2024-10-16 REL PDBe PDBe PDBe 2020-03-06 2020-05-20 2020-05-20 2024-10-16 Netherlands Organisation for Scientific Research (NWO) 731.015.201 Netherlands Cryo-EM structure of trimeric human STEAP1 bound to three Fab120.545 fragments Oosterheert W Gros P Integral membrane protein Heme-binding protein Oxidoreductase Antibody-antigen complex, MEMBRANE PROTEIN Oosterheert W Gros P Cryo-electron microscopy structure and potential enzymatic function of human six-transmembrane epithelial antigen of the prostate 1 (STEAP1). J.Biol.Chem. US 295 9502 9512 2020 32409586 doi:10.1074/jbc.RA120.013690 1083-351X 0071 JBCHA3 6y9b FULLOVERLAP Trimeric human six-transmembrane epithelial antigen of the prostate (STEAP1) bound to three Fab-fragments of antibody mAb120.545 Trimeric human six-transmembrane epithelial antigen of the prostate (STEAP1) bound to three Fab-fragments of antibody mAb120.545 0 1 2 3
STEAP1 was expressed in HEK293 GNT1- cells and purified in digitonin. Fab120.545 was a kind gift from Genmab BV and was produced upon our request.
Homo sapiens plasma membrane 0.134 Fab-fragment 1 of antibody mAb120.545 1 2 3 house mouse secreted Homo-trimeric STEAP1, chain a, b and c. 1 1 Homo sapiens plasma membrane Fab Fragment 2 of antibody mAb120.545 1 2 3 Mus musculus secreted Fab-fragment 3 of antibody mAb120.545 1 2 3 Mus musculus secreted Metalloreductase STEAP1 Homo sapiens 0.044183652 3 Homo sapiens LEVO MGSESRKDITNQEELWKMKPRRNLEEDDYLHKDTGETSMLKRPVLLHLHQTAHADEFDCPSELQHTQELFPQWHLPIKIA AIIASLTFLYTLLREVIHPLATSHQQYFYKIPILVINKVLPMVSITLLALVYLPGVIAAIVQLHNGTKYKKFPHWLDKWM LTRKQFGLLSFFFAVLHAIYSLSYPMRRSYRYKLLNWAYQQVQQNKEDAWIEHDVWRMEIYVSLGIVGLAILALLAVTSI PSVSDSLTWREFHYIQSKLGIVSLLLGTIHALIFAWNKWIDIKQFVWYTPPTFMIAVFLPIVVLIFKSILFLPCLRKKIL KIRHGWEDVTKINKTEICSQLAAAGAAWSHPQFEKGAAWSHPQFEKGAAWSHPQFEKGAA Q9UHE8 1.16.1.- Fab120.545 light chain Mus musculus 0.024344134999999996 3 Homo sapiens LEVO DIVMSQSPSSLAVSVGEKVTMSCKSSQSLLYRSNQKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLT ISSVKAEDLAVYYCQQYYNYPRTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNAL QSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Fab120.545 heavy chain Mus musculus 0.026149922 3 Homo sapiens LEVO DVQVQESGPGLVKPSQSLSLTCTVTGYSITSDYAWNWIRQFPGNKLEWMGYISNSGSTSYNPSLKSRISITRDTSKNQFF LQLISVTTEDTATYYCARERNYDYDDYYYAMDYWGQGTTLTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEP VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTHHHHHH PROTOPORPHYRIN IX CONTAINING FE 0.000616487 3 HEM 1,2-DIMYRISTOYL-SN-GLYCERO-3-PHOSPHATE 0.000591777 3 XP4 singleParticle particle 5.0 7.8 200.0 NaCl sodium chloride 20.0 Tris 0.08 digitonin 1.0 Flavin-Adenine dinucleotide (FAD)
Protein sample was subjected to size-exclusion chromatography in 20 mM Tris pH 7.8, 200 mM NaCl, 0.08% (w/v) digitonin. 1 mM FAD (final concentration) was added to the sample before vitrification.
 Quantifoil R1.2/1.3 GOLD 200 GLOW DISCHARGE ETHANE-PROPANE 100 293 FEI VITROBOT MARK IV
blot 4 seconds, blotforce 0.
STEAP1-Fab120.545 complex purified in digitonin. The sample was monodisperse.
 FEI TALOS ARCTICA FLOOD BEAM BRIGHT FIELD FIELD EMISSION GUN 200 50.0 2.7 1.0 0.4 3.0 3.5 130000.0 NITROGEN GIF Quantum LS 20
Data was collected on a 200 kV Talos Arctica, located at Utrecht University, the Netherlands.
GATAN K2 SUMMIT (4k x 4k) SUPER-RESOLUTION 1-26 1 5325 6.5 49.5 1 616302
Picking was performed in Relion.
 EMD-0199
First 3D classification was performed using the low-pass filtered EM map of STEAP4 with the interacellular domain removed. Classifications performed in Relion.
 C3 2.97 FSC 0.143 CUT-OFF RELION 3.1beta
Final reconstruction was generated in Relion.
169426 MAXIMUM LIKELIHOOD RELION 3.0
All processing performed in Relion.
 MAXIMUM LIKELIHOOD RELION 3.1beta
All processing performed in Relion.
 RELION 3.1beta emd_10735.map.gz 1 IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) 300 300 300 0 0 0 300 300 300 308.55 308.55 308.55 90.0 90.0 90.0 X Y Z -0.037630104 0.0994739 0.00015654201 0.002010027 1.0285 1.0285 1.0285 0.022 AUTHOR Unsharpened cryo-EM map of homotrimeric STEAP1 bound to three Fab-fragments of antibody mAb120.545. The constant region of the Fabs is masked out. 6HCY A 196-454 PDB experimental model RIGID BODY FIT REAL 100.900000000000006 emd_10735_msk_1.map emd_10735_additional_1.map.gz 1 IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) 300 300 300 0 0 0 300 300 300 308.55 308.55 308.55 90.0 90.0 90.0 X Y Z -0.020785382 0.08284458 0.000062289175 0.002355529 1.0285 1.0285 1.0285 AUTHOR Unsharpened, unmasked cryo-EM map of homotrimeric STEAP1 bound to three Fab-fragments of antibody mAb120.545. emd_10735_additional_2.map.gz 1 IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) 300 300 300 0 0 0 300 300 300 308.55 308.55 308.55 90.0 90.0 90.0 X Y Z -0.13612334 0.21794279 0.00015668565 0.0035978141 1.0285 1.0285 1.0285 AUTHOR Sharpened cryo-EM map of homotrimeric STEAP1 bound to three Fab-fragments of antibody mAb120.545. The constant region of the Fabs is masked out. The map is autosharpened (B = -69 Angstrom) in Relion. emd_10735_half_map_1.map.gz 1 IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) 300 300 300 0 0 0 300 300 300 308.55 308.55 308.55 90.0 90.0 90.0 X Y Z -0.025007134 0.09345591 0.00005919613 0.0027577593 1.0285 1.0285 1.0285 AUTHOR Half map 1 of homotrimeric STEAP1 bound to three Fab-fragments of antibody mAb120.545. emd_10735_half_map_2.map.gz 1 IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) 300 300 300 0 0 0 300 300 300 308.55 308.55 308.55 90.0 90.0 90.0 X Y Z -0.025183843 0.09225871 0.000058130263 0.0027580417 1.0285 1.0285 1.0285 AUTHOR Half map 2 of homotrimeric STEAP1 bound to three Fab-fragments of antibody mAb120.545.