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- EMDB-4404: eIF2B:eIF2 complex, phosphorylated on eIF2 alpha serine 52. -

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Basic information

Entry
Database: EMDB / ID: EMD-4404
TitleeIF2B:eIF2 complex, phosphorylated on eIF2 alpha serine 52.
Map dataeIF2B:eIF2 complex with eIF2 alpha phosphorylated on serine 52
Sample
  • Complex: Complex of translation initiation factors eIF2 and eIF2B
    • Protein or peptide: Translation initiation factor eIF-2B subunit alpha
    • Protein or peptide: Translation initiation factor eIF-2B subunit delta
    • Protein or peptide: Translation initiation factor eIF-2B subunit beta
    • Protein or peptide: Translation initiation factor eIF-2B subunit epsilon
    • Protein or peptide: Translation initiation factor eIF-2B subunit gamma
    • Protein or peptide: Eukaryotic translation initiation factor 2 subunit alpha
    • Protein or peptide: Eukaryotic translation initiation factor 2 subunit beta
    • Protein or peptide: Eukaryotic translation initiation factor 2 subunit gamma
Function / homology
Function and homology information


negative regulation of cellular response to amino acid starvation / positive regulation of cellular response to heat / Recycling of eIF2:GDP / Cellular response to mitochondrial stress / eukaryotic translation initiation factor 2B complex / ABC-family proteins mediated transport / methionyl-initiator methionine tRNA binding / eukaryotic translation initiation factor 2 complex / multi-eIF complex / cytoplasmic translational initiation ...negative regulation of cellular response to amino acid starvation / positive regulation of cellular response to heat / Recycling of eIF2:GDP / Cellular response to mitochondrial stress / eukaryotic translation initiation factor 2B complex / ABC-family proteins mediated transport / methionyl-initiator methionine tRNA binding / eukaryotic translation initiation factor 2 complex / multi-eIF complex / cytoplasmic translational initiation / protein-synthesizing GTPase / eukaryotic 43S preinitiation complex / guanyl-nucleotide exchange factor complex / formation of translation preinitiation complex / positive regulation of cellular response to amino acid starvation / eukaryotic 48S preinitiation complex / positive regulation of translational fidelity / regulation of translational initiation / Formation of the ternary complex, and subsequently, the 43S complex / Translation initiation complex formation / Ribosomal scanning and start codon recognition / L13a-mediated translational silencing of Ceruloplasmin expression / positive regulation of translational initiation / enzyme regulator activity / translation initiation factor binding / translation initiation factor activity / guanyl-nucleotide exchange factor activity / translational initiation / cytoplasmic stress granule / ribosome binding / ribosome / GTPase activity / mRNA binding / GTP binding / mitochondrion / RNA binding / metal ion binding / cytosol / cytoplasm
Similarity search - Function
Methylthioribose-1-phosphate isomerase, N-terminal / Translation initiation factor eIF-2B subunit alpha, N-terminal / : / : / Translation initiation factor eIF-2B subunit epsilon, N-terminal / Translation initiation factor eIF-2B subunit epsilon, W2 domain / : / : / Initiation factor 2B-related / Initiation factor 2B-like, C-terminal ...Methylthioribose-1-phosphate isomerase, N-terminal / Translation initiation factor eIF-2B subunit alpha, N-terminal / : / : / Translation initiation factor eIF-2B subunit epsilon, N-terminal / Translation initiation factor eIF-2B subunit epsilon, W2 domain / : / : / Initiation factor 2B-related / Initiation factor 2B-like, C-terminal / Initiation factor 2 subunit family / Translation initiation factor IF2/IF5 domain / Translation initiation factor IF2/IF5, N-terminal / Translation initiation factor IF2/IF5, zinc-binding / Translation initiation factor IF2/IF5 / Domain found in IF2B/IF5 / domain present in translation initiation factor eIF2B and eIF5 / eIF4-gamma/eIF5/eIF2-epsilon / Domain at the C-termini of GCD6, eIF-2B epsilon, eIF-4 gamma and eIF-5 / W2 domain / W2 domain profile. / Translation initiation factor 2, alpha subunit / Translation initiation factor 2, alpha subunit, middle domain superfamily / Translation initiation factor 2, alpha subunit, C-terminal / IF2a, S1-like domain / Eukaryotic translation initiation factor 2 alpha subunit / Initiation factor eIF2 gamma, C-terminal / Initiation factor eIF2 gamma, domain 2 / Initiation factor eIF2 gamma, GTP-binding domain / Initiation factor eIF2 gamma, C terminal / : / Nucleotidyl transferase domain / Nucleotidyl transferase / NagB/RpiA transferase-like / Translation elongation factor EF1A/initiation factor IF2gamma, C-terminal / Trimeric LpxA-like superfamily / S1 domain profile. / Translation elongation factor EFTu-like, domain 2 / Elongation factor Tu domain 2 / Ribosomal protein S1-like RNA-binding domain / S1 RNA binding domain / S1 domain / Translational (tr)-type GTP-binding domain / Elongation factor Tu GTP binding domain / Translational (tr)-type guanine nucleotide-binding (G) domain profile. / Nucleotide-diphospho-sugar transferases / Armadillo-type fold / Translation protein, beta-barrel domain superfamily / Nucleic acid-binding, OB-fold / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Translation initiation factor eIF2B subunit gamma / Eukaryotic translation initiation factor 2 subunit beta / Translation initiation factor eIF2B subunit delta / Translation initiation factor eIF2B subunit alpha / Eukaryotic translation initiation factor 2 subunit alpha / Eukaryotic translation initiation factor 2 subunit gamma / Translation initiation factor eIF2B subunit epsilon / Translation initiation factor eIF2B subunit beta
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast) / Saccharomyces cerevisiae S288C (yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.93 Å
AuthorsAdomavicius T / Roseman AM / Pavitt GD
Funding support United Kingdom, 4 items
OrganizationGrant numberCountry
Biotechnology and Biological Sciences Research CouncilBB/L020157/1 United Kingdom
Biotechnology and Biological Sciences Research CouncilBB/M006565/1 United Kingdom
Biotechnology and Biological Sciences Research CouncilBB/N014049/1 United Kingdom
Biotechnology and Biological Sciences Research CouncilBB/M011208/1 United Kingdom
CitationJournal: Elife / Year: 2017
Title: Fail-safe control of translation initiation by dissociation of eIF2α phosphorylated ternary complexes.
Authors: Martin D Jennings / Christopher J Kershaw / Tomas Adomavicius / Graham D Pavitt /
Abstract: Phosphorylation of eIF2α controls translation initiation by restricting the levels of active eIF2-GTP/Met-tRNAi ternary complexes (TC). This modulates the expression of all eukaryotic mRNAs and ...Phosphorylation of eIF2α controls translation initiation by restricting the levels of active eIF2-GTP/Met-tRNAi ternary complexes (TC). This modulates the expression of all eukaryotic mRNAs and contributes to the cellular integrated stress response. Key to controlling the activity of eIF2 are translation factors eIF2B and eIF5, thought to primarily function with eIF2-GDP and TC respectively. Using a steady-state kinetics approach with purified proteins we demonstrate that eIF2B binds to eIF2 with equal affinity irrespective of the presence or absence of competing guanine nucleotides. We show that eIF2B can compete with Met-tRNAi for eIF2-GTP and can destabilize TC. When TC is formed with unphosphorylated eIF2, eIF5 can out-compete eIF2B to stabilize TC/eIF5 complexes. However when TC/eIF5 is formed with phosphorylated eIF2, eIF2B outcompetes eIF5 and destabilizes TC. These data uncover competition between eIF2B and eIF5 for TC and identify that phosphorylated eIF2-GTP translation initiation intermediate complexes can be inhibited by eIF2B.
History
DepositionNov 6, 2018-
Header (metadata) releaseMay 8, 2019-
Map releaseMay 22, 2019-
UpdateNov 25, 2020-
Current statusNov 25, 2020Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.055
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.055
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6i3m
  • Surface level: 0.055
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_4404.png

