PI3Kγ is a critical immune signaling enzyme activated downstream of diverse cell surface molecules, including Ras, PKCβ activated by the IgE receptor, and Gβγ subunits released from activated GPCRs. PI3Kγ can form two distinct complexes, with the p110γ catalytic subunit binding to either a p101 or p84 regulatory subunit, with these complexes being differentially activated by upstream stimuli. Here, using a combination of cryo electron microscopy, HDX-MS, and biochemical assays, we have identified novel roles of the helical domain of p110γ in regulating lipid kinase activity of distinct PI3Kγ complexes. We defined the molecular basis for how an allosteric inhibitory nanobody potently inhibits kinase activity through rigidifying the helical domain and regulatory motif of the kinase domain. The nanobody did not block either p110γ membrane recruitment or Ras/Gβγ binding, but instead decreased ATP turnover. We also identified that p110γ can be activated by dual PKCβ helical domain phosphorylation leading to partial unfolding of an N-terminal region of the helical domain. PKCβ phosphorylation is selective for p110γ-p84 compared to p110γ-p101, driven by differential dynamics of the helical domain of these different complexes. Nanobody binding prevented PKCβ-mediated phosphorylation. Overall, this work shows an unexpected allosteric regulatory role of the helical domain of p110γ that is distinct between p110γ-p84 and p110γ-p101 and reveals how this can be modulated by either phosphorylation or allosteric inhibitory binding partners. This opens possibilities of future allosteric inhibitor development for therapeutic intervention. The p11The fundamental roles of p110γ in inflammatory processes have made it a therapeutic target in many pathological conditions, including asthma , arthritHere, we report critical roles of the helical domain of p110γ in both activation and inhibition of lipid kinase activity. We characterized an allosteric inhibitory nanobody (NB7) that potently inhibits p110γ activity. Cryo-EM was used to define the inhibitory interface, which is composed of the helical domain, the ABD-RBD linker, and the regulatory motif of the kinase domain of p110γ. The region that the nanobody binds to is in close spatial proximity to a previously identified PKCβ phosphorylation site (S582) in the helical domain, and oncogenic activating mutants in the regulatory motif. We fully characterized the activity and dynamics of stoichiometrically PKCβ phosphorylated p110γ, leading to the discovery of a novel additional phosphorylation site (either S594 or S595). PKCβ phosphorylation was highly selective for p110γ and p110γ-p84, with limited phosphorylation of p110γ-p101. HDX-MS analysis showed that phosphorylation of p110γ leads to unfolding of the N-terminal region of the helical domain, and increased kinase activity. The presence of the inhibitory nanobody significantly blocks PKCβ phosphorylation, while phosphorylation of p110γ prevented binding to NB7. Overall, this work provides unique insight into the critical role of the helical domain in controlling p110γ activity, and how phosphorylation and binding partners can modify this regulation. It also reveals a unique binding site located at the interface of the helical and kinase domain that can be targeted for future allosteric inhibitor design.We previously identified multiple nanobodies that inhibited the activity of p110γ. One from this group (denoted NB7 throughout the manuscript) potently inhibited the membrane-mediated activation of p110γ-p84 by both Ras and Gβγ, with HDX-MS experiments mapping the NB7 binding interface to the RBD, helical and kinase domains . We orig50 measured for the three complexes was different, this is likely mainly due to the dramatic difference in protein required to measure lipid kinase activity in vitro . This suggested that the mechanism of inhibition was not driven by a steric block of Ras association through the RBD, as previously proposed  upon activation by lipidated Gβγ subunits. Intriguingly, we found that all three forms of p110γ were potently inhibited by NB7 . While tproposed . We examproposed . We alsoproposed .To further understand the mechanism by which this nanobody blocked lipid kinase activity we measured the bulk membrane recruitment dynamics of fluorescently labeled Dy647-p84-p110γ on supported lipid bilayers (SLBs) using total internal reflection fluorescence (TIRF) microscopy. We found that the nanobody had no effect on membrane recruitment of p110γ-p84 on bilayers containing membrane-tethered Ras (GTP) and Gβγ . MembranWe wanted to define the molecular mechanism of how nanobody NB7 was a potent allosteric inhibitor of lipid kinase activity. We purified the complex of nanobody NB7 bound to p110γ-p84 to homogeneity by gel filtration. Using this sample, we obtained a cryo-EM reconstruction of the complex of nanobody (NB7)-bound p110γ at 3.0 Å overall resolution from 149,603 particles . The denThe interface between NB7 and p110γ was extensive, with ~1200 Å of buried surface area, with interactions of the ABD-RBD linker, N-terminus of the helical domain, and the regulatory motif at the turn between k8 and k9 (1022-1026aa). This location in the regulatory motif is where both activating oncogenic (R1021C) and inhibitory loss of function mutants have been identified (R1021P) , as wellThe interface of NB7 with p110γ is distant from both the putative membrane binding surface and the catalytic machinery of the kinase domain. To further understand how this nanobody could potently inhibit PI3Kγ activity we examined any other potential modulators of PI3Kγ activity localized in this region. There are two regulatory phosphorylation sites in the helical  and kinaPRKCB2, referred to as PKCβ for simplicity throughout the manuscript)  . S582 isuscript) . To undeuscript) . The moduscript) . Dose-reuscript) , p110γ-puscript) , and p11uscript) . Both p1uscript) . This isTo provide additional insight into the molecular mechanisms underlying p110γ phosphorylation we carried out HDX-MS experiments on p110γ and phosphorylated p110γ  . The fulPIK3CG (When we compared phosphorylated p110γ (>90.8% as measured by mass spectrometry at both sites) to unphosphorylated p110γ we observed extensive increases in dynamics in the C2, helical domain, and kinase domain . The larPIK3CG .2 containing lipid membranes. The experiments in the absence of PIP2 measure turnover of ATP into ADP and phosphate and is a readout of basal catalytic competency. Experiments with PIP2 measured ATP consumed in the generation of PIP3, as well as in non-productive ATP turnover. The p110γ enzyme in the absence of stimulators is very weakly active toward PIP2 substrate with only ~2-fold increased ATP turnover compared to in the absence of membranes. This is consistent with very weak membrane recruitment of p110γ complexes in the absence of lipid activators  (PIK3CD in APDS1)  . This inPIK3CA)  and immun APDS1) . Class In APDS1) . Here, wThe previously identified inhibitory nanobody (NB7)  bound wiPIK3CA, with this being the second most frequently mutated gene in human cancer  and helical domain (E581K) are in close proximity to the nanobody binding site, and both would be expected to disrupt the stability of the helical domain or regulatory motif of the kinase domain , Mus musculus p84 (mouse), Sus scrofa p101 (porcine), and Gβγ were used as previously described  (Plasmids encoding escribed . The plaescribed . The pDOe_23746) . The PKC6 cells/mL were aliquoted into a six-well plate and allowed to attach to form a confluent layer. Transfection reactions were prepared mixing 8–12 μg of bacmid DNA in 100 μL 1×PBS and 12 μg polyethyleneimine  in 100 μL 1×PBS and the reaction was allowed to proceed for 20–30 min before addition to an Sf9 monolayer containing well. Transfections were allowed to proceed for 5–6 days before harvesting virus containing supernatant as a P1 viral stock.The plasmids encoding genes for insect cell expression were transformed into DH10MultiBac cells  to generate bacmid containing the genes of interest. Successful generation was identified by blue-white colony screening and the bacmid was purified using a standard isopropanol-ethanol extraction method. Bacteria were grown overnight (16 hr) in 3–5 mL 2xYT (BioBasic #SD7019). Cells were spun down and the pellet was resuspended in 300 μL of 50 mM Tris-HCl, pH 8.0, 10 mM EDTA, 100 mg/mL RNase A. The pellet was lysed by the addition of 300 μL of 1% sodium dodecyl sulfate (SDS) (wt/vol), 200 mM NaOH, and the reaction was neutralized by addition of 400 μL of 3.0 M potassium acetate, pH 5.5. Following centrifugation at 21130 RCF and 4°C (Rotor #5424R), the supernatant was mixed with 800 μL isopropanol to precipitate bacmid DNA. Following centrifugation, the pelleted bacmid DNA was washed with 500 μL 70% ethanol three times. The pellet was then air dried for 1 min and re-suspended in 50 μL Buffer EB . Purified bacmid was then transfected into Sf9 cells. Two mL of Sf9 cells at 0.6×106 cells/mL (2/100 volume ratio). This amplification was allowed to proceed for 4–5 days and resulted in a P2 stage viral stock that was used in final protein expression. Harvesting of P2 viral stocks was carried out by centrifuging cell suspensions in 50 mL Falcon tubes at 2281 RCF (Beckman GS-15). To the supernatant containing virus, 5–10% inactivated fetal bovine serum  was added and the stock was stored at 4°C.Viral stocks were further amplified by adding P1 to Sf9 cells at ~2 × 10and PKCβ constructs were expressed in Sf9 insect cells using the baculovirus expression system. Following 55 hr after infection with P2 viral stocks, cells were harvested by centrifuging at 1680 RCF (Eppendorf Centrifuge 5810R) and the pellets were snap-frozen in liquid nitrogen. The complex was purified through a combination of nickel affinity, streptavidin affinity, and size-exclusion chromatography.PI3Kγ PI3Kα/β/δ, the purification protocol was performed as described above but instead the protein was eluted in PI3Kα gel filtration buffer . The column was washed with the corresponding gel filtration buffer and loaded with tobacco etch virus protease. After cleavage on the column overnight, the PI3K protein constructs were eluted in gel filtration buffer. The protein was concentrated in a 50,000 MWCO Amicon Concentrator (Millipore) to <1 mL and injected onto a Superdex 200 10/300 GL Increase size-exclusion column  equilibrated in gel filtration buffer. After size exclusion, the protein was concentrated, aliquoted, frozen, and stored at –80°C. For PKCβ, the protein was eluted from the strep column in gel filtration buffer, and the eluate was then loaded on a 1 mL HisTrap FF column to remove his tagged LipTev. The flowthrough was collected, and the column was washed with 2 mL of gel filtration buffer. These fractions were pooled and concentrated and stored at –80°C.Frozen insect cell pellets were resuspended in lysis buffer , protease inhibitor  and sonicated for 2 min . Triton X was added to the lysate to a final concentration of 0.1% and clarified by spinning at 15,000 RCF at 4°C for 45 min (Beckman