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- EMDB-4738: Endogeneous native human 20S proteasome with bound Fabs isolated ... -

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Basic information

Entry
Database: EMDB / ID: EMD-4738
TitleEndogeneous native human 20S proteasome with bound Fabs isolated from less than 1 ul cell lysate
Map dataRELION3 post processed, local resolution filtered.
Sample
  • Complex: Native human 20S proteasome with bound Fabs to the two alpha-4 subunits
    • Protein or peptide: x 14 types
Function / homology
Function and homology information


purine ribonucleoside triphosphate binding / regulation of endopeptidase activity / Regulation of ornithine decarboxylase (ODC) / proteasome core complex / Cross-presentation of soluble exogenous antigens (endosomes) / Somitogenesis / immune system process / myofibril / NF-kappaB binding / proteasome endopeptidase complex ...purine ribonucleoside triphosphate binding / regulation of endopeptidase activity / Regulation of ornithine decarboxylase (ODC) / proteasome core complex / Cross-presentation of soluble exogenous antigens (endosomes) / Somitogenesis / immune system process / myofibril / NF-kappaB binding / proteasome endopeptidase complex / proteasome core complex, beta-subunit complex / proteasome core complex, alpha-subunit complex / threonine-type endopeptidase activity / negative regulation of inflammatory response to antigenic stimulus / response to organonitrogen compound / proteolysis involved in protein catabolic process / proteasome complex / sarcomere / Regulation of activated PAK-2p34 by proteasome mediated degradation / ciliary basal body / Autodegradation of Cdh1 by Cdh1:APC/C / APC/C:Cdc20 mediated degradation of Securin / Asymmetric localization of PCP proteins / SCF-beta-TrCP mediated degradation of Emi1 / NIK-->noncanonical NF-kB signaling / Ubiquitin-dependent degradation of Cyclin D / AUF1 (hnRNP D0) binds and destabilizes mRNA / TNFR2 non-canonical NF-kB pathway / Assembly of the pre-replicative complex / Vpu mediated degradation of CD4 / Degradation of DVL / proteasomal protein catabolic process / P-body / Ubiquitin Mediated Degradation of Phosphorylated Cdc25A / Dectin-1 mediated noncanonical NF-kB signaling / Hh mutants are degraded by ERAD / Cdc20:Phospho-APC/C mediated degradation of Cyclin A / Degradation of AXIN / Defective CFTR causes cystic fibrosis / Degradation of GLI1 by the proteasome / Activation of NF-kappaB in B cells / lipopolysaccharide binding / Hedgehog ligand biogenesis / Negative regulation of NOTCH4 signaling / G2/M Checkpoints / GSK3B and BTRC:CUL1-mediated-degradation of NFE2L2 / Autodegradation of the E3 ubiquitin ligase COP1 / Vif-mediated degradation of APOBEC3G / Hedgehog 'on' state / Regulation of RUNX3 expression and activity / Degradation of GLI2 by the proteasome / GLI3 is processed to GLI3R by the proteasome / MAPK6/MAPK4 signaling / FBXL7 down-regulates AURKA during mitotic entry and in early mitosis / response to virus / APC/C:Cdh1 mediated degradation of Cdc20 and other APC/C:Cdh1 targeted proteins in late mitosis/early G1 / ABC-family proteins mediated transport / Degradation of beta-catenin by the destruction complex / Oxygen-dependent proline hydroxylation of Hypoxia-inducible Factor Alpha / response to organic cyclic compound / CDK-mediated phosphorylation and removal of Cdc6 / CLEC7A (Dectin-1) signaling / SCF(Skp2)-mediated degradation of p27/p21 / nuclear matrix / Regulation of expression of SLITs and ROBOs / FCERI mediated NF-kB activation / Regulation of PTEN stability and activity / Interleukin-1 signaling / Orc1 removal from chromatin / Regulation of RAS by GAPs / Separation of Sister Chromatids / Regulation of RUNX2 expression and activity / UCH proteinases / The role of GTSE1 in G2/M progression after G2 checkpoint / KEAP1-NFE2L2 pathway / Antigen processing: Ubiquitination & Proteasome degradation / Downstream TCR signaling / Neddylation / RUNX1 regulates transcription of genes involved in differentiation of HSCs / positive regulation of NF-kappaB transcription factor activity / peptidase activity / ER-Phagosome pathway / regulation of inflammatory response / postsynapse / proteasome-mediated ubiquitin-dependent protein catabolic process / secretory granule lumen / endopeptidase activity / response to oxidative stress / ficolin-1-rich granule lumen / nuclear body / Ub-specific processing proteases / ribosome / cadherin binding / intracellular membrane-bounded organelle / centrosome / synapse / ubiquitin protein ligase binding / Neutrophil degranulation / mitochondrion / proteolysis
Similarity search - Function
Proteasome subunit alpha 1 / Proteasome beta subunit, C-terminal / Proteasome beta subunits C terminal / Proteasome subunit beta 4 / Proteasome subunit beta 2 / Proteasome beta 3 subunit / Proteasome subunit alpha6 / Proteasome subunit alpha5 / Proteasome beta-type subunits signature. / Peptidase T1A, proteasome beta-subunit ...Proteasome subunit alpha 1 / Proteasome beta subunit, C-terminal / Proteasome beta subunits C terminal / Proteasome subunit beta 4 / Proteasome subunit beta 2 / Proteasome beta 3 subunit / Proteasome subunit alpha6 / Proteasome subunit alpha5 / Proteasome beta-type subunits signature. / Peptidase T1A, proteasome beta-subunit / Proteasome beta-type subunit, conserved site / Proteasome subunit A N-terminal signature / Proteasome alpha-type subunits signature. / Proteasome alpha-subunit, N-terminal domain / Proteasome subunit A N-terminal signature Add an annotation / Proteasome alpha-type subunit / Proteasome alpha-type subunit profile. / Proteasome B-type subunit / Proteasome beta-type subunit profile. / Proteasome subunit / Proteasome, subunit alpha/beta / Nucleophile aminohydrolases, N-terminal
Similarity search - Domain/homology
Proteasome subunit alpha type-7 / Proteasome subunit beta type-1 / Proteasome subunit alpha type-1 / Proteasome subunit alpha type-2 / Proteasome subunit alpha type-3 / Proteasome subunit alpha type-4 / Proteasome subunit alpha type-5 / Proteasome subunit beta type-4 / Proteasome subunit beta type-6 / Proteasome subunit beta type-5 ...Proteasome subunit alpha type-7 / Proteasome subunit beta type-1 / Proteasome subunit alpha type-1 / Proteasome subunit alpha type-2 / Proteasome subunit alpha type-3 / Proteasome subunit alpha type-4 / Proteasome subunit alpha type-5 / Proteasome subunit beta type-4 / Proteasome subunit beta type-6 / Proteasome subunit beta type-5 / Proteasome subunit beta type-3 / Proteasome subunit beta type-2 / Proteasome subunit alpha type-6 / Proteasome subunit beta type-7
Similarity search - Component
Biological speciesHomo sapiens (human) / Human (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsSchmidli C / Albiez S / Rima L / Righetto R / Mohammed I / Oliva P / Kovacik L / Stahlberg H / Braun T
Funding support Switzerland, 2 items
OrganizationGrant numberCountry
Swiss National Science Foundation200021_162521 Switzerland
Swiss National Science Foundation205320_166164 Switzerland
CitationJournal: Proc Natl Acad Sci U S A / Year: 2019
Title: Microfluidic protein isolation and sample preparation for high-resolution cryo-EM.
Authors: Claudio Schmidli / Stefan Albiez / Luca Rima / Ricardo Righetto / Inayatulla Mohammed / Paolo Oliva / Lubomir Kovacik / Henning Stahlberg / Thomas Braun /
Abstract: High-resolution structural information is essential to understand protein function. Protein-structure determination needs a considerable amount of protein, which can be challenging to produce, often ...High-resolution structural information is essential to understand protein function. Protein-structure determination needs a considerable amount of protein, which can be challenging to produce, often involving harsh and lengthy procedures. In contrast, the several thousand to a few million protein particles required for structure determination by cryogenic electron microscopy (cryo-EM) can be provided by miniaturized systems. Here, we present a microfluidic method for the rapid isolation of a target protein and its direct preparation for cryo-EM. Less than 1 μL of cell lysate is required as starting material to solve the atomic structure of the untagged, endogenous human 20S proteasome. Our work paves the way for high-throughput structure determination of proteins from minimal amounts of cell lysate and opens more opportunities for the isolation of sensitive, endogenous protein complexes.
History
DepositionMar 28, 2019-
Header (metadata) releaseJul 3, 2019-
Map releaseJul 3, 2019-
UpdateDec 18, 2019-
Current statusDec 18, 2019Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.025
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.025
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6r70
  • Surface level: 0.025
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_4738.png

