Targeting Aquaporin-4 Subcellular Localization to Treat Central Nervous System Edema

Swelling of the brain or spinal cord (CNS edema) affects millions of people every year. All potential pharmacological interventions have failed in clinical trials, meaning that symptom management is the only treatment option. The water channel protein aquaporin-4 (AQP4) is expressed in astrocytes and mediates water flux across the blood-brain and blood-spinal cord barriers. Here we show that AQP4 cell-surface abundance increases in response to hypoxia-induced cell swelling in a calmodulin-dependent manner. Calmodulin directly binds the AQP4 carboxyl terminus, causing a specific conformational change and driving AQP4 cell-surface localization. Inhibition of calmodulin in a rat spinal cord injury model with the licensed drug trifluoperazine inhibited AQP4 localization to the blood-spinal cord barrier, ablated CNS edema, and led to accelerated functional recovery compared with untreated animals. We propose that targeting the mechanism of calmodulin-mediated cell-surface localization of AQP4 is a viable strategy for development of CNS edema therapies

Vibrio cholerae SAD2090

Vibrio, T4P, competence pilus<strong>Tilt Series Date:</strong> 2020-09-09</p> <strong>Data Taken By:</strong> Stefan Kreida</p> <strong>Species / Specimen:</strong> Vibrio cholerae</p> <strong>Strain:</strong> SAD2090</p> <strong>Tilt Series Settings:</strong> Single Axis, tilt range: (-60.0°, 60.0°), step: 2°, constant angular increment, dosage: 150.0 eV/Ų, defocus: -8.7 μm, magnification: 26000x. </p> <strong>Microscope:</strong> Caltech Titan Krios</p> <strong>Acquisition Software:</strong> Serial EM</p> <strong>Upload Method:</strong> pipeline</p> <strong>Processing Software Used:</strong> Raptor</p> <strong>Collaborators and Roles:</strong> Strain from Ankur B Dalia, [email protected], PhD, Assistant Professor, Indiana University. . Cell growth & preparation, data collection and frame alignment by Stefan Kreida.</p> <strong>Purification / Growth Conditions / Treatment:</strong> 100 uL O/N preculture used to inoculate 3 mL LB, 20 mM MgSO4, 10 mM CaCl2, 100 uM IPTG. . Cells were grown for 7 h (30C) on 170 rpm. 100 uL culture was then spun down at 5,000 x g (5') . and resuspended in 100 uL aquarium salt (Instant Ocean), 7 g/L. </p> <strong>Sample Preparation:</strong> BSA/10 nm gold suspension: 1 mL 10 nm gold suspension mixed with 0.25 mL 5% BSA, spun . at 14,000 rpm (10'). Supernatant removed so that 35 uL remained, which was used to . resuspend the gold particles. 30 uL cells mixed with 5 uL gold suspension. 3 uL sample . applied in Vitrobot at 100% humidity. Grid: Quantifoil R2/2, Cu NH2, LotID 186865.</p>Files available via S3 at https://renc.osn.xsede.org/ini210004tommorrell/tomography_archive/skv2020-09-09-5</p>G12_SAD2090_TS14_ali.mrc, Tilt Series (Pixel Size 0.33 nm), 2.9 GB <a role="button" class="ui compact mini button" href="https://renc.osn.xsede.org/ini210004tommorrell/tomography_archive/skv2020-09-09-5/rawdata/G12_SAD2090_TS14_ali.mrc" > <i class="download icon"></i> Download </a></p> G12_SAD2090_TS14_ali_full.rec, Reconstruction (Pixel Size 1.32 nm), 737.3 MB <a role="button" class="ui compact mini button" href="https://renc.osn.xsede.org/ini210004tommorrell/tomography_archive/skv2020-09-09-5/3dimage_113396/G12_SAD2090_TS14_ali_full.rec" > <i class="download icon"></i> Download </a></p&gt

