EMDB-26519: Human GATOR2 complex

Basic information

Entry

Database: EMDB / ID: EMD-26519

• Title: Human GATOR2 complex
• Map data: COMPOSITE MAP. Sharpened. C2-pseudosymmetric.

Sample

• Complex: Human GATOR2
  • Protein or peptide: GATOR complex protein MIOS
  • Protein or peptide: GATOR complex protein WDR24
  • Protein or peptide: GATOR complex protein WDR59
  • Protein or peptide: Isoform B of Nucleoporin SEH1
  • Protein or peptide: Protein SEC13 homolog
  • Protein or peptide: Unknown
  • Protein or peptide: Unknown
• Ligand: ZINC ION

Function / homology

Function:

GATOR2 complex / Seh1-associated complex / protein exit from endoplasmic reticulum / COPII-coated vesicle budding / COPII-coated vesicle cargo loading / nuclear pore outer ring / nuclear pore organization / COPII vesicle coat / Nuclear Pore Complex (NPC) Disassembly / Amino acids regulate mTORC1 / Transport of Ribonucleoproteins into the Host Nucleus / Regulation of Glucokinase by Glucokinase Regulatory Protein / Defective TPR may confer susceptibility towards thyroid papillary carcinoma (TPC) / Transport of the SLBP independent Mature mRNA / attachment of mitotic spindle microtubules to kinetochore / Transport of the SLBP Dependant Mature mRNA / NS1 Mediated Effects on Host Pathways / SUMOylation of SUMOylation proteins / protein-containing complex localization / Transport of Mature mRNA Derived from an Intronless Transcript / Rev-mediated nuclear export of HIV RNA / SUMOylation of RNA binding proteins / Nuclear import of Rev protein / Transport of Mature mRNA derived from an Intron-Containing Transcript / NEP/NS2 Interacts with the Cellular Export Machinery / tRNA processing in the nucleus / Postmitotic nuclear pore complex (NPC) reformation / nucleocytoplasmic transport / COPII-mediated vesicle transport / Viral Messenger RNA Synthesis / mitotic metaphase chromosome alignment / SUMOylation of ubiquitinylation proteins / Vpr-mediated nuclear import of PICs / cellular response to nutrient levels / SUMOylation of DNA replication proteins / Regulation of HSF1-mediated heat shock response / positive regulation of TOR signaling / mRNA transport / Amplification of signal from unattached kinetochores via a MAD2 inhibitory signal / SUMOylation of DNA damage response and repair proteins / nuclear pore / Mitotic Prometaphase / EML4 and NUDC in mitotic spindle formation / negative regulation of TORC1 signaling / positive regulation of TORC1 signaling / Resolution of Sister Chromatid Cohesion / MHC class II antigen presentation / cellular response to amino acid starvation / SUMOylation of chromatin organization proteins / HCMV Late Events / regulation of autophagy / RHO GTPases Activate Formins / Antigen Presentation: Folding, assembly and peptide loading of class I MHC / Transcriptional regulation by small RNAs / intracellular protein transport / ER to Golgi transport vesicle membrane / ISG15 antiviral mechanism / kinetochore / autophagy / HCMV Early Events / Separation of Sister Chromatids / protein import into nucleus / protein transport / cell junction / nuclear envelope / snRNP Assembly / defense response to Gram-positive bacterium / cell division / lysosomal membrane / intracellular membrane-bounded organelle / endoplasmic reticulum membrane / SARS-CoV-2 activates/modulates innate and adaptive immune responses / structural molecule activity / extracellular exosome / nucleoplasm / identical protein binding / cytoplasm / cytosol

Domain/homology:

