PDB-8eqt: Structure of SARS-CoV-2 Orf3a in plasma membrane-like environment...

Basic information

• Entry: Database: PDB / ID: 8eqt
• Title: Structure of SARS-CoV-2 Orf3a in plasma membrane-like environment, MSP1D1 nanodisc
• Components: ORF3a protein
• Keywords: VIRAL PROTEIN / Membrane protein / SARS-CoV-2

Function / homology

Function:

host cell lysosome / induction by virus of host reticulophagy / Maturation of protein 3a / SARS-CoV-2 modulates autophagy / inorganic cation transmembrane transport / host cell endoplasmic reticulum / voltage-gated calcium channel complex / SARS-CoV-2 targets host intracellular signalling and regulatory pathways / voltage-gated potassium channel complex / molecular function activator activity / : / protein complex oligomerization / monoatomic ion channel activity / host cell endosome / Translation of Structural Proteins / Virion Assembly and Release / Induction of Cell-Cell Fusion / Attachment and Entry / host cell endoplasmic reticulum membrane / SARS-CoV-2 activates/modulates innate and adaptive immune responses / host cell plasma membrane / virion membrane / extracellular region / identical protein binding / plasma membrane

Domain/homology:

Protein 3a, betacoronavirus / 3a-like viroporin, transmembrane domain, alpha/betacoronavirus / 3a-like viroporin, cytosolic domain, alpha/betacoronavirus / Betacoronavirus viroporin / Coronavirus (CoV) 3a-like viroporin trans-membrane (TM) domain profile. / Coronavirus (CoV) 3a-like viroporin cytosolic (CD) domain profile.

Component:

1,2-dioleoyl-sn-glycero-3-phosphoethanolamine / ORF3a protein

• Biological species: Severe acute respiratory syndrome coronavirus 2
• Method: ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.4 Å
• Authors: Miller, A.N. / Houlihan, P.R. / Matamala, E. / Cabezas-Bratesco, D. / Lee, G.Y. / Cristofori-Armstrong, B. / Dilan, T.L. / Sanchez-Martinez, S. / Matthies, D. / Yan, R. / Yu, Z. / Ren, D. / Brauchi, S.E. / Clapham, D.E.

Funding support

United States, 1items

Organization	Grant number	Country
Howard Hughes Medical Institute (HHMI)		United States

Citation

Journal: Elife / Year: 2023
Title: The SARS-CoV-2 accessory protein Orf3a is not an ion channel, but does interact with trafficking proteins.
Authors: Alexandria N Miller / Patrick R Houlihan / Ella Matamala / Deny Cabezas-Bratesco / Gi Young Lee / Ben Cristofori-Armstrong / Tanya L Dilan / Silvia Sanchez-Martinez / Doreen Matthies / Rui Yan / Zhiheng Yu / Dejian Ren / Sebastian E Brauchi / David E Clapham /
Abstract: The severe acute respiratory syndrome associated coronavirus 2 (SARS-CoV-2) and SARS-CoV-1 accessory protein Orf3a colocalizes with markers of the plasma membrane, endocytic pathway, and Golgi apparatus. Some reports have led to annotation of both Orf3a proteins as viroporins. Here, we show that neither SARS-CoV-2 nor SARS-CoV-1 Orf3a form functional ion conducting pores and that the conductances measured are common contaminants in overexpression and with high levels of protein in reconstitution studies. Cryo-EM structures of both SARS-CoV-2 and SARS-CoV-1 Orf3a display a narrow constriction and the presence of a positively charged aqueous vestibule, which would not favor cation permeation. We observe enrichment of the late endosomal marker Rab7 upon SARS-CoV-2 Orf3a overexpression, and co-immunoprecipitation with VPS39. Interestingly, SARS-CoV-1 Orf3a does not cause the same cellular phenotype as SARS-CoV-2 Orf3a and does not interact with VPS39. To explain this difference, we find that a divergent, unstructured loop of SARS-CoV-2 Orf3a facilitates its binding with VPS39, a HOPS complex tethering protein involved in late endosome and autophagosome fusion with lysosomes. We suggest that the added loop enhances SARS-CoV-2 Orf3a's ability to co-opt host cellular trafficking mechanisms for viral exit or host immune evasion.

