Structural basis for targeting human cancer antigen STEAP1 with antibodies

Six-transmembrane epithelial antigen of the prostate (STEAP1) is an integral membrane protein that is highly upregulated on the cell surface of several human cancers, making it a promising therapeutic target. It shares sequence homology with three enzymes (STEAP2-4) that catalyze the NADPH-dependent reduction of iron(III). However, STEAP1 lacks an intracellular NADPH-binding domain and does not exhibit cellular ferric-reductase activity. Thus, both the molecular function of STEAP1 and its role in cancer progression remain elusive. Here, we present a ~3.0 Å cryo-electron microscopy structure of trimeric human STEAP1 bound to three Fab-fragments of the clinically employed antibody mAb120.545. STEAP1 adopts a reductase-like conformation and interacts with the Fabs through its extracellular helices. Enzymatic assays in human cells reveal that STEAP1 promotes iron(III) reduction when fused to the intracellular NADPH-binding domain of its family member STEAP4, implicating STEAP1 as a functional ferric reductase in STEAP hetero-trimers. Our work provides a foundation for deciphering the molecular mechanisms of STEAP1 and will be instrumental in the design of new therapeutic strategies to target STEAP1 in cancer.


Introduction
Since its discovery in 1999 as multi-span membrane protein highly expressed on prostate cancer cells (1), six-transmembrane epithelial antigen of the prostate (STEAP1) emerged as cancer antigen expressed in various human cancers, which include prostate, bladder, colorectal, lung, ovarian, breast carcinoma and Ewing sarcoma. Because its expression in physiological tissue is minimal and mainly confined to the prostate gland (2), STEAP1 represents a potentially attractive therapeutic tool as both cancer biomarker and as a target for anti-cancer therapies (2)(3)(4). Indeed, several strategies for targeting STEAP1 in cancer have been explored; in 2007, a study reported the production and characterization of two monoclonal antibodies (mAb120.545 and mAb92.30) that bind STEAP1 with nanomolar affinity on prostate cancer cells and inhibit the growth of prostate and bladder-tumour xenografts in mice (5). More recently, clinical studies employing humanized variants of mAb120.545 that target STEAP1 were conducted, including: 1) a phase I trial using an antibody-drug-conjugate (termed DSTP3086S or Vandortuzumab Vedotin) to target prostate cancer (6)(7)(8), and 2) a combined Phase I / Phase II trial for the PET-imaging of metastatic castration-resistant prostate cancer patients using Zr 89 labelled antibody (termed [ 89 Zr]Zr-DFO-MSTP2109A) (9)(10)(11). Besides antibody-based strategies, several in vitro and in vivo studies revealed that STEAP1-derived peptides are immunogenic and thus suitable for recognition by cytotoxic T lymphocytes (12)(13)(14)(15)(16), indicating that STEAP1 could represent a potential candidate for the development of anticancer vaccines (4,17). STEAP1 belongs to a protein family that comprises three metalloreductases (18,19) (STEAP2-4, also known as STAMP1-3 (20)(21)(22) ), which reduce iron(III) and copper(II) and are also associated with cancer progression (23)(24)(25). At the molecular level, the four STEAP proteins are predicted to adopt a common architecture with intracellular N-and C-termini, six transmembrane helices and a single heme-b prosthetic group bound in the transmembrane domain (TMD) (26). STEAP2-4 additionally contain an intracellular oxidoreductase domain (OxRD) that binds nicotinamide-adenine dinucleotide phosphate (NADPH) (27,28). The ferric and cupric reductase mechanism of STEAP2-4 is defined by electron transfer from intracellular NADPH through membrane-embedded flavin-adenine dinucleotide (FAD) and heme cofactors to chelated metal-ion complexes at the membrane extracellular side (26,29). Contrary to STEAP2-4, STEAP1 does not exhibit metalloreductase activity when over expressed on mammalian cells (19), suggesting that it may have a distinct, yet unidentified function.
However, a recent study revealed that dithionite-reduced, purified STEAP1 retains heme and is capable of reducing metal-ion complexes and oxygen (30), indicating that the absence of a binding site for an electron-donating substrate like NADPH could explain the lack of reductase activity for STEAP1. It has been proposed that STEAP1 may have a functional role in heterooligomeric complexes with other STEAP paralogs (19,30). In support, its expression often correlates with the expression of STEAP2 in cancers (17) and both proteins co-purify in detergent (30), suggesting they could form a functional complex. Further indications for a functional hetero-trimeric STEAP complex emerged from the recent cryo-EM structures of homo-trimeric human STEAP4 (29), which revealed a domain-swapped architecture, with the intracellular OxRD positioned beneath the TMD of the adjacent protomer. This arrangement supports a model in which the heme in STEAP1 receives electrons from NADPH bound to an adjacent STEAP2/3/4 subunit. However, the in vivo redox activity of STEAP1, in both the absence and presence of other STEAP paralogs, remains to be established. In addition, there are no high-resolution structures available to help distinguish a functional role for STEAP1 as metalloreductase or, as previously proposed, a potential channel or transporter protein (1, 2, 5, 31). Thus, despite that STEAP1 is a populous plasma-membrane component of many different types of cancer cells and hence a promising novel therapeutic target, its structure and function in both health and disease remain unknown.
Here, we present the cryo-EM structure of full-length, trimeric human STEAP1 bound to three Fab fragments of the therapeutically relevant mAb120.545. The Fabs dock on the extracellular helices of STEAP1 through an extensive polar interface. The TMD of STEAP1 resembles the architecture of the STEAP4-TMD, and surprisingly, exhibits cellular ferric reductase activity when fused to the NADPH-binding OxRD of STEAP4.