WDR59, modified RING finger, H2 subclass (C3H3C2-type) / WD repeat protein mio, zinc-ribbon like domain / GATOR complex protein WDR24 / MIOS/Sea4 / Zinc-ribbon like family / RWD domain / RWD domain profile. / RWD / Sec13/Seh1 family / Ubiquitin-conjugating enzyme/RWD-like / G-protein beta WD-40 repeat / WD40 repeat, conserved site / Trp-Asp (WD) repeats signature. / Trp-Asp (WD) repeats profile. / Trp-Asp (WD) repeats circular profile. / WD domain, G-beta repeat / WD40 repeats / WD40 repeat / WD40-repeat-containing domain superfamily / WD40/YVTN repeat-like-containing domain superfamily

Component:

Protein SEC13 homolog / GATOR complex protein WDR59 / Nucleoporin SEH1 / GATOR complex protein WDR24 / GATOR complex protein MIOS

• Biological species: Homo sapiens (human)
• Method: single particle reconstruction / cryo EM / Resolution: 3.66 Å
• Authors: Rogala KB / Valenstein ML / Lalgudi PV

Funding support

United States, 17 items

Organization	Grant number	Country
National Institutes of Health/National Cancer Institute (NIH/NCI)	K99 CA255926	United States
Tuberous Sclerosis Association	2018-F01	United States
Other private	Charles King Trust
Lustgarten Foundation		United States
Department of Defense (DOD, United States)	TS200035	United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)	T32 GM007753	United States
National Institutes of Health/National Cancer Institute (NIH/NCI)	F30 CA228229	United States
National Institutes of Health/National Cancer Institute (NIH/NCI)	F31 CA180271	United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)	F31 GM121093	United States
Other private	Koch Institute Graduate Fellowship
National Institutes of Health/National Cancer Institute (NIH/NCI)	R01 CA103866	United States
National Institutes of Health/National Cancer Institute (NIH/NCI)	R01 CA129105	United States
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)	R01 AI047389	United States
Department of Defense (DOD, United States)	W81XWH-21-1-0260	United States
Other private	Leo Foundation
American Cancer Society		United States
Howard Hughes Medical Institute (HHMI)		United States

• Citation: Journal: Nature / Year: 2022; Title: Structure of the nutrient-sensing hub GATOR2.; Authors: Max L Valenstein / Kacper B Rogala / Pranav V Lalgudi / Edward J Brignole / Xin Gu / Robert A Saxton / Lynne Chantranupong / Jonas Kolibius / Jan-Philipp Quast / David M Sabatini / ; Abstract: Mechanistic target of rapamycin complex 1 (mTORC1) controls growth by regulating anabolic and catabolic processes in response to environmental cues, including nutrients. Amino acids signal to mTORC1 through the Rag GTPases, which are regulated by several protein complexes, including GATOR1 and GATOR2. GATOR2, which has five components (WDR24, MIOS, WDR59, SEH1L and SEC13), is required for amino acids to activate mTORC1 and interacts with the leucine and arginine sensors SESN2 and CASTOR1, respectively. Despite this central role in nutrient sensing, GATOR2 remains mysterious as its subunit stoichiometry, biochemical function and structure are unknown. Here we used cryo-electron microscopy to determine the three-dimensional structure of the human GATOR2 complex. We found that GATOR2 adopts a large (1.1 MDa), two-fold symmetric, cage-like architecture, supported by an octagonal scaffold and decorated with eight pairs of WD40 β-propellers. The scaffold contains two WDR24, four MIOS and two WDR59 subunits circularized via two distinct types of junction involving non-catalytic RING domains and α-solenoids. Integration of SEH1L and SEC13 into the scaffold through β-propeller blade donation stabilizes the GATOR2 complex and reveals an evolutionary relationship to the nuclear pore and membrane-coating complexes. The scaffold orients the WD40 β-propeller dimers, which mediate interactions with SESN2, CASTOR1 and GATOR1. Our work reveals the structure of an essential component of the nutrient-sensing machinery and provides a foundation for understanding the function of GATOR2 within the mTORC1 pathway.

