EMDB-27185: gamma-Arf1 mediated dimeric assembly of AP-1, Arf1, Nef complex w...

Basic information

Entry

Database: EMDB / ID: EMD-27185

• Title: gamma-Arf1 mediated dimeric assembly of AP-1, Arf1, Nef complex within lattice on MHC-I lipopeptide incorporated wide(r) membrane tubes
• Map data: gamma-Arf1 mediated dimeric assembly of AP-1, Arf1, Nef complex within lattice on MHC-I lipopeptide incorporated wide(r) membrane tubes

Sample

• Complex: Complex of AP-1, Arf1, Nef and MHC-I cytosolic tail on a tubulated lipid bilayer
  • Complex: AP-1 heterotetramer
    • Protein or peptide: AP-1 complex subunit beta-1
    • Protein or peptide: AP-1 complex subunit gamma-1
    • Protein or peptide: AP-1 complex subunit mu-1
    • Protein or peptide: AP-1 complex subunit sigma-3
  • Protein or peptide: ADP-ribosylation factor 1
  • Protein or peptide: Protein Nef
  • Protein or peptide: HLA class I histocompatibility antigen, A alpha chain
• Ligand: GUANOSINE-5'-TRIPHOSPHATE
• Ligand: MAGNESIUM ION

• Keywords: nef / AP / trafficking / PROTEIN TRANSPORT

Function / homology

Function:

basolateral protein secretion / : / : / AP-1 adaptor complex / endosome to melanosome transport / Lysosome Vesicle Biogenesis / mitotic cleavage furrow ingression / trans-Golgi Network Vesicle Budding / protein trimerization / platelet dense granule organization / Glycosphingolipid transport / regulation of receptor internalization / symbiont-mediated suppression of host antigen processing and presentation of peptide antigen via MHC class I / melanosome assembly / regulation of Arp2/3 complex-mediated actin nucleation / Intra-Golgi traffic / symbiont-mediated suppression of host antigen processing and presentation of peptide antigen via MHC class II / Golgi Associated Vesicle Biogenesis / symbiont-mediated suppression of host apoptosis / Synthesis of PIPs at the Golgi membrane / clathrin adaptor activity / : / MHC class II antigen presentation / CD4 receptor binding / thioesterase binding / Nef Mediated CD4 Down-regulation / dendritic spine organization / determination of left/right symmetry / clathrin-coated vesicle / long-term synaptic depression / positive regulation of memory T cell activation / T cell mediated cytotoxicity directed against tumor cell target / COPI-dependent Golgi-to-ER retrograde traffic / TAP complex binding / clathrin binding / Golgi medial cisterna / Lysosome Vesicle Biogenesis / positive regulation of CD8-positive, alpha-beta T cell activation / CD8-positive, alpha-beta T cell activation / positive regulation of CD8-positive, alpha-beta T cell proliferation / CD8 receptor binding / Golgi Associated Vesicle Biogenesis / antigen processing and presentation of exogenous peptide antigen via MHC class I / cell leading edge / beta-2-microglobulin binding / MHC class I protein binding / Synthesis of PIPs at the plasma membrane / endoplasmic reticulum exit site / host cell Golgi membrane / antigen processing and presentation of endogenous peptide antigen via MHC class I via ER pathway, TAP-dependent / TAP binding / protection from natural killer cell mediated cytotoxicity / intracellular copper ion homeostasis / antigen processing and presentation of endogenous peptide antigen via MHC class I via ER pathway, TAP-independent / antigen processing and presentation of endogenous peptide antigen via MHC class Ib / protein targeting / T cell receptor binding / COPI-mediated anterograde transport / detection of bacterium / vesicle-mediated transport / clathrin-coated pit / regulation of calcium-mediated signaling / MHC class II antigen presentation / viral life cycle / cytoplasmic vesicle membrane / Neutrophil degranulation / Gene and protein expression by JAK-STAT signaling after Interleukin-12 stimulation / sarcomere / small monomeric GTPase / trans-Golgi network membrane / Nef mediated downregulation of MHC class I complex cell surface expression / kidney development / Endosomal/Vacuolar pathway / lumenal side of endoplasmic reticulum membrane / Antigen Presentation: Folding, assembly and peptide loading of class I MHC / intracellular protein transport / trans-Golgi network / ER to Golgi transport vesicle membrane / peptide antigen assembly with MHC class I protein complex / MHC class I peptide loading complex / T cell mediated cytotoxicity / antigen processing and presentation of endogenous peptide antigen via MHC class I / positive regulation of T cell cytokine production / MHC class I protein complex / peptide antigen binding / SH3 domain binding / virion component / cellular response to virus / positive regulation of T cell mediated cytotoxicity / positive regulation of type II interferon production / recycling endosome membrane / phagocytic vesicle membrane / Immunoregulatory interactions between a Lymphoid and a non-Lymphoid cell / Interferon gamma signaling / Interferon alpha/beta signaling / antibacterial humoral response / synaptic vesicle / E3 ubiquitin ligases ubiquitinate target proteins / T cell receptor signaling pathway / presynapse