Vibrio cholerae SAD2090

Vibrio, T4P, competence pilus<strong>Tilt Series Date:</strong> 2020-09-09</p> <strong>Data Taken By:</strong> Stefan Kreida</p> <strong>Species / Specimen:</strong> Vibrio cholerae</p> <strong>Strain:</strong> SAD2090</p> <strong>Tilt Series Settings:</strong> Single Axis, tilt range: (-60.0°, 60.0°), step: 2°, constant angular increment, dosage: 150.0 eV/Ų, defocus: -8.7 μm, magnification: 26000x. </p> <strong>Microscope:</strong> Caltech Titan Krios</p> <strong>Acquisition Software:</strong> Serial EM</p> <strong>Upload Method:</strong> pipeline</p> <strong>Processing Software Used:</strong> Raptor</p> <strong>Collaborators and Roles:</strong> Strain from Ankur B Dalia, [email protected], PhD, Assistant Professor, Indiana University. . Cell growth & preparation, data collection and frame alignment by Stefan Kreida.</p> <strong>Purification / Growth Conditions / Treatment:</strong> 100 uL O/N preculture used to inoculate 3 mL LB, 20 mM MgSO4, 10 mM CaCl2, 100 uM IPTG. . Cells were grown for 7 h (30C) on 170 rpm. 100 uL culture was then spun down at 5,000 x g (5') . and resuspended in 100 uL aquarium salt (Instant Ocean), 7 g/L. </p> <strong>Sample Preparation:</strong> BSA/10 nm gold suspension: 1 mL 10 nm gold suspension mixed with 0.25 mL 5% BSA, spun . at 14,000 rpm (10'). Supernatant removed so that 35 uL remained, which was used to . resuspend the gold particles. 30 uL cells mixed with 5 uL gold suspension. 3 uL sample . applied in Vitrobot at 100% humidity. Grid: Quantifoil R2/2, Cu NH2, LotID 186865.</p>Files available via S3 at https://renc.osn.xsede.org/ini210004tommorrell/tomography_archive/skv2020-09-09-3</p>G12_SAD2090_TS12_ali.mrc, Tilt Series (Pixel Size 0.33 nm), 2.6 GB <a role="button" class="ui compact mini button" href="https://renc.osn.xsede.org/ini210004tommorrell/tomography_archive/skv2020-09-09-3/rawdata/G12_SAD2090_TS12_ali.mrc" > <i class="download icon"></i> Download </a></p> G12_SAD2090_TS12_ali_full.rec, Reconstruction (Pixel Size 1.32 nm), 737.3 MB <a role="button" class="ui compact mini button" href="https://renc.osn.xsede.org/ini210004tommorrell/tomography_archive/skv2020-09-09-3/3dimage_113394/G12_SAD2090_TS12_ali_full.rec" > <i class="download icon"></i> Download </a></p&gt

Vibrio cholerae SAD2090

Vibrio, T4P, competence pilus<strong>Tilt Series Date:</strong> 2020-09-09</p> <strong>Data Taken By:</strong> Stefan Kreida</p> <strong>Species / Specimen:</strong> Vibrio cholerae</p> <strong>Strain:</strong> SAD2090</p> <strong>Tilt Series Settings:</strong> Single Axis, tilt range: (-60.0°, 60.0°), step: 2°, constant angular increment, dosage: 150.0 eV/Ų, defocus: -8.7 μm, magnification: 26000x. </p> <strong>Microscope:</strong> Caltech Titan Krios</p> <strong>Acquisition Software:</strong> Serial EM</p> <strong>Upload Method:</strong> pipeline</p> <strong>Processing Software Used:</strong> Raptor</p> <strong>Collaborators and Roles:</strong> Strain from Ankur B Dalia, [email protected], PhD, Assistant Professor, Indiana University. . Cell growth & preparation, data collection and frame alignment by Stefan Kreida.</p> <strong>Purification / Growth Conditions / Treatment:</strong> 100 uL O/N preculture used to inoculate 3 mL LB, 20 mM MgSO4, 10 mM CaCl2, 100 uM IPTG. . Cells were grown for 7 h (30C) on 170 rpm. 100 uL culture was then spun down at 5,000 x g (5') . and resuspended in 100 uL aquarium salt (Instant Ocean), 7 g/L. </p> <strong>Sample Preparation:</strong> BSA/10 nm gold suspension: 1 mL 10 nm gold suspension mixed with 0.25 mL 5% BSA, spun . at 14,000 rpm (10'). Supernatant removed so that 35 uL remained, which was used to . resuspend the gold particles. 30 uL cells mixed with 5 uL gold suspension. 3 uL sample . applied in Vitrobot at 100% humidity. Grid: Quantifoil R2/2, Cu NH2, LotID 186865.</p>Files available via S3 at https://renc.osn.xsede.org/ini210004tommorrell/tomography_archive/skv2020-09-09-16</p>G12_SAD2090_TS24_ali.mrc, Tilt Series (Pixel Size 0.33 nm), 2.6 GB <a role="button" class="ui compact mini button" href="https://renc.osn.xsede.org/ini210004tommorrell/tomography_archive/skv2020-09-09-16/rawdata/G12_SAD2090_TS24_ali.mrc" > <i class="download icon"></i> Download </a></p> G12_SAD2090_TS24_ali_part56_5.rec, Reconstruction (Pixel Size 1.32 nm), 737.3 MB <a role="button" class="ui compact mini button" href="https://renc.osn.xsede.org/ini210004tommorrell/tomography_archive/skv2020-09-09-16/3dimage_113404/G12_SAD2090_TS24_ali_part56_5.rec" > <i class="download icon"></i> Download </a></p&gt