History

Deposition	Mar 27, 2022	-
Header (metadata) release	Jul 20, 2022	-
Map release	Jul 20, 2022	-
Update	Aug 3, 2022	-
Current status	Aug 3, 2022	Processing site: RCSB / Status: Released

Map

• File: Download / File: emd_26519.map.gz / Format: CCP4 / Size: 216 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
• Annotation: COMPOSITE MAP. Sharpened. C2-pseudosymmetric.
• Voxel size: X=Y=Z: 1.0922 Å

Density

Contour Level	By AUTHOR: 3.0
Minimum - Maximum	-8.325662 - 78.298676
Average (Standard dev.)	0.02805115 (±1.4866972)

• Symmetry: Space group: 1

Details

EMDB XML:

Map geometry	Axis order Z Y X Origin 0 0 0 Dimensions 384 384 384 Spacing 384 384 384
Cell	A=B=C: 419.40482 Å α=β=γ: 90.0 °

Supplemental data

Mask #1

• File: emd_26519_msk_1.map

Mask #2

• File: emd_26519_msk_2.map

Mask #3

• File: emd_26519_msk_3.map

Mask #4

• File: emd_26519_msk_4.map

Mask #5

• File: emd_26519_msk_5.map

Mask #6

• File: emd_26519_msk_6.map

Additional map: COMPOSITE MAP. Unsharpened. C2-pseudosymmetric. Used for the final...

• File: emd_26519_additional_1.map
• Annotation: COMPOSITE MAP. Unsharpened. C2-pseudosymmetric. Used for the final refinement.

Additional map: LOCAL MAP #4. Sharpened. From C2-symmetry expanded particles.

• File: emd_26519_additional_10.map
• Annotation: LOCAL MAP #4. Sharpened. From C2-symmetry expanded particles.

Additional map: LOCAL MAP #3. Sharpened. From C2-symmetry expanded particles.

• File: emd_26519_additional_11.map
• Annotation: LOCAL MAP #3. Sharpened. From C2-symmetry expanded particles.

Additional map: LOCAL MAP #5. Sharpened.

• File: emd_26519_additional_12.map
• Annotation: LOCAL MAP #5. Sharpened.

Additional map: LOCAL MAP #5. Unsharpened.

• File: emd_26519_additional_13.map
• Annotation: LOCAL MAP #5. Unsharpened.

Additional map: GLOBAL MAP. Sharpened. C2-symmetric.

• File: emd_26519_additional_2.map
• Annotation: GLOBAL MAP. Sharpened. C2-symmetric.

Additional map: GLOBAL MAP. Unsharpened. C2-symmetric.

• File: emd_26519_additional_3.map
• Annotation: GLOBAL MAP. Unsharpened. C2-symmetric.

Additional map: LOCAL MAP #2. Unsharpened. From C2-symmetry expanded particles.

• File: emd_26519_additional_4.map
• Annotation: LOCAL MAP #2. Unsharpened. From C2-symmetry expanded particles.

Additional map: LOCAL MAP #1. Sharpened. From C2-symmetry expanded particles.

• File: emd_26519_additional_5.map
• Annotation: LOCAL MAP #1. Sharpened. From C2-symmetry expanded particles.

Additional map: LOCAL MAP #1. Unsharpened. From C2-symmetry expanded particles.

• File: emd_26519_additional_6.map
• Annotation: LOCAL MAP #1. Unsharpened. From C2-symmetry expanded particles.

Additional map: LOCAL MAP #2. Sharpened. From C2-symmetry expanded particles.

• File: emd_26519_additional_7.map
• Annotation: LOCAL MAP #2. Sharpened. From C2-symmetry expanded particles.

Additional map: LOCAL MAP #3. Unsharpened. From C2-symmetry expanded particles.

• File: emd_26519_additional_8.map
• Annotation: LOCAL MAP #3. Unsharpened. From C2-symmetry expanded particles.

Additional map: LOCAL MAP #4. Unsharpened. From C2-symmetry expanded particles.

• File: emd_26519_additional_9.map
• Annotation: LOCAL MAP #4. Unsharpened. From C2-symmetry expanded particles.

Half map: GLOBAL HALF-MAP B. C2-symmetric.

