNS EQIVEVGEEL INEYASKLGD DIWIIYEQVM IAALDYGRDD LALFCLQELR RQFPGSHRV KRLTGMRFEA MERYDDAIQL YDRILQEDPT NTAARKRKIA IRKAQGKNVE AIRELNEYLE QFVGDQEAWH E LAELYINE HDYAKAAFCL EELMMTNPHN HLYCQQYAEV KYTQGGLENL ELSRKYFAQA LKLNNRNMRA LFGLYMSASH IA SNPKASA KTKKDNMKYA SWAASQINRA YQFAGRSKKE TKYSLKAVED MLETLQITQS

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Macromolecule #3: ER membrane protein complex subunit 3

MacromoleculeName: ER membrane protein complex subunit 3 / type: protein_or_peptide / ID: 3 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
SequenceString: MAGPELLLDS NIRLWVVLPI VIITFFVGMI RHYVSILLQS DKKLTQEQVS DSQVLIRSRV LRENGKYIPK QSFLTRKYYF NNPEDGFFK KTKRKVVPPS PMTDPTMLTD MMKGNVTNVL PMILIGGWIN MTFSGFVTTK VPFPLTLRFK PMLQQGIELL T LDASWVSS ...String:
MAGPELLLDS NIRLWVVLPI VIITFFVGMI RHYVSILLQS DKKLTQEQVS DSQVLIRSRV LRENGKYIPK QSFLTRKYYF NNPEDGFFK KTKRKVVPPS PMTDPTMLTD MMKGNVTNVL PMILIGGWIN MTFSGFVTTK VPFPLTLRFK PMLQQGIELL T LDASWVSS ASWYFLNVFG LRSIYSLILG QDNAADQSRM MQEQMTGAAM AMPADTNKAF KTEWEALELT DHQWALDDVE EE LMAKDLH FEGMFKKELQ TSIF

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Macromolecule #4: ER membrane protein complex subunit 4

MacromoleculeName: ER membrane protein complex subunit 4 / type: protein_or_peptide / ID: 4 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
SequenceString:
MTAQGGLVAN RGRRFKWAIE LSGPGGGSRG RSDRGSGQGD SLYPVGYLDK QVPDTSVQET DRILVEKRCW DIALGPLKQI PMNLFIMYM AGNTISIFPT MMVCMMAWRP IQALMAISAT FKMLESSSQK FLQGLVYLIG NLMGLALAVY KCQSMGLLPT H ASDWLAFI EPPERMEFSG GGLLL

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Macromolecule #5: ER membrane protein complex subunit 5

MacromoleculeName: ER membrane protein complex subunit 5 / type: protein_or_peptide / ID: 5 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
SequenceString:
MAPSLWKGLV GIGLFALAHA AFSAAQHRSY MRLTEKEDES LPIDIVLQTL LAFAVTCYGI VHIAGEFKDM DATSELKNKT FDTLRNHPS FYVFNHRGRV LFRPSDTANS SNQDALSSNT SLKLRKLESL RR

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Macromolecule #6: ER membrane protein complex subunit 6

MacromoleculeName: ER membrane protein complex subunit 6 / type: protein_or_peptide / ID: 6 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
SequenceString:
MAAVVAKREG PPFISEAAVR GNAAVLDYCR TSVSALSGAT AGILGLTGLY GFIFYLLASV LLSLLLILKA GRRWNKYFKS RRPLFTGGL IGGLFTYVLF WTFLYGMVHV Y

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Macromolecule #7: ER membrane protein complex subunit 7