Downloads & links

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Map

FileDownload / File: emd_4738.map.gz / Format: CCP4 / Size: 216 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationRELION3 post processed, local resolution filtered.
Voxel sizeX=Y=Z: 1.015 Å
Density
Contour LevelBy AUTHOR: 0.025 / Movie #1: 0.025
Minimum - Maximum-0.10898206 - 0.18135783
Average (Standard dev.)0.00005549869 (±0.0044360184)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions384384384
Spacing384384384
CellA=B=C: 389.76 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0151.0151.015
M x/y/z384384384
origin x/y/z0.0000.0000.000
length x/y/z389.760389.760389.760
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS384384384
D min/max/mean-0.1090.1810.000

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Supplemental data

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Sample components

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Entire : Native human 20S proteasome with bound Fabs to the two alpha-4 su...

EntireName: Native human 20S proteasome with bound Fabs to the two alpha-4 subunits
Components
  • Complex: Native human 20S proteasome with bound Fabs to the two alpha-4 subunits
    • Protein or peptide: Proteasome subunit beta type-4PSMB4
    • Protein or peptide: Proteasome subunit alpha type-2
    • Protein or peptide: Proteasome subunit alpha type-4
    • Protein or peptide: Proteasome subunit alpha type-7
    • Protein or peptide: Proteasome subunit alpha type-5
    • Protein or peptide: Proteasome subunit alpha type-1
    • Protein or peptide: Proteasome subunit alpha type-3
    • Protein or peptide: Proteasome subunit alpha type-6
    • Protein or peptide: Proteasome subunit beta type-7
    • Protein or peptide: Proteasome subunit beta type-3PSMB3
    • Protein or peptide: Proteasome subunit beta type-2PSMB2
    • Protein or peptide: Proteasome subunit beta type-5PSMB5
    • Protein or peptide: Proteasome subunit beta type-1PSMB1
    • Protein or peptide: Proteasome subunit beta type-6

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Supramolecule #1: Native human 20S proteasome with bound Fabs to the two alpha-4 su...

SupramoleculeName: Native human 20S proteasome with bound Fabs to the two alpha-4 subunits
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: Fab fragment generated by proteolytic cleavage of monoclonal IgG1 antibody.
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: Proteasome subunit beta type-4

MacromoleculeName: Proteasome subunit beta type-4 / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO / EC number: proteasome endopeptidase complex
Source (natural)Organism: Human (human)
Molecular weightTheoretical: 24.081402 KDa
SequenceString: TQNPMVTGTS VLGVKFEGGV VIAADMLGSY GSLARFRNIS RIMRVNNSTM LGASGDYADF QYLKQVLGQM VIDEELLGDG HSYSPRAIH SWLTRAMYSR RSKMNPLWNT MVIGGYADGE SFLGYVDMLG VAYEAPSLAT GYGAYLAQPL LREVLEKQPV L SQTEARDL ...String:
TQNPMVTGTS VLGVKFEGGV VIAADMLGSY GSLARFRNIS RIMRVNNSTM LGASGDYADF QYLKQVLGQM VIDEELLGDG HSYSPRAIH SWLTRAMYSR RSKMNPLWNT MVIGGYADGE SFLGYVDMLG VAYEAPSLAT GYGAYLAQPL LREVLEKQPV L SQTEARDL VERCMRVLYY RDARSYNRFQ IATVTEKGVE IEGPLSTETN WDIAHMIS

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Macromolecule #2: Proteasome subunit alpha type-2

MacromoleculeName: Proteasome subunit alpha type-2 / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO / EC number: proteasome endopeptidase complex
Source (natural)Organism: Human (human)
Molecular weightTheoretical: 25.525068 KDa
SequenceString: RGYSFSLTTF SPSGKLVQIE YALAAVAGGA PSVGIKAANG VVLATEKKQK SILYDERSVH KVEPITKHIG LVYSGMGPDY RVLVHRARK LAQQYYLVYQ EPIPTAQLVQ RVASVMQEYT QSGGVRPFGV SLLICGWNEG RPYLFQSDPS GAYFAWKATA M GKNYVNGK ...String:
RGYSFSLTTF SPSGKLVQIE YALAAVAGGA PSVGIKAANG VVLATEKKQK SILYDERSVH KVEPITKHIG LVYSGMGPDY RVLVHRARK LAQQYYLVYQ EPIPTAQLVQ RVASVMQEYT QSGGVRPFGV SLLICGWNEG RPYLFQSDPS GAYFAWKATA M GKNYVNGK TFLEKRYNED LELEDAIHTA ILTLKESFEG QMTEDNIEVG ICNEAGFRRL TPTEVKDYLA AI

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Macromolecule #3: Proteasome subunit alpha type-4

MacromoleculeName: Proteasome subunit alpha type-4 / type: protein_or_peptide / ID: 3 / Number of copies: 2 / Enantiomer: LEVO / EC number: proteasome endopeptidase complex
Source (natural)Organism: Human (human)
Molecular weightTheoretical: 27.859896 KDa
SequenceString: SRRYDSRTTI FSPEGRLYQV EYAMEAIGHA GTCLGILAND GVLLAAERRN IHKLLDEVFF SEKIYKLNED MACSVAGITS DANVLTNEL RLIAQRYLLQ YQEPIPCEQL VTALCDIKQA YTQFGGKRPF GVSLLYIGWD KHYGFQLYQS DPSGNYGGWK A TCIGNNSA ...String:
SRRYDSRTTI FSPEGRLYQV EYAMEAIGHA GTCLGILAND GVLLAAERRN IHKLLDEVFF SEKIYKLNED MACSVAGITS DANVLTNEL RLIAQRYLLQ YQEPIPCEQL VTALCDIKQA YTQFGGKRPF GVSLLYIGWD KHYGFQLYQS DPSGNYGGWK A TCIGNNSA AAVSMLKQDY KEGEMTLKSA LALAIKVLNK TMDVSKLSAE KVEIATLTRE NGKTVIRVLK QKEVEQLIKK HE EEEAKAE R

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Macromolecule #4: Proteasome subunit alpha type-7

MacromoleculeName: Proteasome subunit alpha type-7 / type: protein_or_peptide / ID: 4 / Number of copies: 2 / Enantiomer: LEVO / EC number: proteasome endopeptidase complex
Source (natural)Organism: Human (human)
Molecular weightTheoretical: 26.623291 KDa
SequenceString: SYDRAITVFS PDGHLFQVEY AQEAVKKGST AVGVRGRDIV VLGVEKKSVA KLQDERTVRK ICALDDNVCM AFAGLTADAR IVINRARVE CQSHRLTVED PVTVEYITRY IASLKQRYTQ SNGRRPFGIS ALIVGFDFDG TPRLYQTDPS GTYHAWKANA I GRGAKSVR ...String:
SYDRAITVFS PDGHLFQVEY AQEAVKKGST AVGVRGRDIV VLGVEKKSVA KLQDERTVRK ICALDDNVCM AFAGLTADAR IVINRARVE CQSHRLTVED PVTVEYITRY IASLKQRYTQ SNGRRPFGIS ALIVGFDFDG TPRLYQTDPS GTYHAWKANA I GRGAKSVR EFLEKNYTDE AIETDDLTIK LVIKALLEVV QSGGKNIELA VMRRDQSLKI LNPEEIEKYV AEIEKEKEE

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Macromolecule #5: Proteasome subunit alpha type-5

MacromoleculeName: Proteasome subunit alpha type-5 / type: protein_or_peptide / ID: 5 / Number of copies: 2 / Enantiomer: LEVO / EC number: proteasome endopeptidase complex
Source (natural)Organism: Human (human)
Molecular weightTheoretical: 25.406785 KDa
SequenceString: DRGVNTFSPE GRLFQVEYAI EAIKLGSTAI GIQTSEGVCL AVEKRITSPL MEPSSIEKIV EIDAHIGCAM SGLIADAKTL IDKARVETQ NHWFTYNETM TVESVTQAVS NLALQFGEED ADPGAMSRPF GVALLFGGVD EKGPQLFHMD PSGTFVQCDA R AIGSASEG ...String:
DRGVNTFSPE GRLFQVEYAI EAIKLGSTAI GIQTSEGVCL AVEKRITSPL MEPSSIEKIV EIDAHIGCAM SGLIADAKTL IDKARVETQ NHWFTYNETM TVESVTQAVS NLALQFGEED ADPGAMSRPF GVALLFGGVD EKGPQLFHMD PSGTFVQCDA R AIGSASEG AQSSLQEVYH KSMTLKEAIK SSLIILKQVM EEKLNATNIE LATVQPGQNF HMFTKEELEE VIKDI

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Macromolecule #6: Proteasome subunit alpha type-1

MacromoleculeName: Proteasome subunit alpha type-1 / type: protein_or_peptide / ID: 6 / Number of copies: 2 / Enantiomer: LEVO / EC number: proteasome endopeptidase complex
Source (natural)Organism: Human (human)
Molecular weightTheoretical: 26.807551 KDa
SequenceString: FRNQYDNDVT VWSPQGRIHQ IEYAMEAVKQ GSATVGLKSK THAVLVALKR AQSELAAHQK KILHVDNHIG ISIAGLTADA RLLCNFMRQ ECLDSRFVFD RPLPVSRLVS LIGSKTQIPT QRYGRRPYGV GLLIAGYDDM GPHIFQTCPS ANYFDCRAMS I GARSQSAR ...String:
FRNQYDNDVT VWSPQGRIHQ IEYAMEAVKQ GSATVGLKSK THAVLVALKR AQSELAAHQK KILHVDNHIG ISIAGLTADA RLLCNFMRQ ECLDSRFVFD RPLPVSRLVS LIGSKTQIPT QRYGRRPYGV GLLIAGYDDM GPHIFQTCPS ANYFDCRAMS I GARSQSAR TYLERHMSEF MECNLNELVK HGLRALRETL PAEQDLTTKN VSIGIVGKDL EFTIYDDDDV SPFLEGLEER

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Macromolecule #7: Proteasome subunit alpha type-3

