2020-11-25 REL PDBe PDBe PDBe 2019-09-18 2020-07-29 2020-07-29 2020-11-25 Biotechnology and Biological Sciences Research Council BRIC PhD, BBSRC Reference BB/K02034X/1 United Kingdom Hepatitis B virus core protein virus-like particle displaying the antigen: extra cellular adhesion domain NadA from Neisseria meningitidis. Collins RF Roseman AM Derrick JP Aston-Deaville S Carlsson E Saleem M Thistlethwaite A Chan H Maharjan S Facchetti A Feavers IM Alistair Siebert C Collins RF Roseman A Derrick JP An assessment of the use of Hepatitis B Virus core protein virus-like particles to display heterologous antigens from Neisseria meningitidis. Vaccine NE 38 3201 3209 2020 32178907 doi:10.1016/j.vaccine.2020.03.001 1873-2518 EMD-10316 associated EM volume VLP fusion sequence expressed in E.coli. VLP fusion sequence expressed in E.coli. 0 1
HBcS-NadA-CFHbp Fusion protein with linker regions. HBc - Ordered core of VLP: from HBV core protein (NCBI taxonomy ID 10407). NadA - Partially ordered surface displayed domain: extracellular domain of NadA from Neisseria meningitidis (NCBI taxonomy ID 487). CFHbp - Unresolved/disordered component : C-terminal domain of factor H binding protein from Neisseria meningitidis (NCBI taxonomy ID 487).
Neisseria meningitidis Escherichia coli BL21 pET-17b 14.9 HBcS-NadA-CFHbp
expressed fusion chimera protein
LEVO MDIDPYKEFGATVELLSFLPSDFFPSVRDLLDTASALYREALESPEHCSPHHTALRQAI LCWGELMTLATWVGNNLEDGGGGSGGGGSATSDDDVKKAATVAIVAAYNNGQEINGFKAG ETIYDIGEDGTITQKDATAADVEADDFKGLGLKKVVTNLTKTVNENKQNVDAKVKAAESE IEKLTTKLADTDAALADTDAALDETTNALNKLGENITTFAEETKTNIVKIDEKLEAVADT VDKHAEAFNDIADSLDETNTKADEAVKTANEAKQTAEETKQNVDAKVKAAETAAGKAEAA AGTANTAADKAEAVAAKVTDIKADIATNKADIAKNSARIDSLDKNVANLRKETRQGLAEQ AALSGLFQPYNVGEFGGGGSGGGGSRDLVVNYVNTNMGLKIRQLLWFHISCLTFGRETVL EYLVSFGVWIRTPPAYRPPNAPILSTLPETTVVGSGGGTHTSFDKLPEGGRATYRGTAFG SDDAGGKLTYTIDFAAKQGNGKIEHLKSPELNVDLAAADIKPDGKRHAVISGSVLYNQAE KGSYSLGIFGGKAQEVAGSAEVKTVNGIRHIGLAAKQKLGGGWSHPQFEK singleParticle particle 1 8
PBS
 Quantifoil R2/2 COPPER 400 GLOW DISCHARGE AIR 101.325 ETHANE 90 295 FEI VITROBOT MARK IV
3 ul applied for 30s at room temperature, then 4-5 s blot..
FEI TITAN KRIOS FLOOD BEAM BRIGHT FIELD FIELD EMISSION GUN 300 70.0 2.7 0.001 0.424 0.0025 3.84 104167.0 100719.0 FEI TITAN KRIOS AUTOGRID HOLDER NITROGEN FEI FALCON III (4k x 4k) INTEGRATING 4096 4096 14.0 3-36 1 2367 4.0 79.0 1 10119
Initially ~44,000 particles were initially picked and extracted in EMAN. Approx 12,000 were shell fragments or incomplete. Others were intact but lower resolution, so damaged or affected by charging, or other issue. After filtering/cleaning by EMAN2 2D classification 10119 particles were taken forwards for initial 3D reconstruction in EMAN2. This set of 10119 was then 2D cleaned by CryoSPARC to give 9145 particles.
 EMAN2 2.01 then CTF applied by CryoSPARC I 3.4 FSC 0.143 CUT-OFF cryoSPARC v0.65 8598 OTHER cryoSPARC v0.65 MAXIMUM LIKELIHOOD cryoSPARC v0.65 3 cryoSPARC v0.65
model #0 94.2 % mode l#1 4.6 % model #2 1.2%
 emd_10316.map.gz 1 IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) 512 512 512 0 0 0 512 512 512 711.68 711.68 711.68 90.0 90.0 90.0 X Y Z -1.1796018 4.9631643 0.011435016 0.23314595 1.39 1.39 1.39 1.5 AUTHOR Hepatitis B virus core protein with the extracellular domain of NadA inserted at the tip of the spikes of the VLP/capsid spikes. emd_10316_additional_1.map.gz 1 IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) 512 512 512 0 0 0 512 512 512 711.68 711.68 711.68 90.0 90.0 90.0 X Y Z -0.59937704 2.8089573 0.011435016 0.18962559 1.39 1.39 1.39 AUTHOR Unsharpened map emd_10316_additional_2.map.gz 1 IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) 512 512 512 0 0 0 512 512 512 711.68 711.68 711.68 90.0 90.0 90.0 X Y Z -0.59937704 2.8089573 0.011435016 0.18962559 1.39 1.39 1.39 AUTHOR Mask for FSC calculation emd_10316_half_map_1.map.gz 1 IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) 512 512 512 0 0 0 512 512 512 711.68 711.68 711.68 90.0 90.0 90.0 X Y Z -0.64066875 2.8184156 0.007166497 0.19254555 1.39 1.39 1.39 AUTHOR Half map A. emd_10316_half_map_2.map.gz 1 IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) 512 512 512 0 0 0 512 512 512 711.68 711.68 711.68 90.0 90.0 90.0 X Y Z -0.64951 2.833559 0.0068834 0.19220723 1.39 1.39 1.39 AUTHOR Half map B.