Coulter JA-20 rotor). The supernatant was loaded onto a 5 mL HisTrap FF crude column  equilibrated in NiNTA A buffer . The column was washed with high salt NiNTA A buffer , NiNTA A buffer, 6% NiNTA B buffer  and the protein was eluted with 100% NiNTA B. The eluent was loaded onto a 5 mL StrepTrap HP column  equilibrated in gel filtration buffer  phosphine [TCEP]). To purify 2, and 1 mM ATP and allowed to incubate on ice for 4 hr. The protein was eluted by adding 7 mL of gel filtration buffer and treated with a second dose of PKCβ (same ratio as above) and allowed to incubate on ice for another 3 hr. For non-phosphorylated p110γ, same protocol was followed with the exception in the addition of PKCβ. Both the proteins were concentrated in a 50,000 MWCO Amicon Concentrator (Millipore) to <1 mL and injected onto a Superdex 200 10/300 GL Increase size-exclusion column  equilibrated in gel filtration buffer. The final phosphorylation level of the two sites was characterized by mass spectrometry, with these values being 92% and 90.8%, for S582 and S594/S595, respectively. After size exclusion, the protein was concentrated, aliquoted, frozen, and stored at –80°C.To purify phosphorylated p110γ, the purification protocol as described above was performed but PKCβ was added to the strep column at a molar ratio of 1:3 (PKCβ:p110γ) along with LipTEV, 20 mM MgClGβγ (Gβ1γ2) was expressed in Sf9 insect cells and purified as described previously. After 65 hr of expression, cells were harvested, and the pellets were frozen as described above. Pellets were resuspended in lysis buffer  and sonicated for 2 min . The lysate was spun at 500 RCF (Eppendorf Centrifuge 5810R) to remove intact cells and the supernatant was centrifuged again at 25,000 RCF for 1 hr (Beckman Coulter JA-20 rotor). The pellet was resuspended in lysis buffer and sodium cholate was added to a final concentration of 1% and stirred at 4°C for 1 hr. The membrane extract was clarified by spinning at 10,000 RCF for 30 min (Beckman Coulter JA-20 rotor). The supernatant was diluted three times with NiNTA A buffer  and loaded onto a 5 mL HisTrap FF crude column  equilibrated in the same buffer. The column was washed with NiNTA A, 6% NiNTA B buffer  and the protein was eluted with 100% NiNTA B. The eluent was loaded onto HiTrap Q HP anion exchange column equilibrated in Hep A buffer . A gradient was started with Hep B buffer  and the protein was eluted in ~50% Hep B buffer. The eluent was concentrated in a 30,000 MWCO Amicon Concentrator (Millipore) to <1 mL and injected onto a Superdex 75 10/300 GL size-exclusion column  equilibrated in Gel Filtration buffer . Fractions containing protein were pooled, concentrated, aliquoted, frozen, and stored at –80°C.Full-length, lipidated human E. coli. A 1 L culture was grown to OD600 of 0.7 in Terrific Broth containing 0.1% glucose and 2 mM MgCl2 in the presence of 100 μg/mL ampicillin and was induced with 0.5 mM isopropyl-β-D-thiogalactoside (IPTG). Cells were harvested the following day by centrifuging at 2500 RCF (Eppendorf Centrifuge 5810R) and the pellet was snap-frozen in liquid nitrogen. The frozen pellet was resuspended in 15 mL of TES buffer containing 200 mM Tris pH 8.0, 0.5 mM ethylenediaminetetraacetic acid (EDTA), and 500 mM sucrose and was mixed for 45 min at 4°C. To this mixture, 30 mL of TES buffer diluted four times in water was added and mixed for 45 min at 4°C to induce osmotic shock. The lysate was clarified by centrifuging at 14,000 rpm for 15 min (Beckman Coulter JA-20 rotor). Imidazole was added to the supernatant to final concentration of 10 mM loaded onto a 5 mL HisTrap FF crude column  equilibrated in NiNTA A buffer . The column was washed with high salt NiNTA A buffer , followed by 100% NiNTA A buffer, then a 6% NiNTA B wash buffer  and the protein was eluted with 100% NiNTA B. The eluent was concentrated in a 10,000 MWCO Amicon Concentrator (Millipore) to <1 mL and injected onto a Superdex 75 10/300 GL Increase size-exclusion column  equilibrated in gel filtration buffer . Following size exclusion, the protein was concentrated, frozen, and stored at –80°C.Nanobody NB7-PIK3CG with a C-terminal 6× His tag was expressed from a pMESy4 vector in the periplasm of WK6 Lipid vesicles containing 5% brain phosphatidylinositol 4,5-bisphosphate (PIP2) and 95% brain phosphatidylserine (PS) were prepared by mixing the lipids solutions in organic solvent. The solvent was evaporated in a stream of argon following which the lipid film was desiccated in a vacuum for 45 min. The lipids were resuspended in lipid buffer  and the solution was vortexed for 5 min followed by sonication for 15 min. The vesicles were then subjected to 10 freeze thaw cycles and extruded 11 times through a 100 nm filter (T&T Scientific: TT-002-0010). The extruded vesicles were sub-aliquoted and stored at –80°C. Final vesicle concentration was 2 mg/mL.2 and 95% PS at a final concentration of 0.5 mg/mL.All kinase assays were done using Transcreener ADP2 Fluorescence Intensity (FI) assays (Bellbrook labs) which measures ADP production. All assays contained ATP at a final concentration of 100 μM, and those with membranes used vesicles containing 5% PIP2, 0.03% CHAPS, 2 mM TCEP, and 1 mM EGTA) and allowed to sit on ice for 15 min. Two μL of protein mix was mixed with 2 μL of lipid solution containing Gβγ , ATP (100 μM), PIP2 lipid vesicles , and lipid buffer  and incubated at 20°C for 60 min.For assays measuring the inhibition by nanobody, 4× kinase  was mixed with varying 4× concentrations of nanobody  or kinase buffer , or both nanobody and lipid. The reaction was incubated at 20°C for 60 min.For assays comparing the difference in activation between phosphorylated and non-phosphorylated p110γ, 2× kinase  was mixed with 2× lipid solutions containing ATP (100 μM), and lipid buffer and either nanobody , PIPAfter the 60 min incubation, all reactions were stopped with 4 μL of 2× stop and detect solution containing Stop and Detect buffer , 8 nM ADP Alexa Fluor 594 Tracer and 93.7 μg/mL ADP2 Antibody IRDye QC-1, covered and incubated at 20°C for 1 hr before reading the fluorescence. The fluorescence intensity was measured using a SpectraMax M5 plate reader at excitation 590 nm and emission 620 nm. All data was normalized against the appropriate measurements obtained for 100 μM ATP and 100 μM ADP with no kinase. The percent ATP turnover was interpolated using a standard curve (0.1–100 μM ADP). Interpolated values were then used to calculate the specific activity of the enzyme.All BLI experiments were performed using the Octet K2 instrument (Fortebio Inc). For all experiments His-tagged nanobody (500 nM) was immobilized on an Anti-Penta-His biosensor for 600 s, and the sensor was dipped into varying concentrations of the protein complex being measured. A dose response was carried out for p110γ, p110γ-p84, and p110γ-p101 (50 nM – 1.9 nM), with association occurring for 600 s, followed by a 1200s dissociation in Octet Buffer . Experiments comparing class IA PI3K versus class IB PI3Kγ used 50 nM of each class IA PI3K.D (dissociation constant) for the different p110γ complexes was calculated from the binding curves based on their global fit to a 1:1 binding model using ForteBio data analysis 12.0 (Fortebio Inc).When comparing nanobody binding to phosphorylated and unphosphorylated p110γ, we used a final concentration of 25 nM for both phosphorylated and non-phosphorylated p110γ with association occurring for 600 s, followed by a 600 s dissociation. The Ksn-glycero-3-phosphocholine , 1,2-dioleoyl-sn-glycero-3-phospho-L-serine , 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[4-cyclohexane-carboxamide] . Lipid compositions reported in figure legends represent the molar percentage of each lipid species.The membrane binding dynamics of Dy647-p84-p110γ were measured in the absence and presence of nanobody 7 (NB7) using TIRF microscopy. As previously described , SLBs we2, 50 µM GDP, and 0.1 mM TCEP for 2 hr. The membrane conjugation reaction was terminated after 2 hr with 1× PBS (pH 7.4) containing 5 mM β-ME. Membranes were then washed and stored in 1× PBS (pH 7.4) until performing the TIRF microscopy (TIRF-M) membrane binding experiments. H-Ras was purified as previously described  and deposited on Piranha etched glass coverslips (25×75 mm) adhered to an IBIDI chamber. After a 30 min incubation, membranes were washed with 4 mL of 1× PBS (pH 7.4) and then blocked for 10 min with 1 mg/mL beta casein  in 1× PBS (pH 7.4) . To conjugate H-Ras to maleimide lipids (MCC-PE), blocked membranes were incubated with 30 µM H-Ras (GDP) in buffer containing 1× PBS (pH 7.4), 1 mM MgClescribed .Gβγ was equilibrated into the supported membranes for 30 min. In parallel, nucleotide exchange of H-Ras (GDP) was performed by adding 50 nM guanine nucleotide exchange factor (SosCat) in 1× PBS (pH 7.4), 1 mM MgCl2, 50 µM GDP. To measure membrane binding, Dy647-p84-p110γ was diluted into the following buffer: 20 mM HEPES (pH 7.0), 150 mM NaCl, 50 µM GTP, 1 mM ATP, 5 mM MgCl2, 0.5 mM EGTA, 20 mM glucose, 200 µg/mL beta casein , 20 mM BME, 320 µg/mL glucose oxidase , 50 µg/mL catalase , and 2 mM Trolox. Trolox was prepared as previously described  oil immersion objective. The x-axis and y-axis positions were controlled using a Nikon motorized stage. Dy647-p84-p110γ was excited with a 637 nm diode laser  controlled with an acousto-optic tunable filter and laser launch built by Vortran . The power output measured through the objective for single particle imaging was 1–3 mW. Excitation light passing through quad multi-pass dichroic filter cube (Semrock). Fluorescence emission passed through Nikon emission filter wheel containing the following 25 mm ET700/75 M emission filters (Semrock) before being detected on iXion Life 897 EMCCD camera . All TIRF-M experiments were performed at room temperature (23°C). Microscope hardware was controlled using Nikon NIS elements. Data analysis was performed using ImageJ/Fiji and Prism graphing program.