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- EMDB-28608: Cryo-EM structure of CH848 10.17DT DS-SOSIP-2P Env -

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Basic information

Entry
Database: EMDB / ID: EMD-28608
TitleCryo-EM structure of CH848 10.17DT DS-SOSIP-2P Env
Map dataFull map used for model refinement. Calculated in cryoSPARC and sharpened using DeepEMhancer.
Sample
  • Complex: CH848 10.17DT DS-SOSIP-2P
    • Protein or peptide: CH848 10.17DT SOSIP Envelope glycoprotein gp160
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
Function / homology
Function and homology information


positive regulation of plasma membrane raft polarization / positive regulation of receptor clustering / positive regulation of establishment of T cell polarity / virus-mediated perturbation of host defense response / host cell endosome membrane / clathrin-dependent endocytosis of virus by host cell / viral protein processing / fusion of virus membrane with host plasma membrane / fusion of virus membrane with host endosome membrane / viral envelope ...positive regulation of plasma membrane raft polarization / positive regulation of receptor clustering / positive regulation of establishment of T cell polarity / virus-mediated perturbation of host defense response / host cell endosome membrane / clathrin-dependent endocytosis of virus by host cell / viral protein processing / fusion of virus membrane with host plasma membrane / fusion of virus membrane with host endosome membrane / viral envelope / virion attachment to host cell / host cell plasma membrane / virion membrane / structural molecule activity / plasma membrane
Similarity search - Function
Envelope glycoprotein Gp160 / Retroviral envelope protein / Retroviral envelope protein GP41-like / Gp120 core superfamily / Envelope glycoprotein GP120 / Human immunodeficiency virus 1, envelope glycoprotein Gp120
Similarity search - Domain/homology
Envelope glycoprotein gp160
Similarity search - Component
Biological speciesHuman immunodeficiency virus 1
Methodsingle particle reconstruction / cryo EM / Resolution: 3.73 Å
AuthorsWrapp D / Acharya P / Haynes BF
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)5UM1AI144371 United States
CitationJournal: J Virol / Year: 2023
Title: Structure-Based Stabilization of SOSIP Env Enhances Recombinant Ectodomain Durability and Yield.
Authors: Daniel Wrapp / Zekun Mu / Bhishem Thakur / Katarzyna Janowska / Oluwatobi Ajayi / Maggie Barr / Robert Parks / Katayoun Mansouri / Robert J Edwards / Beatrice H Hahn / Priyamvada Acharya / ...Authors: Daniel Wrapp / Zekun Mu / Bhishem Thakur / Katarzyna Janowska / Oluwatobi Ajayi / Maggie Barr / Robert Parks / Katayoun Mansouri / Robert J Edwards / Beatrice H Hahn / Priyamvada Acharya / Kevin O Saunders / Barton F Haynes /
Abstract: The envelope glycoprotein (Env) is the main focus of human immunodeficiency virus type 1 (HIV-1) vaccine development due to its critical role in viral entry. Despite advances in protein engineering, ...The envelope glycoprotein (Env) is the main focus of human immunodeficiency virus type 1 (HIV-1) vaccine development due to its critical role in viral entry. Despite advances in protein engineering, many Env proteins remain recalcitrant to recombinant expression due to their inherent metastability, making biochemical and immunological experiments impractical or impossible. Here, we report a novel proline stabilization strategy to facilitate the production of prefusion Env trimers. This approach, termed "2P," works synergistically with previously described SOSIP mutations and dramatically increases the yield of recombinantly expressed Env ectodomains without altering the antigenic or conformational properties of near-native Env. We determined that the 2P mutations function by enhancing the durability of the prefusion conformation and that this stabilization strategy is broadly applicable to evolutionarily and antigenically diverse Env constructs. These findings provide a new Env stabilization platform to facilitate biochemical research and expand the number of Env variants that can be developed as future HIV-1 vaccine candidates. Recent estimates have placed the number of new human immunodeficiency virus type 1 (HIV-1) infections at approximately 1.5 million per year, emphasizing the ongoing and urgent need for an effective vaccine. The envelope (Env) glycoprotein is the main focus of HIV-1 vaccine development, but, due to its inherent metastability, many Env variants are difficult to recombinantly express in the relatively large quantities that are required for biochemical studies and animal trials. Here, we describe a novel structure-based stabilization strategy that works synergistically with previously described SOSIP mutations to increase the yield of prefusion HIV-1 Env.
History
DepositionOct 18, 2022-
Header (metadata) releaseJan 4, 2023-
Map releaseJan 4, 2023-
UpdateFeb 8, 2023-
Current statusFeb 8, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_28608.png

Downloads & links

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Map

FileDownload / File: emd_28608.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationFull map used for model refinement. Calculated in cryoSPARC and sharpened using DeepEMhancer.
Voxel sizeX=Y=Z: 1.08 Å
Density
Contour LevelBy AUTHOR: 0.1
Minimum - Maximum-0.016316876 - 2.0112488
Average (Standard dev.)0.0018591227 (±0.030748026)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 276.48 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: Full map calculated in cryoSPARC (unsharpened).