• File: emd_26519_half_map_1.map
• Annotation: GLOBAL HALF-MAP B. C2-symmetric.

Half map: GLOBAL HALF-MAP A. C2-symmetric.

• File: emd_26519_half_map_2.map
• Annotation: GLOBAL HALF-MAP A. C2-symmetric.

Sample components

Entire : Human GATOR2

• Entire: Name: Human GATOR2

Components

• Complex: Human GATOR2
  • Protein or peptide: GATOR complex protein MIOS
  • Protein or peptide: GATOR complex protein WDR24
  • Protein or peptide: GATOR complex protein WDR59
  • Protein or peptide: Isoform B of Nucleoporin SEH1
  • Protein or peptide: Protein SEC13 homolog
  • Protein or peptide: Unknown
  • Protein or peptide: Unknown
• Ligand: ZINC ION

Supramolecule #1: Human GATOR2

• Supramolecule: Name: Human GATOR2 / type: complex / Chimera: Yes / ID: 1 / Parent: 0 / Macromolecule list: #1-#7; Details: Purified five-component GATOR2 complex via a Flag-tag on MIOS.
• Source (natural): Organism: Homo sapiens (human)
• Recombinant expression: Organism: Homo sapiens (human) / Recombinant cell: HEK-293T
• Molecular weight: Theoretical: 1.1 MDa

Macromolecule #1: GATOR complex protein MIOS

• Macromolecule: Name: GATOR complex protein MIOS / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
• Source (natural): Organism: Homo sapiens (human)
• Molecular weight: Theoretical: 100.633383 KDa
• Recombinant expression: Organism: Homo sapiens (human)

Sequence

String:

MEMDYKDDDD KGGGGGASTM SGTKPDILWA PHHVDRFVVC DSELSLYHVE STVNSELKAG SLRLSEDSAA TLLSINSDTP YMKCVAWYL NYDPECLLAV GQANGRVVLT SLGQDHNSKF KDLIGKEFVP KHARQCNTLA WNPLDSNWLA AGLDKHRADF S VLIWDICS KYTPDIVPME KVKLSAGETE TTLLVTKPLY ELGQNDACLS LCWLPRDQKL LLAGMHRNLA IFDLRNTSQK MF VNTKAVQ GVTVDPYFHD RVASFYEGQV AIWDLRKFEK PVLTLTEQPK PLTKVAWCPT RTGLLATLTR DSNIIRLYDM QHT PTPIGD ETEPTIIERS VQPCDNYIAS FAWHPTSQNR MIVVTPNRTM SDFTVFERIS LAWSPITSLM WACGRHLYEC TEEE NDNSL EKDIATKMRL RALSRYGLDT EQVWRNHILA GNEDPQLKSL WYTLHFMKQY TEDMDQKSPG NKGSLVYAGI KSIVK SSLG MVESSRHNWS GLDKQSDIQN LNEERILALQ LCGWIKKGTD VDVGPFLNSL VQEGEWERAA AVALFNLDIR RAIQIL NEG ASSEKGDLNL NVVAMALSGY TDEKNSLWRE MCSTLRLQLN NPYLCVMFAF LTSETGSYDG VLYENKVAVR DRVAFAC KF LSDTQLNRYI EKLTNEMKEA GNLEGILLTG LTKDGVDLME SYVDRTGDVQ TASYCMLQGS PLDVLKDERV QYWIENYR N LLDAWRFWHK RAEFDIHRSK LDPSSKPLAQ VFVSCNFCGK SISYSCSAVP HQGRGFSQYG VSGSPTKSKV TSCPGCRKP LPRCALCLIN MGTPVSSCPG GTKSDEKVDL SKDKKLAQFN NWFTWCHNCR HGGHAGHMLS WFRDHAECPV SACTCKCMQL DTTGNLVPA ETVQP

Macromolecule #2: GATOR complex protein WDR24

• Macromolecule: Name: GATOR complex protein WDR24 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
• Source (natural): Organism: Homo sapiens (human)
• Molecular weight: Theoretical: 88.326953 KDa
• Recombinant expression: Organism: Homo sapiens (human)