Domain/homology:

AP-1 complex subunit sigma / Adaptor protein complex AP-1, gamma subunit / Gamma-adaptin ear (GAE) domain / Gamma-adaptin ear (GAE) domain profile. / HIV-1 Nef protein, anchor domain superfamily / Clathrin adaptor, beta-adaptin, appendage, Ig-like subdomain / Beta2-adaptin appendage, C-terminal sub-domain / HIV negative factor Nef / AP-1/2/4 complex subunit beta / Adaptor protein complex, sigma subunit / HIV-1 Nef protein, core domain superfamily / Negative factor, (F-Protein) or Nef / : / Beta-adaptin appendage, C-terminal subdomain / Beta2-adaptin appendage, C-terminal sub-domain / ADP-ribosylation factor 1-5 / AP complex subunit beta / Clathrin adaptor complex, small chain / Clathrin adaptor complexes small chain signature. / Clathrin adaptor complexes medium chain signature 1. / Clathrin adaptor, mu subunit / Clathrin adaptor, mu subunit, conserved site / Clathrin adaptor complexes medium chain signature 2. / : / Coatomer/calthrin adaptor appendage, C-terminal subdomain / Clathrin adaptor, alpha/beta/gamma-adaptin, appendage, Ig-like subdomain / Adaptin C-terminal domain / Adaptin C-terminal domain / AP complex, mu/sigma subunit / Clathrin adaptor complex small chain / Adaptor complexes medium subunit family / AP-2 complex subunit mu, C-terminal superfamily / Clathrin/coatomer adaptor, adaptin-like, N-terminal / Adaptin N terminal region / Mu homology domain / Mu homology domain (MHD) profile. / Clathrin adaptor, appendage, Ig-like subdomain superfamily / Small GTPase superfamily, ARF type / small GTPase Arf family profile. / Sar1p-like members of the Ras-family of small GTPases / Small GTPase superfamily, ARF/SAR type / ADP-ribosylation factor family / Longin-like domain superfamily / ARF-like small GTPases; ARF, ADP-ribosylation factor / TBP domain superfamily / Armadillo/beta-catenin-like repeats / Armadillo / MHC class I, alpha chain, C-terminal / MHC_I C-terminus / MHC class I alpha chain, alpha1 alpha2 domains / Class I Histocompatibility antigen, domains alpha 1 and 2 / : / MHC class I-like antigen recognition-like / MHC class I-like antigen recognition-like superfamily / Rab subfamily of small GTPases / MHC classes I/II-like antigen recognition protein / Armadillo-like helical / Small GTP-binding protein domain / Immunoglobulin/major histocompatibility complex, conserved site / Immunoglobulins and major histocompatibility complex proteins signature. / Immunoglobulin C-Type / Immunoglobulin C1-set / Immunoglobulin C1-set domain / Armadillo-type fold / Ig-like domain profile. / Immunoglobulin-like domain / Immunoglobulin-like domain superfamily / Immunoglobulin-like fold / P-loop containing nucleoside triphosphate hydrolase

Component:

HLA class I histocompatibility antigen, A alpha chain / AP-1 complex subunit gamma-1 / AP-1 complex subunit mu-1 / ADP-ribosylation factor 1 / AP-1 complex subunit beta-1 / Protein Nef / AP-1 complex subunit sigma-3

• Biological species: Homo sapiens (human) / Mus musculus (house mouse) / Human immunodeficiency virus 1
• Method: subtomogram averaging / cryo EM / Resolution: 11.6 Å
• Authors: Hooy RM / Hurley JH

Funding support

United States, 3 items

Organization	Grant number	Country
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)	F32 AI152971	United States
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)	R01 AI120691	United States
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)	P50 AI150476	United States