Downloads & links

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Map

FileDownload / File: emd_4404.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationeIF2B:eIF2 complex with eIF2 alpha phosphorylated on serine 52
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.34 Å/pix.
x 320 pix.
= 428.8 Å		1.34 Å/pix.
x 320 pix.
= 428.8 Å		1.34 Å/pix.
x 320 pix.
= 428.8 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.34 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.055 / Movie #1: 0.055
Minimum - Maximum-0.18108554 - 0.3041722
Average (Standard dev.)0.00024270266 (±0.008404886)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 428.80002 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.341.341.34
M x/y/z320320320
origin x/y/z0.0000.0000.000
length x/y/z428.800428.800428.800
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ513513513
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS320320320
D min/max/mean-0.1810.3040.000

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Supplemental data

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Mask #1

Fileemd_4404_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: LocScale version of main map

Fileemd_4404_additional.map
AnnotationLocScale version of main map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: relion half-map 1

Fileemd_4404_half_map_1.map
Annotationrelion half-map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: relion half-map 2

Fileemd_4404_half_map_2.map
Annotationrelion half-map 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Complex of translation initiation factors eIF2 and eIF2B

EntireName: Complex of translation initiation factors eIF2 and eIF2B
Components
  • Complex: Complex of translation initiation factors eIF2 and eIF2B
    • Protein or peptide: Translation initiation factor eIF-2B subunit alpha
    • Protein or peptide: Translation initiation factor eIF-2B subunit delta
    • Protein or peptide: Translation initiation factor eIF-2B subunit beta
    • Protein or peptide: Translation initiation factor eIF-2B subunit epsilon
    • Protein or peptide: Translation initiation factor eIF-2B subunit gamma
    • Protein or peptide: Eukaryotic translation initiation factor 2 subunit alpha
    • Protein or peptide: Eukaryotic translation initiation factor 2 subunit beta
    • Protein or peptide: Eukaryotic translation initiation factor 2 subunit gamma

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Supramolecule #1: Complex of translation initiation factors eIF2 and eIF2B

SupramoleculeName: Complex of translation initiation factors eIF2 and eIF2B
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightExperimental: 838 KDa

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Macromolecule #1: Translation initiation factor eIF-2B subunit alpha

MacromoleculeName: Translation initiation factor eIF-2B subunit alpha / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae S288C (yeast)
Molecular weightTheoretical: 34.062027 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast)
SequenceString: MSEFNITETY LRFLEEDTEM TMPIAAIEAL VTLLRIKTPE TAAEMINTIK SSTEELIKSI PNSVSLRAGC DIFMRFVLRN LHLYGDWEN CKQHLIENGQ LFVSRAKKSR NKIAEIGVDF IADDDIILVH GYSRAVFSLL NHAANKFIRF RCVVTESRPS K QGNQLYTL ...String:
MSEFNITETY LRFLEEDTEM TMPIAAIEAL VTLLRIKTPE TAAEMINTIK SSTEELIKSI PNSVSLRAGC DIFMRFVLRN LHLYGDWEN CKQHLIENGQ LFVSRAKKSR NKIAEIGVDF IADDDIILVH GYSRAVFSLL NHAANKFIRF RCVVTESRPS K QGNQLYTL LEQKGIPVTL IVDSAVGAVI DKVDKVFVGA EGVAESGGII NLVGTYSVGV LAHNARKPFY VVTESHKFVR MF PLSSDDL PMAGPPLDFT RRTDDLEDAL RGPTIDYTAQ EYITALITDL GVLTPSAVSE ELIKMWYD

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Macromolecule #2: Translation initiation factor eIF-2B subunit delta

