Biocrystallography: Past, present, future

Giegé, Richard; Sauter, Claude

doi:10.2976/1.3369281

Cited by 19 publications

(15 citation statements)

References 119 publications

(121 reference statements)

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“…The use of capillaries for crystallization of large macromolecular complexes has been underexploited in the recent past, even though it is known to work well for smaller proteins ( 53 , 54 ). This work shows that unconventional methods like these can be helpful for initial screening of crystallization conditions, especially for inherently challenging samples and provide first hits to be further pursued in thicker capillaries or crystallization drops.…”

Section: Discussionmentioning

confidence: 99%

Purification, characterization and crystallization of the human 80S ribosome

Khatter

Myasnikov

Mastio

et al. 2014

Nucleic Acids Research

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Ribosomes are key macromolecular protein synthesis machineries in the cell. Human ribosomes have so far not been studied to atomic resolution because of their particularly complex structure as compared with other eukaryotic or prokaryotic ribosomes, and they are difficult to prepare to high homogeneity, which is a key requisite for high-resolution structural work. We established a purification protocol for human 80S ribosomes isolated from HeLa cells that allows obtaining large quantities of homogenous samples as characterized by biophysical methods using analytical ultracentrifugation and multiangle laser light scattering. Samples prepared under different conditions were characterized by direct single particle imaging using cryo electron microscopy, which helped optimizing the preparation protocol. From a small data set, a 3D reconstruction at subnanometric resolution was obtained showing all prominent structural features of the human ribosome, and revealing a salt concentration dependence of the presence of the exit site tRNA, which we show is critical for obtaining crystals. With these well-characterized samples first human 80S ribosome crystals were obtained from several crystallization conditions in capillaries and sitting drops, which diffract to 26 Å resolution at cryo temperatures and for which the crystallographic parameters were determined, paving the way for future high-resolution work.

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Section: Discussionmentioning

confidence: 99%

Purification, characterization and crystallization of the human 80S ribosome

Khatter

Myasnikov

Mastio

et al. 2014

Nucleic Acids Research

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“…Since its birth in the 1960s, biocrystallography has been a primary source of structural information, contributing more than 90% of 3D structures accessible in the Protein Data Bank [1] and remains a central player in structural biology, alongside NMR and CryoEM. Over the past decade, new experimental setups have been introduced that widen its applicability and transform the daily practice of crystal growers and crystallographers.…”

Section: Introductionmentioning

confidence: 99%

Monitoring the Production of High Diffraction-Quality Crystals of Two Enzymes in Real Time Using In Situ Dynamic Light Scattering

et al. 2020

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The reproducible preparation of well-diffracting crystals is a prerequisite for every structural study based on crystallography. An instrument called XtalController has recently been designed that allows the monitoring of crystallization assays using dynamic light scattering and microscopy, and integrates piezo pumps to alter the composition of the mother liquor during the experiment. We have applied this technology to study the crystallization of two enzymes, the CCA-adding enzyme of the psychrophilic bacterium Planococcus halocryophilus, and the lysozyme from hen egg white in the presence of a synthetic chemical nucleant. We were able to (i) detect early nucleation events and (ii) drive the crystallization system (through cycles of dissolution/crystallization) toward growth conditions yielding crystals with excellent diffraction properties. This technology opens a way to the rational production of samples for crystallography, ranging from nanocrystals for electron diffraction, microcrystals for serial or conventional X-ray diffraction, to larger crystals for neutron diffraction.

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“…Since its birth in the sixties, biocrystallography has been a primary source of structural information, contributing more than 90% of 3D structures accessible in the Protein Databank [1] and remains a central player in structural biology, alongside NMR and CryoEM. Over the last decade, new experimental setups have been introduced that widen its applicability and transform the daily practice of crystal growers and crystallographers.…”

Section: Introductionmentioning

confidence: 99%

Monitoring the production of high diffraction-quality crystals of two enzymes in real time usingin situdynamic light scattering

Wijn

Rollet

Engilberge

et al. 2020

Preprint

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The reproducible preparation of well diffracting crystals is a prerequisite for every structural study based on crystallography. An instrument called the XtalController has recently been designed that allows the monitoring of crystallization assays using dynamic light scattering and microscopy, and integrates piezo pumps to alter the composition of the mother liquor during the experiment. We have applied this technology to study the crystallization of two enzymes, the CCA-adding enzyme of the psychrophilic bacterium Planococcus halocryophilus and the hen egg white lysozyme in the presence of a synthetic chemical nucleant. We were able to i) detect early nucleation events and ii) drive the crystallization system (through cycles of dissolution/crystallization) towards growth conditions yielding crystals with excellent diffraction properties. This technology opens a way to the rational production of samples for crystallography, ranging from nanocrystals for electron diffraction, microcrystals for serial or conventional X-ray diffraction, to larger crystals for neutron diffraction.de Wijn et al.

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Biocrystallography: Past, present, future

Cited by 19 publications

References 119 publications

Purification, characterization and crystallization of the human 80S ribosome

Purification, characterization and crystallization of the human 80S ribosome

Monitoring the Production of High Diffraction-Quality Crystals of Two Enzymes in Real Time Using In Situ Dynamic Light Scattering

Monitoring the production of high diffraction-quality crystals of two enzymes in real time usingin situdynamic light scattering

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