• Citation: Journal: Sci Adv / Year: 2022; Title: Self-assembly and structure of a clathrin-independent AP-1:Arf1 tubular membrane coat.; Authors: Richard M Hooy / Yuichiro Iwamoto / Dan A Tudorica / Xuefeng Ren / James H Hurley / ; Abstract: The adaptor protein (AP) complexes not only form the inner layer of clathrin coats but also have clathrin-independent roles in membrane traffic whose mechanisms are unknown. HIV-1 Nef hijacks AP-1 to sequester major histocompatibility complex class I (MHC-I), evading immune detection. We found that AP-1:Arf1:Nef:MHC-I forms a coat on tubulated membranes without clathrin and determined its structure. The coat assembles via Arf1 dimer interfaces. AP-1-positive tubules are enriched in cells upon clathrin knockdown. Nef localizes preferentially to AP-1 tubules in cells, explaining how Nef sequesters MHC-I. Coat contact residues are conserved across Arf isoforms and the Arf-dependent AP complexes AP-1, AP-3, and AP-4. Thus, AP complexes can self-assemble with Arf1 into tubular coats without clathrin or other scaffolding factors. The AP-1:Arf1 coat defines the structural basis of a broader class of tubulovesicular membrane coats as an intermediate in clathrin vesicle formation from internal membranes and as an MHC-I sequestration mechanism in HIV-1 infection.

History

Deposition	Jun 1, 2022	-
Header (metadata) release	Jun 14, 2023	-
Map release	Jun 14, 2023	-
Update	Jun 12, 2024	-
Current status	Jun 12, 2024	Processing site: RCSB / Status: Released

Map

• File: Download / File: emd_27185.map.gz / Format: CCP4 / Size: 11.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
• Annotation: gamma-Arf1 mediated dimeric assembly of AP-1, Arf1, Nef complex within lattice on MHC-I lipopeptide incorporated wide(r) membrane tubes
• Voxel size: X=Y=Z: 2.1 Å

Density

Contour Level	By AUTHOR: 2.5
Minimum - Maximum	-11.100322999999999 - 13.121973000000001
Average (Standard dev.)	0.056348052 (±1.4032784)

• Symmetry: Space group: 1

Details

EMDB XML:

Map geometry	Axis order X Y Z Origin 0 0 0 Dimensions 144 144 144 Spacing 144 144 144
Cell	A=B=C: 302.4 Å α=β=γ: 90.0 °

Supplemental data

Mask #1

• File: emd_27185_msk_1.map

Half map: Half Map 1

• File: emd_27185_half_map_1.map
• Annotation: Half Map 1

Half map: Half Map 2

• File: emd_27185_half_map_2.map
• Annotation: Half Map 2

Sample components

Entire : Complex of AP-1, Arf1, Nef and MHC-I cytosolic tail on a tubulate...

• Entire: Name: Complex of AP-1, Arf1, Nef and MHC-I cytosolic tail on a tubulated lipid bilayer

Components

• Complex: Complex of AP-1, Arf1, Nef and MHC-I cytosolic tail on a tubulated lipid bilayer
  • Complex: AP-1 heterotetramer
    • Protein or peptide: AP-1 complex subunit beta-1
    • Protein or peptide: AP-1 complex subunit gamma-1
    • Protein or peptide: AP-1 complex subunit mu-1
    • Protein or peptide: AP-1 complex subunit sigma-3
  • Protein or peptide: ADP-ribosylation factor 1
  • Protein or peptide: Protein Nef
  • Protein or peptide: HLA class I histocompatibility antigen, A alpha chain
• Ligand: GUANOSINE-5'-TRIPHOSPHATE
• Ligand: MAGNESIUM ION

Supramolecule #1: Complex of AP-1, Arf1, Nef and MHC-I cytosolic tail on a tubulate...