MacromoleculeName: Translation initiation factor eIF-2B subunit delta / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae S288C (yeast)
Molecular weightTheoretical: 70.945195 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast)
SequenceString: MSESEAKSRS ATPPSKAKQA TPTTTAAANG EKKLTNKELK ELKKQEKAAK RAAMKQANGI SIEQQQQQAQ MKKEKKQLQR EQQQKREQK QKNANKKKQN ERNVKKSTLF GHLETTEERR ATILALTSAV SSPKTSRITA AGLMVPVVAS ALSGSNVLTA S SLMPVGPN ...String:
MSESEAKSRS ATPPSKAKQA TPTTTAAANG EKKLTNKELK ELKKQEKAAK RAAMKQANGI SIEQQQQQAQ MKKEKKQLQR EQQQKREQK QKNANKKKQN ERNVKKSTLF GHLETTEERR ATILALTSAV SSPKTSRITA AGLMVPVVAS ALSGSNVLTA S SLMPVGPN ASSTVSASAP ASTTTTLPAS SAALSAGTSS ASTNTPTAIQ QEIASSNASD VAKTLASISL EAGEFNVIPG IS SVIPTVL EQSFDNSSLI SSVKELLLNK DLIHPSILLL TSHLAHYKIV GSIPRCIAML EVFQIVIKDY QTPKGTTLSR NLT SYLSHQ IDLLKKARPL SVTMGNAIRW LKQEISLIDP STPDKAAKKD LCEKIGQFAK EKIELADQLI IDNASTQIEE STTI VTYGS SKVLTELLLH NAISLKKNIK VIVVDSRPLF EGRKMAETLR NAGVNVMYAL ITSLDTIFNM DVDYVFLGAH SILSN GFLY SRAGTAMLAM SAKRRNIPVL VCCESLKFSQ RVQLDSVTFN ELADPNDLVN IDYENPVERR GNKGALLNQF IKERKF EKK KLAMENKPKG NKIGGKKGSE GESKDASNEE DSNSKNILDG WQELPSLNIV NILYDLTPPE YIKKVITEFG ALPPSSV PV ILREYKGSA

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Macromolecule #3: Translation initiation factor eIF-2B subunit beta

MacromoleculeName: Translation initiation factor eIF-2B subunit beta / type: protein_or_peptide / ID: 3 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae S288C (yeast)
Molecular weightTheoretical: 42.621441 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast)
SequenceString: MSSQAFTSVH PNAATSDVNV TIDTFVAKLK RRQVQGSYAI ALETLQLLMR FISAARWNHV NDLIEQIRDL GNSLEKAHPT AFSCGNVIR RILAVLRDEV EEDTMSTTVT STSVAEPLIS SMFNLLQKPE QPHQNRKNSS GSSSMKTKTD YRQVAIQGIK D LIDEIKNI ...String:
MSSQAFTSVH PNAATSDVNV TIDTFVAKLK RRQVQGSYAI ALETLQLLMR FISAARWNHV NDLIEQIRDL GNSLEKAHPT AFSCGNVIR RILAVLRDEV EEDTMSTTVT STSVAEPLIS SMFNLLQKPE QPHQNRKNSS GSSSMKTKTD YRQVAIQGIK D LIDEIKNI DEGIQQIAID LIHDHEILLT PTPDSKTVLK FLITARERSN RTFTVLVTEG FPNNTKNAHE FAKKLAQHNI ET LVVPDSA VFALMSRVGK VIIGTKAVFV NGGTISSNSG VSSVCECARE FRTPVFAVAG LYKLSPLYPF DVEKFVEFGG SQR ILPRMD PRKRLDTVNQ ITDYVPPENI DIYITNVGGF NPSFIYRIAW DNYKQIDVHL DKNKA

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Macromolecule #4: Translation initiation factor eIF-2B subunit epsilon

