Full-length αIIbβ3 CryoEM structure reveals intact integrin initiate-activation intrinsic architecture

Summary

Integrin αIIbβ3 is the key receptor regulating platelet retraction and accumulation and a proven drug-target for antithrombotic therapies. Here we resolve the cryoEM structures of the full-length αIIbβ3, which covers three distinct states along the activation pathway. Firstly, we obtain the αIIbβ3 structure at 3Å resolution in the inactive state, revealing the overall topology of the heterodimer with the transmembrane (TM) helices and the ligand-binding domain tucked in a specific angle proximity to the TM region. After the addition of a Mn²⁺ agonist, we resolve two coexisting structures representing two new states between inactive and active state. Our structures show conformational changes of the αIIbβ3 activating trajectory and a unique twisting of the integrin legs, which is required for platelets accumulation. Our structure provides direct structural evidence for how the lower legs are involved in full-length integrin activation mechanisms and offers a new strategy to target the αIIbβ3 lower leg.

Graphical Abstract

eTOC Blurb

Tong et al. determine the cryo-EM structures of full-length integrin αIIbβ3 in different states, which facilitates the understanding of the activation mechanism of the integrin αIIbβ3.

Introduction

Platelets are fundamental to preventing hemorrhaging at sites of vascular injuries through hemostasis, a healthy response to injury intended to stop and prevent further bleeding. However, their functions are tightly controlled since abnormal thrombosis can cause life-threatening health problems when a clot obstructs blood flow through healthy blood vessels in the circulatory system.¹ Integrins are a major family of cell surface receptors which are essential to platelet activation.^2–6 Integrins are multifunctional, since they mediate the adhesion of cells to the extracellular matrix and to other cells and participate intracellular and extracellular signaling in diverse cellular processes .^5–8

αIIbβ3 is the major integrin expressed on platelets.^5,6 Both the αIIb and β3 protomers are single-pass transmembrane proteins,⁸ (also known as a bitopic protein) each with a short cytoplasmic tail and a large extracellular domain responsible for heterodimer and ligand interactions. Integrin αIIbβ3 plays an integral role in thrombosis.^9,10 Functionally, integrin αIIbβ3 transmits bidirectional signals across the cell membrane, in both “outside-in” and “inside-out” directions.¹¹ Upon receiving extracellular or intracellular signals, αIIbβ3 undergoes conformational changes to achieve an activated state. The activated αIIbβ3 serves as one of the key elements in platelet activation and thus plays a pivotal role in thrombosis.¹² αIIbβ3 direct involvement in the regulation of thrombosis makes it an appealing target for therapeutic strategy development.

Prior structural studies on integrin αIIbβ3 and its homologs have focused on individual regions namely the ectodomain and the headpiece.^13–16 The flexibility of the linkers between the ectodomain and transmembrane domain has posed challenges in elucidating certain aspects of the full-length structure. Specifically, details regarding the connection between the ectodomain and transmembrane (TM) domain, as well as the overall orientation of the integrin on the cell membrane, have remained elusive. This limitation has constrained our comprehensive understanding of the precise arrangement of each domain in relation to one another. The orientations between the ectodomain and the TM helices are fundamental to fully understanding the integrin conformational changes on the cell surface. Such knowledge will provide crucial insights into the mechanism of ligand binding modulation and of coupling between extra- and intra-cellular domains, which allows the signal to be transduced across the membrane. In the process of signal transduction, integrins need to undergo a conformational transition from a bent form to an open form. Previous research has discovered that integrin αIIbβ3 in nanodisc had four states during the activation process induced by RGD peptide, which demonstrates the conformational change from a bent form to an upright form.¹⁷ It was also proposed that Mn²⁺ can activate the integrins by perturbing the conformation of the extracellular domains and thus mimic the signaling process.^18–20 Several intermediate states between bent and open form were found based on the addition of Mn²⁺ by using SAXS or crystallography.^21,22 However, previous studies on the structural determination of integrin αIIbβ3 in open state either were restricted to the head region²³ or only yielded low resolution structures by electron microscopy of negatively stained protein.^15,17,24 While the current understanding of the integrin activation process suggests a substantial conformational change from the inactive to active states, obtaining detailed structural information about these intermediate states is crucial.^25,26 In this study, we present the structure of full-length integrin αIIbβ3 derived from platelets, which offers the opportunity to examine the integrin in its native context with physiologically relevant glycosylation. Our structure would provide new insights into the mechanism of integrin activation and its role in thrombosis.

Result

Overall structure of full-length integrin αIIbβ3 in its inactive state

We solved three unique structures of αIIbβ3 induced by different combinations of divalent cations: state A (5 mM Mg²⁺/1 mM Ca²⁺); state B (1 mM Mn²⁺/0.2 mM Ca²⁺); and state C (1 mM Mn²⁺/0.2 mM Ca²⁺), with states B and C co-existing, but separable from cryo-EM data.

In the absence of an exogenous activator, such as RGD peptide or Mn²⁺,²⁷ purified integrin αIIbβ3 exhibited a bent form, indicating the inactive state (state A) based on the overall features (Fig. 1 and 2, Supplemental Video 1).⁸ The head and leg region form a sharp angle (~70°) between each other, showing the orientation of the whole extracellular domain and characterizing the bent form of integrin αIIbβ3. The calf-2 domain from αIIb and tail domain from β3 connected to the corresponding single transmembrane helix anchoring on the detergent shell that mimics the cell membrane. The leg region of αIIbβ3 was found to form an angle of approximately 60 degrees to the horizontal direction, suggesting an orientation of αIIbβ3 on the cell surface if the TM helices vertically trans-through membrane. Due to no evidence for a specific orientation before, previous schematic models proposed in papers^28,29 depicted a vertically standing integrin with its head region facing the membrane by default. However, our structure showed αIIbβ3 adopted a tilted orientation that consequently would make the head region and the ligand binding site more accessible.

Figure 1. Cryo-EM single-particle reconstruction of inactive integrin αIIbβ3.

A) CryoEM map of αIIbß3 heterodimer. B) The structure of αIIbß3 heterodimer. The structure is composed of αIIb and ß3 molecules. Both integrin molecules within the αIIbß heterodimer, αIIb and ß3, are clearly identifiable and well resolved. All five αIIb domains (ß-propeller, thigh, Calf-1, Calf-2, and TM) and all nine ß3 domains (ßI, Hybrid, PSI, EGF-1,2,3,4, Tail, and TM) are clearly assigned. C) Domain demarcation along the integrin amino acid sequence based on the structural annotation. Each domain was colored differently in the map and structure, with the same color scheme used across all figures in this paper.

Figure 2. Structure of inactive integrin αIIbβ3.

A) Regions of integrin. Based on the domain organization, the whole integrin structure could be split into three regions: Head, Leg, and TM. The integrin stands on the cell membrane in a titled orientation, with a ~70° angle between the Head and Leg regions, and ~60° between the Leg and supposed cell membrane. B) The inset figures show the densities corresponding to the ion binding at MIDAS (Mg²⁺), ADMIDAS (Ca²⁺), SyMBS (Ca²⁺), Asn320 glycosylation and the TM region. (corresponding color in A: red, yellow, purple)

The ion binding pockets including SyMBS (synergistic metal ion-binding site), MIDAS (metal ion-dependent adhesion site), and ADMIDAS (adjacent to MIDAS) residing in the head region are well-resolved in our structure and the coordinated ions could also be identified. (Fig. 2). We also observed in the distal location of the ligand-binding site for the β-propeller, four Ca²⁺ ions coordinated with adjacent residues to further stabilize the β-sheets, which is in line with previously reported structures(Supplemental Figure. 2).

Since the relative orientation between the TM region and extracellular domain had yet to be determined before this study, excluding some low resolution structures by negative staining,³⁰ the structure resolved here that showed the structural connections between TM region and extracellular domain would help explain how these two domains are involved in the activation process. To deal with the low signal-to-noise ratio (SNR) and TM region flexibility, we employed 3D focus classification. The new map allowed us to effectively sort out particles and successfully resolve two helices from αIIb and β3, as well as the linker region between the Calf-2 domain and the αIIb helix. The two helices adopted a twisted conformation and went across each other forming a clasp at the membrane proximal site. The loop linker between the TM region and whole extracellular domain renders the potential flexibility for the integrin to separate the transmembrane helices in the presence of Mn²⁺.²⁰

