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- EMDB-41094: CryoEM structure of the HisRS-like domain of human GCN2 -

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Basic information

Entry
Database: EMDB / ID: EMD-41094
TitleCryoEM structure of the HisRS-like domain of human GCN2
Map datamain map from homogeneous refinement non sharpened
Sample
  • Complex: dimeric HisRS-like domain of human GCN2
    • Protein or peptide: eIF-2-alpha kinase GCN2
KeywordsGCN2 / HisRS / pseudoenzyme / translation / TRANSFERASE
Function / homology
Function and homology information


regulation of translational initiation by eIF2 alpha phosphorylation / positive regulation of translational initiation in response to starvation / negative regulation of CREB transcription factor activity / eiF2alpha phosphorylation in response to endoplasmic reticulum stress / GCN2-mediated signaling / negative regulation of translational initiation in response to stress / negative regulation by host of viral genome replication / positive regulation of adaptive immune response / regulation of feeding behavior / T cell activation involved in immune response ...regulation of translational initiation by eIF2 alpha phosphorylation / positive regulation of translational initiation in response to starvation / negative regulation of CREB transcription factor activity / eiF2alpha phosphorylation in response to endoplasmic reticulum stress / GCN2-mediated signaling / negative regulation of translational initiation in response to stress / negative regulation by host of viral genome replication / positive regulation of adaptive immune response / regulation of feeding behavior / T cell activation involved in immune response / regulation of translational initiation / neuron projection extension / cellular response to cold / Response of EIF2AK4 (GCN2) to amino acid deficiency / negative regulation of neuron differentiation / long-term memory / negative regulation of translational initiation / positive regulation of defense response to virus by host / cytosolic ribosome / cellular response to amino acid starvation / DNA damage checkpoint signaling / learning / positive regulation of long-term synaptic potentiation / cellular response to UV / protein autophosphorylation / defense response to virus / viral translation / adaptive immune response / eukaryotic translation initiation factor 2alpha kinase activity / tRNA binding / 3-phosphoinositide-dependent protein kinase activity / DNA-dependent protein kinase activity / ribosomal protein S6 kinase activity / histone H3S10 kinase activity / histone H2AXS139 kinase activity / histone H3S28 kinase activity / histone H4S1 kinase activity / histone H2BS14 kinase activity / histone H3T3 kinase activity / histone H2AS121 kinase activity / Rho-dependent protein serine/threonine kinase activity / histone H2BS36 kinase activity / histone H3S57 kinase activity / histone H2AT120 kinase activity / AMP-activated protein kinase activity / histone H2AS1 kinase activity / histone H3T6 kinase activity / histone H3T11 kinase activity / histone H3T45 kinase activity / non-specific serine/threonine protein kinase / protein phosphorylation / protein serine kinase activity / protein serine/threonine kinase activity / ATP binding / nucleus / cytosol / cytoplasm
Similarity search - Function
eIF-2-alpha kinase Gcn2 / Histidyl tRNA synthetase-related domain / Anticodon binding domain of tRNAs / RWD domain / RWD domain profile. / RWD / RWD domain / Class II Histidinyl-tRNA synthetase (HisRS)-like catalytic core domain / Histidyl-tRNA synthetase / : ...eIF-2-alpha kinase Gcn2 / Histidyl tRNA synthetase-related domain / Anticodon binding domain of tRNAs / RWD domain / RWD domain profile. / RWD / RWD domain / Class II Histidinyl-tRNA synthetase (HisRS)-like catalytic core domain / Histidyl-tRNA synthetase / : / Anticodon-binding domain superfamily / Ubiquitin-conjugating enzyme/RWD-like / Class II Aminoacyl-tRNA synthetase/Biotinyl protein ligase (BPL) and lipoyl protein ligase (LPL) / Serine/threonine-protein kinase, active site / Serine/Threonine protein kinases active-site signature. / Protein kinase domain / Serine/Threonine protein kinases, catalytic domain / Protein kinase, ATP binding site / Protein kinases ATP-binding region signature. / Protein kinase domain profile. / Protein kinase domain / Protein kinase-like domain superfamily
Similarity search - Domain/homology
eIF-2-alpha kinase GCN2
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsYin JZ / Keszei AFA / Mazhab-Jafari MT / Sicheri F
Funding support Canada, 1 items
OrganizationGrant numberCountry
Canadian Institutes of Health Research (CIHR) Canada
CitationJournal: Structure / Year: 2024
Title: The HisRS-like domain of GCN2 is a pseudoenzyme that can bind uncharged tRNA.
Authors: Jay Z Yin / Alexander F A Keszei / Scott Houliston / Frantisek Filandr / Jonah Beenstock / Salima Daou / Julia Kitaygorodsky / David C Schriemer / Mohammad T Mazhab-Jafari / Anne-Claude ...Authors: Jay Z Yin / Alexander F A Keszei / Scott Houliston / Frantisek Filandr / Jonah Beenstock / Salima Daou / Julia Kitaygorodsky / David C Schriemer / Mohammad T Mazhab-Jafari / Anne-Claude Gingras / Frank Sicheri /
Abstract: GCN2 is a stress response kinase that phosphorylates the translation initiation factor eIF2α to inhibit general protein synthesis when activated by uncharged tRNA and stalled ribosomes. The presence ...GCN2 is a stress response kinase that phosphorylates the translation initiation factor eIF2α to inhibit general protein synthesis when activated by uncharged tRNA and stalled ribosomes. The presence of a HisRS-like domain in GCN2, normally associated with tRNA aminoacylation, led to the hypothesis that eIF2α kinase activity is regulated by the direct binding of this domain to uncharged tRNA. Here we solved the structure of the HisRS-like domain in the context of full-length GCN2 by cryoEM. Structure and function analysis shows the HisRS-like domain of GCN2 has lost histidine and ATP binding but retains tRNA binding abilities. Hydrogen deuterium exchange mass spectrometry, site-directed mutagenesis and computational docking experiments support a tRNA binding model that is partially shifted from that employed by bona fide HisRS enzymes. These results demonstrate that the HisRS-like domain of GCN2 is a pseudoenzyme and advance our understanding of GCN2 regulation and function.
History
DepositionJun 21, 2023-
Header (metadata) releaseMar 13, 2024-
Map releaseMar 13, 2024-
UpdateSep 25, 2024-
Current statusSep 25, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_41094.png