Vibrio cholerae SAD2090

Vibrio, T4P, competence pilus<strong>Tilt Series Date:</strong> 2020-09-09</p> <strong>Data Taken By:</strong> Stefan Kreida</p> <strong>Species / Specimen:</strong> Vibrio cholerae</p> <strong>Strain:</strong> SAD2090</p> <strong>Tilt Series Settings:</strong> Single Axis, tilt range: (-60.0°, 60.0°), step: 2°, constant angular increment, dosage: 150.0 eV/Ų, defocus: -8.7 μm, magnification: 26000x. </p> <strong>Microscope:</strong> Caltech Titan Krios</p> <strong>Acquisition Software:</strong> Serial EM</p> <strong>Upload Method:</strong> pipeline</p> <strong>Processing Software Used:</strong> Raptor</p> <strong>Collaborators and Roles:</strong> Strain from Ankur B Dalia, [email protected], PhD, Assistant Professor, Indiana University. . Cell growth & preparation, data collection and frame alignment by Stefan Kreida.</p> <strong>Purification / Growth Conditions / Treatment:</strong> 100 uL O/N preculture used to inoculate 3 mL LB, 20 mM MgSO4, 10 mM CaCl2, 100 uM IPTG. . Cells were grown for 7 h (30C) on 170 rpm. 100 uL culture was then spun down at 5,000 x g (5') . and resuspended in 100 uL aquarium salt (Instant Ocean), 7 g/L. </p> <strong>Sample Preparation:</strong> BSA/10 nm gold suspension: 1 mL 10 nm gold suspension mixed with 0.25 mL 5% BSA, spun . at 14,000 rpm (10'). Supernatant removed so that 35 uL remained, which was used to . resuspend the gold particles. 30 uL cells mixed with 5 uL gold suspension. 3 uL sample . applied in Vitrobot at 100% humidity. Grid: Quantifoil R2/2, Cu NH2, LotID 186865.</p>Files available via S3 at https://renc.osn.xsede.org/ini210004tommorrell/tomography_archive/skv2020-09-09-2</p>G12_SAD2090_TS11_ali.mrc, Tilt Series (Pixel Size 0.33 nm), 2.8 GB <a role="button" class="ui compact mini button" href="https://renc.osn.xsede.org/ini210004tommorrell/tomography_archive/skv2020-09-09-2/rawdata/G12_SAD2090_TS11_ali.mrc" > <i class="download icon"></i> Download </a></p> G12_SAD2090_TS11_ali_part60_1.rec, Reconstruction (Pixel Size 1.32 nm), 737.3 MB <a role="button" class="ui compact mini button" href="https://renc.osn.xsede.org/ini210004tommorrell/tomography_archive/skv2020-09-09-2/3dimage_113393/G12_SAD2090_TS11_ali_part60_1.rec" > <i class="download icon"></i> Download </a></p&gt