Sequence

String:

MEKMSRVTTA LGGSVLTGRT MHCHLDAPAN AISVCRDAAQ VVVAGRSIFK IYAIEEEQFV EKLNLRVGRK PSLNLSCADV VWHQMDENL LATAATNGVV VTWNLGRPSR NKQDQLFTEH KRTVNKVCFH PTEAHVLLSG SQDGFMKCFD LRRKDSVSTF S GQSESVRD VQFSIRDYFT FASTFENGNV QLWDIRRPDR CERMFTAHNG PVFCCDWHPE DRGWLATGGR DKMVKVWDMT TH RAKEMHC VQTIASVARV KWRPECRHHL ATCSMMVDHN IYVWDVRRPF VPAAMFEEHR DVTTGIAWRH PHDPSFLLSG SKD SSLCQH LFRDASQPVE RANPEGLCYG LFGDLAFAAK ESLVAAESGR KPYTGDRRHP IFFKRKLDPA EPFAGLASSA LSVF ETEPG GGGMRWFVDT AERYALAGRP LAELCDHNAK VARELGRNQV AQTWTMLRII YCSPGLVPTA NLNHSVGKGG SCGLP LMNS FNLKDMAPGL GSETRLDRSK GDARSDTVLL DSSATLITNE DNEETEGSDV PADYLLGDVE GEEDELYLLD PEHAHP EDP ECVLPQEAFP LRHEIVDTPP GPEHLQDKAD SPHVSGSEAD VASLAPVDSS FSLLSVSHAL YDSRLPPDFF GVLVRDM LH FYAEQGDVQM AVSVLIVLGE RVRKDIDEQT QEHWYTSYID LLQRFRLWNV SNEVVKLSTS RAVSCLNQAS TTLHVNCS H CKRPMSSRGW VCDRCHRCAS MCAVCHHVVK GLFVWCQGCS HGGHLQHIMK WLEGSSHCPA GCGHLCEYS

Macromolecule #3: GATOR complex protein WDR59

• Macromolecule: Name: GATOR complex protein WDR59 / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
• Source (natural): Organism: Homo sapiens (human)
• Molecular weight: Theoretical: 109.938391 KDa
• Recombinant expression: Organism: Homo sapiens (human)

Sequence

String:

MAARWSSENV VVEFRDSQAT AMSVDCLGQH AVLSGRRFLY IVNLDAPFEG HRKISRQSKW DIGAVQWNPH DSFAHYFAAS SNQRVDLYK WKDGSGEVGT TLQGHTRVIS DLDWAVFEPD LLVTSSVDTY IYIWDIKDTR KPTVALSAVA GASQVKWNKK N ANCLATSH DGDVRIWDKR KPSTAVEYLA AHLSKIHGLD WHPDSEHILA TSSQDNSVKF WDYRQPRKYL NILPCQVPVW KA RYTPFSN GLVTVMVPQL RRENSLLLWN VFDLNTPVHT FVGHDDVVLE FQWRKQKEGS KDYQLVTWSR DQTLRMWRVD SQM QRLCAN DILDGVDEFI ESISLLPEPE KTLHTEDTDH QHTASHGEEE ALKEDPPRNL LEERKSDQLG LPQTLQQEFS LINV QIRNV NVEMDAADRS CTVSVHCSNH RVKMLVKFPA QYPNNAAPSF QFINPTTITS TMKAKLLKIL KDTALQKVKR GQSCL EPCL RQLVSCLESF VNQEDSASSN PFALPNSVTP PLPTFARVTT AYGSYQDANI PFPRTSGARF CGAGYLVYFT RPMTMH RAV SPTEPTPRSL SALSAYHTGL IAPMKIRTEA PGNLRLYSGS PTRSEKEQVS ISSFYYKERK SRRWKSKREG SDSGNRQ IK AAGKVIIQDI ACLLPVHKSL GELYILNVND IQETCQKNAA SALLVGRKDL VQVWSLATVA TDLCLGPKSD PDLETPWA R HPFGRQLLES LLAHYCRLRD VQTLAMLCSV FEAQSRPQGL PNPFGPFPNR SSNLVVSHSR YPSFTSSGSC SSMSDPGLN TGGWNIAGRE AEHLSSPWGE SSPEELRFGS LTYSDPRERE RDQHDKNKRL LDPANTQQFD DFKKCYGEIL YRWGLREKRA EVLKFVSCP PDPHKGIEFG VYCSHCRSEV RGTQCAICKG FTFQCAICHV AVRGSSNFCL TCGHGGHTSH MMEWFRTQEV C PTGCGCHC LLESTF