-/Å2 over 50 frames.Three µL of purified nanobody-bound p110γ at 0.45 mg/mL was adsorbed onto C-Flat 2/2-T grids that were glow discharged for 25 s at 15 mA. Grids were then plunged into liquid ethane using a Vitrobot Mark IV (Thermo Fisher Scientific) with the following settings: –5 blot force, 1.5 s blot time, 100% humidity, and 4°C. Vitrified specimens were screened for ice and particle quality at the UBC high-resolution macromolecular electron microscopy (HRMEM) facility using a 200 kV Glacios transmission electron microscope equipped with a Falcon 3EC direct electron detector (DED). Clipped grids were sent to the Pacific Northwest Cryo-EM Center (PNCC) where 7322 movies were collected using a Titan Krios equipped with a Gatan K3 DED and a BioQuantum K3 energy filter with a slit width of 20 eV. The movies were collected at a physical pixel size of 0.830 Å/pix and a total dose of 50 eThe data were processed using cryoSPARC v.3.3.2 . The movThe previous structural model of full-length p110γ from the complex of p110γ-p101 (PDB: 7MEZ)  was fit 2 (20 mM), and various amounts of PKCβ . Reactions were incubated for 3 hr on ice and quenched with 50 µL of ice-cold acidic quench buffer , followed by immediate freezing using liquid nitrogen and storage at −80°C.For the dose-response phosphorylation of p110γ, p110γ/p84, and p110γ/p101, each protein or complex (750 nM) was mixed with ATP (200 µM), GFB , MgCl2(20 mM), with nanobody and PKCβ present at 1200 nM and 500 nM, respectively. Reactions were incubated for 1 hr at room temperature and quenched with 54 µL of ice-cold acidic quench buffer  followed by immediate freezing using liquid nitrogen and storage at −80°C.For the experiment studying the effect of nanobody on phosphorylation, p110γ or p110γ/p101 (500 nM) was mixed with ATP (1 mM), GFB , MgClPhosphorylation of all proteins was confirmed using mass spectrometry and PEAKS7 analysis. The LC-MS analysis of these samples was carried out using the same pipeline as used in the HDX-MS section. The phosphorylated and non-phosphorylated peptide ratios were determined by generating extracted ion chromatograms for each phosphorylated or non-phosphorylated peptide using their molecular formula and charge state in the Bruker Compass Data Analysis software. The area under each extracted curve was then extracted. The full MS quantification of each of the phosphorylated and non-phosphorylated peptide is provided in 2O buffer  to the 2 µL protein for a final D2O concentration of 75.4%. Exchange was carried out over five time points  and the reaction was quenched with addition of 60 µL of ice-cold acidic quench buffer . After quenching, samples were immediately frozen in liquid nitrogen and stored at –80°C. All reactions were carried out in triplicate.Exchange reactions to assess differences in p110γ upon phosphorylation were carried out at 20°C in 10 µL volumes with final concentrations of 1.6 µM for both apo and phosphorylated p110γ. A total of two conditions were assessed: p110γ apo and PKCβ phosphorylated p110γ. The H/D exchange reaction was initiated by the addition of 8 µL D2 C18 UPLC column (Waters 186002352), using a gradient of 3–35% B  over 11 min immediately followed by a gradient of 35–80% B over 5 min. MS experiments acquired over a mass range from 150 to 2200 mass/charge ratio (m/z) using an electrospray ionization source operated at a temperature of 200°C and a spray voltage of 4.5 kV.Protein samples for both HDX-MS and phosphorylation analysis were analyzed using the same LC-MS setup. Samples were rapidly thawed and injected onto an integrated fluidics system containing an HDx-3 PAL liquid handling robot and climate-controlled chromatography system (LEAP Technologies), a Dionex Ultimate 3000 UHPLC system, as well as an Impact HD QTOF Mass spectrometer (Bruker). The protein was run over two immobilized pepsin columns  at 200 μL/min for 3 min. The resulting peptides were collected and desalted on a C18 trap column . The trap was subsequently eluted in line with an ACQUITY 1.7 µm particle, 100×1 mmPeptides were identified using data-dependent acquisition following tandem MS/MS experiments . MS/MS datasets were analyzed using PEAKS7 (PEAKS), and a false discovery rate was set at 0.1% using a database of purified proteins and known contaminants. The same approach was used to identify phosphorylated and non-phosphorylated peptides for our in vitro phosphorylation experiments, with variable phosphorylation of STY residues was added to the search. The search parameters were set with a precursor tolerance of 20 parts per million, fragment mass error 0.02 Da, and charge states from 1 to 8, with a selection criterion of peptides that had a −10logp score of >24.03 for phosphorylated and >23.05 for non-phosphorylated. The MS/MS spectra of the PKCβ phosphorylated peptides are included in HD‐Examiner Software  was used to automatically calculate the level of deuterium incorporation into each peptide. All peptides were manually inspected for correct charge state, correct retention time, and appropriate selection of isotopic distribution. Deuteration levels were calculated using the centroid of the experimental isotope clusters. HDX-MS results are presented with no correction for back exchange shown in  fundamental new insight into the regulatory apparatus of PI3Kgamma; an important kinase in signaling pathways that control the immune response and cancer. A suite of biophysical and biochemical approaches provide convincing evidence for new sites of allosteric control over enzyme activity. The rigorous findings provide structure and dynamic information that may be exploited in efforts to control PI3Kgamma activity in a therapeutic setting.This study presents  The authors examine signaling factors that differentiate parallel routes to activating phosphoinositide 3-kinase gamma (PI3Kγ). Dissecting the convergent pathways that control PI3Kγ activity is critical because PI3Kγ is a therapeutic target for treating inflammatory disease and cancer. Here, the authors employ a multipronged approach to reveal new aspects for how p84 and p101 pair with p110γ to activate the PI3Kγ heterodimer. The key instigator to this study is a previously reported inhibitory Nanobody, NB7. The hypothesized mechanism for NB7 allosteric inhibition of p84- p110γ was previously proposed to involve blockage of the Ras-binding domain. The authors revise the allosteric inhibition model based on meticulous profiling of various PI3Kγ complex interactions with NB7. In parallel, a cryo-EM-derived model of NB7 bound to the p110γ subunit convincingly reveals a Nanobody interaction pocket involving the helical domain and regulatory motifs of the kinase domain. This revelation shifts the focus to the helical domain, a known target of PKC phosphorylation. While the connections between NB7 interactions and the effects of PKC phosphorylation are sometimes tenuous, it could be argued that the Nanobody served as a tool to reveal the importance of the helical domain to p110γ regulation.The sites of PKC-mediated p110γ helical domain phosphorylation were unexpectedly inaccessible in the available structural models. Nevertheless, mass spectrometry (MS)-based phosphorylation profiling indicates that PKC can phosphorylate the helical domain of p110γ and p84/p110γ (but not p101/p110γ) in vitro. The authors hypothesize that helical domain dynamics dictate susceptibility to PKC phosphorylation. To explore this notion, carefully executed, rigorous H/D exchange MS (HDX-MS) experiments were performed comparing phosphorylated vs. unphosphorylated p110γ. Notably, this design reveals more about the consequences of p110γ phosphorylation, rather than the mechanisms of p84/p101 promoting/resisting phosphorylation. Nevertheless, HDX-MS is very well suited to exploring secondary structure dynamics, and helical domain phosphorylation strikingly increases dynamics consistent with increased regional accessibility. The increased dynamics also nicely map to the pocket enveloped by the inhibitory NB7 Nanobody.Ultimately, this study reveals an unexpected p110γ pocket that allows an engineered Nanobody to allosterically inhibit PI3Kγ complexes. The cryo-EM characterization of the interaction inspired an HDX-MS investigation of known sites of phosphorylation in the region. These insights could be linked to differences/convergences of p84 and p101 complex formation and activation of PI3Kγ, and future work may clarify these mechanisms further. The data presented herein will also be useful for broadening the target surface for future therapeutic developments. New allosteric connections between effector binding sites and post-translational modifications are always welcome. Harris et al. have described the cryo-EM structure of PI3K p110gamma in a complex with a nanobody that inhibits the enzyme. This provided the first structure of full-length of PI3Kgamma in the absence of a regulatory subunit. This nanobody is a potent allosteric inhibitor of the enzyme, and might provide a starting point for developing allosteric, isotype-specific inhibitors of the enzyme. One distinct effect of the nanobody is to greatly decrease the dynamics of the enzyme as shown by HDX-MS, which is consistent with a growing body of observations suggesting that for the whole PI3K superfamily, enzyme activators increase enzyme dynamics.The most remarkable outcome of the study is that upon observing the site of nanobody binding, the authors searched the literature and found that there was a previous report of a PKCbeta phosphorylation of PI3Kgamma in the helical domain that is near the nanobody binding site. This led the authors to re-examine the consequence of the phosphorylation armed with better structural models and the tools to study the effects of this phosphorylation on enzyme dynamics. They found that the site of phosphorylation is buried in the helical domain, suggesting that a large conformational change would have to take place to enable the phosphorylation. HDX-MS showed that phosphorylation at three sites clustered in the helical domain generate a distinctly different conformation with rapid deuterium exchange. This suggests that the phosphorylation locks the enzyme in a more dynamic state. Their enzyme kinetics show that the phosphorylated, dynamic enzyme is activated.While this phosphorylation was reported before, the authors have provided a mechanism for why this activates the enzyme, and they have shown why binders that stabilise the helical domain (such as binding to the p101 regulatory subunit and the nanobody) prevent the phosphorylation. It is this insight into the dynamics of the PI3Kgamma that will likely be the long-lasting influence of the work.The paper is well written and the methods are clear. The following is the authors' response to the original reviews.We appreciate the in depth review of our manuscript, and the excellent suggestions from the two reviewers. We have addressed all concerns as described in the point by point response below. We have also added all of these changes to a revised version posted to biorxiv on May 23rd 2023 (BIORXIV/2023/536585).Reviewer #1 (Recommendations For The Authors):It is sometimes difficult to connect the rationalizations behind the transitions between NB7 binding interaction, the compare/contrast of p84 and p101 effectors, and the synergy with phosphorylation. More explanation of the rationalizations behind these transitions in the Results would be helpful.We agree that the manuscript would benefit from better transitions between the sections. We have added a new paragraph in the final section describing the nanobody structure before the helical domain phosphorylation that fully describes the rationale for how both inform on the critical role of helical domain dynamics in kinase activity. This paragraph is shown below.is the dynamics of this kinase-helical interface. To fully define the role of NB7 in altering the dynamics of the helical domain we needed to study other modulators of helical domain dynamics.’_‘The interface of NB7 with p110_g _is distant from both the putative membrane binding surface, as well as the catalytic machinery of the kinase domain. To further understand how this nanobody could so potently inhibit PI3K activity we examined any other potential modulators of PI3K activity localised in this region. There are two regulatory phosphorylation sites in the helical  and kinase domain  localised at the NB7 interface. This is intriguing as helical domain phosphorylation is activating, and kinase domain phosphorylation is inhibitory. This suggested a critical role in the regulation of p110_g The Methods section would benefit from careful copy editing for clarity and consistency.We have gone through the methods section and edited for clarity and consistency throughout.There's a minor ambiguity throughout when referring to the phosphorylation of S594/S595. Although close inspection makes it clear that this refers to the monophosphorylation of either site S594 or S595, there are several references to "S594/S595" that could be interpreted as phosphorylation of both residues.We agree that this was ambiguous in the original text. We have added an explicit statement describing this as a single phosphorylation event.‘The modification at this site results in a single phosphorylation event , but due to CID MS/MS fragmentation we cannot determine which site is modified, and will be described as S594/S595 throughout the manuscript.’In Figure 2B, the authors show the cryo-EM density map and the structural model based on this map. It would be helpful to also include an image of the structural model fit in the density map to allow readers to evaluate the quality of the map and model. The 2F panel provides an important view of this fit, but CD3 models are difficult to discern.We agree that this would help interpret model quality. We have added a new supplemental figure showing the fit of both p110 and NB7 into this Cryo EM density .Paragraph starting at line 258: The shift to monitoring ATPase activity is confusing here. ATPase activity indicates production of ADP + phosphate (rather than ADP + PIP3). However, an explanation is provided that states that measuring ADP production serves as a surrogate for measuring PIP3 production. The apparent absence of membrane PIP2 substrate in Figure 4E (left) suggests that there is a true ATPase background activity in this kinase. If so, does the increase in ADP production in Fig. 4E reflect the inclusion of PIP2 substrate, increased background ATPase activity, or both?We agree that this was worded confusingly in the original version. We have now clarified exactly what we are observing in these ATPase assays. The new paragraph is appended below‘To further explore the potential role of phosphorylation in mediating p110g activity, we examined the kinase activity of p110g under two conditions: basal ATP turnover, and with PIP2 containing lipid membranes. The experiments in the absence of PIP2 measure turnover of ATP into ADP and phosphate, and is a readout of basal catalytic competency. Experiments with PIP2, measured ATP consumed in the generation of PIP3, as well as in non-productive ATP turnover. The p110g enzyme in the absence of stimulators is very weakly active towards PIP2 substrate with only ~2 fold increased ATP turnover compared to in the absence of membranes. This is consistent with very weak membrane recruitment of p110g complexes in the absence of lipid activators . PKCb-mediated phosphorylation enhanced the ATPase activity of p110g ~2-fold in both the absence and presence of membranes . This suggests that the effect of phosphorylation is to change the intrinsic catalytic efficiency of phosphorylated p110g, with limited effect on membrane binding.’In the section "Nanobody blocks p110gamma phosphorylation," it's not entirely convincing that "the presence of NB7 showed even lower phosphorylation than p110gamma-p101." This does not seem to be subject to a significance test in Figure 5A/B. The follow-up point about "complete abrogation of phosphorylation," however, is readily apparent.We agree that we could have been more precise with our language, as this is not a complete block of phosphorylation, it is merely a significant decrease in phosphorylation. We have removed the comparison to p110-p101, and also removed the statement about complete abrogation of phosphorylation. This is now reworded to‘The presence of NB7 showed a significant decrease in p110g phosphorylation at both sites .’Figure 1: Legend needs to include more detail to define the data. (1A) Representations of variance need to be clarified . Consider "Normalized lipid kinase activity" as a y-axis label and expand on the activity measurement and normalization in the legend. (1B) How was error calculated? (1C) Mislabeled as 1B? Also, consider clarifying the first title highlighting the comparison to class IA PI3Ks. (1D) Typo: "Y647-p84/p110gamma." Also, would it not be more accurate to say "effect of nanobody NB7 on PI3K displacement..." for this experiment?We apologise for these oversights. See details on what has been changed in Fig 1A, 1B, 1C, and 1D.For Fig 1A we now show the data where each replicate is indicated in the graph in the absence of error bars, and have also more clearly expanded on this activity measurement in the figure legend and also stated the replicate number.For Fig 1B we now clearly state how the error was generated.For Fig 1C we have fixed the typoFor Fig 1D, we have fixed this typo and also changed the sub-heading as suggested.New figure legend is below as wellFigure 1. The inhibitory nanobody NB7 binds tightly to all p110γ complexes and inhibits kinase activity, but does not prevent membrane bindingA.Cartoon schematic depicting nanobody inhibition of activation by lipidated Gβγ . Lipid kinase assays show a potent inhibition of lipid kinase activity with increasing concentrations of NB7 (3-3000 nM) for the different complexes. Experiments are carried out in triplicate (n=3) with each replicate shown. The y-axis shows lipid kinase activity normalised for each complex activated by Gβγ in the absence of nanobody. Concentrations of each protein were selected to give a lipid kinase value in the detectable range of the ATPase transcreener assay. The protein concentration of p110γ (300 nM), p110γ-p84 (330 nM) and p110γ-p101 (12 nM) was different due to intrinsic differences of each complex to be activated by lipidated Gβγ, and is likely mainly dependent for the difference seen in NB7 response.B. Association and dissociation curves for the dose response of His-NB7 binding to p110γ, p110γ-p84 and p110γ-p101 (50 – 1.9 nM) is shown. A cartoon schematic of BLI analysis of the binding of immobilized His-NB7 to p110γ is shown on the left. Dissociation constants (KD) were calculated based on a global fit to a 1:1 model for the top three concentrations and averaged with error shown. Error was calculated from the association and dissociation value (n=3) with standard deviation shown. Full details are present in the source data.C. Association and dissociation curves for His-NB7 binding to p110γ, p110a-p85a, p110b-p85b, and p110d-p85b. Experiments were performed in duplicate with a final concentration of 50 nM of each class I PI3K complex.D. Effect of NB7 on PI3K recruitment to supported lipid bilayers containing H-Ras(GTP) and farnesyl-Gbg as measured by Total Internal Reflection Fluorescence Microscopy (TIRF-M). DY647-p84/p110g displays rapid equilibration kinetics and is insensitive to the addition of 500 nM nanobody  on supported lipid bilayers containing H-Ras(GTP) and farnesyl-Gbg.E. Kinetics of 50 nM DY647-p84/p110g membrane recruitment appears indistinguishable in the absence and presence of nanobody. Prior to sample injection, DY647-p84/p110g was incubated for 10 minutes with 500 nM nanobody.F. Representative TIRF-M images showing the localization of 50 nM DY647-p84/p110g visualized in the absence or presence of 500 nM nanobody (+NB7). Membrane composition for panels C-E: 93% DOPC, 5% DOPS, 2% MCC-PE, Ras(GTP) covalently attached to MCC-PE, and 200 nM farnesyl-Gbg.Figure 2: (1A) For consistency with the rest of the paper, p110g can be updated with the Greek character. (1B) This may have been intentional to attract attention to subdomains interacting with NB7, but "colored according to the schematic" omits the purple RBD. (2F) the figure legend should specify whether p110gamma surfaces depicted are the cryo-EM density or a surface rendition of the structural model.We agree and have fixed the p110 typo, and have also colored the schematic the same as shown in the cartoon model.The data shown in Fig 1B is indeed the Cryo EM density and this is now clearly indicated in the legend.Figure 3: (3B) Specifying the [M+H] as [M+2H]2+ and [M+4H]4+ would help the reader understand the delta mass for monophosphorylation here. Given the broad readership of this journal, it would be useful to define 't' and 'e' as 'theoretical' and 'experimental' in the legend. It may also help to be explicit about the meaning of the red spectra and residues in the legend. (3C-E) autocorrect typo for "(C)" and an opportunity to update "b" for Greek character beta.We agree that clearly defining the charge state of each spectra will make it more obvious that we are dealing with a mono-phosphorylation and have made this change as suggested in the figure. We have also clearly define m/z t and m/z e in the figure legend, as well as the black and red lines, and characters. Finally we have added PKCb for all descriptions of PKC treatment in the figure, and fixed the incorrect PKC’b’ in the legend.Figure 4: (4C) Given the common use of "ND" for other terms, it would be useful to spell out "no deuterium" or "undeuterated." (4E) the parenthetical "" could be clarified. How are the (presumably p100gamma) concentration ranges reflected in the three plotted data points per treatment? See also Figure 5E.We agree and have redefined ND as undeuterated. We apologise for the typo in the figure legend, as the concentrations of p110 gamma were the same for both phosphorylated and non-phosphorylated, with this being a typo . This has been changed here and in Fig 5E.Figure 5: (5A/B) Some clarification that we're looking at extracted ion chromatograms would be very useful in this figure legend. On a related note, the experimental details on the LC-MS methodology for this data appear to be split between two sections of the Methods: the "Phosphorylation analysis" paragraph (line 526) and the HDX-MS section. Some more explicit cross-referencing would clarify this experiment. (5E) Clarify inclusion of PIP2 membranes here.We have clearly described that we are looking at extracted ion chromatograms in both panel A and B. We also have normalized the experimental methods in the LC-MS as these used exactly the same procedure. Finally, we now clearly describe the assays shown in Fig 5E were performed in the absence of PIP2 membranes.Miscellaneous typos:Line 205: reference omitted for "Previous study.."We have added this referenceLine 196: "unambiguous"Fixed to unambiguouslyReviewer #2 (Recommendations For The Authors):The only mistake I spotted was that on line 729 there is a reference to Fig 3C that should actually be Fig. 4CWe have changed this to the correct Fig 4C.


To investigate the association of genetically proxied (using a surrogate biomarker) inhibition of phosphodiesterase 5 (PDE5), an established drug target for erectile dysfunction, with fertility, sexual behaviour, and subjective wellbeing.Two sample cis-mendelian randomisation study.Summary data on genetic associations obtained from the International Consortium for Blood Pressure and UK Biobank.Individuals of European ancestry from the International Consortium for Blood Pressure (n=757 601) for estimating PDE5 inhibition (using the surrogate biomarker of diastolic blood pressure reduction), and UK Biobank (n=211 840) for estimating the fertility, sexual behaviour, and subjective wellbeing outcomes in male participants.Genetically proxied PDE5 inhibition.Number of children fathered, number of sexual partners, probability of never having had sexual intercourse, and subjective wellbeing.Genetically proxied PDE5 inhibition was associated with male participants having 0.28  more children . This association was not identified in female participants. No evidence was found of an association between genetically proxied PDE5 inhibition and number of sexual partners, probability of never having had sexual intercourse, or self-reported wellbeing in male participants.The findings of this study provide genetic support for PDE5 inhibition potentially increasing the number of children fathered by male individuals. Absence of this association in female participants supports increased propensity for sustained and robust penile erections as a potential underlying mechanism. Further studies are required to confirm this, however, and these findings should not promote indiscriminate use of PDE5 inhibitors, which can also have harmful adverse effects. Phosphodiesterase 5 (PDE5) inhibitors such as sildenafil, vardenafil, tadalafil, and avanafil are commonly used for the treatment of erectile dysfunction and pulmonary hypertension.Although randomised clinical trials provide vital data on drug efficacy, safety, and adverse effects, the limited duration of use does not always permit investigation of longer term outcomes. For PDE5 inhibitors, longer term outcomes could include effects on fertility, sexual behaviour, and subjective wellbeing. As PDE5 inhibitors are available to buy over the counter in countries such as the UK, it is important to understand their potential application for improving fertilityInvestigating such effects using traditional observational studies is undermined by confounding from environmental factors and reverse causation. Mendelian randomisation is an alternative epidemiological approach for strengthening causal inference in observational study designs.Given that most drug targets are proteins and that genes encode proteins, mendelian randomisation has been paradigmatically extended to study the effects of perturbing specific drug targets.Given the known effects of PDE5 inhibitors on promoting sustained and robust penile erections, and the ability of this physiological state to facilitate fulfilling sexual intercourse, we hypothesised that PDE5 inhibition may have effects on male fertility, sexual behaviour, and subjective wellbeing. We therefore performed cis-mendelian randomisation to investigate associations of genetically proxied PDE5 inhibition with each of these three outcomes.-mendelian randomisation to explore the association of genetically proxied PDE5 inhibition with number of children fathered, number of sexual partners, probability of never having had sexual intercourse, and self-reported wellbeing. The main analyses were performed in male participants, with follow-up analyses performed in female participants to explore whether any identified associations were related to the presence of a penis (the erection of which might be facilitated by PDE5 inhibition). We used cis2, sex, and body mass index, and the UK Biobank sample was additionally corrected for drug use. Further information on participant and genotype quality control checks can be found in the original publication.Estimates on the association between variant and blood pressure were extracted from a genome-wide association study of diastolic blood pressure.Variant-outcome associations were derived from UK Biobank, a large population cohort study of UK residents of predominantly European ancestry born between 1934 and 1971 and with about 500 000 participants.The supplementary methods provide details on data sources used for instrument selection, including expression quantitative trait loci. The expression quantitative trait loci and diastolic blood pressure data included both male and female participants.−8). To ensure that the variants used as instruments in mendelian randomisation are not highly correlated with each other, we then ranked them in order of the P values of their associations with diastolic blood pressure and pruned to linkage disequilibrium correlation r2<0.35 and distance threshold 10 000 kilobases. When accounting for correlation between variants, the inclusion of mildly correlated variants can improve power compared to selecting strictly independent variants. Mendelian randomisation estimators can, however, become unstable when variants are too highly correlated.2 threshold was chosen to balance these two issues. As PDE5 inhibitors are used in the clinical management of erectile dysfunction and pulmonary hypertension,To select genetic variants for studying the effect of PDE5 inhibition, we considered single nucleotide polymorphisms within the PDE5 gene (GRCh37/hg19 chromosome 4 position 120 415 550–120 550 146) that associated with expression of the gene  in blood at genome-wide significance  supports the presence of a shared causal variant underlying both traits, whereas a high posterior probability for the fourth hypothesis (>0.8) supports the presence of distinct causal variants underlying each trait, and thus indicates confounding by linkage disequilibrium in the corresponding mendelian randomisation association . In the presence of a statistically significant mendelian randomisation association that is not a false positive finding, if the posterior probability for both the fourth and fifth hypotheses are <0.8 this would suggest that the colocalisation analysis is likely underpowered to discriminate between whether the mendelian randomisation association is attributable to a shared causal variant or a confounding variant in linkage disequilibrium .We replicated our primary mendelian randomisation analyses where we identified statistically significant associations using genome-wide association study summary data restricted to female participants, to investigate the penis dependence of our findings. In other words, we aimed to investigate whether effects related to the penis (and its propensity to become erect) may potentially explain any beneficial effects of PDE5 inhibition in male participants. The supplementary methods provide details on how we ran the outcome genome-wide association studies in female participants. Since these genome-wide association studies had a similar sample size to those performed in male participants, they should have similar statistical power.−5, selected following Bonferroni correction for the number of traits in PhenoScanner.-mendelian randomisation to adjust our mendelian randomisation estimates for any effect mediated by these traits (see supplementary figure S1). Two step cis-mendelian randomisation uses a two step mediation approach, similar to two step network mendelian randomisation, to adjust variant-outcome associations for potential pleiotropic pathways or confounding by linkage disequilibrium.-mendelian randomisation to adjust for body mass index to verify that its inclusion as a covariate in the diastolic blood pressure genome-wide association study had not induced collider bias.Horizontal pleiotropy occurs when a genetic variant influences the outcome through pathways other than the phenotype being studied, thereby violating a requisite assumption of mendelian randomisation (see supplementary methods). To explore potential horizontal pleiotropic effects, we searched PhenoScanner,Mendelian randomisation analyses in this paper were run using the TwoSampleMR, TwoStepCisMR, MRPopTest, and meta R packages.The first author (BW) has been prescribed PDE5 inhibitors for pulmonary hypertension. Although BW is currently childless, personal experience of the broad beneficial effects of PDE5 inhibitors on erectile or other physiological and psychological variables may or may not have inspired BW to undertake this study.After clumping, we identified five variants to serve as the genetic instrument for PDE5 inhibition . The leaWe found no strong evidence of an association between genetically proxied inhibition of PDE5 and any of the other outcomes considered in male participants: number of sexual partners , false discovery rate corrected P>0.99), probability of never having had sexual intercourse , false discovery rate corrected P>0.99), or self-reported wellbeing , false discovery rate corrected P>0.99). As is apparent from the wide confidence interval, the null finding for number of sexual partners may be underpowered.We did not find evidence of an association between genetically proxied PDE5 inhibition and number of children in female participants . Supplementary table 2 presents the associations between genetically proxied PDE5 inhibition and the other outcomes in female participants. Little evidence of an association was found for any of the considered outcomes.Sensitivity analyses using systolic blood pressure to weight mendelian randomisation estimates instead of diastolic blood pressure produced similar results (see supplementary table 3). We also found a similar result for the association between genetically predicted PDE5 inhibition and number of children for male participants when excluding the variant with the weakest association with diastolic blood pressure and using only the four variants with F statistics >10  (0.28 mo−5) with at least one of the variants included in the PDE5 inhibition genetic instrument (see supplementary table 4). The conclusions of the main analysis did not change when adjusting for potential pleiotropic bias from these associations using two step cis-mendelian randomisation (see supplementary table 5). This suggests it is unlikely that the genetic variants explain any clinically relevant variation in health related outcomes that is not reported.PhenoScanner identified 12 traits that associated (P<1×10In this study, we investigated the association of genetically proxied PDE5 inhibition with measures of fertility, sexual behaviour, and wellbeing. We did not find evidence of an effect of PDE5 inhibition on number of sexual partners, probability of never having had sexual intercourse, or wellbeing in either male or female participants. We did, however, identify genetic evidence that lifelong PDE5 inhibition may increase the number of children had by male patients. Similar evidence was not identified in female patients, consistent with the notion that any effects of PDE5 inhibition on fertility in male patients may be attributable to penile mechanisms.Although epidemiological evidence that PDE5 inhibition may have beneficial effects on fertility in female patients and reproductive outcomes exists,The potential implication of our research is that use of PDE5 inhibitors could improve fertility in male patients, particularly when this is related to erectile dysfunction. Further clinical study is, however, necessary to validate these findings. Consistent with our null finding in people who do not have penises, the effect of PDE5 inhibitors on fertility in male patients may be through effects on erectile function. Of relevance, random samples of general populations in the UK generally report higher age specific estimates of erectile dysfunction than the UK Biobank, where prevalence is less than 3%.Because fertility is declining in many countries,-mendelian randomisation to investigate the causal effects of PDE5 inhibition. The novelty of our research question is strengthened by our analytical approach, which is more robust to the influence of confounding and reverse causation compared with traditional observational research methods. We showed the validity of our instrument with two positive controls, erectile dysfunction and pulmonary hypertension. Furthermore, use of two step cis-mendelian randomisation to adjust for potential biasing pleiotropic pathways provides further assurance that the mendelian randomisation assumptions are valid. By integrating biological knowledge to guide the study design, our findings are amenable to clinical contextualisation towards informing further research. This is paramount given the growing availability of large scale genetic association data, which may promote a temptation towards mis-specified application of mendelian randomisation for injudiciously contemplated research questions.This study leveraged cisA potential limitation of our study is the apparent failure in the colocalisation analysis to support the association between genetically proxied PDE5 inhibition and number of children fathered. One possible explanation is limited statistical power, which may also have conceivably resulted in false negative findings for some of our other analyses. A second and important limitation is generalisability. Since genetic variants are inherited at conception and PDE5 inhibitors are typically used post-puberty, the mendelian randomisation estimates derived here may not be representative of PDE5 inhibitor use in practice. Furthermore, our study comprised participants of European ancestry and so we cannot be certain that our results would generalise to other populations. Finally, our mendelian randomisation model assumes that the effects of PDE5 inhibition are linear across the dose-response range. Of note, the genetic variants used as a proxy for the effect of PDE5 inhibition predicted less than a 1 mm Hg lower diastolic blood pressure, and thus our mendelian randomisation estimates may well not extrapolate to the effect of PDE5 inhibitors used in practice.We found genetic evidence to support the hypothesis that PDE5 inhibition may result in male patients fathering more children. This suggests that use of PDE5 inhibitors, and perhaps improved sexual performance in male patients more generally, might potentially help alleviate the declining fertility rates observed in many countries. However, further studies are required to confirm this, and we absolutely do not advocate indiscriminate use of PDE5 inhibitors—although relatively rare, PDE5 inhibitors can have harmful adverse effects.PDE5 inhibitors are a drug class commonly used for the treatment of erectile dysfunction, but their effects on fertility, sexual behaviour, and subjective wellbeing in male patients are not knownDrug target mendelian randomisation is a quasi-experimental method that uses genetic variants as instrumental variables for studying the effects of drug target perturbationEvidence from drug target mendelian randomisation supports the potential for PDE5 inhibition to increase the number of children fathered by male patients, but with no evidence of such an effect in female patientsNo strong evidence was found for PDE5 inhibitors affecting number of sexual partners, probability of never having had sexual intercourse, or subjective wellbeing in either male or female patients


Spheroidal carbonaceous particles (SCPs) are a component of fly-ash, the particulate by-product of industrial high temperature combustion of fuel-oil and coal-series fuels. We provide the first evidence that these indelible markers of industrialisation have been deposited in Antarctic ice, thousands of kilometres from any potential source. The earliest observed particle was deposited in an ice layer from 1936 CE. While depositional fluxes are low, chemical analysis of individual SCPs indicates a coal combustion origin. These properties allow SCPs to be extracted from, and hence identified within, natural archives such as lake and marine sediments and peat sequences. Therefore, they are an unambiguous environmental indicator of industrialisation. The global scale of SCP emission provides a clear stratigraphic signal, representing a primary driving force (fossil-fuel combustion) of global anthropogenic change. Consequently, it has been suggested that SCPs may provide a useful marker for a mid-twentieth century Global boundary Stratigraphic Section and Point (GSSP) for the proposed Anthropocene Epoch. Stratigraphic records of SCP concentrations and fluxes show a major increase in response to the upsurge in demand for electricity following the Second World War, met by elevated coal combustion and the introduction of oil-fired power stations2.SCPs have no natural sources, and unlike other combustion-derived components of black carbon are not produced by any other anthropogenic sources. They are primarily composed of elemental carbon making them resistant to chemical attack and are morphologically distinct under light and scanning electron microscopy3. In the Arctic, SCPs have been recorded in cryoconite holes on the Russell glacier in southwest Greenland4, and in a single, coarse-resolution (7 years) ice core from Lomonosovfonna ice cap, Svalbard6. In the Southern Ocean, SCPs were detected in sediments from coastal and sub-Antarctic lakes7 and from near-shore marine sediments in Admiralty Bay, King George Island, Antarctica8. However, to our knowledge, SCPs have never been observed from an Antarctic ice core.Most historical records of SCPs within radiometrically dated sequences have been produced from lake sediments and these have been reported from every continent on Earth. Dated SCP records from marine sediments and peats have also been published but are much scarcerIn this article, we present SCP data from annually resolved samples between 1900 and 2011 taken from an ice core collected from the Palmer Land region of the Antarctic Peninsula   chemical dissolution and light microscope detection and (2) continuous filtering and scanning electron microscope detection. The oldest potential evidence of SCPs at Palmer is found in the sample corresponding to 1930–1937 CE 9, demonstrate that most air-masses reaching the Palmer site originate from the Bellingshausen Sea, with a smaller contribution from the Weddell Sea and the Antarctic continent. Forward-trajectory modelling10, from the Southern Hemisphere landmasses, suggest that South America is the dominant source region of 5-day trajectories reaching the Antarctic Peninsula. However, less frequent incursions from New Zealand and Australia are also observed. There is no evidence of air-masses from South Africa reaching the Antarctic Peninsula, at least not within the 5-day window10. This is supported by dust provenance studies which suggest that South America is the largest dust contributor to the Antarctic continent11. While the Antarctic Peninsula receives a mixture of dust from Australia and South America12, there is no evidence of dust derived from southern Africa.While SCPs have been observed at many sites across the globe, few sites are more than a few thousand kilometres from the nearest industrial combustion source13. Three points were analysed for each SCP. In addition to carbon and oxygen, the main peaks for all SCPs analysed were found to be Al, Si and S with less frequent peaks of P, Mg, Ca and K  lower than peak fluxes reported for the Lomosovfonna ice core from Svalbard4. Despite evidence of increased coal-fired plants being commissioned in the Southern Hemisphere during the mid-twentieth century, we do not observe the characteristic acceleration in SCPs from the 1950s onwards as observed at other locations2. Indeed, there are no SCPs observed between 1960–1981 CE and this absence may indicate the importance of atmospheric circulation in fly-ash transport and deposition to the core site. The dominant mode of variability in the Southern Hemisphere is the Southern Annular Mode (SAM), which reflects the zonal pressure difference between the mid- and high latitudes. SAM governs the strength and position of the Southern Hemisphere westerly winds. During the 1960s the SAM was in its negative phase, identified as a period of weaker westerly winds, that were closer to the equator17. Thus, the absence of SCPs during this period may reflect a weakening in the transport regime, or a northward shift of the winds, rather than a change in the source emissions. From the 1980s onward, the westerly winds increased in strength and shifted closer to the Antarctic continent18. This mechanism has been attributed to the observed increase in snow accumulation in the Antarctica Peninsula19 and may explain the re-appearance of SCPs since 1980 and the apparent increase in 1997 CE, a period of documented strong westerly winds associated with the onset of the 1997/1998 El Niño20. By contrast, we attribute the absence of SCPs in the ice core prior to 1936 CE to lower levels of SCP emission and deposition combined with current limitations of analytical detection.While their presence here is clear, the peak SCP fluxes recorded in the Palmer ice core of 3060 m21, and thus we might expect to see increased deposition of fly-ash particles  in Antarctica in the future.Here we provide the first, accurately dated, evidence of fly-ash deposition in an Antarctic ice core. The low sample concentrations, due to the small sample size, prevent a definitive attribution to a source location and we propose that this could be better addressed in a further study based on multiple Antarctic ice core locations and larger sample volumes. Our data indicate that the SCPs we have identified throughout the ice core are derived from coal combustion. Therefore, we hypothesise that the most probable source of the SCPs is Australian coal-fired power plants, with potential additional input from South America. Atmospheric circulation and transport processes have probably influenced the deposition of SCPs in Antarctica throughout the twentieth century, especially related to the strength and position of the Southern Hemisphere westerly winds. The westerly winds are predicted to increase in strength during the twenty-first century22, based on the seasonal deposition of non-sea-salt sulphate (nssSO42−), hydrogen peroxide (H2O2), water-stable isotopes (δ18O) and methane sulfonic acid (MSA)23. The full core covers the period 1621–2012 CE. The estimated error in annual layer counting during the twentieth century (the focus period of this study) is less than 1-year.The ice core was drilled in December 2012 (1897 m above sea level), using an electromechanical dry drill to a depth of 133.45 m. The cores were cut to 85 cm length and wrapped in layflat tubing. The ice core was dated using annual layer counting14. Between acid treatments, the samples were washed with distilled water, centrifuged, and decanted to remove any remaining acid. The final suspension was evaporated onto multiple coverslips and mounted onto microscope slides where the number of SCPs was counted using a light microscope at × 400 magnification using the criteria described in1. The SCP count was converted to a flux, based on the known ice core sample area of 11.4 cm2. All digestion apparatus was rigorously cleaned prior to use and filtration equipment was washed with deionised water in between each sample. All digestion steps from filtration and digestion through to slide preparation were undertaken in a laboratory clean room, inside a fume extraction cupboard. In between stages, all samples were kept covered even within the fume extraction cupboard.The SCPs were analysed using two independent methods. The first method was based on discrete samples cut to annual resolution between 1900 to 1998 CE. The outer 5 mm of the ice core was removed prior to sub-sampling, to avoid any potential contamination from field handling, transport, or storage. The frozen samples were melted in sealed sterile low-density polyethylene  bottles and filtered through Whatman™ glass microfibre filters. Due to the varying snow accumulation at Palmer, the annual liquid sample volumes ranged from 64 to 430 mL per year. SCPs are composed mostly of elemental carbon and are chemically robust. Thus, other particulate material and the filter paper were removed, using a series of acid treatments including hydrofluoric acid followed by hydrochloric acid24. The conversion to concentration was based on an estimated ice core surface volume through the melt-head of 6.53 cm2. In both methods, analytical blanks were included during the melting and filtration process. No SCPs were observed in the blanks of either method.The second method sampled SCPs from the meltwater collected during the continuous flow analysis (CFA). The CFA section is taken from the central part of the ice core to avoid contamination. Melt water was collected in sealed low-density polyethylene  bottles, sampled at approximately annual resolution during the period between 1980–2011 CE. Samples corresponding to years 1900–1979 CE were sampled at approximately five to eight-year resolution (16 samples). Sample volumes ranged between 44 and 927 mL. The meltwater was filtered through 13 mm diameter, 1.0 μm pore size Whatman™ polycarbonate membrane filters, inside clean polypropylene Swinnnex™ filter holders. Each filter was then mounted onto an aluminium stub for analyses on a Quanta-650F Scanning Electron Microscope (SEM) using back scattered electrons (BSE) on a low-vacuum mode. Each filter was imaged at × 800 magnification for SCPs identificationSCP surface chemistry analyses were conducted using a Quanta-650F Scanning Electron Microscope (SEM) equipped with two Bruker XFlash 6130 detectors for energy dispersive X-ray spectroscopy (EDS). The SEM/EDS setup was configured to work at 15 kV on a low-vacuum mode. Three spot analyses were performed on each SCP.


Two novel structural modelsare presented based on single-crystal diffraction analysis, namely,cubic c-MTBC-M6 and trigonal tr-MTBC-M6, which comprise 12-coordinated clusters and 4-coordinatedtetrahedral linkers. Notably, the cubic phase features a new architecturebased on orientational cluster disorder, which is essential for itsformation and has been analyzed by a combination of average structurerefinements and diffuse scattering analysis from both powder and single-crystalX-ray diffraction data. The trigonal phase also features structuredisorder, although involving both linkers and secondary building units.In both phases, remarkable geometrical distortion of the MTBC linkersillustrates how linker flexibility is also essential for their formationand expands the range of achievable topologies in Zr-based MOFs andits analogues.The outstanding diversity of Zr-based frameworks is inherentlylinked to the variable coordination geometry of Zr-oxo clusters andthe conformational flexibility of the linker, both of which allowfor different framework topologies based on the same linker–clustercombination. In addition, intrinsic structural disorder provides alargely unexplored handle to further expand the accessibility of novelmetal–organic framework (MOF) structures that can be formed.In this work, we report the concomitant synthesis of three topologicallydifferent MOFs based on the same M This can occur during theformation of a framework, when some of its inorganic nodes acceptlinker coordination with a geometry that is not compatible with aunique orientation of the metal cluster, thus resulting in multiplecluster orientations bearing analogous—if not equivalent—energies.23 It is worthwhile keeping in mind that the structural complexityof such disordered MOFs can complicate the interpretation of crystallographicdata and even result in misleading structural interpretation.24 For example, it was recently shown that cubicZr8O6 clusters appearing via structure refinementsfrom single-crystal X-ray diffraction (SCXRD) of PCN-221 actuallyresulted from the superposition of disordered Zr6O4(OH)4 SBUs.13 Analogousoctanuclear clusters were also reported in Zr-MTBC,25 a MOF based on 4-connected MTBC linkers and a combinationof 12-connected cubic Zr8O8(OH)4 andoctahedral Zr6O4(OH)4 SBUs. Here,we show that observation of these cubic clusters is, similar to dPCN-224,a marker for orientational disorder, which allows the formation ofthis particular framework topology. We thus present a new model todescribe the structure, herein abbreviated as c-MTBC-Zr6, based on disordered Zr6O4(OH)4 SBUs, which shares analogous unit cell parameters and buh topology with Zr-MTBC. We further describe the complexvariety of other phases obtained by the combination of octahedralclusters of the type M6O4(OH)4  and MTBC linkers, which include an unreported MOF with trigonalsymmetry, tr-MTBC-M6, featuring a complex disorderedstructure. By these means, we highlight a new approach to investigate,resolve, and structurally define disorder as a new design tool forZr-and Hf-based MOFs toward unexpected structural motifs. This poorlyexplored phenomenon and the overlooked degrees of freedom are likelypresent in many other reported structures.26 In addition, our results present disorder as a strategy to circumventstructural frustration that may lead to novel properties in theseframeworks.The topological diversityof Zr-based MOFs is generally limitedto combinations of linkers and clusters whose geometries are compatiblewith the formation of two- or three-dimensional periodic architectures.In this regard, linker flexibility can be key to expanding the numberof topologies by mitigating moderate geometrical frustration of theMOF;6 wasachieved by reacting ZrCl4 and MTBC in diethylformamide(DEF) with benzoic acid as modulator, following a reported procedure(Supporting Information Section S1.2).25 Under these conditions, crystals with sizesin the range of 2–5 μm were obtained as truncated octahedra. The experimentalpowder X-ray diffraction (PXRD) pattern of the product agrees wellwith the simulated pattern of the published Zr-MTBC structure, whichwas reported with both cubic Zr8O6 and octahedralZr6O4(OH)4 SBUs  analysis,which suggests the presence of Zr6O4(OH)4 SBUs only 4 SBUs.13Solvothermal synthesis of c-MTBC-ZrH)4 SBUs 1a.25 To BUs only 1b. This TO) morphology . Structure refinements usingSCXRD data revealed a cubic framework where two distinct cluster sitesare present, both accepting 12 carboxylate groups of 12 linkers exhibitinga moderate distortion (100–123°) from their ideal tetrahedralgeometry (109.5°). As far as the SBUs are concerned, c-MTBC-Zr6 features two crystallographically distinct sites hostingZr6O4(OH)4 12-connected SBUs: oneperfectly ordered and one where they are disordered in four alternativepositions (6O4(OH)4 environment found in UiO-66.27 The geometricaldistortion for the other site is, however, severe enough to causemultiple degenerate orientations of the cluster (6O4(OH)4 SBUs adopt one of four possible orientations, resulting ina framework with well-defined linker positions, but disordered, 12-foldcoordinated clusters. The coordination environment of these clustersis essentially identical to the one found in the isoreticular frameworkNPF-200.28 Its structure features the sametopology and space group symmetry as that of c-MTBC-Zr6 and, in the sites where we report disordered Zr6O4(OH)4 octahedra, cubic Zr8(μ2-O(H))12 clusters were assigned to the observedelectron densities. While this was proposed as second evidence ofthe existence of cubic Zr8-type clusters after their firstclaim in PCN221, also in this case, refinements were based on diffractiondata of relatively poor quality (reported Rint ≈ 11% at 1.0 Å resolution). This might have hamperedthe observation of electron density residues consistent with the presenceof disordered Zr6O4(OH)4.We next optimized the synthesis for increasingthe crystal size.By conducting the reaction without stirring to favor heterogeneousover homogeneous nucleation (as supersaturation decreases in proximityof nucleation sites when diffusion layers are not eliminated by mixing),we obtained single crystals of up to 200 μm in size with thesame truncated octahedral .While the crystal size remained similar (≈200 μm), cube-shapedc-MTBC-Zr6 features a significantly different orientationaldisorder of the cluster, as additional electron density maxima canbe observed from difference electron density maps. These were successfullymodeled as additional metal positions consistent with an overall superpositionof six Zr6 octahedra instead of four (Supporting Information for further details). A deconstruction of theiroverlapped structure is displayed in the Supporting Information file, Figure S6.2.12. Concomitantly, these cube-shapedcrystals show, compared to their TO analogues, sharpBragg peaks up to considerably higher spatial frequencies (linkedto higher diffraction angle in the experiments) due to a higher frameworkperiodicity MTBC-Zrrphology S3.2.1c.rphology S3.2.1c.rphology S3.2.1c.rphology S3.2.1c.6 are linked to synthetic conditions, some diffuse scatteringcharacteristics are systematically found in both TO andcube-shaped crystals (6O4(OH)4 SBUs from our refinements (3.5001(9)Å). Furthermore, the diffuse-to-Bragg intensity ratio visiblyincreases from the origin of reciprocal space outward, and, in thecase of TO crystals, diffuse scattering remains the onlytype of intensity present until the edge of the sampled reciprocalspace. As no notable intensity maxima are present between Bragg reflections,the overall picture points toward a major displacive disorder contributionto the observed diffuse intensities,31 without anyindication of missing cluster or missing linker defects—whichmight be present in low, undetectable amounts. To understand the originof the observed diffuse intensities, we calculated the diffractionpattern of c-MTBC-Zr6 model crystals featuring onlyrandom cluster disorder, based on the average structure refined from TO crystals . The computed diffractionpattern shows diffuse intensity clouds arranged with a significantlydifferent distribution with respect to the experimental patterns (h00 and 0k0 in the hk0 plane). This is because none of thedifferent cluster orientations in the disordered sites includes Zr–Zrcorrelations along the main crystal directions . The ordered clusters, on the other hand, contain such correlations,and a simulated diffraction pattern from a single unit cell basedexclusively on the ordered structure (no disordered clusters) showsan overall good match with the diffuse intensity distributions ofthe experimental patterns MTBC-Zrcrystals 4a,b. Fircrystals 4a, whichcrystals 4d. These,12MTBC-Zpatterns 4c. This 6 can be found in the three-dimensional(3D) PDF (3DPDF) andin the 3D delta PDF (3DΔPDF).32 Theformer can be obtained by direct Fourier transform (FT) of the totalexperimental intensities obtained from single-crystal diffractionmeasurements, the latter by first removing the Bragg reflections fromthe reconstructed 3D reciprocal space and then by applying FT (Supporting Information Section S7.1).33 The result is a 3D map displaying the differencebetween the interatomic correlations in the real structure of c-MTBC-Zr6 and those belonging to the average structure—subtractedby the Bragg peaks’ removal. In these maps from both cube-shapedand TO crystals, the absence of isolated negative correlationpeaks within 1 unit cell distance indicates that no vacancies suchas missing linkers or clusters are detectable. Indeed, vacancies wouldproduce characteristic negative maxima without surrounding positivesignals of comparable intensity. On the contrary, the 3DΔPDFis dominated by positive correlation peaks with proximal negativeoscillations, which signal the presence of displacive disorder. Thepositive components of the 3DΔPDF share similar positions, atshort distances, with the total 3D PDF MTBC-Zr6 crystals originates fromcorrelated framework distortion. Its origin can be reasonably attributedto the alternative orientations of the disordered clusters and theresulting adaptation of surrounding linkers, which creates a static“wave” of deformation across the framework. This interpretationis consistent with the alternation of positive and negative correlationpeaks in the 3DΔPDF,36 indicating that displacive disorder is the main deviationfrom periodicity in both cube-shaped and TO crystals.As for the link between their differences in morphology, defect structure,and diffuse intensities, it is not possible to define a rationalethat is reliably supported by experimental data. There is, however,a clear link between lower reactants’ concentration in thesynthesis, faster growth of {111} crystal facets with respect to {100}ones (resulting in a cubic morphology), and an overall higher crystallinity.The latter is also consistent with the isotropic Zr atoms’mean-square displacements ,which range from 0.0505(1) to 0.0526(1) Å2 in cube-shapedcrystals and from 0.1135(2) to 0.1318(3) Å2 in TO crystals.Overall, the available evidence indicates thatthe observed diffusescattering from c-MTBC-Zr6 single crystals did not yield a phase-pure product (Supporting Information Section S1.4–1.5)but instead resulted in phase mixtures where two additional compoundswere found as elongated octahedral crystals and hexagonal platelets.The first was identified by SCXRD as the tetragonal MOF PCN-521 -flu,20 while the latter revealedanother yet unreported MTBC-Zr6 framework with the sameMTBC-Zr6 connectivity, but with a trigonal symmetry(space group P3̅c1) and unprecedentedtopology: tr-MTBC-Zr6. Compared to the cubic trinodal-buh of c-MTBC-Zr6, the trigonalphase features a new unreported tetranodal  net (Supporting Information Section S8).The optimized synthesis of c-MTBC-Zr6 clusters . Nonetheless, the latter appears to be inconsistent with a well-definedvalue of the c-axis and suggests the presence ofa complex structure modulation whose analysis is beyond the scopeof this study. Interestingly, the linkers belonging to the framework’supshifted variant bind the ordered clusters with a typical bridgingcoordination, where each oxygen binds to a different Zr center. Conversely,the other fraction of disordered linkers—downshifted variant—bindthese clusters with a monodentate η1-coordinationMTBC-Zr6 variants, but its formation may be kineticallyor entropically challenging.Thetrigonal structure features a complex combination of mutually exclusivenetworks originating from correlated disorder of the building blocks6. In parirection S6.2.10. irection S6.2.10. 6 product, wetested several conditions including variation of the reaction time,temperature, concentration of reactants, solvent, and water contentduring synthesis from ZrCl4 (Supporting Information Sections S1.4 and S1.5). Changes in temperatureand concentrations of reactants marginally affect the overall phasecomposition as analyzed by PXRD and mostly yield the previously describedc-MTBC-Zr6. Increasing the reaction time shifts theyield more in favor of tetragonal PCN-521 and trigonal tr-MTBC-Zr6, and a smaller percentage of the cubic phase is obtained.When employing dimethylacetamide (DMA) as the solvent instead of DEFto disfavor solvent decomposition, cubic c-MTBC-Zr6 was not formed. Here, 24 h reaction times yielded a mixture of thetrigonal and tetragonal phase, whereas reaction for 3 days considerablyshifted the yield in favor of the tetragonal MOF, suggesting thatthe trigonal phase might transform into the tetragonal phase underthese conditions. Key for shifting phase selectivity has been conductingthe synthesis with a controlled amount of water. We indeed screenedsynthetic conditions by employing anhydrous ZrCl4 thathas been opened and stored under dry conditions in a glovebox andtested the addition of 0, 4, 8, and 12 μL of H2Owhile using identical amounts of reagents dissolved in 3.2 mL of DMA(Supporting Information Table S1.5.1).Reaction under the addition of 4 μL of water yielded hexagonalparticles, while synthesis with 8 or 12 μL of water yieldedmixtures of hexagonal and octahedral particles, characteristic forthe trigonal and tetragonal phases, respectively. Also, in these cases,reaction has a strong effect on phase composition, as phase homogeneity was only observed for reactions involving4 μL of water lasting 1 and 2 days, whereas extending reactiontimes to 3 days results in the appearance of octahedral crystals,which become predominant after 4 days and the only product after 6and 12 days . Unfortunately, poor crystallinity and the problematic presenceof amorphous content hampered unambiguous phase identification byPXRD . Therefore, the observation of hexagonal platelets shouldnot be linked to the formation of tr-MTBC-Zr6 withabsolute certainty as the presence of unknown MTBC–Zr phaseswith hexagonal platelet morphology cannot be excluded. The decisiverole of water in defining the outcome of the MTBC–Zr systemdraws interesting parallels to the chemistry of Zr-porphyrinic MOFs,which we recently showed as being highly dependent on water content.40 This suggests that water is not only an essentialreagent but also a key player in the formation kinetics for the broaderZr MOF family.Aiming to obtain a pure tr-MTBC-Zr4 in the synthesis, thereby yielding cubic c-MTBC-Hf6, trigonal tr-MTBC-Hf6, and tetragonal PCN-523. Similar to c-MTBC-Zr6, PDF analysis of c-MTBC-Hf6 revealed thepresence of octahedral Hf6O4(OH)4 clusters only, which suggests that reported cubic Hf8 clusters25 also result from an incorrectmodeling of what in fact is the superposition of orientational disorderedHf6O4(OH)4 clusters MTBC-Zr6 crystals MTBC-Zr6 exemplifies this concept. Here, the geometry of the largeMTBC linker strongly deviates from an ideal tetrahedron as the meanangle between the central sp3 carbon (linker node) andthe terminal sp2 carbons ranges approximately from 88 to123°. Such rather extreme deviations from the ideal 109.5°,necessary for the framework’s architecture, result from a combinationof distorted bonding geometry of the sp3 carbon and bendingof the aromatic arms of the linker. This flexibility naturally increaseswhen using larger linkers in synthesizing isoreticular frameworks,thus suggesting that expanding the linkers’ size while maintainingtheir geometry could lead to the occurrence of more diverse phasemixtures and possibly unknown polymorphic behaviors.The multiphasebehavior in Zr-MTBC chemistry is not specific to their chemical composition.Indeed, the Hf-based isostructural analogues of the three discussedZr-MTBC phases could be obtained by simply changing the metal precursorto HfCl PCN-523S3.2.1d,e PCN-523S3.2.1d,e6/Hf6 and tr-MTBC-Zr6/Hf6. Interestingly, in these new frameworks, distincttypes of disorder are present, which complicate the rationalizationof the MOF synthesis and its optimization toward phase purity. Whilesuch disordered states will affect the properties of the material,they can be critical in allowing the formation of such highly connectedcrystalline structures in the first place. In fact, we showed thatthese can be achieved through the combination of linker flexibilityand cluster disorder, enabling the formation of MOFs with topologiesotherwise prohibited by geometrical frustration.The family of Zr-based MOFs presents extremelydiverse structuralpossibilities, particularly with respect to synthesizing differentframeworks sharing the same molecular building blocks. In this regard,the work herein presents the multiphase behavior of Zr- and Hf-MTBC,where identical synthesis conditions yield a phase mixture comprisingup to three different phases, including the two previously unknownstructures c-MTBC-Zr6, this leads not only to one networkbut also to virtually infinite variations thereof, depending on howalternative kinds of framework connectivity are distributed in space,which could be, in principle, controlled upon varying synthetic conditions.Lastly, detailed structural information on framework disorder is stillrelatively rare. The current understanding relies on powder diffractionand spectroscopy data, which are often complex to interpret and yieldonly spatially averaged information of the material structure. Whilethe importance of acquiring reliable insights into polycrystallinesamples is paramount, a more detailed 3D picture of the real structureof MOFs can be extracted from single-crystal diffuse scattering data.This practice is currently rare but highly needed, particularly inthe context of reticular chemistry and defect engineering, as it willallow for better understanding of MOF formation when disorder is notinduced to create variants of an ordered MOF but is intrinsic to thestructure and essential for its formation. Further development andestablishment of single-crystal diffraction techniques for both averageand local structure analysis, combined with more extended and automatedsynthetic screenings, will be key to effectively chart the vast compositionaland structural landscape of Zr- and Hf-MTBC frameworks as well asmany other analogous systems.These considerationslead to three main conclusions. First, a broaderexploration of synthetic variables such as solvents, methodologies,additives, etc. is needed to achieve selectivity towards the variousZr/Hf-MTBC phases. Second, the new structures we reported highlighthow building block disorder can be of crucial benefit in MOF chemistryas a solution to the problem of geometrical frustration and a usefulsource of new, high-connected framework topologies. In the case oftr-MTBC-Zr/Hf