#1: Journal: bioRxiv / Year: 2022
Title: The SARS-CoV-2 accessory protein Orf3a is not an ion channel, but does interact with trafficking proteins.
Authors: Alexandria N Miller / Patrick R Houlihan / Ella Matamala / Deny Cabezas-Bratesco / Gi Young Lee / Ben Cristofori-Armstrong / Tanya L Dilan / Silvia Sanchez-Martinez / Doreen Matthies / Rui Yan / Zhiheng Yu / Dejian Ren / Sebastian E Brauchi / David E Clapham
Abstract: The severe acute respiratory syndrome associated coronavirus 2 (SARS-CoV-2) and SARS-CoV-1 accessory protein Orf3a colocalizes with markers of the plasma membrane, endocytic pathway, and Golgi apparatus. Some reports have led to annotation of both Orf3a proteins as a viroporin. Here we show that neither SARS-CoV-2 nor SARS-CoV-1 form functional ion conducting pores and that the conductances measured are common contaminants in overexpression and with high levels of protein in reconstitution studies. Cryo-EM structures of both SARS-CoV-2 and SARS-CoV-1 Orf3a display a narrow constriction and the presence of a basic aqueous vestibule, which would not favor cation permeation. We observe enrichment of the late endosomal marker Rab7 upon SARS-CoV-2 Orf3a overexpression, and co-immunoprecipitation with VPS39. Interestingly, SARS-CoV-1 Orf3a does not cause the same cellular phenotype as SARS-CoV-2 Orf3a and does not interact with VPS39. To explain this difference, we find that a divergent, unstructured loop of SARS-CoV-2 Orf3a facilitates its binding with VPS39, a HOPS complex tethering protein involved in late endosome and autophagosome fusion with lysosomes. We suggest that the added loop enhances SARS-CoV-2 Orf3a ability to co-opt host cellular trafficking mechanisms for viral exit or host immune evasion.

History

Deposition	Oct 9, 2022	Deposition site: RCSB / Processing site: RCSB
Revision 1.0	Feb 8, 2023	Provider: repository / Type: Initial release

Assembly

Deposited unit

A: ORF3a protein

B: ORF3a protein

hetero molecules

Summary:

• 76 kDa, 2 polymers

Component details:

	Theoretical mass	Number of molelcules
Total (without water)	75,955	6
Polymers	72,979	2
Non-polymers	2,976	4
Water	0

1

Summary:

• Idetical with deposited unit
• defined by author
• Evidence: cross-linking

Symmetry operations:

Type	Name	Symmetry operation	Number
identity operation	1_555		1

Components

#1: Protein

A B ORF3a protein / ORF3a / Accessory protein 3a / Protein 3a / Protein U274 / Protein X1

Details:

Mass: 36489.445 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Severe acute respiratory syndrome coronavirus 2
Gene: 3a / Production host: Homo sapiens (human) / References: UniProt: P0DTC3

#2: Chemical

A-401 A-402 B-401 B-402
ChemComp-PEE / 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine / DOPE / Discrete optimized protein energy

Details:

Mass: 744.034 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C₄₁H₇₈NO₈P / Comment: DOPE, phospholipid*YM

• Has ligand of interest: N

Experimental details

Experiment

• Experiment: Method: ELECTRON MICROSCOPY
• EM experiment: Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

Sample preparation

• Component: Name: Structure of SARS-CoV2 Orf3a in plasma membrane-like environment, MSP1D1 nanodisc; Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
• Molecular weight: Experimental value: NO
• Source (natural): Organism: Severe acute respiratory syndrome coronavirus 2
• Source (recombinant): Organism: Homo sapiens (human)
• Buffer solution: pH: 7.5
• Specimen: Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
• Vitrification: Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE

Electron microscopy imaging

• Experimental equipment: Model: Titan Krios / Image courtesy: FEI Company
• Microscopy: Model: FEI TITAN KRIOS
• Electron gun: Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
• Electron lens: Mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2000 nm / Nominal defocus min: 800 nm
• Image recording: Electron dose: 50 e/Ų / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 15637
• EM imaging optics: Energyfilter name: GIF Bioquantum

Processing

EM software

ID	Name	Version	Category
1	Topaz	0.9	particle selection
4	CTFFIND	4	CTF correction
9	cryoSPARC	3	initial Euler assignment
10	RELION	3.1	final Euler assignment

• CTF correction: Type: NONE
• Symmetry: Point symmetry: C₂ (2 fold cyclic)
• 3D reconstruction: Resolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 125625 / Num. of class averages: 1 / Symmetry type: POINT
• Atomic model building: Protocol: AB INITIO MODEL