EMDB-27190: Subtomogram average of AP-1, Arf1 and Nef complexes on wide(r) me...

Basic information

Entry

Database: EMDB / ID: EMD-27190

• Title: Subtomogram average of AP-1, Arf1 and Nef complexes on wide(r) membrane tubes centered on beta-Arf1 dimers

Sample

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

• Keywords: nef / AP / HIV / trafficking / VIRAL PROTEIN

Function / homology

Function:

basolateral protein secretion / perturbation by virus of host immune response / negative regulation of CD4 production / symbiont-mediated suppression of host T-cell mediated immune response / AP-1 adaptor complex / endosome to melanosome transport / positive regulation of natural killer cell degranulation / Lysosome Vesicle Biogenesis / platelet dense granule organization / protein trimerization / melanosome assembly / symbiont-mediated suppression of host antigen processing and presentation of peptide antigen via MHC class I / Golgi to lysosome transport / Golgi to vacuole transport / symbiont-mediated suppression of host antigen processing and presentation of peptide antigen via MHC class II / Golgi Associated Vesicle Biogenesis / GTP-dependent protein binding / suppression by virus of host autophagy / clathrin adaptor activity / MHC class II antigen presentation / melanosome organization / thioesterase binding / CD4 receptor binding / determination of left/right symmetry / clathrin-coated vesicle / Lysosome Vesicle Biogenesis / clathrin binding / Golgi Associated Vesicle Biogenesis / positive regulation of natural killer cell mediated cytotoxicity / host cell Golgi membrane / kinesin binding / T cell mediated cytotoxicity directed against tumor cell target / antigen processing and presentation of endogenous peptide antigen via MHC class I via ER pathway, TAP-dependent / positive regulation of memory T cell activation / TAP complex binding / antigen processing and presentation of exogenous peptide antigen via MHC class I / Golgi medial cisterna / 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 / protein targeting / CD8 receptor binding / MHC class I protein binding / endoplasmic reticulum exit site / beta-2-microglobulin binding / TAP binding / regulation of calcium-mediated signaling / clathrin-coated pit / protection from natural killer cell mediated cytotoxicity / vesicle-mediated transport / viral life cycle / 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 / MHC class II antigen presentation / detection of bacterium / Neutrophil degranulation / T cell receptor binding / trans-Golgi network membrane / Nef mediated downregulation of MHC class I complex cell surface expression / kidney development / virion component / lumenal side of endoplasmic reticulum membrane / Endosomal/Vacuolar pathway / Antigen Presentation: Folding, assembly and peptide loading of class I MHC / intracellular protein transport / cytoplasmic vesicle membrane / 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 / recycling endosome / antigen processing and presentation of endogenous peptide antigen via MHC class I / small GTPase binding / positive regulation of T cell cytokine production / MHC class I protein complex / SH3 domain binding / positive regulation of T cell mediated cytotoxicity / recycling endosome membrane / phagocytic vesicle membrane / peptide antigen binding / Immunoregulatory interactions between a Lymphoid and a non-Lymphoid cell / Interferon gamma signaling / Interferon alpha/beta signaling / positive regulation of type II interferon production / protein transport / E3 ubiquitin ligases ubiquitinate target proteins / heart development / ATPase binding / ER-Phagosome pathway / antibacterial humoral response / T cell receptor signaling pathway / early endosome membrane / postsynaptic density / early endosome / defense response to Gram-positive bacterium / immune response / lysosomal membrane / external side of plasma membrane / Golgi membrane

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 / ADP-ribosylation factor 1-5 / Adaptor protein complex, sigma subunit / Clathrin adaptor, beta-adaptin, appendage, Ig-like subdomain / Beta-adaptin appendage, C-terminal subdomain / AP-1/2/4 complex subunit beta / Beta2-adaptin appendage, C-terminal sub-domain / Beta2-adaptin appendage, C-terminal sub-domain / HIV negative factor Nef / HIV-1 Nef protein, core domain superfamily / Negative factor, (F-Protein) or Nef / 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 / Adaptor complexes medium subunit family / AP complex, mu/sigma subunit / Clathrin adaptor complex small chain / AP-2 complex subunit mu, C-terminal superfamily / Mu homology domain / Mu homology domain (MHD) profile. / Clathrin adaptor, alpha/beta/gamma-adaptin, appendage, Ig-like subdomain / Adaptin C-terminal domain / Adaptin C-terminal domain / Clathrin/coatomer adaptor, adaptin-like, N-terminal / Adaptin N terminal region / small GTPase Arf family profile. / Clathrin adaptor, appendage, Ig-like subdomain superfamily / 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 / Rab subfamily of small GTPases / MHC class I-like antigen recognition-like / MHC class I-like antigen recognition-like superfamily / 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:

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

• Biological species: Homo sapiens (human)
• Method: subtomogram averaging / cryo EM / Resolution: 20.0 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	Nov 9, 2022	-
Map release	Nov 9, 2022	-
Update	Jan 17, 2024	-
Current status	Jan 17, 2024	Processing site: RCSB / Status: Released

Map

• File: Download / File: emd_27190.map.gz / Format: CCP4 / Size: 5.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
• Voxel size: X=Y=Z: 4.2 Å

Density

Contour Level	By AUTHOR: 2.0
Minimum - Maximum	-14.842743 - 17.319690000000001
Average (Standard dev.)	0.1680531 (±1.8374624)

• Symmetry: Space group: 1

Details

EMDB XML:

Map geometry	Axis order X Y Z Origin 0 0 0 Dimensions 112 112 112 Spacing 112 112 112
Cell	A=B=C: 470.39996 Å α=β=γ: 90.0 °

Supplemental data

Mask #1

• File: emd_27190_msk_1.map

Half map: #1

• File: emd_27190_half_map_1.map

Half map: #2

• File: emd_27190_half_map_2.map

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

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-#8; Details: Subtomogram average encompasses multiple beta-Arf1 linked AP-1 dimers
• Source (natural): Organism: Homo sapiens (human)

Supramolecule #2: AP-1 heterotetramer

• Supramolecule: Name: AP-1 heterotetramer / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #4-#5, #7-#8; Details: All four subunits are co-expressed from the same plasmid. Assembly occurs in situ during expression.
• Source (natural): Organism: Homo sapiens (human)

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
• Electron beam: Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
• Electron optics: Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 4.5 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 42000
• Specialist optics: Energy filter - Slit width: 25 eV
• Sample stage: Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
• 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
• Experimental equipment: Model: Titan Krios / Image courtesy: FEI Company

Image processing

• Extraction: Number tomograms: 17 / Number images used: 4208 / Reference model: Reference-free / Software - Name: Dynamo (ver. 1.1532); 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 axially sampling.
• Final angle assignment: Type: NOT APPLICABLE
• Final reconstruction: Applied symmetry - Point group: C₁ (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 20.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1.1) / Number subtomograms used: 2719
• Details: The images were gain-normalized

Atomic model buiding 1

• Refinement: Protocol: RIGID BODY FIT

Output model

PDB-8d9s:
AP-1, Arf1, Nef lattice on MHC-I lipopeptide incorporated wide membrane tubes, centered on beta-Arf1