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Yorodumi- EMDB-22967: Negative stain electron microscopy reconstruction of 2P SARS-CoV-... -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-22967 | |||||||||
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Title | Negative stain electron microscopy reconstruction of 2P SARS-CoV-2 spike ectodomain in the 3-RBD-down state after cold-denaturation at 4 degrees C for one week followed by re-naturation at 37 degrees C for 3 hours. | |||||||||
Map data | Final sharpened map | |||||||||
Sample |
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Biological species | Severe acute respiratory syndrome coronavirus 2 | |||||||||
Method | single particle reconstruction / negative staining / Resolution: 16.8 Å | |||||||||
Authors | Edwards RJ / Mansouri K | |||||||||
Funding support | United States, 1 items
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Citation | Journal: bioRxiv / Year: 2020 Title: Cold sensitivity of the SARS-CoV-2 spike ectodomain. Authors: Robert J Edwards / Katayoun Mansouri / Victoria Stalls / Kartik Manne / Brian Watts / Rob Parks / Katarzyna Janowska / Sophie M C Gobeil / Megan Kopp / Dapeng Li / Xiaozhi Lu / Zekun Mu / ...Authors: Robert J Edwards / Katayoun Mansouri / Victoria Stalls / Kartik Manne / Brian Watts / Rob Parks / Katarzyna Janowska / Sophie M C Gobeil / Megan Kopp / Dapeng Li / Xiaozhi Lu / Zekun Mu / Margaret Deyton / Thomas H Oguin / Jordan Sprenz / Wilton Williams / Kevin Saunders / David Montefiori / Gregory D Sempowski / Rory Henderson / Munir Alam / Barton F Haynes / Priyamvada Acharya Abstract: The SARS-CoV-2 spike (S) protein, a primary target for COVID-19 vaccine development, presents its Receptor Binding Domain in two conformations: receptor-accessible "up" or receptor-inaccessible ...The SARS-CoV-2 spike (S) protein, a primary target for COVID-19 vaccine development, presents its Receptor Binding Domain in two conformations: receptor-accessible "up" or receptor-inaccessible "down" conformations. Here, we report that the commonly used stabilized S ectodomain construct "2P" is sensitive to cold temperature, and that this cold sensitivity is resolved in a "down" state stabilized spike. Our results will impact structural, functional and vaccine studies that use the SARS-CoV-2 S ectodomain. | |||||||||
History |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | EM map: SurfViewMolmilJmol/JSmol |
Supplemental images |
-Downloads & links
-EMDB archive
Map data | emd_22967.map.gz | 3.1 MB | EMDB map data format | |
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Header (meta data) | emd-22967-v30.xml emd-22967.xml | 22.7 KB 22.7 KB | Display Display | EMDB header |
FSC (resolution estimation) | emd_22967_fsc.xml | 3.6 KB | Display | FSC data file |
Images | emd_22967.png | 57 KB | ||
Masks | emd_22967_msk_1.map | 3.4 MB | Mask map | |
Others | emd_22967_additional_1.map.gz emd_22967_half_map_1.map.gz emd_22967_half_map_2.map.gz | 2.4 MB 2.4 MB 2.4 MB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-22967 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-22967 | HTTPS FTP |
-Validation report
Summary document | emd_22967_validation.pdf.gz | 78.2 KB | Display | EMDB validaton report |
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Full document | emd_22967_full_validation.pdf.gz | 77.3 KB | Display | |
Data in XML | emd_22967_validation.xml.gz | 495 B | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-22967 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-22967 | HTTPS FTP |
-Related structure data
Related structure data | C: citing same article (ref.) |
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Similar structure data |
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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-Map
File | Download / File: emd_22967.map.gz / Format: CCP4 / Size: 3.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | Final sharpened map | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Projections & slices | Image control
Images are generated by Spider. | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 4.02 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Mask #1
File | emd_22967_msk_1.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Additional map: Unsharpened map
File | emd_22967_additional_1.map | ||||||||||||
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Annotation | Unsharpened map | ||||||||||||
Projections & Slices |
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Density Histograms |
-Half map: Half-map 1
File | emd_22967_half_map_1.map | ||||||||||||
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Annotation | Half-map 1 | ||||||||||||
Projections & Slices |
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Density Histograms |
-Half map: Half-map2
File | emd_22967_half_map_2.map | ||||||||||||