Downloads & links

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Map

FileDownload / File: emd_41094.map.gz / Format: CCP4 / Size: 30.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationmain map from homogeneous refinement non sharpened
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.35 Å/pix.
x 200 pix.
= 270. Å		1.35 Å/pix.
x 200 pix.
= 270. Å		1.35 Å/pix.
x 200 pix.
= 270. Å

Surface

Projections

Slices (1/3)

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Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.35 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.22
Minimum - Maximum-0.42584234 - 1.0154661
Average (Standard dev.)0.00046816628 (±0.028361958)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions200200200
Spacing200200200
CellA=B=C: 270.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_41094_msk_1.map
Projections & Slices
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Histogram (log scale)

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Mask #2

Fileemd_41094_msk_2.map
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Additional map: additional map from non uniform refinement non sharpened

Fileemd_41094_additional_1.map
Annotationadditional map from non uniform refinement non sharpened
Projections & Slices
AxesZYX

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Histogram

Histogram (log scale)

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Half map: half map A of main map from homogeneous refinement

Fileemd_41094_half_map_1.map
Annotationhalf map A of main map from homogeneous refinement
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half map B of main map from homogeneous refinement

Fileemd_41094_half_map_2.map
Annotationhalf map B of main map from homogeneous refinement
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AxesZYX

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Histogram

Histogram (log scale)

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Sample components

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Entire : dimeric HisRS-like domain of human GCN2

EntireName: dimeric HisRS-like domain of human GCN2
Components
  • Complex: dimeric HisRS-like domain of human GCN2
    • Protein or peptide: eIF-2-alpha kinase GCN2

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Supramolecule #1: dimeric HisRS-like domain of human GCN2

SupramoleculeName: dimeric HisRS-like domain of human GCN2 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: eIF-2-alpha kinase GCN2