Vibrio cholerae SAD2090

Vibrio, T4P, competence pilus<strong>Tilt Series Date:</strong> 2020-09-09</p> <strong>Data Taken By:</strong> Stefan Kreida</p> <strong>Species / Specimen:</strong> Vibrio cholerae</p> <strong>Strain:</strong> SAD2090</p> <strong>Tilt Series Settings:</strong> Single Axis, tilt range: (-60.0°, 60.0°), step: 2°, constant angular increment, dosage: 150.0 eV/Ų, defocus: -8.7 μm, magnification: 26000x. </p> <strong>Microscope:</strong> Caltech Titan Krios</p> <strong>Acquisition Software:</strong> Serial EM</p> <strong>Upload Method:</strong> pipeline</p> <strong>Processing Software Used:</strong> Raptor</p> <strong>Collaborators and Roles:</strong> Strain from Ankur B Dalia, [email protected], PhD, Assistant Professor, Indiana University. . Cell growth & preparation, data collection and frame alignment by Stefan Kreida.</p> <strong>Purification / Growth Conditions / Treatment:</strong> 100 uL O/N preculture used to inoculate 3 mL LB, 20 mM MgSO4, 10 mM CaCl2, 100 uM IPTG. . Cells were grown for 7 h (30C) on 170 rpm. 100 uL culture was then spun down at 5,000 x g (5') . and resuspended in 100 uL aquarium salt (Instant Ocean), 7 g/L. </p> <strong>Sample Preparation:</strong> BSA/10 nm gold suspension: 1 mL 10 nm gold suspension mixed with 0.25 mL 5% BSA, spun . at 14,000 rpm (10'). Supernatant removed so that 35 uL remained, which was used to . resuspend the gold particles. 30 uL cells mixed with 5 uL gold suspension. 3 uL sample . applied in Vitrobot at 100% humidity. Grid: Quantifoil R2/2, Cu NH2, LotID 186865.</p>Files available via S3 at https://renc.osn.xsede.org/ini210004tommorrell/tomography_archive/skv2020-09-09-18</p>G12_SAD2090_TS3_ali.mrc, Tilt Series (Pixel Size 0.33 nm), 2.9 GB <a role="button" class="ui compact mini button" href="https://renc.osn.xsede.org/ini210004tommorrell/tomography_archive/skv2020-09-09-18/rawdata/G12_SAD2090_TS3_ali.mrc" > <i class="download icon"></i> Download </a></p> G12_SAD2090_TS3_ali_full.rec, Reconstruction (Pixel Size 1.32 nm), 737.3 MB <a role="button" class="ui compact mini button" href="https://renc.osn.xsede.org/ini210004tommorrell/tomography_archive/skv2020-09-09-18/3dimage_113407/G12_SAD2090_TS3_ali_full.rec" > <i class="download icon"></i> Download </a></p&gt

Regulation of Aquaporins by Proteins, Metal ions and Phosphate groups

Aquaporins (AQPs) are membrane-bound intrinsic water channels that allow the passive diffusion of water and small solutes across cellular membranes. There are 13 highly conserved isoforms of these channels in human, which differ in specificity, regulation and localisation in cells and tissues. The work in this thesis focuses on AQP0 and AQP2; water-specific aquaporins that are known to interact with regulatory proteins via their respective C-termini and which both contain multiple phosphorylation sites within this region. AQP0 is the main aquaporin of the eye lens. It binds calmodulin (CaM); the ubiquitous calcium-sensing protein, and this interaction has been proposed to block the water pore. Using microscale thermophoresis and fluorescence anisotropy we characterised the interaction between CaM and AQP0 in different phosphorylation states. Our results show that CaM binds full-length AQP0 with positive cooperativity, which is not seen when AQP0 C-terminal peptides are used. Moreover, the phospho-mimicking mutants AQP0-S229D and S235D blocks binding to CaM, whereas S231D can bind with similar affinity as wild type but in a different way. Functional water conductance assays confirm previous studies that CaM blocks the channel in a calcium-dependent manner whereas all mutants are insensitive to CaM/Ca and locked in an open state. Using a combination of fluorescence anisotropy, chemical crosslinking and small-angle X-ray scattering, we further show that zinc binding induces a conformational change in CaM, thereby affecting its interaction with AQP0. This may be of physiological importance for CaM-mediated regulation of AQP0 in the eye. AQP2 is regulated by vasopressin-dependent trafficking in the kidney collecting duct, which involves phosphorylation of the AQP2 C-terminus. Here we investigate how phosphorylation affects the interaction with LIP5, an ESCRT-III/Vps4 adaptor protein which plays a role in targeting AQP2 to multivesicular bodies (MVB) and subsequent lysosomal degradation. Our results from far-western blot and MST show that the phospho-mimicking mutants S256E, S261E, T269E and S256E-T269E impair LIP5 binding, whereas S264E does not significantly affect the affinity to LIP5. Removal of all phosphorylation sites through truncation while retaining the LIP5 binding site reduced the affinity further, suggesting that phosphorylation of AQP2 allosterically controls its interaction with LIP5. Our results fit well with the suggested roles of the respective phosphorylation sites in AQP2 trafficking and suggests that LIP5 may have a more direct role in MVB cargo sorting than previously understood