MacromoleculeName: Proteasome subunit alpha type-3 / type: protein_or_peptide / ID: 7 / Number of copies: 2 / Enantiomer: LEVO / EC number: proteasome endopeptidase complex
Source (natural)Organism: Human (human)
Molecular weightTheoretical: 26.813619 KDa
SequenceString: TGYDLSASTF SPDGRVFQVE YAMKAVENSS TAIGIRCKDG VVFGVEKLVL SKLYEEGSNK RLFNVDRHVG MAVAGLLADA RSLADIARE EASNFRSNFG YNIPLKHLAD RVAMYVHAYT LYSAVRPFGC SFMLGSYSVN DGAQLYMIDP SGVSYGYWGC A IGKARQAA ...String:
TGYDLSASTF SPDGRVFQVE YAMKAVENSS TAIGIRCKDG VVFGVEKLVL SKLYEEGSNK RLFNVDRHVG MAVAGLLADA RSLADIARE EASNFRSNFG YNIPLKHLAD RVAMYVHAYT LYSAVRPFGC SFMLGSYSVN DGAQLYMIDP SGVSYGYWGC A IGKARQAA KTEIEKLQMK EMTCRDIVKE VAKIIYIVHD EVKDKAFELE LSWVGELTNG RHEIVPKDIR EEAEKYAKES LK

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Macromolecule #8: Proteasome subunit alpha type-6

MacromoleculeName: Proteasome subunit alpha type-6 / type: protein_or_peptide / ID: 8 / Number of copies: 2 / Enantiomer: LEVO / EC number: proteasome endopeptidase complex
Source (natural)Organism: Human (human)
Molecular weightTheoretical: 27.186174 KDa
SequenceString: SRGSSAGFDR HITIFSPEGR LYQVEYAFKA INQGGLTSVA VRGKDCAVIV TQKKVPDKLL DSSTVTHLFK ITENIGCVMT GMTADSRSQ VQRARYEAAN WKYKYGYEIP VDMLCKRIAD ISQVYTQNAE MRPLGCCMIL IGIDEEQGPQ VYKCDPAGYY C GFKATAAG ...String:
SRGSSAGFDR HITIFSPEGR LYQVEYAFKA INQGGLTSVA VRGKDCAVIV TQKKVPDKLL DSSTVTHLFK ITENIGCVMT GMTADSRSQ VQRARYEAAN WKYKYGYEIP VDMLCKRIAD ISQVYTQNAE MRPLGCCMIL IGIDEEQGPQ VYKCDPAGYY C GFKATAAG VKQTESTSFL EKKVKKKFDW TFEQTVETAI TCLSTVLSID FKPSEIEVGV VTVENPKFRI LTEAEIDAHL VA LAER

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Macromolecule #9: Proteasome subunit beta type-7

MacromoleculeName: Proteasome subunit beta type-7 / type: protein_or_peptide / ID: 9 / Number of copies: 2 / Enantiomer: LEVO / EC number: proteasome endopeptidase complex
Source (natural)Organism: Human (human)
Molecular weightTheoretical: 23.745256 KDa
SequenceString: TTIAGVVYKD GIVLGADTRA TEGMVVADKN CSKIHFISPN IYCCGAGTAA DTDMTTQLIS SNLELHSLST GRLPRVVTAN RMLKQMLFR YQGYIGAALV LGGVDVTGPH LYSIYPHGST DKLPYVTMGS GSLAAMAVFE DKFRPDMEEE EAKNLVSEAI A AGIFNDLG ...String:
TTIAGVVYKD GIVLGADTRA TEGMVVADKN CSKIHFISPN IYCCGAGTAA DTDMTTQLIS SNLELHSLST GRLPRVVTAN RMLKQMLFR YQGYIGAALV LGGVDVTGPH LYSIYPHGST DKLPYVTMGS GSLAAMAVFE DKFRPDMEEE EAKNLVSEAI A AGIFNDLG SGSNIDLCVI SKNKLDFLRP YTVPNKKGTR LGRYRCEKGT TAVLTEKITP LE

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Macromolecule #10: Proteasome subunit beta type-3

MacromoleculeName: Proteasome subunit beta type-3 / type: protein_or_peptide / ID: 10 / Number of copies: 2 / Enantiomer: LEVO / EC number: proteasome endopeptidase complex
Source (natural)Organism: Human (human)
Molecular weightTheoretical: 22.841701 KDa
SequenceString: SIMSYNGGAV MAMKGKNCVA IAADRRFGIQ AQMVTTDFQK IFPMGDRLYI GLAGLATDVQ TVAQRLKFRL NLYELKEGRQ IKPYTLMSM VANLLYEKRF GPYYTEPVIA GLDPKTFKPF ICSLDLIGCP MVTDDFVVSG TCAEQMYGMC ESLWEPNMDP D HLFETISQ ...String:
SIMSYNGGAV MAMKGKNCVA IAADRRFGIQ AQMVTTDFQK IFPMGDRLYI GLAGLATDVQ TVAQRLKFRL NLYELKEGRQ IKPYTLMSM VANLLYEKRF GPYYTEPVIA GLDPKTFKPF ICSLDLIGCP MVTDDFVVSG TCAEQMYGMC ESLWEPNMDP D HLFETISQ AMLNAVDRDA VSGMGVIVHI IEKDKITTRT LKARMD

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Macromolecule #11: Proteasome subunit beta type-2

MacromoleculeName: Proteasome subunit beta type-2 / type: protein_or_peptide / ID: 11 / Number of copies: 2 / Enantiomer: LEVO / EC number: proteasome endopeptidase complex
Source (natural)Organism: Human (human)
Molecular weightTheoretical: 22.365721 KDa
SequenceString: MEYLIGIQGP DYVLVASDRV AASNIVQMKD DHDKMFKMSE KILLLCVGEA GDTVQFAEYI QKNVQLYKMR NGYELSPTAA ANFTRRNLA DCLRSRTPYH VNLLLAGYDE HEGPALYYMD YLAALAKAPF AAHGYGAFLT LSILDRYYTP TISRERAVEL L RKCLEELQ ...String:
MEYLIGIQGP DYVLVASDRV AASNIVQMKD DHDKMFKMSE KILLLCVGEA GDTVQFAEYI QKNVQLYKMR NGYELSPTAA ANFTRRNLA DCLRSRTPYH VNLLLAGYDE HEGPALYYMD YLAALAKAPF AAHGYGAFLT LSILDRYYTP TISRERAVEL L RKCLEELQ KRFILNLPTF SVRIIDKNGI HDLDNISF

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Macromolecule #12: Proteasome subunit beta type-5

MacromoleculeName: Proteasome subunit beta type-5 / type: protein_or_peptide / ID: 12 / Number of copies: 2 / Enantiomer: LEVO / EC number: proteasome endopeptidase complex
Source (natural)Organism: Human (human)
Molecular weightTheoretical: 22.199072 KDa
SequenceString: TTTLAFKFRH GVIVAADSRA TAGAYIASQT VKKVIEINPY LLGTMAGGAA DCSFWERLLA RQCRIYELRN KERISVAAAS KLLANMVYQ YKGMGLSMGT MICGWDKRGP GLYYVDSEGN RISGATFSVG SGSVYAYGVM DRGYSYDLEV EQAYDLARRA I YQATYRDA ...String:
TTTLAFKFRH GVIVAADSRA TAGAYIASQT VKKVIEINPY LLGTMAGGAA DCSFWERLLA RQCRIYELRN KERISVAAAS KLLANMVYQ YKGMGLSMGT MICGWDKRGP GLYYVDSEGN RISGATFSVG SGSVYAYGVM DRGYSYDLEV EQAYDLARRA I YQATYRDA YSGGAVNLYH VREDGWIRVS SDNVADLHEK YSG

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Macromolecule #13: Proteasome subunit beta type-1

MacromoleculeName: Proteasome subunit beta type-1 / type: protein_or_peptide / ID: 13 / Number of copies: 2 / Enantiomer: LEVO / EC number: proteasome endopeptidase complex
Source (natural)Organism: Human (human)
Molecular weightTheoretical: 23.578986 KDa
SequenceString: RFSPYVFNGG TILAIAGEDF AIVASDTRLS EGFSIHTRDS PKCYKLTDKT VIGCSGFHGD CLTLTKIIEA RLKMYKHSNN KAMTTGAIA AMLSTILYSR RFFPYYVYNI IGGLDEEGKG AVYSFDPVGS YQRDSFKAGG SASAMLQPLL DNQVGFKNMQ N VEHVPLSL ...String:
RFSPYVFNGG TILAIAGEDF AIVASDTRLS EGFSIHTRDS PKCYKLTDKT VIGCSGFHGD CLTLTKIIEA RLKMYKHSNN KAMTTGAIA AMLSTILYSR RFFPYYVYNI IGGLDEEGKG AVYSFDPVGS YQRDSFKAGG SASAMLQPLL DNQVGFKNMQ N VEHVPLSL DRAMRLVKDV FISAAERDVY TGDALRICIV TKEGIREETV SLRKD

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Macromolecule #14: Proteasome subunit beta type-6

MacromoleculeName: Proteasome subunit beta type-6 / type: protein_or_peptide / ID: 14 / Number of copies: 2 / Enantiomer: LEVO / EC number: proteasome endopeptidase complex
Source (natural)Organism: Human (human)
Molecular weightTheoretical: 21.753643 KDa
SequenceString: TTIMAVQFDG GVVLGADSRT TTGSYIANRV TDKLTPIHDR IFCCRSGSAA DTQAVADAVT YQLGFHSIEL NEPPLVHTAA SLFKEMCYR YREDLMAGII IAGWDPQEGG QVYSVPMGGM MVRQSFAIGG SGSSYIYGYV DATYREGMTK EECLQFTANA L ALAMERDG ...String:
TTIMAVQFDG GVVLGADSRT TTGSYIANRV TDKLTPIHDR IFCCRSGSAA DTQAVADAVT YQLGFHSIEL NEPPLVHTAA SLFKEMCYR YREDLMAGII IAGWDPQEGG QVYSVPMGGM MVRQSFAIGG SGSSYIYGYV DATYREGMTK EECLQFTANA L ALAMERDG SSGGVIRLAA IAESGVERQV LLGDQIPKFA VATLP

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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: 7.4
Component:
ConcentrationFormulaName
150.0 mMNaClSodium chlorideSodium Chloride
1.5 mMKH2PO4Monopotassium phosphate
8.1 mMNa2HPO4Disodium phosphate
2.6 mMKClPotassium chloride

Details: PBS was purchased from Sigma # D8537, Switzerland.
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE
Details: CryoWriter. (see https://doi.org/10.1016/j.jsb.2016.11.002 for more information).
Details20S proteasome was purified using Fabs and eluted in a buffer containing 1mg/ml tobacco mosaic virus (TMV). The sample contains also unbound Fabs.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 QUANTUM (4k x 4k) / Detector mode: SUPER-RESOLUTION / Digitization - Dimensions - Width: 7676 pixel / Digitization - Dimensions - Height: 7420 pixel / Digitization - Frames/image: 0-30 / Number grids imaged: 1 / Number real images: 523 / Average exposure time: 6.0 sec. / Average electron dose: 72.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 523
CTF correctionSoftware - Name: CTFFIND (ver. 4)
Startup modelType of model: OTHER / Details: cryoSPARC v2 reconstruction
Initial angle assignmentType: NOT APPLICABLE
Final 3D classificationSoftware - Name: RELION (ver. 3) / Software - details: beta
Final angle assignmentType: NOT APPLICABLE
Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3) / Software - details: beta / Number images used: 16015
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

5le5

Output model

PDB-6r70:
Endogeneous native human 20S proteasome

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