MacromoleculeName: Translation initiation factor eIF-2B subunit epsilon / type: protein_or_peptide / ID: 4 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae S288C (yeast)
Molecular weightTheoretical: 81.249062 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast)
SequenceString: MAGKKGQKKS GLGNHGKNSD MDVEDRLQAV VLTDSYETRF MPLTAVKPRC LLPLANVPLI EYTLEFLAKA GVHEVFLICS SHANQINDY IENSKWNLPW SPFKITTIMS PEARCTGDVM RDLDNRGIIT GDFILVSGDV LTNIDFSKML EFHKKMHLQD K DHISTMCL ...String:
MAGKKGQKKS GLGNHGKNSD MDVEDRLQAV VLTDSYETRF MPLTAVKPRC LLPLANVPLI EYTLEFLAKA GVHEVFLICS SHANQINDY IENSKWNLPW SPFKITTIMS PEARCTGDVM RDLDNRGIIT GDFILVSGDV LTNIDFSKML EFHKKMHLQD K DHISTMCL SKASTYPKTR TIEPAAFVLD KSTSRCIYYQ DLPLPSSREK TSIQIDPELL DNVDEFVIRN DLIDCRIDIC TS HVPLIFQ ENFDYQSLRT DFVKGVISSD ILGKHIYAYL TDEYAVRVES WQTYDTISQD FLGRWCYPLV LDSNIQDDQT YSY ESRHIY KEKDVVLAQS CKIGKCTAIG SGTKIGEGTK IENSVIGRNC QIGENIRIKN SFIWDDCIIG NNSIIDHSLI ASNA TLGSN VRLNDGCIIG FNVKIDDNMD LDRNTKISAS PLKNAGSRMY DNESNEQFDQ DLDDQTLAVS IVGDKGVGYI YESEV SDDE DSSTEACKEI NTLSNQLDEL YLSDDSISSA TKKTKKRRTM SVNSIYTDRE EIDSEFEDED FEKEGIATVE RAMENN HDL DTALLELNTL RMSMNVTYHE VRIATITALL RRVYHFIATQ TLGPKDAVVK VFNQWGLLFK RQAFDEEEYI DLMNIIM EK IVEQSFDKPD LILFSALVSL YDNDIIEEDV IYKWWDNVST DPRYDEVKKL TVKWVEWLQN ADEESSSEEE

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Macromolecule #5: Translation initiation factor eIF-2B subunit gamma

MacromoleculeName: Translation initiation factor eIF-2B subunit gamma / type: protein_or_peptide / ID: 5 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae S288C (yeast)
Molecular weightTheoretical: 65.76832 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast)
SequenceString: MSIQAFVFCG KGSNLAPFTQ PDFPFQTQNK DSTAATSGDK LNELVNSALD STVINEFMQH STRLPKALLP IGNRPMIEYV LDWCDQADF KEISVVAPVD EIELIESGLT SFLSLRKQQF ELIYKALSNS NHSHHLQDPK KINFIPSKAN STGESLQKEL L PRINGDFV ...String:
MSIQAFVFCG KGSNLAPFTQ PDFPFQTQNK DSTAATSGDK LNELVNSALD STVINEFMQH STRLPKALLP IGNRPMIEYV LDWCDQADF KEISVVAPVD EIELIESGLT SFLSLRKQQF ELIYKALSNS NHSHHLQDPK KINFIPSKAN STGESLQKEL L PRINGDFV ILPCDFVTDI PPQVLVDQFR NRDDNNLAMT IYYKNSLDSS IDKKQQQKQK QQQFFTVYSE NEDSERQPIL LD VYSQRDV TKTKYLQIRS HLLWNYPNLT VSTKLLNSFI YFCSFELCQL LKLGPQSMSR QASFKDPFTG NQQQQNPPTT DDD EDRNHD DDDDYKPSAT SIQPTYFKKK NDLILDPINC NKSLSKVFRD LSRRSWQHSK PREPIGIFIL PNETLFIRAN NLNA YMDAN RFVLKIKSQT MFTKNIQIQS AAIGADAIVD PKCQISAHSN VKMSVLGTQA NIGSRCRVAG SLLFPGVHLG DEVIL ENCI IGPMAKIGSK CKLSNCYIEG HYVVEPKNNF KGETLANVYL DEDEEDELIY DDSVIAGESE IAEETDSDDR SDEDSD DSE YTDEYEYEDD GLFER

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Macromolecule #6: Eukaryotic translation initiation factor 2 subunit alpha

