Robotic sample changers for macromolecular X-ray crystallography and biological small-angle X-ray scattering at the National Synchrotron Light Source II

Lazo, Edwin; Antonelli, Stephen; Aishima, Jun; Bernstein, H. J.; Bhogadi, Dileep; Fuchs, Martin R.; Guichard, Nicolas; McSweeney, Seán; Myers, Stuart; Qian, Kun; Schneider, Dieter K.; Shea-McCarthy, Grace; Skinner, John M.; Sweet, Robert M.; Yang, Lin; Jakoncic, Jean

doi:10.1107/s1600577521007578

“…SEC-SAXS was performed at NSLS-II using their mail-in service on the Life Sciences X-ray Scattering 16-ID beamline 83 – 85 . The SPACA6 ectodomain was dialyzed into commercially prepared 1X PBS (Millipore Sigma) and concentrated to ~6.5 mg mL −1 prior to shipment on ice.…”

Section: Methodsmentioning

confidence: 99%

SPACA6 ectodomain structure reveals a conserved superfamily of gamete fusion-associated proteins

Vance

¹

,

Yip

²

,

Jiménez

³

et al. 2022

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SPACA6 is a sperm-expressed surface protein that is critical for gamete fusion during mammalian sexual reproduction. Despite this fundamental role, little is known about how SPACA6 specifically functions. We elucidated the crystal structure of the SPACA6 ectodomain at 2.2-Å resolution, revealing a two-domain protein containing a four-helix bundle and Ig-like β-sandwich connected via a quasi-flexible linker. This structure is reminiscent of IZUMO1, another gamete fusion-associated protein, making SPACA6 and IZUMO1 founding members of a superfamily of fertilization-associated proteins, herein dubbed the IST superfamily. The IST superfamily is defined structurally by its distorted four-helix bundle and a pair of disulfide-bonded CXXC motifs. A structure-based search of the AlphaFold human proteome identified more protein members to this superfamily; remarkably, many of these proteins are linked to gamete fusion. The SPACA6 structure and its connection to other IST-superfamily members provide a missing link in our knowledge of mammalian gamete fusion.

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“…Green represents reliability expressed as the percentage of successfully mounted samples. For a detailed definition of reliability, see Lazo et al (2021).…”

Section: Experimental Endstationmentioning

confidence: 99%

“…The six-axis robotic arm is outfitted with an inhouse gripper based on an original design from the Advanced Light Source, USA. The gripper is significantly modified and functionalized with several sensors(Lazo et al, 2021) including a force torque real time sensor. Machine vision applications detect the presence and position of the sample holder in the gripper and the goniometer.…”

mentioning

confidence: 99%

AMX – the highly automated macromolecular crystallography (17-ID-1) beamline at the NSLS-II

Schneider¹,

Soares²,

Lazo³

et al. 2022

J Synchrotron Radiat

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The highly automated macromolecular crystallography beamline AMX/17-ID-1 is an undulator-based high-intensity (>5 × 1012 photons s−1), micro-focus (7 µm × 5 µm), low-divergence (1 mrad × 0.35 mrad) energy-tunable (5–18 keV) beamline at the NSLS-II, Brookhaven National Laboratory, Upton, NY, USA. It is one of the three life science beamlines constructed by the NIH under the ABBIX project and it shares sector 17-ID with the FMX beamline, the frontier micro-focus macromolecular crystallography beamline. AMX saw first light in March 2016 and started general user operation in February 2017. At AMX, emphasis has been placed on high throughput, high capacity, and automation to enable data collection from the most challenging projects using an intense micro-focus beam. Here, the current state and capabilities of the beamline are reported, and the different macromolecular crystallography experiments that are routinely performed at AMX/17-ID-1 as well as some plans for the near future are presented.

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“…Small angle X-ray scattering SEC-SAXS was performed at NSLS-II using their mail-in service on the Life Sciences X-ray Scattering 16-ID beamline (79)(80)(81). The SPACA6 ectodomain was dialyzed into commercially prepared 1X PBS (Millipore Sigma) and concentrated to ~6.5 mg mL −1 prior to shipment on ice.…”

Section: Pull Down Assaymentioning

confidence: 99%

SPACA6 structure reveals a conserved superfamily of gamete fusion-associated proteins

Vance

¹

,

Yip

²

,

Jiménez

³

et al. 2022

Preprint

2

0

View full text Add to dashboard Cite

SPACA6 is a sperm-expressed surface protein that is critical for gamete fusion during mammalian sexual reproduction. Despite this fundamental role, little is known about how SPACA6 specifically functions. We elucidated the crystal structure of SPACA6 at 2.2-Å resolution, revealing a two-domain protein containing a four-helix bundle and Ig-like β-sandwich connected via a quasi-flexible linker. Based on the structural analysis, we propose SPACA6 is a founding member of a superfamily of gamete fusion-associated proteins, herein dubbed the IST superfamily. The IST superfamily is defined structurally by its distorted four-helix bundle and a pair of disulfide-bonded CXXC motifs. A structure-based search of the AlphaFold human proteome identified more protein members to this superfamily; remarkably, many of these proteins are linked to gamete fusion. The SPACA6 structure and its connection to other IST-superfamily members provide a missing link in our knowledge of mammalian gamete fusion.Significance StatementSPACA6 is a human sperm protein vital for the fusion of gametes, though its exact function remains a mystery. We present the first solved structure of SPACA6: a two-domain fold comprised of an Ig-like domain and a distorted four-helix bundle. Dali searches of the PDB and AlphaFold reveal a family of structurally related proteins, several of which are also known to play a role in gamete fusion; as such, SPACA6 is a founding member of a conserved protein superfamily, dubbed the IST superfamily. Evolutionary analysis to ascertain functionally relevant structural elements in SPACA6 show a conservation of flexibility between the two domains and several conserved surfaces that could function as protein-protein interfaces.

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Robotic sample changers for macromolecular X-ray crystallography and biological small-angle X-ray scattering at the National Synchrotron Light Source II

Cited by 14 publications

References 13 publications

SPACA6 ectodomain structure reveals a conserved superfamily of gamete fusion-associated proteins

SPACA6 ectodomain structure reveals a conserved superfamily of gamete fusion-associated proteins

AMX – the highly automated macromolecular crystallography (17-ID-1) beamline at the NSLS-II

SPACA6 structure reveals a conserved superfamily of gamete fusion-associated proteins

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