Macromolecule #4: Isoform B of Nucleoporin SEH1

• Macromolecule: Name: Isoform B of Nucleoporin SEH1 / type: protein_or_peptide / ID: 4 / Number of copies: 3 / Enantiomer: LEVO
• Source (natural): Organism: Homo sapiens (human)
• Molecular weight: Theoretical: 46.636289 KDa
• Recombinant expression: Organism: Homo sapiens (human)

Sequence

String:

MFVARSIAAD HKDLIHDVSF DFHGRRMATC SSDQSVKVWD KSESGDWHCT ASWKTHSGSV WRVTWAHPEF GQVLASCSFD RTAAVWEEI VGESNDKLRG QSHWVKRTTL VDSRTSVTDV KFAPKHMGLM LATCSADGIV RIYEAPDVMN LSQWSLQHEI S CKLSCSCI SWNPSSSRAH SPMIAVGSDD SSPNAMAKVQ IFEYNENTRK YAKAETLMTV TDPVHDIAFA PNLGRSFHIL AI ATKDVRI FTLKPVRKEL TSSGGPTKFE IHIVAQFDNH NSQVWRVSWN ITGTVLASSG DDGCVRLWKA NYMDNWKCTG ILK GNGSPV NGSSQQGTSN PSLGSTIPSL QNSLNGSSAG RYFFTPLDSP RAGSRWSSYA QLLPPPPPPL VEHSCDADTA NLQY PHPRR RYLSRPLNPL PENEGI

Macromolecule #5: Protein SEC13 homolog

• Macromolecule: Name: Protein SEC13 homolog / type: protein_or_peptide / ID: 5 / Number of copies: 1 / Enantiomer: LEVO
• Source (natural): Organism: Homo sapiens (human)
• Molecular weight: Theoretical: 35.578438 KDa
• Recombinant expression: Organism: Homo sapiens (human)

Sequence

String:

MVSVINTVDT SHEDMIHDAQ MDYYGTRLAT CSSDRSVKIF DVRNGGQILI ADLRGHEGPV WQVAWAHPMY GNILASCSYD RKVIIWREE NGTWEKSHEH AGHDSSVNSV CWAPHDYGLI LACGSSDGAI SLLTYTGEGQ WEVKKINNAH TIGCNAVSWA P AVVPGSLI DHPSGQKPNY IKRFASGGCD NLIKLWKEEE DGQWKEEQKL EAHSDWVRDV AWAPSIGLPT STIASCSQDG RV FIWTCDD ASSNTWSPKL LHKFNDVVWH VSWSITANIL AVSGGDNKVT LWKESVDGQW VCISDVNKGQ GSVSASVTEG QQN EQ

Macromolecule #6: Unknown

• Macromolecule: Name: Unknown / type: protein_or_peptide / ID: 6 / Number of copies: 1 / Enantiomer: LEVO
• Source (natural): Organism: Homo sapiens (human)
• Molecular weight: Theoretical: 1.294587 KDa
• Recombinant expression: Organism: Homo sapiens (human)

Sequence

String:

(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)

Macromolecule #7: Unknown

• Macromolecule: Name: Unknown / type: protein_or_peptide / ID: 7 / Number of copies: 1 / Enantiomer: LEVO
• Source (natural): Organism: Homo sapiens (human)
• Molecular weight: Theoretical: 698.854 Da
• Recombinant expression: Organism: Homo sapiens (human)