• Supramolecule: Name: Complex of AP-1, Arf1, Nef and MHC-I cytosolic tail on a tubulated lipid bilayer; type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#7; Details: gamma-Arf1 linked AP-1/Arf1/Nef complexes within the lattice on wide(r) membrane tubes
• Source (natural): Organism: Homo sapiens (human)

Supramolecule #2: AP-1 heterotetramer

• Supramolecule: Name: AP-1 heterotetramer / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1, #3, #5-#6; Details: All four subunits are co-expressed from the same plasmid. Assembly occurs in bacteria during expression.
• Source (natural): Organism: Homo sapiens (human)

Macromolecule #1: AP-1 complex subunit beta-1

• Macromolecule: Name: AP-1 complex subunit beta-1 / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
• Source (natural): Organism: Homo sapiens (human)
• Molecular weight: Theoretical: 104.736461 KDa
• Recombinant expression: Organism: Escherichia coli BL21(DE3) (bacteria)

Sequence

String:

MTDSKYFTTT KKGEIFELKA ELNSDKKEKK KEAVKKVIAS MTVGKDVSAL FPDVVNCMQT DNLELKKLVY LYLMNYAKSQ PDMAIMAVN TFVKDCEDPN PLIRALAVRT MGCIRVDKIT EYLCEPLRKC LKDEDPYVRK TAAVCVAKLH DINAQLVEDQ G FLDTLKDL ISDSNPMVVA NAVAALSEIA ESHPSSNLLD LNPQSINKLL TALNECTEWG QIFILDCLAN YMPKDDREAQ SI CERVTPR LSHANSAVVL SAVKVLMKFM EMLSKDLDYY GTLLKKLAPP LVTLLSAEPE LQYVALRNIN LIVQKRPEIL KHE MKVFFV KYNDPIYVKL EKLDIMIRLA SQANIAQVLA ELREYATEVD VDFVRKAVRA IGRCAIKVEQ SAERCVSTLL DLIQ TKVNY VVQEAIVVIK DIFRKYPNKY ESVIATLCEN LDSLDEPEAR AAMIWIVGEY AERIDNADEL LESFLEGFHD KSTQV QLQL LTAIVKLFLK KPTETQELVQ QVLSLATQDS DNPDLRDRGY IYWRLLSTDP VAAKEVVLAE KPLISEETDL IEPTLL DEL ICYIGTLASV YHKPPSAFVE GGRGVVHKSL PPRTASSESA ESPETAPTGA PPGEQPDVIP AQGDLLGDLL NLDLGPP VS GPPLATSSVQ MGAVDLLGGG LDSLMGDEPE GIGGTNFVAP PTAAVPANLG APIGSGLSDL FDLTSGVGTL SGSYVAPK A VWLPAMKAKG LEISGTFTRQ VGSISMDLQL TNKALQVMTD FAIQFNRNSF GLAPAAPLQV HAPLSPNQTV EISLPLSTV GSVMKMEPLN NLQVAVKNNI DVFYFSTLYP LHILFVEDGK MDRQMFLATW KDIPNENEAQ FQIRDCPLNA EAASSKLQSS NIFTVAKRN VEGQDMLYQS LKLTNGIWVL AELRIQPGNP SCTDLELSLK CRAPEVSQHV YQAYETILKN

UniProtKB: AP-1 complex subunit beta-1

Macromolecule #2: ADP-ribosylation factor 1

• Macromolecule: Name: ADP-ribosylation factor 1 / type: protein_or_peptide / ID: 2 / Details: N-terminal myristoylation / Number of copies: 6 / Enantiomer: LEVO
• Source (natural): Organism: Homo sapiens (human)
• Molecular weight: Theoretical: 20.721742 KDa
• Recombinant expression: Organism: Escherichia coli BL21(DE3) (bacteria)

Sequence

String:

MGNIFANLFK GLFGKKEMRI LMVGLDAAGK TTILYKLKLG EIVTTIPTIG FNVETVEYKN ISFTVWDVGG QDKIRPLWRH YFQNTQGLI FVVDSNDRER VNEAREELMR MLAEDELRDA VLLVFANKQD LPNAMNAAEI TDKLGLHSLR HRNWYIQATC A TSGDGLYE GLDWLSNQLR NQK

UniProtKB: ADP-ribosylation factor 1

Macromolecule #3: AP-1 complex subunit gamma-1

• Macromolecule: Name: AP-1 complex subunit gamma-1 / type: protein_or_peptide / ID: 3 / Number of copies: 2 / Enantiomer: LEVO
• Source (natural): Organism: Mus musculus (house mouse)
• Molecular weight: Theoretical: 68.194094 KDa
• Recombinant expression: Organism: Escherichia coli BL21(DE3) (bacteria)

Sequence

String:

MPAPIRLREL IRTIRTARTQ AEEREMIQKE CAAIRSSFRE EDNTYRCRNV AKLLYMHMLG YPAHFGQLEC LKLIASQKFT DKRIGYLGA MLLLDERQDV HLLMTNCIKN DLNHSTQFVQ GLALCTLGCM GSSEMCRDLA GEVEKLLKTS NSYLRKKAAL C AVHVIRKV PELMEMFLPA TKNLLNEKNH GVLHTSVVLL TEMCERSPDM LAHFRKLVPQ LVRILKNLIM SGYSPEHDVS GI SDPFLQV RILRLLRILG RNDDDSSEAM NDILAQVATN TETSKNVGNA ILYETVLTIM DIKSESGLRV LAINILGRFL LNN DKNIRY VALTSLLKTV QTDHNAVQRH RSTIVDCLKD LDVSIKRRAM ELSFALVNGN NIRGMMKELL YFLDSCEPEF KADC ASGIF LAAEKYAPSK RWHIDTIMRV LTTAGSYVRD DAVPNLIQLI TNSVEMHAYT VQRLYKAILG DYSQQPLVQV AAWCI GEYG DLLVSGQCEE EEPIQVTEDE VLDILESVLI SNMSTSVTRG YALTAIMKLS TRFTCTVNRI KKVVSIYGSS IDVELQ QRA VEYNALFKKY DHMRSALLER MPVMEKVTTN GPENLYFQ

UniProtKB: AP-1 complex subunit gamma-1

Macromolecule #4: Protein Nef

• Macromolecule: Name: Protein Nef / type: protein_or_peptide / ID: 4 / Details: N-terminal myristoylation / Number of copies: 4 / Enantiomer: LEVO
• Source (natural): Organism: Human immunodeficiency virus 1
• Molecular weight: Theoretical: 24.285244 KDa
• Recombinant expression: Organism: Escherichia coli BL21(DE3) (bacteria)

Sequence

String:

MGGKWSKSSV IGWPAVRERM RRAEPAADGV GAVSRDLEKH GAITSSNTAA NNAACAWLEA QEEEEVGFPV TPQVPLRPMT YKAAVDLSH FLKEKGGLEG LIHSQRRQDI LDLWIYHTQG YFPDWQNYTP GPGVRYPLTF GWCYKLVPVE PDKVEEANKG E NTSLLHPV SLHGMDDPER EVLEWRFDSR LAFHHVAREL HPEYFKNCGH HHHHH

UniProtKB: Protein Nef

Macromolecule #5: AP-1 complex subunit mu-1

• Macromolecule: Name: AP-1 complex subunit mu-1 / type: protein_or_peptide / ID: 5 / Number of copies: 2 / Enantiomer: LEVO
• Source (natural): Organism: Mus musculus (house mouse)
• Molecular weight: Theoretical: 48.60673 KDa
• Recombinant expression: Organism: Escherichia coli BL21(DE3) (bacteria)

Sequence

String:

MSASAVYVLD LKGKVLICRN YRGDVDMSEV EHFMPILMEK EEEGMLSPIL AHGGVRFMWI KHNNLYLVAT SKKNACVSLV FSFLYKVVQ VFSEYFKELE EESIRDNFVI IYELLDELMD FGYPQTTDSK ILQEYITQEG HKLETGAPRP PATVTNAVSW R SEGIKYRK NEVFLDVIEA VNLLVSANGN VLRSEIVGSI KMRVFLSGMP ELRLGLNDKV LFDNTGRGKS KSVELEDVKF HQ CVRLSRF ENDRTISFIP PDGEFELMSY RLNTHVKPLI WIESVIEKHS HSRIEYMVKA KSQFKRRSTA NNVEIHIPVP NDA DSPKFK TTVGSVKWVP ENSEIVWSVK SFPGGKEYLM RAHFGLPSVE AEDKEGKPPI SVKFEIPYFT TSGIQVRYLK IIEK SGYQA LPWVRYITQN GDYQLRTQ

UniProtKB: AP-1 complex subunit mu-1

Macromolecule #6: AP-1 complex subunit sigma-3

• Macromolecule: Name: AP-1 complex subunit sigma-3 / type: protein_or_peptide / ID: 6 / Number of copies: 2 / Enantiomer: LEVO
• Source (natural): Organism: Homo sapiens (human)
• Molecular weight: Theoretical: 18.305273 KDa
• Recombinant expression: Organism: Escherichia coli BL21(DE3) (bacteria)

