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. 2021 Jan 7;81(1):88–103.e6. doi: 10.1016/j.molcel.2020.10.031
REAGENT or RESOURCE SOURCE IDENTIFIER
Antibodies
Mouse monoclonal anti-FLAG M2 Sigma Aldrich RRID:AB_262044
Mouse monoclonal anti-puromycin Schmidt et al., 2009 RRID:AB_2566826
Rabbit monoclonal anti-phospho-eIF2alpha-Ser51 AbCam RRID: AB_732117
Mouse monoclonal anti-eIF2alpha (total) Scorsone et al., 1987 N/A
Bacterial and Virus Strains
T7 Express Competent E. coli New England Biolabs C2566I
T7 Express lysY/Iq competent E. coli New England Biolabs C3013
Chemicals, Peptides, and Recombinant Proteins
trans-ISRIB Sekine et al., 2015 Gift of Peter Fischer U. Nottingham
FAM-ISRIB Zyryanova et al., 2018 Gift of Peter Fischer U. Nottingham
Human eIF2 trimer Kashiwagi et al., 2019 and this study N/A
Human eIF2B decamer Kashiwagi et al., 2019 and this study N/A
Human eIF2α-NTD This study N/A
BODIPY-FL-GDP Invitrogen G22360
Deposited Data
Cryo-EM map of the αP1 complex This study EMDB: EMD-30570
Cryo-EM map of the αP2 complex This study EMDB: EMD-30569
Cryo-EM map of the αPγ complex This study EMDB: EMD-30568
Cryo-EM map of eIF2B apo This study EMDB: EMD-30571
Coordinates of the αP1 complex This study PDB: 7D45
Coordinates of the αP2 complex This study PDB: 7D44
Coordinates of the αPγ complex This study PDB: 7D43
Coordinates of the eIF2B apo structure This study PDB: 7D46
Experimental Models: Cell Lines
CHO-S21 dual reporter [CHOP::GFP; Xbp1::Turquoise] Sekine et al., 2016 N/A
CHO-S51A dual reporter ISR-insensitive eIF2αS51A Sekine et al., 2016 N/A
CHO-S21 [Eif2b4_L316N] ISR-insensitive This study N/A
CHO-S21 [Eif2b4_E312K; L316V] ISR-insensitive This study N/A
CHO-C30 CHOP::GFP-reporter eIF2Bγ-3xFlag-tagged Zyryanova et al., 2018 N/A
HeLa [3 X Flag-EIF2B2] Sekine et al., 2015 N/A
FreeStyle 293-F cells Thermo Fisher Scientific R79007
For full list see Table S1 N/A N/A
Oligonucleotides
Oligo2213_CHO_eIF2B4_Exon10_ssODN_L310X: GGTTTTTCAGTCAGGTATTCACCATACCATCCAT
ATACCAGGATCACGTCCCCGTCACTGATCTTC
TTAGAGGCAAACCGTGAAATTGCTTGAGCTGCN
NNCACAATCTTCTCTTGTACATACCGATCAATG
GCTTCTCTAAGTTCTGACTTTGCCTAAATGTTG
AGAGAACAGTGATATAATTCACCC		 Integrated DNA Technologies N/A
Oligo2214_CHO_eIF2B4_Exon10_ssODN_E306K_L310X: GGTTTTTCAGTCAGGTATTCACCATACCATCCATATACC
AGGATCACGTCCCCGTCACTGATCTTCTTAGAGGCAAA
CCGTGAAATTGCTTGAGCTGCNNNCACAATCTTCTTTT
GTACATACCGATCAATGGCTTCTCTAAGTTCTGACTTT
GCCTAAATGTTGAGAGAACAGTGATATAATTCACCC		 Integrated DNA Technologies N/A
For full list see Table S3 This study N/A
Recombinant DNA
UK2731_heIF2a_2-187_pSUMO3 This study N/A
UK2733_heIF2a_2-187_WT_AviTag_H6_pET-30a(+) This study N/A
pETDuet-2B4-2B2_dE310K This study N/A
pETDuet-2B4-2B2_dL314Q This study N/A
pEBMulti-Neo-human-eIF2alpha-PA This study N/A
pEBMulti-Neo-human-eIF2alpha-S52A-PA This study N/A
pEBMulti-Neo Fuji Film Wako 057-08131
For full list see Table S2 This study N/A
Software and Algorithms
GraphPad Prism version 8 https://www.graphpad.com:443/ N/A
Fiji (ImageJ 1.53c NIH) Schindelin et al., 2012 N/A
RELION 3.0 Zivanov et al., 2018 N/A
Gautomatch https://www.mrc-lmb.cam.ac.uk/kzhang/ N/A
EMAN2 Tang et al., 2007 N/A
Coot Emsley et al., 2010 N/A
PHENIX Adams et al., 2010 N/A
FlowJo https://www.flowjo.com N/A