Sequence

String:

(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)

Macromolecule #8: ZINC ION

• Macromolecule: Name: ZINC ION / type: ligand / ID: 8 / Number of copies: 16 / Formula: ZN
• Molecular weight: Theoretical: 65.409 Da

Experimental details

Structure determination

• Method: cryo EM
• Processing: single particle reconstruction
• Aggregation state: particle

Sample preparation

• Concentration: 3.0 mg/mL

Buffer

pH: 7.5
Component:

Concentration	Name
50.0 mM	HEPES
150.0 mM	sodium chloride
0.1 %	CHAPS
50.0 mM	L-arginine
50.0 mM	L-glutamate
2.0 mM	magnesium chloride
2.0 mM	dithiothreitol

• Grid: Model: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 1e-05 kPa
• Vitrification: Cryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
• Details: 95% pure, monodisperse protein complex. Partial loss of a few subunits observed. Contaminating CCT chaperonin present.

Electron microscopy

• Microscope: FEI TITAN KRIOS
• Specialist optics: Energy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
• Image recording: #0 - Image recording ID: 1 / #0 - Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / #0 - Number grids imaged: 1 / #0 - Number real images: 4103 / #0 - Average exposure time: 2.4 sec. / #0 - Average electron dose: 44.0 e/Ų; #0 - Details: Movie stacks were recorded in super-resolution counting mode at the UMass Medical School's Cryo-EM Core Facility in Worcester, Massachusetts, USA. 30 frames per stack.; #1 - Image recording ID: 2 / #1 - Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / #1 - Number grids imaged: 2 / #1 - Number real images: 28792 / #1 - Average exposure time: 4.0 sec. / #1 - Average electron dose: 47.0 e/Ų; #1 - Details: Movie stacks were recorded in super-resolution counting mode at MIT.nano in Cambridge, Massachusetts, USA. 30 frames per stack.
• Electron beam: Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
• Electron optics: C2 aperture diameter: 50.0 µm / Calibrated magnification: 45779 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 81000
• Sample stage: Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
• Experimental equipment: Model: Titan Krios / Image courtesy: FEI Company

Image processing

• Image recording ID: 2
• Details: Movie frames were weighted according to electron dose and particle movement. Please note that the two datasets collected at (1) UMass and (2) MIT.nano were eventually pixel-size-matched and merged into a single dataset.
• Particle selection: Number selected: 7237873 ; Details: The reported particle number is the sum of particles from two datasets, including parallel picking strategies. See details in the methods section of Valenstein and Rogala et al (2022).
• CTF correction: Software - Name: Gctf (ver. 1.06)
• Startup model: Type of model: OTHER; Details: Ab initio map was generated using stochastic gradient descent methods.
• Final reconstruction: Number classes used: 5 / Applied symmetry - Point group: C₂ (2 fold cyclic) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.66 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.2.0) / Details: Duplicate particles were removed. / Number images used: 784651
• Initial angle assignment: Type: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
• Final angle assignment: Type: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
• Final 3D classification: Number classes: 16 / Avg.num./class: 253807 / Software - Name: RELION (ver. 3.1); Details: Four parallel particle sets were classified in 3D (4 classes per set). Only classes containing the fully assembled GATOR2 complex were processed further.

Atomic model buiding 1

Initial model

PDB ID	Chain
4l9o	chain_id: B, residue_range: 1043-2298
3jrp	chain_id: A, residue_range: 3-1179
3f3f	chain_id: A, residue_range: 1-346
3f3f	chain_id: C, residue_range: 47-99

• Details: Most of the structure was built de novo in Coot. WD40 propellers were rebuilt after fitting either deposited PDB coordinates of homologous structures or predicted models from RoseTTAFold.
• Refinement: Space: REAL / Protocol: OTHER / Overall B value: 182

Output model

PDB-7uhy:
Human GATOR2 complex