MacromoleculeName: ER membrane protein complex subunit 7 / type: protein_or_peptide / ID: 7 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
SequenceString: MAAALWGFFP VLLLLLLSGD VQSSEVPGAA AEGSGGSGVG IGDRFKIEGR AVVPGVKPQD WISAARVLVD GEEHVGFLKT DGSFVVHDI PSGSYVVEVV SPAYRFDPVR VDITSKGKMR ARYVNYIKTS EVVRLPYPLQ MKSSGPPSYF IKRESWGWTD F LMNPMVMM ...String:
MAAALWGFFP VLLLLLLSGD VQSSEVPGAA AEGSGGSGVG IGDRFKIEGR AVVPGVKPQD WISAARVLVD GEEHVGFLKT DGSFVVHDI PSGSYVVEVV SPAYRFDPVR VDITSKGKMR ARYVNYIKTS EVVRLPYPLQ MKSSGPPSYF IKRESWGWTD F LMNPMVMM MVLPLLIFVL LPKVVNTSDP DMRREMEQSM NMLNSNHELP DVSEFMTRLF SSKSSGKSSS GSSKTGKSGA GK RR

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Macromolecule #8: ER membrane protein complex subunit 8

MacromoleculeName: ER membrane protein complex subunit 8 / type: protein_or_peptide / ID: 8 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
SequenceString: MPGVKLTTQA YCKMVLHGAK YPHCAVNGLL VAEKQKPRKE HLPLGGPGAH HTLFVDCIPL FHGTLALAPM LEVALTLIDS WCKDHSYVI AGYYQANERV KDASPNQVAE KVASRIAEGF SDTALIMVDN TKFTMDCVAP TIHVYEHHEN RWRCRDPHHD Y CEDWPEAQ ...String:
MPGVKLTTQA YCKMVLHGAK YPHCAVNGLL VAEKQKPRKE HLPLGGPGAH HTLFVDCIPL FHGTLALAPM LEVALTLIDS WCKDHSYVI AGYYQANERV KDASPNQVAE KVASRIAEGF SDTALIMVDN TKFTMDCVAP TIHVYEHHEN RWRCRDPHHD Y CEDWPEAQ RISASLLDSR SYETLVDFDN HLDDIRNDWT NPEINKAVLH LC

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Macromolecule #9: ER membrane protein complex subunit 10

MacromoleculeName: ER membrane protein complex subunit 10 / type: protein_or_peptide / ID: 9 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
SequenceString: MAAASAGATR LLLLLLMAVA APSRARGSGC RAGTGARGAG AEGREGEACG TVGLLLEHSF EIDDSANFRK RGSLLWNQQD GTLSLSQRQ LSEEERGRLR DVAALNGLYR VRIPRRPGAL DGLEAGGYVS SFVPACSLVE SHLSDQLTLH VDVAGNVVGV S VVTHPGGC ...String:
MAAASAGATR LLLLLLMAVA APSRARGSGC RAGTGARGAG AEGREGEACG TVGLLLEHSF EIDDSANFRK RGSLLWNQQD GTLSLSQRQ LSEEERGRLR DVAALNGLYR VRIPRRPGAL DGLEAGGYVS SFVPACSLVE SHLSDQLTLH VDVAGNVVGV S VVTHPGGC RGHEVEDVDL ELFNTSVQLQ PPTTAPGPET AAFIERLEME QAQKAKNPQE QKSFFAKYWM YIIPVVLFLM MS GAPDTGG QGGGGGGGGG GGSGR

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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

Concentration0.5 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
50.0 mMHEPESN-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid
200.0 mMNaClSodium chloride
2.0 mMMgAc2magnesium acetate
1.0 mMDTTDL-Dithiothreito
0.05 % (w/v)GDNSynthetic substitute for Digitonin
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 279 K / Instrument: FEI VITROBOT MARK IV
DetailsSample solubilized and purified in GDN

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF Quantum LS / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 2 / Number real images: 11822 / Average exposure time: 2.66 sec. / Average electron dose: 60.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: SPOT SCAN / Imaging mode: DARK FIELD / Cs: 2.7 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 130000
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: 1034250
Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.0) / Number images used: 193900
Initial angle assignmentType: NOT APPLICABLE
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.3)
Final 3D classificationNumber classes: 4 / Software - Name: cryoSPARC (ver. 4.0)

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