Vibrio cholerae SAD2090

Vibrio, T4P, competence pilus<strong>Tilt Series Date:</strong> 2020-09-09</p> <strong>Data Taken By:</strong> Stefan Kreida</p> <strong>Species / Specimen:</strong> Vibrio cholerae</p> <strong>Strain:</strong> SAD2090</p> <strong>Tilt Series Settings:</strong> Single Axis, tilt range: (-60.0°, 60.0°), step: 2°, constant angular increment, dosage: 150.0 eV/Ų, defocus: -8.7 μm, magnification: 26000x. </p> <strong>Microscope:</strong> Caltech Titan Krios</p> <strong>Acquisition Software:</strong> Serial EM</p> <strong>Upload Method:</strong> pipeline</p> <strong>Processing Software Used:</strong> Raptor</p> <strong>Collaborators and Roles:</strong> Strain from Ankur B Dalia, [email protected], PhD, Assistant Professor, Indiana University. . Cell growth & preparation, data collection and frame alignment by Stefan Kreida.</p> <strong>Purification / Growth Conditions / Treatment:</strong> 100 uL O/N preculture used to inoculate 3 mL LB, 20 mM MgSO4, 10 mM CaCl2, 100 uM IPTG. . Cells were grown for 7 h (30C) on 170 rpm. 100 uL culture was then spun down at 5,000 x g (5') . and resuspended in 100 uL aquarium salt (Instant Ocean), 7 g/L. </p> <strong>Sample Preparation:</strong> BSA/10 nm gold suspension: 1 mL 10 nm gold suspension mixed with 0.25 mL 5% BSA, spun . at 14,000 rpm (10'). Supernatant removed so that 35 uL remained, which was used to . resuspend the gold particles. 30 uL cells mixed with 5 uL gold suspension. 3 uL sample . applied in Vitrobot at 100% humidity. Grid: Quantifoil R2/2, Cu NH2, LotID 186865.</p>Files available via S3 at https://renc.osn.xsede.org/ini210004tommorrell/tomography_archive/skv2020-09-09-8</p>G12_SAD2090_TS17_ali.mrc, Tilt Series (Pixel Size 0.33 nm), 2.5 GB <a role="button" class="ui compact mini button" href="https://renc.osn.xsede.org/ini210004tommorrell/tomography_archive/skv2020-09-09-8/rawdata/G12_SAD2090_TS17_ali.mrc" > <i class="download icon"></i> Download </a></p> G12_SAD2090_TS17_ali_part53_2.rec, Reconstruction (Pixel Size 1.32 nm), 737.3 MB <a role="button" class="ui compact mini button" href="https://renc.osn.xsede.org/ini210004tommorrell/tomography_archive/skv2020-09-09-8/3dimage_113397/G12_SAD2090_TS17_ali_part53_2.rec" > <i class="download icon"></i> Download </a></p&gt

Vibrio cholerae SAD2090

Vibrio, T4P, competence pilus<strong>Tilt Series Date:</strong> 2020-09-09</p> <strong>Data Taken By:</strong> Stefan Kreida</p> <strong>Species / Specimen:</strong> Vibrio cholerae</p> <strong>Strain:</strong> SAD2090</p> <strong>Tilt Series Settings:</strong> Single Axis, tilt range: (-60.0°, 60.0°), step: 2°, constant angular increment, dosage: 150.0 eV/Ų, defocus: -8.7 μm, magnification: 26000x. </p> <strong>Microscope:</strong> Caltech Titan Krios</p> <strong>Acquisition Software:</strong> Serial EM</p> <strong>Upload Method:</strong> pipeline</p> <strong>Processing Software Used:</strong> Raptor</p> <strong>Collaborators and Roles:</strong> Strain from Ankur B Dalia, [email protected], PhD, Assistant Professor, Indiana University. . Cell growth & preparation, data collection and frame alignment by Stefan Kreida.</p> <strong>Purification / Growth Conditions / Treatment:</strong> 100 uL O/N preculture used to inoculate 3 mL LB, 20 mM MgSO4, 10 mM CaCl2, 100 uM IPTG. . Cells were grown for 7 h (30C) on 170 rpm. 100 uL culture was then spun down at 5,000 x g (5') . and resuspended in 100 uL aquarium salt (Instant Ocean), 7 g/L. </p> <strong>Sample Preparation:</strong> BSA/10 nm gold suspension: 1 mL 10 nm gold suspension mixed with 0.25 mL 5% BSA, spun . at 14,000 rpm (10'). Supernatant removed so that 35 uL remained, which was used to . resuspend the gold particles. 30 uL cells mixed with 5 uL gold suspension. 3 uL sample . applied in Vitrobot at 100% humidity. Grid: Quantifoil R2/2, Cu NH2, LotID 186865.</p>Files available via S3 at https://renc.osn.xsede.org/ini210004tommorrell/tomography_archive/skv2020-09-09-15</p>G12_SAD2090_TS23_ali.mrc, Tilt Series (Pixel Size 0.33 nm), 2.9 GB <a role="button" class="ui compact mini button" href="https://renc.osn.xsede.org/ini210004tommorrell/tomography_archive/skv2020-09-09-15/rawdata/G12_SAD2090_TS23_ali.mrc" > <i class="download icon"></i> Download </a></p> G12_SAD2090_TS23_ali_part61_7.rec, Reconstruction (Pixel Size 1.32 nm), 737.3 MB <a role="button" class="ui compact mini button" href="https://renc.osn.xsede.org/ini210004tommorrell/tomography_archive/skv2020-09-09-15/3dimage_113405/G12_SAD2090_TS23_ali_part61_7.rec" > <i class="download icon"></i> Download </a></p&gt

Vibrio cholerae SAD2090

Vibrio, T4P, competence pilus<strong>Tilt Series Date:</strong> 2020-09-09</p> <strong>Data Taken By:</strong> Stefan Kreida</p> <strong>Species / Specimen:</strong> Vibrio cholerae</p> <strong>Strain:</strong> SAD2090</p> <strong>Tilt Series Settings:</strong> Single Axis, tilt range: (-60.0°, 60.0°), step: 2°, constant angular increment, dosage: 150.0 eV/Ų, defocus: -8.7 μm, magnification: 26000x. </p> <strong>Microscope:</strong> Caltech Titan Krios</p> <strong>Acquisition Software:</strong> Serial EM</p> <strong>Upload Method:</strong> pipeline</p> <strong>Processing Software Used:</strong> Raptor</p> <strong>Collaborators and Roles:</strong> Strain from Ankur B Dalia, [email protected], PhD, Assistant Professor, Indiana University. . Cell growth & preparation, data collection and frame alignment by Stefan Kreida.</p> <strong>Purification / Growth Conditions / Treatment:</strong> 100 uL O/N preculture used to inoculate 3 mL LB, 20 mM MgSO4, 10 mM CaCl2, 100 uM IPTG. . Cells were grown for 7 h (30C) on 170 rpm. 100 uL culture was then spun down at 5,000 x g (5') . and resuspended in 100 uL aquarium salt (Instant Ocean), 7 g/L. </p> <strong>Sample Preparation:</strong> BSA/10 nm gold suspension: 1 mL 10 nm gold suspension mixed with 0.25 mL 5% BSA, spun . at 14,000 rpm (10'). Supernatant removed so that 35 uL remained, which was used to . resuspend the gold particles. 30 uL cells mixed with 5 uL gold suspension. 3 uL sample . applied in Vitrobot at 100% humidity. Grid: Quantifoil R2/2, Cu NH2, LotID 186865.</p>Files available via S3 at https://renc.osn.xsede.org/ini210004tommorrell/tomography_archive/skv2020-09-09-12</p>G12_SAD2090_TS20_ali.mrc, Tilt Series (Pixel Size 0.33 nm), 2.6 GB <a role="button" class="ui compact mini button" href="https://renc.osn.xsede.org/ini210004tommorrell/tomography_archive/skv2020-09-09-12/rawdata/G12_SAD2090_TS20_ali.mrc" > <i class="download icon"></i> Download </a></p> G12_SAD2090_TS20_ali_part56_12.rec, Reconstruction (Pixel Size 1.32 nm), 737.3 MB <a role="button" class="ui compact mini button" href="https://renc.osn.xsede.org/ini210004tommorrell/tomography_archive/skv2020-09-09-12/3dimage_113401/G12_SAD2090_TS20_ali_part56_12.rec" > <i class="download icon"></i> Download </a></p&gt