MacromoleculeName: eIF-2-alpha kinase GCN2 / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO / EC number: non-specific serine/threonine protein kinase
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 187.216578 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: GMAGGRGAPG RGRDEPPESY PQRQDHELQA LEAIYGADFQ DLRPDACGPV KEPPEINLVL YPQGLTGEEV YVKVDLRVKC PPTYPDVVP EIELKNAKGL SNESVNLLKS RLEELAKKHC GEVMIFELAY HVQSFLSEHN KPPPKSFHEE MLERRAQEEQ Q RLLEAKRK ...String:
GMAGGRGAPG RGRDEPPESY PQRQDHELQA LEAIYGADFQ DLRPDACGPV KEPPEINLVL YPQGLTGEEV YVKVDLRVKC PPTYPDVVP EIELKNAKGL SNESVNLLKS RLEELAKKHC GEVMIFELAY HVQSFLSEHN KPPPKSFHEE MLERRAQEEQ Q RLLEAKRK EEQEQREILH EIQRRKEEIK EEKKRKEMAK QERLEIASLS NQDHTSKKDP GGHRTAAILH GGSPDFVGNG KH RANSSGR SRRERQYSVC NSEDSPGSCE ILYFNMGSPD QLMVHKGKCI GSDEQLGKLV YNALETATGG FVLLYEWVLQ WQK KMGPFL TSQEKEKIDK CKKQIQGTET EFNSLVKLSH PNVVRYLAMN LKEQDDSIVV DILVEHISGV SLAAHLSHSG PIPV HQLRR YTAQLLSGLD YLHSNSVVHK VLSASNVLVD AEGTVKITDY SISKRLADIC KEDVFEQTRV RFSDNALPYK TGKKG DVWR LGLLLLSLSQ GQECGEYPVT IPSDLPADFQ DFLKKCVCLD DKERWSPQQL LKHSFINPQP KMPLVEQSPE DSEGQD YVE TVIPSNRLPS AAFFSETQRQ FSRYFIEFEE LQLLGKGAFG AVIKVQNKLD GCCYAVKRIP INPASRQFRR IKGEVTL LS RLHHENIVRY YNAWIERHER PAGPGTPPPD SGPLAKDDRA ARGQPASDTD GLDSVEAAAP PPILSSSVEW STSGERSA S ARFPATGPGS SDDEDDDEDE HGGVFSQSFL PASDSESDII FDNEDENSKS QNQDEDCNEK NGCHESEPSV TTEAVHYLY IQMEYCEKST LRDTIDQGLY RDTVRLWRLF REILDGLAYI HEKGMIHRDL KPVNIFLDSD DHVKIGDFGL ATDHLAFSAD SKQDDQTGD LIKSDPSGHL TGMVGTALYV SPEVQGSTKS AYNQKVDLFS LGIIFFEMSY HPMVTASERI FVLNQLRDPT S PKFPEDFD DGEHAKQKSV ISWLLNHDPA KRPTATELLK SELLPPPQME ESELHEVLHH TLTNVDGKAY RTMMAQIFSQ RI SPAIDYT YDSDILKGNF SIRTAKMQQH VCETIIRIFK RHGAVQLCTP LLLPRNRQIY EHNEAALFMD HSGMLVMLPF DLR IPFARY VARNNILNLK RYCIERVFRP RKLDRFHPKE LLECAFDIVT STTNSFLPTA EIIYTIYEII QEFPALQERN YSIY LNHTM LLKAILLHCG IPEDKLSQVY IILYDAVTEK LTRREVEAKF CNLSLSSNSL CRLYKFIEQK GDLQDLMPTI NSLIK QKTG IAQLVKYGLK DLEEVVGLLK KLGIKLQVLI NLGLVYKVQQ HNGIIFQFVA FIKRRQRAVP EILAAGGRYD LLIPQF RGP QALGPVPTAI GVSIAIDKIS AAVLNMEESV TISSCDLLVV SVGQMSMSRA INLTQKLWTA GITAEIMYDW SQSQEEL QE YCRHHEITYV ALVSDKEGSH VKVKSFEKER QTEKRVLETE LVDHVLQKLR TKVTDERNGR EASDNLAVQN LKGSFSNA S GLFEIHGATV VPIVSVLAPE KLSASTRRRY ETQVQTRLQT SLANLHQKSS EIEILAVDLP KETILQFLSL EWDADEQAF NTTVKQLLSR LPKQRYLKLV CDEIYNIKVE KKVSVLFLYS YRDDYYRILF

UniProtKB: eIF-2-alpha kinase GCN2

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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: 8
VitrificationCryogen name: ETHANE / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 80.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: OTHER / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.0 µm
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: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 229225
Initial angle assignmentType: OTHER / Software - Name: cryoSPARC
Final angle assignmentType: OTHER / Software - Name: cryoSPARC
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelChain - Source name: AlphaFold / Chain - Initial model type: in silico model
RefinementSpace: REAL / Protocol: OTHER
Output model

PDB-8t7t:
CryoEM structure of the HisRS-like domain of human GCN2

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