Fileemd_28608_additional_1.map
AnnotationFull map calculated in cryoSPARC (unsharpened).
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half-map B

Fileemd_28608_half_map_1.map
AnnotationHalf-map B
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half-map A

Fileemd_28608_half_map_2.map
AnnotationHalf-map A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : CH848 10.17DT DS-SOSIP-2P

EntireName: CH848 10.17DT DS-SOSIP-2P
Components
  • Complex: CH848 10.17DT DS-SOSIP-2P
    • Protein or peptide: CH848 10.17DT SOSIP Envelope glycoprotein gp160
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose

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Supramolecule #1: CH848 10.17DT DS-SOSIP-2P

SupramoleculeName: CH848 10.17DT DS-SOSIP-2P / type: complex / ID: 1 / Chimera: Yes / Parent: 0 / Macromolecule list: #1
Details: Prefusion conformation of HIV-1 Env (homotrimer of gp120/gp41 heterodimers)
Source (natural)Organism: Human immunodeficiency virus 1
Molecular weightTheoretical: 233 KDa

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Macromolecule #1: CH848 10.17DT SOSIP Envelope glycoprotein gp160

MacromoleculeName: CH848 10.17DT SOSIP Envelope glycoprotein gp160 / type: protein_or_peptide / ID: 1 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Human immunodeficiency virus 1
Molecular weightTheoretical: 77.69507 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MPMGSLQPLA TLYLLGMLVA SVLAAENLWV TVYYGVPVWK EAKTTLFCAS DARAYEKEVH NVWATHACVP TDPSPQELVL GNVTENFNM WKNDMVDQMH EDIISLWDQS LKPCVKLTPL CVTLICSDAT VKTGTVEEMK NCSFNTTTEI RDKEKKEYAL F YKPDIVPL ...String:
MPMGSLQPLA TLYLLGMLVA SVLAAENLWV TVYYGVPVWK EAKTTLFCAS DARAYEKEVH NVWATHACVP TDPSPQELVL GNVTENFNM WKNDMVDQMH EDIISLWDQS LKPCVKLTPL CVTLICSDAT VKTGTVEEMK NCSFNTTTEI RDKEKKEYAL F YKPDIVPL SETNNTSEYR LINCNTSACT QACPKVTFEP IPIHYCAPAG YAILKCNDET FNGTGPCSNV STVQCTHGIR PV VSTQLLL NGSLAEKEIV IRSENLTNNA KIIIVHLHTP VEIVCTRPNN NTRKSVRIGP GQTFYATGDI IGDIKQAHCN ISE EKWNDT LQKVGIELQK HFPNKTIKYN QSAGGDMEIT THSFNCGGEF FYCNTSNLFN GTYNGTYIST NSSANSTSTI TLQC RIKQI INMWQGVGRC MYAPPIAGNI TCRSNITGLL LTRDGGTNSN ETETFRPAGG DMRDNWRSEL YKYKVVKIEP LGVAP TRCK RRVVGRRRRR RAVGIGAVFL GFLGAAGSTM GAASMTLTVQ ARNLLSGIVQ QQSNLLRAPE AQQHLLKPPV WGIKQL QAR VLAVERYLRD QQLLGIWGCS GKLICCTNVP WNSSWSNRNL SEIWDNMTWL QWDKEISNYT QIIYGLLEES QNQQEKN EQ DLLALDGSLE VLFQGPGHHH HHHHHSAWSH PQFEKGGGSG GGGSGGSAWS HPQFEK

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Macromolecule #3: 2-acetamido-2-deoxy-beta-D-glucopyranose

MacromoleculeName: 2-acetamido-2-deoxy-beta-D-glucopyranose / type: ligand / ID: 3 / Number of copies: 27 / Formula: NAG
Molecular weightTheoretical: 221.208 Da
Chemical component information

ChemComp-NAG:
2-acetamido-2-deoxy-beta-D-glucopyranose / N-Acetylglucosamine

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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

Concentration1.80 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
10.0 mMC4H11NO3Tris
200.0 mMNaClSodium chloridesodium chloride
0.02 %NaN3sodium azide
0.085 mMC24H46O11DDM
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Support film - Material: CARBON / Support film - topology: HOLEY ARRAY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 10 sec.
Details: Glow discharged using an easiGlow Glow Discharge Cleaning System (PELCO).
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Instrument: LEICA EM GP

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2.4 µm / Nominal defocus min: 0.8 µm
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 61.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionApplied symmetry - Point group: C3 (3 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 3.73 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 111026
FSC plot (resolution estimation)

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