Sequence

String:

MIHFILLFSR QGKLRLQKWY ITLPDKERKK ITREIVQIIL SRGHRTSSFV DWKELKLVYK RYASLYFCCA IENQDNELLT LEIVHRYVE LLDKYFGNVC ELDIIFNFEK AYFILDEFII GGEIQETSKK IAVKAIEDSD MLQEVSTVSQ TMGER

UniProtKB: AP-1 complex subunit sigma-3

Macromolecule #7: HLA class I histocompatibility antigen, A alpha chain

• Macromolecule: Name: HLA class I histocompatibility antigen, A alpha chain / type: protein_or_peptide / ID: 7 / Number of copies: 2 / Enantiomer: LEVO
• Source (natural): Organism: Homo sapiens (human)
• Molecular weight: Theoretical: 4.139429 KDa
• Recombinant expression: Organism: Escherichia coli BL21(DE3) (bacteria)

Sequence

String:

CRKSSDRKGG SYSQAAGSDS AQSSDVSLTA AKVHHHHHH

UniProtKB: HLA class I histocompatibility antigen, A alpha chain

Macromolecule #8: GUANOSINE-5'-TRIPHOSPHATE

• Macromolecule: Name: GUANOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 8 / Number of copies: 6 / Formula: GTP
• Molecular weight: Theoretical: 523.18 Da

Chemical component information

ChemComp-GTP:
GUANOSINE-5'-TRIPHOSPHATE / GTP, energy-carrying molecule*YM

Macromolecule #9: MAGNESIUM ION

• Macromolecule: Name: MAGNESIUM ION / type: ligand / ID: 9 / Number of copies: 6 / Formula: MG
• Molecular weight: Theoretical: 24.305 Da

Experimental details

Structure determination

• Method: cryo EM
• Processing: subtomogram averaging
• Aggregation state: particle

Sample preparation

• Concentration: 0.2 mg/mL

Buffer

pH: 7.2
Component:

Concentration	Name	Formula
20.0 mM	HEPES
125.0 mM	potassium acetate	KOAc
2.0 mM	magnesium chloride
1.0 mM	DTT

Details: HEPES/KOAc concentrated stocks are diluted to their final concentrations then pH'd to 7.2 with KOH prior to use in experiments.

• Grid: Model: EMS Lacey Carbon / Support film - Material: CARBON / Support film - topology: LACEY / Support film - Film thickness: 50
• Vitrification: Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 298 K / Instrument: FEI VITROBOT MARK IV; Details: 60 second wait, 3-5 second blot, 597 filter paper, 0.5 second drain. Sample was supplemented with 10nm BSA-gold fiducials. 3.5ul of the mixture was double-side blotted..

Electron microscopy

• Microscope: FEI TITAN KRIOS
• Specialist optics: Energy filter - Slit width: 25 eV
• Image recording: Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Digitization - Dimensions - Width: 5760 pixel / Digitization - Dimensions - Height: 4092 pixel / Number grids imaged: 1 / Average exposure time: 3.0 sec. / Average electron dose: 3.0 e/Ų; Details: Tilt images were collected in movie-mode. Each movie/tilt consisted of 3-4 frames each
• Electron beam: Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
• Electron optics: Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 4.5 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 42000
• 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

• Details: The images were gain-normalized
• Final reconstruction: Applied symmetry - Point group: C₂ (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 11.6 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1.1) / Number subtomograms used: 5094
• Extraction: Number tomograms: 13 / Number images used: 8601 / Reference model: Reference-free / Software - Name: Dynamo (ver. 1.1.532); Details: Tubes were annotated by tracing the center of the tube in Dynamo and recording the average apparent diameter. Initial subtomogram positions were picked using uniform radial and axial sampling.
• Final angle assignment: Type: NOT APPLICABLE

Atomic model buiding 1

Initial model

PDB ID	Chain
6cm9	source_name: PDB, initial_model_type: experimental model
4en2	chain_id: B, source_name: PDB, initial_model_type: experimental model
4en2	chain_id: E, source_name: PDB, initial_model_type: experimental model

• Refinement: Protocol: RIGID BODY FIT

Output model

PDB-8d4g:
gamma-Arf1 mediated dimeric assembly of AP-1, Arf1, Nef complex within lattice on MHC-I lipopeptide incorporated wide(r) membrane tubes