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Annotation | Half-map2 | ||||||||||||
Projections & Slices |
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Density Histograms |
-Sample components
-Entire : 2P SARS-CoV-2 spike ectodomain trimer
Entire | Name: 2P SARS-CoV-2 spike ectodomain trimer |
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Components |
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-Supramolecule #1: 2P SARS-CoV-2 spike ectodomain trimer
Supramolecule | Name: 2P SARS-CoV-2 spike ectodomain trimer / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all |
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Source (natural) | Organism: Severe acute respiratory syndrome coronavirus 2 |
Recombinant expression | Organism: Homo sapiens (human) / Recombinant cell: 293F / Recombinant plasmid: p-alpha-H |
Molecular weight | Theoretical: 481.8 KDa |
-Macromolecule #1: 2P SARS-CoV-2 spike ectodomain
Macromolecule | Name: 2P SARS-CoV-2 spike ectodomain / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO |
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Source (natural) | Organism: Severe acute respiratory syndrome coronavirus 2 |
Recombinant expression | Organism: Homo sapiens (human) |
Sequence | String: MFVFLVLLPL VSSQCVNLTT RTQLPPAYTN SFTRGVYYPD KVFRSSVLHS TQDLFLPFFS NVTWFHAIHV SGTNGTKRFD NPVLPFNDGV YFASTEKSNI IRGWIFGTTL DSKTQSLLIV NNATNVVIKV CEFQFCNDPF LGVYYHKNNK SWMESEFRVY SSANNCTFEY ...String: MFVFLVLLPL VSSQCVNLTT RTQLPPAYTN SFTRGVYYPD KVFRSSVLHS TQDLFLPFFS NVTWFHAIHV SGTNGTKRFD NPVLPFNDGV YFASTEKSNI IRGWIFGTTL DSKTQSLLIV NNATNVVIKV CEFQFCNDPF LGVYYHKNNK SWMESEFRVY SSANNCTFEY VSQPFLMDLE GKQGNFKNLR EFVFKNIDGY FKIYSKHTPI NLVRDLPQGF SALEPLVDLP IGINITRFQT LLALHRSYLT PGDSSSGWTA GAAAYYVGYL QPRTFLLKYN ENGTITDAVD CALDPLSETK CTLKSFTVEK GIYQTSNFRV QPTESIVRFP NITNLCPFGE VFNATRFASV YAWNRKRISN CVADYSVLYN SASFSTFKCY GVSPTKLNDL CFTNVYADSF VIRGDEVRQI APGQTGKIAD YNYKLPDDFT GCVIAWNSNN LDSKVGGNYN YLYRLFRKSN LKPFERDIST EIYQAGSTPC NGVEGFNCYF PLQSYGFQPT NGVGYQPYRV VVLSFELLHA PATVCGPKKS TNLVKNKCVN FNFNGLTGTG VLTESNKKFL PFQQFGRDIA DTTDAVRDPQ TLEILDITPC SFGGVSVITP GTNTSNQVAV LYQDVNCTEV PVAIHADQLT PTWRVYSTGS NVFQTRAGCL IGAEHVNNSY ECDIPIGAGI CASYQTQTNS PGSASSVASQ SIIAYTMSLG AENSVAYSNN SIAIPTNFTI SVTTEILPVS MTKTSVDCTM YICGDSTECS NLLLQYGSFC TQLNRALTGI AVEQDKNTQE VFAQVKQIYK TPPIKDFGGF NFSQILPDPS KPSKRSFIED LLFNKVTLAD AGFIKQYGDC LGDIAARDLI CAQKFNGLTV LPPLLTDEMI AQYTSALLAG TITSGWTFGA GAALQIPFAM QMAYRFNGIG VTQNVLYENQ KLIANQFNSA IGKIQDSLSS TASALGKLQD VVNQNAQALN TLVKQLSSNF GAISSVLNDI LSRLDPPEAE VQIDRLITGR LQSLQTYVTQ QLIRAAEIRA SANLAATKMS ECVLGQSKRV DFCGKGYHLM SFPQSAPHGV VFLHVTYVPA QEKNFTTAPA ICHDGKAHFP REGVFVSNGT HWFVTQRNFY EPQIITTDNT FVSGNCDVVI GIVNNTVYDP LQPELDSFKE ELDKYFKNHT SPDVDLGDIS GINASVVNIQ KEIDRLNEVA KNLNESLIDL QELGKYEQGS GYIPEAPRDG QAYVRKDGEW VLLSTFLGRS LEVLFQGPGH HHHHHHHSAW SHPQFEKGGG SGGGGSGGSA WSHPQFEK |
-Experimental details
-Structure determination
Method | negative staining |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 3 mg/mL | ||||||||||||
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Buffer | pH: 8 Component:
Details: Samples were stored in buffer at 4 degrees C for 1 week, then moved to a 37 degrees C heat block for 3 hours prior to dilution and preparation of negatively stained grids. | ||||||||||||
Staining | Type: NEGATIVE / Material: Uranyl Formate Details: Samples were diluted to 0.1 mg/mL in 20 mM HEPES buffer, pH 7.4, with 5% glycerol, 150 mM NaCl, and 7.5 mM glutaraldehyde. After 5 minute incubation at room temperature, sufficient 1 M Tris ...Details: Samples were diluted to 0.1 mg/mL in 20 mM HEPES buffer, pH 7.4, with 5% glycerol, 150 mM NaCl, and 7.5 mM glutaraldehyde. After 5 minute incubation at room temperature, sufficient 1 M Tris stock, pH 7.4, was added to a final concentration of 75 mM Tris to quench unreacted glutaraldehyde, and was then incubated 5 minutes. Sample was then applied to a carbon film over 400 mesh copper EM grids that had been glow-discharged, incubated 1 minute, and then stained with 2% uranyl formate. | ||||||||||||
Grid | Model: Homemade / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Support film - Film thickness: 0.05 nm / Pretreatment - Type: GLOW DISCHARGE |
-Electron microscopy
Microscope | FEI/PHILIPS EM420 |
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Image recording | Film or detector model: OTHER / Digitization - Dimensions - Width: 2048 pixel / Digitization - Dimensions - Height: 2048 pixel / Digitization - Sampling interval: 33.0 µm / Number grids imaged: 1 / Number real images: 124 / Average exposure time: 0.5 sec. / Average electron dose: 32.0 e/Å2 |
Electron beam | Acceleration voltage: 120 kV / Electron source: LAB6 |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.0 mm / Nominal defocus max: 1.5 µm / Nominal defocus min: 0.4 µm / Nominal magnification: 82000 |
Sample stage | Specimen holder model: SIDE ENTRY, EUCENTRIC |