MacromoleculeName: Eukaryotic translation initiation factor 2 subunit alpha
type: protein_or_peptide / ID: 6 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae S288C (yeast)
Molecular weightTheoretical: 34.843633 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast)
SequenceString: MSTSHCRFYE NKYPEIDDIV MVNVQQIAEM GAYVKLLEYD NIEGMILLSE L(SEP)RRRIRSIQ KLIRVGKNDV AVVLRV DKE KGYIDLSKRR VSSEDIIKCE EKYQKSKTVH SILRYCAEKF QIPLEELYKT IAWPLSRKFG HAYEAFKLSI IDETVWE GI EPPSKDVLDE ...String:
MSTSHCRFYE NKYPEIDDIV MVNVQQIAEM GAYVKLLEYD NIEGMILLSE L(SEP)RRRIRSIQ KLIRVGKNDV AVVLRV DKE KGYIDLSKRR VSSEDIIKCE EKYQKSKTVH SILRYCAEKF QIPLEELYKT IAWPLSRKFG HAYEAFKLSI IDETVWE GI EPPSKDVLDE LKNYISKRLT PQAVKIRADV EVSCFSYEGI DAIKDALKSA EDMSTEQMQV KVKLVAAPLY VLTTQALD K QKGIEQLESA IEKITEVITK YGGVCNITMP PKAVTATEDA ELQALLESKE LDNRSDSEDD EDESDDE

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Macromolecule #7: Eukaryotic translation initiation factor 2 subunit beta

MacromoleculeName: Eukaryotic translation initiation factor 2 subunit beta
type: protein_or_peptide / ID: 7 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae S288C (yeast)
Molecular weightTheoretical: 31.631309 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast)
SequenceString: MSSDLAAELG FDPALKKKKK TKKVIPDDFD AAVNGKENGS GDDLFAGLKK KKKKSKSVSA DAEAEKEPTD DIAEALGELS LKKKKKKTK DSSVDAFEKE LAKAGLDNVD AESKEGTPSA NSSIQQEVGL PYSELLSRFF NILRTNNPEL AGDRSGPKFR I PPPVCLRD ...String:
MSSDLAAELG FDPALKKKKK TKKVIPDDFD AAVNGKENGS GDDLFAGLKK KKKKSKSVSA DAEAEKEPTD DIAEALGELS LKKKKKKTK DSSVDAFEKE LAKAGLDNVD AESKEGTPSA NSSIQQEVGL PYSELLSRFF NILRTNNPEL AGDRSGPKFR I PPPVCLRD GKKTIFSNIQ DIAEKLHRSP EHLIQYLFAE LGTSGSVDGQ KRLVIKGKFQ SKQMENVLRR YILEYVTCKT CK SINTELK REQSNRLFFM VCKSCGSTRS VSSIKTGFQA TVGKRRRM

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Macromolecule #8: Eukaryotic translation initiation factor 2 subunit gamma

MacromoleculeName: Eukaryotic translation initiation factor 2 subunit gamma
type: protein_or_peptide / ID: 8 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae S288C (yeast)
Molecular weightTheoretical: 57.942699 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast)
SequenceString: MSDLQDQEPS IIINGNLEPV GEPDIVEETE VVAQETQETQ DADKPKKKVA FTGLEEDGET EEEKRKREFE EGGGLPEQPL NPDFSKLNP LSAEIINRQA TINIGTIGHV AHGKSTVVRA ISGVQTVRFK DELERNITIK LGYANAKIYK CQEPTCPEPD C YRSFKSDK ...String:
MSDLQDQEPS IIINGNLEPV GEPDIVEETE VVAQETQETQ DADKPKKKVA FTGLEEDGET EEEKRKREFE EGGGLPEQPL NPDFSKLNP LSAEIINRQA TINIGTIGHV AHGKSTVVRA ISGVQTVRFK DELERNITIK LGYANAKIYK CQEPTCPEPD C YRSFKSDK EISPKCQRPG CPGRYKLVRH VSFVDCPGHD ILMSTMLSGA AVMDAALLLI AGNESCPQPQ TSEHLAAIEI MK LKHVIIL QNKVDLMREE SALEHQKSIL KFIRGTIADG APIVPISAQL KYNIDAVNEF IVKTIPVPPR DFMISPRLIV IRS FDVNKP GAEIEDLKGG VAGGSILNGV FKLGDEIEIR PGIVTKDDKG KIQCKPIFSN IVSLFAEQND LKFAVPGGLI GVGT KVDPT LCRADRLVGQ VVGAKGHLPN IYTDIEINYF LLRRLLGVKT DGQKQAKVRK LEPNEVLMVN IGSTATGARV VAVKA DMAR LQLTSPACTE INEKIALSRR IEKHWRLIGW ATIKKGTTLE PIA

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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

Concentration0.25 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
100.0 mMKClpotassium chloride
20.0 mMTris
1.0 mMTCEP

Details: Solutions for sample preparation were made fresh, filter sterilized, and degassed.
GridModel: Homemade / Material: COPPER / Mesh: 400 / Support film - #0 - Film type ID: 1 / Support film - #0 - Material: CARBON / Support film - #0 - topology: LACEY / Support film - #1 - Film type ID: 2 / Support film - #1 - Material: CARBON / Support film - #1 - topology: CONTINUOUS / Support film - #1 - Film thickness: 3.0 nm / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 101.325 kPa
Details: Ultra-thin carbon support film, 3nm - on lacey carbon grids from Agar Scientific were used for 35 degree tilted data collection. For zero tilt data collection, 200 mesh Au Quantifoil, R2/2 grids were used.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 294 K / Instrument: FEI VITROBOT MARK III / Details: Blot for 2 seconds before plunging.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF Quantum LS / Energy filter - Slit width: 20 eV
DetailsTilt at zero and 35 degrees.
Image recordingFilm or detector model: GATAN K2 QUANTUM (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 3838 pixel / Digitization - Dimensions - Height: 3710 pixel / Digitization - Sampling interval: 5.0 µm / Digitization - Frames/image: 2-48 / Number grids imaged: 2 / Number real images: 4533 / Average exposure time: 12.0 sec. / Average electron dose: 40.0 e/Å2
Details: 2 separate data collections for zero degree (2255 images) and tilted (2278 images) specimen. Both on Titan Krios
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated defocus max: 5.5 µm / Calibrated defocus min: 1.0 µm / Calibrated magnification: 37313 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 4.0 µm / Nominal defocus min: 1.5 µm
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: 655000
Details: Initially, approximately 3000 particles were picked manually and used for 2D classification. Then 4 best looking 2D class average images were used as references for automated particle ...Details: Initially, approximately 3000 particles were picked manually and used for 2D classification. Then 4 best looking 2D class average images were used as references for automated particle picking in RELION. For the zero tilt data collection, 406000 particles were picked. For the 35 degree tilt data collection, 249000 particles were picked
CTF correctionSoftware - Name: Gctf
Details: CTF determination was performed per micrograph initially. After particle picking and initial reconstruction, per particle CTF determination was performed.
Startup modelType of model: OTHER / Details: Initial model was generated in Cryosparc.
Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 3.93 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 2.1) / Number images used: 64541
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 2.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 2.1)
Final 3D classificationNumber classes: 1 / Software - Name: RELION (ver. 2.1)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model
PDB IDChain

5b04


3jap

chain_id: j, residue_range: 3-174
DetailsModeller was used to build homology model of S. cerevisiae eIF2B structure based on S. pombe crystal structure. Subunits of the homology model, along with eIF2 alpha domains 1 and 2, were then rigid body fitted into our map using UCSF Chimera. The model then was refined using phenix and manually adjusted in Coot.
RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Target criteria: correlation coeficient
Output model

PDB-6i3m:
eIF2B:eIF2 complex, phosphorylated on eIF2 alpha serine 52.

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Atomic model buiding 2

Initial model
PDB IDChain

3jap

chain_id: j, residue_range: 182-265

3jap

chain_id: k

3jap

chain_id: l, residue_range: 127-143
DetailseIF2 alpha domain 3, eIF2 gamma, and eIF2 beta (3JAP) were rigid body fitted into our map using Chimera.
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Target criteria: Cross-correlation coefficient
Output model

PDB-6i3m:
eIF2B:eIF2 complex, phosphorylated on eIF2 alpha serine 52.

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