Biochemical characterization of STEAP1
A previous pioneering study reported the biophysical and electrochemical characterization of N-terminally truncated rabbit STEAP1, purified from insect cells in lauryl maltose neopentyl glycol (LMNG) detergent (30). Our initial attempts to purify full-length, human STEAP1 from mammalian HEK-cells using a similar protocol were hampered by the loss of the noncovalently bound heme-b cofactor during the purification, suggesting that STEAP1 was not natively folded. We therefore screened several other detergents for the solubilization of STEAP1 and identified digitonin as a suitable replacement for LMNG. In digitonin, the purified protein retained its heme cofactor (Fig. 1A) and eluted as a monodisperse peak in size exclusion chromatography (SEC) experiments (Fig. 1B, D). In addition, thermostability assays revealed a melting temperature (TM) of ~55.5 o C for STEAP1 in digitonin (Fig. 1D, E), indicating that the protein was stable outside its native membrane environment. To assess if purified STEAP1 adopted a physiological-relevant conformation, we tested the binding of STEAP1 to the Fab fragment of monoclonal antibody mAb120.545, which exhibits 1 nM affinity for STEAP1 on cells (5). Size-exclusion chromatography assays revealed a smaller elution volume for STEAP1 when it was premixed with Fab120.545 (Fig. 1B), suggesting the formation of a complex, which was then confirmed by SDS page gel analysis of the eluted sample (Fig. 1C). Thus, the conformation of the STEAP1-epitope recognized by the Fab fragment on cells is preserved during the detergent solubilization and purification of STEAP1.

Cryo-EM structure determination
To gain insights into the molecular architecture of STEAP1, we set out to obtain a structural model of the protein using single-particle cryo-EM. Full-length, trimeric STEAP1 proved to be a challenging sample for EM due to its small size (<120 kDa) and the absence of folded domains protruding from the membrane region. To create a larger particle with more extramembrane features to facilitate EM-image processing, we opted to determine the structure of STEAP1 purified in complex with Fab120.545. The complementarity-determining regions (CDRs) of Fab120.545 are identical to those present in STEAP1-antibodies used in clinical trials ( Fig. S1), indicating that the structure of the STEAP1-Fab120.545 complex could also be instrumental in engineering antibodies and other molecules that target STEAP1 in cancer.
Micrographs collected on a 200 kV Talos Arctica microscope showed non-aggregated particles distributed in vitreous ice (Fig. S2A). Subsequent 2D classification experiments yielded class averages with clear secondary structure elements and furthermore revealed that more than one Fab fragment is bound to micelle-embedded STEAP1 ( Fig. 2A, Fig. S2B). Image processing in Relion (32) finally resulted in a reconstructed cryo-EM-density map at ~3.0 Å resolution ( Fig. 2B, Fig. S2C-F). The map displayed well-defined sidechain density for the TMD of STEAP1 and the variable regions of the Fab (Fig. S3). The model for STEAP1 was built with the TMD of STEAP4 as the template (29) (pdb 6HCY), whereas the starting model for the Fab was generated through the PIGS homology server (33). The refined structure has acceptable stereochemistry and exhibits high correlation to the cryo-EM density map within the determined resolution (Fig. S3G, H, Table 1).

Architecture
The cryo-EM structure reveals a one-to-one stoichiometry of the STEAP1-Fab120.545 complex, with three STEAP1 protomers interacting with three Fab molecules ( Fig. 2B-D). The Fabs bind at the extracellular region of STEAP1, consistent with the observation that the antibody targets STEAP1 expressed on intact cancer cells. The intracellular loops of STEAP1 extend ~18 Å from the membrane region into the cytoplasm, whereas the Fabs protrude up to ~75 Å into the extracellular space. STEAP1 adopts a trimeric arrangement that is strikingly similar to the one of its family member STEAP4 (41% amino-acid sequence identity, rmsd 0.8 Å for 640 Cα atoms, Fig. 3). Each STEAP1 subunit comprises six membrane-spanning αhelices (h1 -h6) that define the TMD of the protein. A single b-type heme cofactor is sandwiched between helices h2 -h5 at the extracellular membrane leaflet (Fig. S3B). Strictly conserved histidine residues H175 and H268 coordinate the central iron moiety of the heme prosthetic group, thereby resembling the hexa-coordinated heme arrangement of STEAP4. At the intracellular membrane leaflet side of the TMD, we observed weak density not corresponding to any protein residues. An overlay with the structure of STEAP4 revealed that the observed density overlaps with the flavin ring of the FAD-binding site in STEAP4 (Fig.   S3D). The FAD-interacting residues in the TMD of STEAP3 and 4 are conserved in STEAP1 and the STEAP1-Fab120.545 cryo-EM sample was supplemented with 1 mM FAD before grid freezing. However, STEAP3 and 4 additionally interact with the adenine-moiety of FAD via their intracellular OxRD (26,29) (Fig. 3B, Fig. S3D), which is missing in STEAP1. In line with this, STEAP3 and 4 exhibit a low micromolar affinity for FAD (Kd = ~1 µM) (26,29), whereas the affinity of STEAP1 for FAD is much weaker (Kd = 34 µM) (30). The cryo-EM density in this region could therefore correspond to a loosely bound FAD cofactor, although the weak density does not allow for modelling the complete cofactor. Instead of an OxRD of ~175 amino acids in length, STEAP1 contains a 69 residue N-terminal intracellular tail with no predicted domain architecture. Indeed, we observed no density for the first 65 intracellularamino acids of STEAP1, indicating that these residues are flexible, which is consistent with insilico disorder predictions using RONN (34). Thus, contrary to other STEAP family members, the homo-trimeric human STEAP1 structure does not harbor a folded N-terminal intracellular domain.

STEAP1-Fab120.545 interface
We next analyzed the interface between STEAP1 and Fab120.545. The epitope recognized by the Fab spans the first and second extracellular regions (EC1 and EC2, respectively) of STEAP1, which bridge membrane helices h1-h2 and h3-h4. The total interface formed between the STEAP1 trimer and three Fabs comprises ~5,730 Å 2 of buried surface area and is arranged so that a single Fab molecule interacts with all three STEAP1 protomers. The interface is stabilized by a network of polar and hydrophobic interactions and can largely be described by two 'interaction hotspots' on the EC2 of STEAP1 (Fig. 4A, B). The first hotspot involves the extracellular helix of STEAP1 (residues 186 -201), which extends from membrane helix h3.
Y190, N194, W195 and Q198 interact with Fab heavy and light chain residues Y102H, Y104H, Y108H, Y31L and S33L (Fab chain identifier in subscript) (Fig. 4A). The carboxyl group of S33L furthermore forms a hydrogen bond with the sidechain of Y107 of the EC1, whereas Y104H bridges two STEAP1 protomers by interacting with both Y190 and W195 from different chains. The second hotspot comprises residues Q201, Q202, N203, Q205 and D206, which reside in the loop that connects the extracellular helix of STEAP1 to membrane helix h4.
Hotspot-two consists of numerous polar interactions, including a salt bridge between D206 and R32L. Other Fab residues involved in binding to hotspot-two are Y51H, S57H, T58H, S59H, Q27L, S33L,N99L and Y100L (Fig. 4B). Besides the Fab residues in close vicinity (<4 Å) to STEAP1, we identified three aspartate residues (D103H, D105H, D106H) in the Fab heavy chain that orient towards the basic amino-acid ring above the heme (Fig. 4C). These aspartates are at least 4.5 Å separated from any STEAP1-residues and could participate in long-range electrostatic interactions with the basic ring of STEAP1. Interestingly, in STEAP4 the corresponding basic amino-acids comprise the substrate-binding site (Fig. 4D). To experimentally verify the STEAP1-Fab interface observed in the structure, we generated several mutants of Fab120.545 and tested their ability to bind purified STEAP1 using sizeexclusion chromatography assays (Fig. S4). Mutants R32LE and N99LD were designed to create charge repulsions in hotspot-2 between STEAP1 and Fab120.545. As expected, we did not observe binding events for these two mutants ( Fig. S4A-C). Intriguingly, Fab mutants D103HN,D105HN,D106HN (Fab-NNN) and D103HA,D105HA,D106HA (Fab-AAA) similarly did not interact with purified STEAP1 (Fig. S4A, D, E), indicating that the long-range electrostatic interactions between the three Fab-aspartates and STEAP1 are essential for maintaining a high-affinity antibody-antigen complex.

Generation of a functional STEAP4/1 fusion protein
The reductase-like architecture of STEAP1 (Fig. 3) and its heme-redox potential of -114 to -118 mV (30), indicate that the protein could be functional in reducing metal-ion complexes in vivo. However, to the best of our knowledge there is currently no experimental data available that shows that STEAP1 is capable of reducing ferric iron in a physiological setting. Although STEAP1 may form relevant heterotrimeric complexes with other STEAP homologs (30), coexpressions of different STEAP family members will likely result in a mixed population of homo-and heterotrimers, making cellular ferric-reductase experiments difficult to interpret. To overcome these hurdles and to assess if the TMD of STEAP1 can direct electron transport across mammalian cell membranes, we aimed to design a construct in which the STEAP1-TMD is fused to the intracellular OxRD of another STEAP homolog. To this end, a sequence alignment of all human STEAP proteins revealed that STEAP4 and STEAP1 share a common three-amino acid LFP-motif at the start of membrane helix h1. Additionally, an overlay of their cryo-EM structures did not show any obvious clashes between the OxRD of STEAP4 and the TMD of STEAP1. Thus, we generated a construct that spans residues M1 -Q195 of STEAP4, the shared LFP motif and residues Q69 -L339 of STEAP1, which we termed STEAP4/1chimera ( Fig. 5A, B). We then expressed STEAP4/1chimera in HEK293 cells and compared its cellular reductase activity to cells expressing STEAP1 or STEAP4, using the physiologically relevant ferric-citrate as a substrate. Consistent with a previous study (19), over-expression of STEAP1 did not result in measurable cell-surface ferric-reductase activity compared to the empty-vector control, whereas cells expressing STEAP4 reduced ~57 pmol Fe 3+ per minute per well (Fig.   5C). Intriguingly, the STEAP4/1chimera expressing cells also showed a highly significant reductase activity of ~43 pmol Fe 3+ per minute per well (Fig. 5C). To verify that the observed activity was dependent on transmembrane-electron transport through the TMD of STEAP1, we additionally tested STEAP4/1chimera mutants R161E and H175A (STEAP1 numbering) in which, respectively, the FAD and heme-binding sites in the TMD are abolished. Cells expressing STEAP4/1chimera-R161E and STEAP4/1chimera-H175A did not exhibit any ferric reductase activity (Fig. 5C), indicating that the STEAP1-TMD of the chimera is indeed crucial for cell-surface iron reduction. Confocal microscopy experiments subsequently confirmed that all expressed proteins except for STEAP4/1chimera-H175A localize to the plasma membrane ( Fig. S5). Taken together, our cell-based experiments reveal that STEAP1 adopts a conformation that facilitates transmembrane-electron transport to reduce ferric citrate at the membrane extracellular side. Therefore, the lack of reductase activity of the protein can be explained by the absence of a binding site for an electron-donating substrate in homo-trimeric STEAP1.

Fab120.545 inhibits ferric reductase activity of STEAP4/1chimera
Because Fab120.545 binds in close proximity to the putative substrate-binding site in STEAP1 ( Fig. 4C, D), we assessed if Fab binding could influence the ferric reductase activity of STEAP4/1chimera. Indeed, the addition of 4 µM Fab120.545 to cells expressing STEAP4/1chimera led to a significant decrease in iron-citrate reduction from ~43 to ~18 pmol Fe 3+ per minute per well (Fig. 5C). Conversely, the activity of STEAP4 was unchanged, indicating that the Fab specifically recognizes an extracellular epitope of STEAP1. We further characterized the inhibitory properties of the Fab by incubating cells expressing STEAP4 or STEAP4/1chimera with different Fab concentrations. This revealed a Fab-concentration dependent effect on the inhibition of the ferric reductase activity of STEAP4/1chimera, while the amount of iron(III) reduced by STEAP4 remained unaltered over the entire concentration range tested (Fig. 5D).

Discussion
Based on its amino-acid sequence and subcellular localization, STEAP1 was previously predicted to function as a channel or transporter protein (1, 2, 5, 31). The cryo-EM structure of STEAP1 bound to antibody-fragment Fab120.545 revealed a trimeric arrangement similar to the one of its family member STEAP4 (Fig. 3), showing no obvious structural features of ion channels or transporters. Instead, the 1) strictly conserved FAD-binding residues and putative loosely-bound FAD at the inner-membrane leaflet of the TMD (Fig. S3D), 2) heme-binding site at the outer-membrane leaflet (Fig. S3B) and 3) basic amino-acid ring above the heme (Fig.   4C), all imply that STEAP1 may function as a transmembrane oxidoreductase. In our previous work, we showed that substrate iron(III), complexed to a negatively-charged chelator like citrate, binds in the basic ring of STEAP4 (Fig. 4D) and we proposed that this ring of positive amino acids may polarize the iron(III)-chelator complex to facilitate the iron-reduction reaction (29). The presence of a comparable positively charged ring (Fig. 4C, D) indicates that STEAP1 harbors a similar protein environment for the reduction of iron(III).
We investigated whether the TMD of STEAP1 is capable of directing transmembrane-electron transport by generating a fusion construct between the intracellular region of STEAP4 and the TMD of STEAP1, termed STEAP4/1chimera (Fig. 5A, B). Cell-surface expressed STEAP4/1chimera catalyzed the reduction of iron-citrate, providing evidence that STEAP1 is a functional reductase, albeit only when a binding site for intracellular electron-donating substrate NADPH is available (Fig. 5C, D). The lack of enzymatic activity of STEAP4/1chimera-R161E (Fig. 5C), that localizes to the plasma membrane in HEK-cells (Fig. S5D), confirmed that the TMD of STEAP1 enables transmembrane-electron transport. We also showed that STEAP4/1chimera-H175A exhibited no significant ferric-reductase activity (Fig. 5C). However, fluorescence microscopy experiments revealed that this mutant resides almost exclusively in intracellular compartments (Fig. S5E), suggesting that the protein misfolds when the hemecofactor binding site is abolished.
The addition of Fab120.545 resulted in a concentration-dependent decrease of ferric reductase activity of STEAP4/1chimera-expressing cells, but not of STEAP4-expressing-cells (Fig. 5C, D).
This demonstrates that the Fab likely does not interact with STEAP4, which can be explained by the observation that the glycan on STEAP4-residue N323 would clash with the Fab light chain. In contrast, the residue at the equivalent position in STEAP1 (N194) is not glycosylated and forms a hydrogen bond with S33L (Fig. 4A).
How does Fab120.545 inhibit the cell-surface ferric reductase activity of STEAP4/1chimera? The conformations of the extracellular regions of STEAP4 and Fab-bound STEAP1 are similar ( Fig. 3), so the binding of Fab120.545 is not expected to induce large conformational changes in STEAP1. Instead, the Fab partially blocks access to the putative substrate-binding site in the basic ring above the heme (Fig. 4C). Alternatively, Fab residues D103H, D105H and D106H might neutralize the positive-charged substrate-binding site and thereby prevent substrate polarization. However, mutagenesis of these aspartates to either asparagines (Fab-NNN) or alanines (Fab-AAA) resulted in a loss of binding of Fab120.545 to purified STEAP1 (Fig. S4A, D, E), hence this hypothesis could not be tested. Nevertheless, our results suggest that antibodies can be employed as tools to inhibit the ferric-reductase activity of STEAP enzymes.
Additionally, the 3:3 STEAP1:Fab120.545 stoichiometry (Fig. 2B, C) indicates that full-length antibodies may crosslink STEAP1 trimers into higher-order assemblies on cell membranes. A similar antibody-induced crosslinking mechanism has recently been reported for the therapeutic antibody rituximab binding to its dimeric target protein CD20 (35).
In conclusion, the study presented here describes the first structure-function analysis of the human cancer antigen STEAP1. Our results support a model in which STEAP1 forms heteromeric assemblies with partner proteins that recruit and orient intracellular electrondonating substrates towards the TMD of STEAP1, enabling transmembrane-electron transport and the reduction of extracellular metal-ion complexes. This model warrants further investigations into the physiological function of STEAP1; for example, the incorporation of STEAP1 into STEAP heterotrimers might moderate iron(III)-reduction rates locally and thereby prevent deleterious reactions associated with iron overload. We therefore envision that it will be of great interest to focus future research endeavors on these putative assemblies of STEAP1 with STEAP2-4 family members and other unidentified accessory proteins in relevant cancer tissues. Ultimately, understanding the molecular principles that underly the function of STEAP1 will guide the design of anti-STEAP1 focused cancer therapies, thereby exploiting the protein's high expression in cancer and minimal presence in healthy cells.

