The New Era of Microcrystallography

Banerjee, Sanchari; Montaville, Pierre; Chavas, Leonard M. G.; Ramaswamy, S.

doi:10.1007/s41745-018-0086-0

Cited by 4 publications

(2 citation statements)

References 40 publications

(57 reference statements)

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“…Insect cells were chosen as the primary expression system for this pipeline because of their relatively high protein production level and broad eukaryotic protein processing abilities 17 . In vivo protein crystallization has been observed in nature in all kingdoms of life, where it is often functionally associated with protein storage, protection, and stabilization 5 , 37 . While native crystal formation usually provides advantageous functions for the organism, some accidental intracellular crystallization, induced by small environmental or structural changes, is considered to be related to certain diseases 5 , 6 .…”

Section: Discussionmentioning

confidence: 99%

Construction of gateway-compatible baculovirus expression vectors for high-throughput protein expression and in vivo microcrystal screening

Tang

Saul

Nagaratnam

et al. 2020

Sci Rep

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Baculovirus mediated-insect cell expression systems have been widely used for producing heterogeneous proteins. However, to date, there is still the lack of an easy-to-manipulate system that enables the high-throughput protein characterization in insect cells by taking advantage of large existing Gateway clone libraries. To resolve this limitation, we have constructed a suite of Gateway-compatible pIEx-derived baculovirus expression vectors that allow the rapid and cost-effective construction of expression clones for mass parallel protein expression in insect cells. This vector collection also supports the attachment of a variety of fusion tags to target proteins to meet the needs for different research applications. We first demonstrated the utility of these vectors for protein expression and purification using a set of 40 target proteins of various sizes, cellular localizations and host organisms. We then established a scalable pipeline coupled with the SONICC and TEM techniques to screen for microcrystal formation within living insect cells. Using this pipeline, we successfully identified microcrystals for ~ 16% of the tested protein set, which can be potentially used for structure elucidation by X-ray crystallography. In summary, we have established a versatile pipeline enabling parallel gene cloning, protein expression and purification, and in vivo microcrystal screening for structural studies.

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Section: Discussionmentioning

confidence: 99%

Construction of gateway-compatible baculovirus expression vectors for high-throughput protein expression and in vivo microcrystal screening

Tang

Saul

Nagaratnam

et al. 2020

Sci Rep

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“…A typical standard in vitro crystallization pipeline involves protein expression, purification, crystallization optimization, and crystal harvesting and cryoprotection. However, in vivo crystallization enables crystal growth in the cells that express the protein, bypassing the protein purification and crystallization steps (Banerjee et al, 2018;Boudes et al, 2016). Cells are lysed and crystals are then harvested and cryoprotected for data collection.…”

Section: Introductionmentioning

confidence: 99%

Enhanced X-ray diffraction of in vivo-grown μNS crystals by viscous jets at XFELs

Nagaratnam¹,

Tang²,

Botha³

et al. 2020

Acta Cryst Sect F

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NS is a 70 kDa major nonstructural protein of avian reoviruses, which cause significant economic losses in the poultry industry. They replicate inside viral factories in host cells, and the NS protein has been suggested to be the minimal viral factor required for factory formation. Thus, determining the structure of NS is of great importance for understanding its role in viral infection. In the study presented here, a fragment consisting of residues 448-605 of NS was expressed as an EGFP fusion protein in Sf9 insect cells. EGFP-NS crystallization in Sf9 cells was monitored and verified by several imaging techniques. Cells infected with the EGFP-NS (448-605) baculovirus formed rodshaped microcrystals (5-15 mm in length) which were reconstituted in highviscosity media (LCP and agarose) and investigated by serial femtosecond X-ray diffraction using viscous jets at an X-ray free-electron laser (XFEL). The crystals diffracted to 4.5 Å resolution. A total of 4227 diffraction snapshots were successfully indexed into a hexagonal lattice with unit-cell parameters a = 109.29, b = 110.29, c = 324.97 Å . The final data set was merged and refined to 7.0 Å resolution. Preliminary electron-density maps were obtained. While more diffraction data are required to solve the structure of NS , the current experimental strategy, which couples high-viscosity crystal delivery at an XFEL with in cellulo crystallization, paves the way towards structure determination of the NS protein. methods communications Acta Cryst. (2020). F76, 278-289 Nagaratnam et al. SFX diffraction of in vivo-grown NS crystals 279

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Polyhedra, spindles, phage nucleus and pyramids: Structural biology of viral superstructures

Coulibaly

2019

Advances in Virus Research

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The New Era of Microcrystallography

Abstract: | The function of a protein dictates its physical state in a cell. Evolution has imparted selection pressure on proteins to maximize their function and minimize cell death. Most of the proteins exist in their soluble form inside or outside the cells. However, a small fraction of proteins

Cited by 4 publications

References 40 publications

Construction of gateway-compatible baculovirus expression vectors for high-throughput protein expression and in vivo microcrystal screening

Construction of gateway-compatible baculovirus expression vectors for high-throughput protein expression and in vivo microcrystal screening

Enhanced X-ray diffraction of in vivo-grown μNS crystals by viscous jets at XFELs

Polyhedra, spindles, phage nucleus and pyramids: Structural biology of viral superstructures

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