Transition from inactive to a proposed intermediate state

The replacement of Mg²⁺ with Mn²⁺ would increase the ligand binding affinity of αIIbβ3 and triggers the activation of αIIbβ3, as deduced from recognition of Mn²⁺ induced conformation-specific antibodies.²⁷ We used Mn²⁺ during the purification and determined the αIIbβ3 structure in the presence of Mn²⁺. We found two new conformations (state B and C) with a particle ratio of 1:1, exhibiting domain movement and a partially opening. The structures were determined at 3.09 Å (state B) and 5.46 Å (state C) resolutions respectively. Previous structural studies of Mn²⁺ bound integrin in the open state, which were limited to a negative stained TEM result, provided no high resolution details.³¹ However, we proposed that the two structures obtained in this study would be representing two steps during activation.

Since this new structure in state B cannot be assigned to any known state, we proposed that this state could be an “intermediate state”, compared with the inactive state. Substantial conformational changes between the intermediate and inactive state of αIIbβ3 were found between these two structures. Readily discernible conformational changes and domain movements were found, even though both still adopt a bent orientation. To better elaborate the allosteric changes between these two states, β-propeller domains from inactive and intermediate states were superimposed and resultant relative orientations of other domains were examined (Fig. 3, Supplemental Video 2). Big movements mainly appear at the leg region including the Calf-1 (α-Calf-1) and Calf-2 (α-Calf-2) domains from αIIb, and the EGF-4 (β-EGF-4) and Tail (β-Tail) domain from β3 indicated by the RMSD values for the domains ranging from 2.7 to 8.4 Å (Fig. 3, Supplemental Video 3).

Figure 3. Transition from state A (inactive state) to state B (proposed intermediate state).

Domain shifts between state A and B are shown in the picture. The structure in state B is marked with colors, while the inactive state A is gray. The ß-propeller domain is superimposed to investigate shifts occurring on other domains, and C-alpha RMSD for each pair of domains is reported in the inset table. Regions I,II, and III recapitulate the movements the structure undergoes.

The movements appeared mainly in three regions (I, II, and III in Fig. 3). The region I showed a movement of α1 helix represented by a 2.6 Å shift in backbone at Asp126 from the βI domain, indicating the head region in the β3 molecule underwent a separation from the leg region. The region II has a split effect at the leg region, which is pulling apart the α-Calf-2 and β-Tail domain. Both the α-Calf-2 and β-Tail swing outwards separating the two legs, which is exemplified by the distance between α-Pro778 and β-Asn654 changing from 10.7 Å to 12.7 Å. The region III, mainly located at the β-EGF-4 domain, generated a shift moving away from the β3 head region as well as αIIb leg region. In summary, when integrin αIIbβ3 was treated with Mn²⁺/Ca²⁺ instead of Mg²⁺/Ca²⁺, αIIbβ3 exhibited domain movement shown as a separation between head region and leg region and thus a potential to open or extend. All three regions of movements happened simultaneously to potentially open the αIIbβ3. Region I, II, and III exhibited the potential to turn the head region, to separate the leg region, and to break the connection between the head and leg regions, respectively. Consequently, TM helices were also separated by the aforementioned movement, making the signal too weak to resolve the individual transmembrane helix in our final structure of this state.

A proposed pre-active form captured after the intermediate form

Given the conformation of the Mn²⁺ bound integrin αIIbβ3 is highly dynamic, as shown previously²¹ as well as by this study, we demonstrated both conformations coexist simultaneously in the same ratio. In our study, in addition to the intermediate form of αIIbβ3, another new form was also found during data processing, providing a new view of the molecular mechanism underlying the activation of αIIbβ3 (Fig. 4) in presence of Mn²⁺.²⁷ Compared to the state B (intermediate state), domain movement and rearrangement became more obvious at regions I, II, and III in state C, indicating this is a resultant or subsequent state after the intermediate state. We proposed state C could be a “pre-active” state between the intermediate and active state.

Figure 4. Transition from state B (proposed intermediate state) to state C (proposed preactive state).

Domain shifts between the state B and C are shown in the picture. The structure and label for state C is marked with colors, while state B is in gray. At regions I and II, α-Calf-2 includes two superimposed states, which shows a turning head region and simultaneous leg region separation. At region III, ßI and the hybrid domains in ß3 are superimposed to demonstrate the ß3 leg region swinging away.

When α-Calf-2 domains from state B and C are superimposed, variations at region I in the head region are the most obvious movement if viewed in the front (Fig. 4). As the β-βI domain separates from leg region, it forms a conformation that significantly turns in the counterclockwise direction (as the same direction shown in Fig. 4), which makes the head region move farther away from the membrane. At region II, the legs were separated wider than all other states (represented by movement of β-EGF4 domain), and interactions between the α-Calf-2 and β-Tail domain could have already been disrupted at this distance (Supplemental Figure 7). In addition, separation between the head and leg region also became more obvious in region III. The leg region, including β-EGF-2, 3, and 4, swung away from and thus formed a bigger angle with the head region. In summary, the pre-active state exhibited a subsequent conformation of the intermediate state reflected by the continuous movement occurring at regions I, II, and III. The resultant effect made the integrin αIIbβ3 undergo both intra-subunit separation (head and leg domain in β3) and inter-subunit separation (leg regions from αIIb and β3).

Discussion

Detergent-solubilized integrin αIIbβ3 has been extensively studied for cryo-EM SPA,³² tomography,³³ SANS (Small angle neutron scattering)³⁴ and negative staining EM^30,31,35. The activity and conformational change induced by Mn²⁺ were also validated by antibody-binding and SAXS data,³⁵ which demonstrates that detergent micelle is less likely to restrain the activation of integrin. The detergents used in this study, Triton X-100 and DDM, have been found optimal for stabilization and activity for intact αIIbβ3.³⁵

In this study, we resolve the structure of the integrin αIIbβ3 with the TM region in the inactive state, which indicates the possible orientation of αIIbβ3 in the physiological environment. The leg region is found to form a 60° angle with the supposed cell membrane, which resembles another newly reported full-length integrin αIIbβ3 structure in nanoparticles³⁶(Fig. 5a). When comparing our αIIbβ3 structure solved in detergent with the other structure solved in nanoparticles, we found that the leg region has conformational shift at Calf-2 domain and Tail domain when head region is superimposed (Fig. 5a). In addition, the transmembrane helices from the two structures also adopt two distinct conformations. Unlike the parallel and separate helices found in the nanoparticle structure, the helices in our structure still adopt a “crossing” conformation which is in line with the previous proposed inactive model.³⁷ It is possible that such shifts and different conformations are caused by different approaches employed to stabilize the transmembrane domain, however, it indicates the flexibility of the TM region and how it affects the leg region of integrin.

Figure 5. TM helix orientation and proposed model for activation transition.

A) TM region comparison between the structure of inactive state in this study (indicated by α2b, β3) and full-length structure in nanoparticles (indicated by α2b’, β3’ from PDB: 8T2V). Head region is superimposed and omitted. Orientational difference at TM region and conformational shift at α-Calf-2 and β-tail domain were shown. B) α1 helix movement during activation indicated by comparison with previous reported closed state (gray, PDB:3T3P) and open state (gold, PDB:2VDR)) and intermediate state in this study (cyan). C) Model for activation transition. The state B and C, both of which show the inter- and intra-molecular separation potential, would exist before the active state adopts a fully open form. Main movements start in state B at regions I, II, and III, while continuing through state C. The bent form with separated legs indicates that the leg motion precedes the head lift-up, which explains how the inside out signal transmits.

As previously described, the bent form of integrin would have a breathing motion and may shift the conformational equilibrium from bent state toward an open state.¹⁵ The coiled loop linking the extracellular domain and TM region renders the flexibility of the integrin so that the TM helices could have conformational change during activation. In our model, the distance from membrane to the “head” region is around 52.5 Å compared to 24.1 Å showed in previous suggested models,^38,39 which adopts similar orientation that found in the in situ study.³⁷ (Supplemental Figure 4) Since the head region, where the RGD-motif binds, is important in terms of ligand-binding, the longer distance to the platelet membrane would make the head region more accessible for the large population of integrin ligand.

As a cell membrane glycoprotein, integrin αIIbβ3 carries several N-glycans in the extracellular domain. However, it remains unclear how the glycans involved in integrin heterodimeric interaction and potential function during activation, it is tempting to speculate the N-glycans would facilitate structural changes during the activation of integrins.^40,41 In this study, we extracted the integrin αIIbβ3 directly from platelets to keep the structure close to the physiological state, especially for the glycosylation.^39,42,43 Without predictions or partially resolved,^{15,22,40,44,45} our structure resolves eight glycosylation sites in the intact integrin (Supplemental Figure. 3). We observed a previously proposed but never resolved glycan site Asn931. Extra density was found connected to Asn931 indicating that N-linked glycans are attached to this site (Supplemental Figure 3), though it was formerly predicted as a glycosylation site.⁴⁰ In addition, we observed the density for glycosylation at Asn320 from β3. Previously resolved structures either identified only two glycans in an ectodomain structure or five glycans in a head region only structure, which made it hard to clearly demonstrate how the glycans of Asn320 participates in the activation of the integrin. In contrast, in our full-length structure, there are five glycan residues that could be seen attached to Asn320. In this structure, we found connecting density between αIIb and β3-Asn320 glycans, and tentatively introduced a water molecule at this position to indicate the potential interactions. (Fig.2b) It is possible that in the context of blood circulation molecules other than water would be more favorable for this position. However, our structure exhibits the potential that the glycans of Asn320 may be involved in the αIIb and β3 interactions. The dimerization of αIIb and β3, which is the prerequisite to becoming a functional unit, is likely to be reinforced by this glycan-induced interaction.

In our study, we also resolved two co-existing structures in the presence of Mn²⁺, which was proposed to activate the integrin.^27,46,47 However, the density of Mn²⁺ ion is not distinguishable from that of Mg²⁺ due to the resolution limitation. Instead of an open form of integrin, both forms found in our study adopt the conformation between an inactive and fully active state, which indicated an “intermediate” and “pre-active” state. Previously, structural mapping focused on head piece revealed that eight different steps might exist during the activation process³⁸, demonstrated by the movement of α1 helix. Such proposed activation process, especially the shift that α1 helix undergoes, has yet to be fully validated in full-length integrins. We map our structure in state B to the proposed eight steps and found that it falls into the proposed moving trajectory (approximated to the proposed position 4 or 5²², see details in Fig. 5b). One previous study on full-length αIIbβ3 in nanodisc also reveals that it exists in a continuous conformational equilibrium composed by four main states.¹⁷ Induced by RGD peptide and Talin, αIIbβ3 would mainly adopt the extended and upright conformation. In contrast, we used Mn²⁺ to induce the activation and obtained two structures neither of which is in extended conformation. Instead of fully activating the integrin to an extended state, addition of Mn²⁺ detailed conformational change of head region before extension appears. Through comparison of all three structures solved in our study, we hypothesized they could represent a series of states before the active state (Fig. 5c). When treated with Mn²⁺, the integrin αIIbβ3 has the potential to be activated, which is hinted by the motion of leg region and head region. A further state may also exist before fully open form, showing a wider separation between the leg region and head region of αIIbβ3.

Upon activation, previous structure of β3-only fragments (contains the headpiece and β-knee regions but lacks the α unit) reported an extended conformation at the I-EGF1/2 junction making the leg almost form a right angle with the head region.^23,48 The increment of angle between the head and leg regions is also found in our pre-active structure, however, the angle is not as large as it was in the β3-only structure. Assuming the leg and head region move away from each other from an acute angle to a straight orientation during activation, it is possible the conformation found in our study is an initial activation state prior to the proposed activated state. Since Mn²⁺ has the potential to trigger the activation of αIIbβ3,^47,49–52 the bent form with separated leg region obtained in our study is in line with the previous hypothetical model,²² but provides a substantial and detailed structural basis. We discern two potential conformational states of integrin in the activation process, utilizing a universal Mn²⁺ activation buffer. However, it is imperative to note that the occurrence of this activation states in the platelet cell membrane (or under other recombinant near-physiological conditions) necessitates further test.

STAR Methods

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Zhao Wang (zhaow@bcm.edu)

Materials availability

This study did not generate new unique reagents.

Data and code availability

• All data are available either in this paper or in the public database. The final maps and models were submitted to the Electron Microscopy Data Bank (EMDB) (accession no. EMD-29931 , no. EMD-29932 and EMD-43046) and PDB (accession no.: 8GCD and 8GCE).

• This paper does not report the original code.

• Any additional information required to reanalyze the data reported is available from the lead contact upon request.

EXPERIMENTAL MODEL AND STUDY PARTICIPANT DETAILS

Platelet Sample

Human platelets used in this study were obtained from the Gulf Coast Regional Blood Center (Houston, TX, USA).

METHOD DETAILS

Protein extraction and purification

The integrin αIIbβ3 was initially extracted from human platelets obtained from the Gulf Coast Regional Blood Center (Houston, TX, USA). We purified WT full length integrin αIIbβ3 from human platelets by adapting a protocol described previously²¹. Cells were spun down at 1,200 rpm for 5 min and then resuspended in the CGS buffer (13.4 mM sodium citrate, 33.3 mM dextrose, 123 mM NaCl, pH 7.0) for several rounds until red blood cells were fully removed. The final supernatant was collected and centrifuged at 1200 x g for 30 min, and the resultant platelet pellet was resuspended using a TBS buffer. After several rounds of homogenization using a Dounce B set, the platelet lysis was spun down at 780 x g for 10 min to remove the cell debris followed by another centrifuge at 256,000 x g for 1 hour to collect the cell membrane fraction. The cell membrane fraction was solubilized at 4°C overnight in the 2% (w/v) Triton X-100 supplemented TBS buffer containing 10 mM Tris-HCl pH 7.4, 150 mM NaCl, 5 mM MgCl₂, and 1 mM CaCl₂. The solubilized membrane protein was collected from the supernatant after centrifugation at 256,000 x g for 1 hour. The supernatant was then applied to concanavalin-A, an affinity column (Con A Sepharose 4B), and eluted with a buffer containing methyl-α-D-mannopyranoside and 0.01%-0.02% (w/v) Triton X-100. Ion exchange chromatography (MonoQ) and size-exclusion chromatography (Superose 6) were completed using a buffer containing 20 mM Hepes, 150 mM NaCl, DDM (2x-4x CMC), 5 mM MgCl₂, and 1 mM CaCl₂. Peak fraction was collected for the cryoEM specimen preparation (Supplemental Figure 1). For integrin αIIbβ3 in the Mn²⁺ condition, the buffer was supplemented with 1 mM MnCl₂ and 0.2 mM CaCl₂ instead of Mg²⁺/Ca²⁺, while using the same purification protocols.

CryoEM sample preparation and data collection

For cryo-EM sample preparation, a 3 μl aliquot of purified integrin αIIbβ3 was applied onto a 200-mesh R3.5/1 Quantifoil 2nm-Cfilm grid. After applying the sample, the grid was blotted for 3 s and rapidly frozen in liquid ethane using a Vitrobot IV (FEI), with constant temperature and humidity during the process of blotting. The grid was stored in liquid nitrogen before imaging.

Movie stacks were collected at 300 kV on a Krios electron microscope (FEI) with an in-column energy filter (30 eV width) equipped with a direct electron detector K2 Summit camera (Gatan). Images were collected semi automatically by EPU (Thermo Fisher Scientific) in the dose fractionation super-resolution counting mode at a calibrated physical pixel size of 1.07 Å. The images were collected with a defocus range from −1.0 to −2.6 μm. The total exposure time for the dataset was 7 s, leading to a total accumulated dose of 50 electrons Ų on the specimen. Each image stack was fractionated into 35 subframes, each with an accumulation time of 0.2 s per frame. The final frame average was computed from averages of every three consecutive frames to correct beam-induced motion during exposure by MotionCor2.⁵³ The image in each frame was weighted according to radiation damage. CTF (Contrast Transfer Function) parameters of the particles in each frame average were determined by the program Patch CTF in cryoSPARC.⁵⁴

In total, 2,758,651 particle images were automatically boxed out by autopicking in cryoSPARC with a box size of 256 × 256 pixels using an averaged sum of 35 raw frames per specimen area. Two-dimensional (2D) reference-free class averages were computed using cryoSPARC. Initial models for every reconstruction were generated from scratch using selected good quality 2D averages with C1 symmetry based on the 2D averaged results. This initial model was low-pass–filtered to 60 Å, and refinements were carried out using cryoSPARC. After several rounds of homogeneous refinement and local refinement, the resolution achieved 3 A but with a weak signal in the TM region. To improve the quality of the TM region, aligned particles were transferred to RELION⁵⁵ for further processing. To deal with the low signal-to-noise ratio (SNR) and flexibility of the TM region, we employed 3D focus classification to further sort out particles. A spherical mask was created focusing on the TM region and applied to the 3D classification in RELION (Supplemental Figure 5). The final map was constructed by the combination of the extracellular domain and TM region.

For the map in the Mn²⁺ condition, the cryoSPARC processing workflow is similar to that for native structure (Supplemental Figure 6). In total, 1,701,301 particles were obtained after iterative 2D classification, and ~10% of particles were used for the initial model reconstruction. Each conformation was obtained from subclasses in the same Mn²⁺ dataset, and corresponding particles were separated for further processing. Two maps showing significant differences were obtained with the particle ratio 1:1.02. For state C, the EM map was refined to a proper resolution where domain information could be seen, while details for the secondary structure were still missing (Fig. 4). Individual domain from integrin αIIbβ3 was rigid-docking in the density. After individual refinement for each particle set, the two maps were finally refined to 3.09Å and 5.46 Å respectfully.

Cryo-EM model building and refinement

We started model building for full-length inactive integrin αIIbβ3 by docking the extracellular domain (PDBID:3FCS)¹⁵ and TM region (PDBID:2KNC).³⁷ The model was refined against the corresponding map using PHENIX⁵⁶ in real space with the secondary structure and geometry restraints. Since the EM density map is not confidently resolvable for every amino acid at the leg and TM regions due to the flexibility, we carried out rigid fitting of structures of the Calf-2 domain from αIIb, the Tail domain from β3, and the TM region. We used previously published TM region structure determined by Nuclear Magnetic Resonance (NMR) (2KNC) to build these parts of the model, instead of utilizing real-space refinement. The model was then manually refined and adjusted in Coot.⁵⁷ The model quality was validated using PHENIX and the local resolution was estimated using cryoSPARC. The aforementioned workflow applied to structure refinement in the inactive form and intermediate form, while the pre-active form was only applied with the rigid body-fitting for each domain due to low resolution.

QUANTIFICATION AND STATISTICAL ANALYSIS

Cryo-EM data collection and refinement statistics are shown in Table S1.

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Biological samples
Human platelets	Gulf Coast Regional Blood Center (Houston, TX, USA)	N/A
Chemicals, peptides, and recombinant proteins
Sodium citrate	Sigma Aldrich	Catalog Number: 71402
Dextrose	Sigma Aldrich	Catalog Number: G7879
Sodium chloride	Sigma Aldrich	Catalog Number: S6546
Triton X-100	Sigma Aldrich	Catalog Number: T8787
Tris-HCl	Fisher Scientific	Catalog Number: 15-567-027
MgCl2	Sigma Aldrich	Catalog Number: 68475
CaCl2	Fisher Scientific	Catalog Number: C79-500
DDM	Anatrace	Catalog Number: D310S
MnCl2	Thermo Fisher	Catalog Number: 011563-36
Hepes	Sigma Aldrich	Catalog Number: 83264-500ML-F
Deposited data
Inactive αIIbβ3 EM Map	This manuscript	EMDB- 29931
Intermediate αIIbβ3 EM Map	This manuscript	EMDB- 29932
Inactive αIIbβ3 Model	This manuscript	PDB- 8GCD
Intermediate αIIbβ3 Model	This manuscript	PDB- 8GCE
Pre-active αIIbβ3 EM Map	This manuscript	EMD-43046
Software and algorithms
cryoSPARC	Ali Punjani et al.54	https://guide.cryosparc.com/
Relion	Sjors Scheres et al.55	https://relion.readthedocs.io/en/release-5.0/
PHENIX	Afonine, P. V. et al.56	https://phenix-online.org/
COOT	Emsley, P et al.57	https://www2.mrc-lmb.cam.ac.uk/
Other
Con A Sepharose 4B column	Sigma Aldrich	Catalog Number: GE17-0440-03
MonoQ column	Cytiva	Catalog Number: 17115301
Superose 6 column	Cytiva	Catalog Number: 29091596

Highlights.

• Cryo-EM structures of full-length integrin αIIbβ3 purified from human platelets

• Three integrin αIIbβ3 states are revealed that are involved in the activation process

• Allosteric structural variations are likely important for the activation mechanism