Chemicals
All chemicals were purchased from Sigma-Aldrich unless specified otherwise.

Constructs
Codon-optimized DNA for mammalian cell expression encoding for human STEAP1 and STEAP4 was purchased from Geneart. The full-length STEAP1 construct used for structure determination was cloned in a pUPE expression vector (U-Protein Express BV) with Cterminal Strep3 tag. The STEAP4/1chimera construct was generated through Gibson-assembly cloning (NEB). For functional assays in HEK-cells, all constructs were cloned in a pUPE expression vector with a C-terminal GFP-Strep3 tag with a TEV-protease site. Mutagenesis of STEAP construct was performed using the Q5 Site-Directed Mutagenesis Kit (NEB). The primers used in this study are listed in Table S1. In the manuscript, the amino-acid numbering of STEAP1 was used for mutations introduced in the STEAP4/1chimera (R161E and H175A), because the mutated amino acids reside in the STEAP1-domain of the chimera. These residues correspond to R290 and H304 in both STEAP4/1chimera and STEAP4.

Protein expression and purification
The protein production protocol was adapted from the previously described protocol for STEAP4 (29). Full-length STEAP1 with a C-terminal Strep3 tag was expressed in HEK293

EM data collection
Movie collection was performed on a 200 kV Talos Arctica microscope (Thermo Fisher Scientific) equipped with a K2 summit detector (Gatan) and a post column 20 eV energy filter.
Using EPU (Thermo Fisher Scientific) in superresolution/counting mode (pixel size 0.514 Å), movies were collected for 6.5 s in 26 frames with a dose of 1.905 e -/Å 2 /frame (measured in an empty hole without ice), corresponding to a total-electron exposure of 49.5 e -/Å 2 . Defocus values for collection in EPU were set between -1 and -3 µm, but varied between -0.4 and -3.5 µm during data collection.

Image processing
5,352 movies were imported in the Relion3.0 pipeline. The movies recorded in super-resolution mode were binned 2x (resulting pixel size 1.03 Å) and motion corrected using UCSF MotionCor2 (36), followed by CTF estimation using GCTF (37). 695 movies were subsequently discarded based on their poor CTF spectra, resulting in 4,657 movies (87% of total) for further processing. 1,791 particles were picked manually and 2D classified. The generated classes were used as templates for autopicking in Relion (38)

Model building and refinement
To build the model for STEAP1, the TMD structure of human STEAP4 (residues 196 -454) was rigid-body fitted into the cryo-EM map. For one chain, all STEAP4 residues were changed to the corresponding STEAP1 residue using the 'mutate residue range' option in Coot (40), after which the model was manually inspected, adjusted and refined in Coot. The model of this chain was copied and fitted in the density of the other two subunits. The starting model for the variable regions of Fab120.545 was obtained through the PIGS homology server (33) by uploading the heavy and light chain sequences. This model was rigid-body fitted in the cryo-EM map and the CDR regions were manually built in Coot. Then, the STEAP1-Fab120.545 model was iteratively refined using Coot (manually) and Phenix real-space refine (41) with geometric restraints and non-crystallographic symmetry (NCS) constraints. Final refinements were performed using the non-sharpened cryo-EM map, in which the constant region of the Fab was masked out. The non-sharpened map revealed sufficient side-chain detail for modelling. The final model uploaded to the pdb comprises residues 67 -312 of STEAP1, residues 1 -112 of the Fab120.545 light chain and residues 2 -122 of the Fab120.545 heavy chain.