+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-28608 | |||||||||
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Title | Cryo-EM structure of CH848 10.17DT DS-SOSIP-2P Env | |||||||||
Map data | Full map used for model refinement. Calculated in cryoSPARC and sharpened using DeepEMhancer. | |||||||||
Sample |
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Keywords | HIV-1 / Env / viral fusion protein / glycoprotein / VIRAL PROTEIN | |||||||||
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 / host cell endosome membrane / clathrin-dependent endocytosis of virus by host cell / viral protein processing / fusion of virus membrane with host plasma membrane / virus-mediated perturbation of host defense response / 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 / host cell endosome membrane / clathrin-dependent endocytosis of virus by host cell / viral protein processing / fusion of virus membrane with host plasma membrane / virus-mediated perturbation of host defense response / fusion of virus membrane with host endosome membrane / viral envelope / virion attachment to host cell / apoptotic process / host cell plasma membrane / virion membrane / structural molecule activity / plasma membrane Similarity search - Function | |||||||||
Biological species | Human immunodeficiency virus 1 | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 3.73 Å | |||||||||
Authors | Wrapp D / Acharya P / Haynes BF | |||||||||
Funding support | United States, 1 items
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Citation | Journal: 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 |
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-Structure visualization
Supplemental images |
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-Downloads & links
-EMDB archive
Map data | emd_28608.map.gz | 55 MB | EMDB map data format | |
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Header (meta data) | emd-28608-v30.xml emd-28608.xml | 19 KB 19 KB | Display Display | EMDB header |
FSC (resolution estimation) | emd_28608_fsc.xml | 8.5 KB | Display | FSC data file |
Images | emd_28608.png | 201.3 KB | ||
Filedesc metadata | emd-28608.cif.gz | 6.4 KB | ||
Others | emd_28608_additional_1.map.gz emd_28608_half_map_1.map.gz emd_28608_half_map_2.map.gz | 31.7 MB 59.3 MB 59.3 MB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-28608 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-28608 | HTTPS FTP |
-Validation report
Summary document | emd_28608_validation.pdf.gz | 774.1 KB | Display | EMDB validaton report |
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Full document | emd_28608_full_validation.pdf.gz | 773.7 KB | Display | |
Data in XML | emd_28608_validation.xml.gz | 16 KB | Display | |
Data in CIF | emd_28608_validation.cif.gz | 21.1 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-28608 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-28608 | HTTPS FTP |
-Related structure data
Related structure data | 8eu8MC M: atomic model generated by this map C: citing same article (ref.) |
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Similar structure data | Similarity search - Function & homologyF&H Search |
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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Related items in Molecule of the Month |
-Map
File | Download / File: emd_28608.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||
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Annotation | Full map used for model refinement. Calculated in cryoSPARC and sharpened using DeepEMhancer. | ||||||||||||||||||||||||||||||||||||
Projections & slices | Image control
Images are generated by Spider. | ||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 1.08 Å | ||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
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-Supplemental data
-Additional map: Full map calculated in cryoSPARC (unsharpened).
File | emd_28608_additional_1.map | ||||||||||||
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Annotation | Full map calculated in cryoSPARC (unsharpened). | ||||||||||||
Projections & Slices |
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Density Histograms |
-Half map: Half-map B
File | emd_28608_half_map_1.map | ||||||||||||
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Annotation | Half-map B | ||||||||||||
Projections & Slices |
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Density Histograms |
-Half map: Half-map A
File | emd_28608_half_map_2.map | ||||||||||||
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Annotation | Half-map A | ||||||||||||
Projections & Slices |
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Density Histograms |
-Sample components
-Entire : CH848 10.17DT DS-SOSIP-2P
Entire | Name: CH848 10.17DT DS-SOSIP-2P |
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Components |
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-Supramolecule #1: CH848 10.17DT DS-SOSIP-2P
Supramolecule | Name: CH848 10.17DT DS-SOSIP-2P / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1 Details: Prefusion conformation of HIV-1 Env (homotrimer of gp120/gp41 heterodimers) |
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Source (natural) | Organism: Human immunodeficiency virus 1 |
Molecular weight | Theoretical: 233 KDa |
-Macromolecule #1: CH848 10.17DT SOSIP Envelope glycoprotein gp160
Macromolecule | Name: CH848 10.17DT SOSIP Envelope glycoprotein gp160 / type: protein_or_peptide / ID: 1 / Number of copies: 3 / Enantiomer: LEVO |
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Source (natural) | Organism: Human immunodeficiency virus 1 |
Molecular weight | Theoretical: 77.69507 KDa |
Recombinant expression | Organism: Homo sapiens (human) |
Sequence | String: 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 UniProtKB: Envelope glycoprotein gp160 |
-Macromolecule #3: 2-acetamido-2-deoxy-beta-D-glucopyranose
Macromolecule | Name: 2-acetamido-2-deoxy-beta-D-glucopyranose / type: ligand / ID: 3 / Number of copies: 27 / Formula: NAG |
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Molecular weight | Theoretical: 221.208 Da |
Chemical component information | ChemComp-NAG: |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 1.80 mg/mL | |||||||||||||||
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Buffer | pH: 8 Component:
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Grid | Model: 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). | |||||||||||||||
Vitrification | Cryogen name: ETHANE / Chamber humidity: 95 % / Instrument: LEICA EM GP |
-Electron microscopy
Microscope | FEI TITAN KRIOS |
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Image recording | Film or detector model: GATAN K3 (6k x 4k) / Average electron dose: 61.0 e/Å2 |
Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.4 µm / Nominal defocus min: 0.8 µm |
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |