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Sub-atomic resolution X-ray crystallography and neutron crystallography: promise, challenges and potential.

Blakeley MP, Hasnain SS, Antonyuk SV - IUCrJ (2015)

Bottom Line: Although the development of neutron macromolecular crystallography over the years has been far less pronounced, and its application much less widespread, the availability of new and improved instrumentation, combined with dedicated deuteration facilities, are beginning to transform the field.Sub-mm(3) crystals are now regularly being used for data collection, structures have been determined to atomic resolution for a few small proteins, and much larger unit-cell systems (cell edges >100 Å) are being successfully studied.New results from two metalloproteins, copper nitrite reductase and cytochrome c', are also included, which illustrate the type of information that can be obtained from sub-atomic-resolution (∼0.8 Å) X-ray structures, while also highlighting the need for complementary neutron studies that can provide details of H atoms not provided by X-ray crystallography.

View Article: PubMed Central - HTML - PubMed

Affiliation: Large-Scale Structures Group, Institut Laue-Langevin , 71 Avenue des Martyrs, Grenoble 38000, France.

ABSTRACT
The International Year of Crystallography saw the number of macromolecular structures deposited in the Protein Data Bank cross the 100000 mark, with more than 90000 of these provided by X-ray crystallography. The number of X-ray structures determined to sub-atomic resolution (i.e. ≤1 Å) has passed 600 and this is likely to continue to grow rapidly with diffraction-limited synchrotron radiation sources such as MAX-IV (Sweden) and Sirius (Brazil) under construction. A dozen X-ray structures have been deposited to ultra-high resolution (i.e. ≤0.7 Å), for which precise electron density can be exploited to obtain charge density and provide information on the bonding character of catalytic or electron transfer sites. Although the development of neutron macromolecular crystallography over the years has been far less pronounced, and its application much less widespread, the availability of new and improved instrumentation, combined with dedicated deuteration facilities, are beginning to transform the field. Of the 83 macromolecular structures deposited with neutron diffraction data, more than half (49/83, 59%) were released since 2010. Sub-mm(3) crystals are now regularly being used for data collection, structures have been determined to atomic resolution for a few small proteins, and much larger unit-cell systems (cell edges >100 Å) are being successfully studied. While some details relating to H-atom positions are tractable with X-ray crystallography at sub-atomic resolution, the mobility of certain H atoms precludes them from being located. In addition, highly polarized H atoms and protons (H(+)) remain invisible with X-rays. Moreover, the majority of X-ray structures are determined from cryo-cooled crystals at 100 K, and, although radiation damage can be strongly controlled, especially since the advent of shutterless fast detectors, and by using limited doses and crystal translation at micro-focus beams, radiation damage can still take place. Neutron crystallography therefore remains the only approach where diffraction data can be collected at room temperature without radiation damage issues and the only approach to locate mobile or highly polarized H atoms and protons. Here a review of the current status of sub-atomic X-ray and neutron macromolecular crystallography is given and future prospects for combined approaches are outlined. New results from two metalloproteins, copper nitrite reductase and cytochrome c', are also included, which illustrate the type of information that can be obtained from sub-atomic-resolution (∼0.8 Å) X-ray structures, while also highlighting the need for complementary neutron studies that can provide details of H atoms not provided by X-ray crystallography.

No MeSH data available.


Related in: MedlinePlus

Neutron and joint X-ray/neutron structures of macromolecules deposited in the PDB since 2010, including data collection details and crystallographic parameters for each. The structures are ordered in terms of the ratio of the crystal volume to the asymmetric unit volume, from lowest to highest. Those with the lowest ratios can be considered the most challenging. Highlighted in red is the study of HIV-1 protease with the antiretroviral drug amprenavir bound (Weber et al., 2013 ▸) that has the lowest ratio of the crystal volume to the asymmetric unit volume. Highlighted in orange is the study of inorganic pyrophosphatase from Thermococcus thioreducens (I-PPase; Hughes et al., 2012 ▸) that currently is the largest unit cell and asymmetric unit volume to be studied. Highlighted in blue is a study of rubredoxin from Pyrococcus furiosus (RdPf; Munshi et al., 2012 ▸) that is the fastest data collection to date at 14 h. Highlighted in green is another study of RdPf (Cuypers et al., 2013 ▸) which is currently the highest resolution study at 1.05 Å. Highlighted in purple are neutron cryo-crystallography studies performed at 100 K for cytochrome c peroxidase (MW ∼34 kDa) (Casadei et al., 2014 ▸) and β-lactamase (MW ∼28 kDa) (Coates et al., 2014 ▸), and highlighted in yellow are the studies of Cu nitrite reductase (MW ∼37 kDa) from Achromobacter cycloclastes (AcNiR) and cytochrome c′ (MW ∼14 kDa) from Alcaligenes xylosoxidans (AxCytCp) presented here.
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fig1: Neutron and joint X-ray/neutron structures of macromolecules deposited in the PDB since 2010, including data collection details and crystallographic parameters for each. The structures are ordered in terms of the ratio of the crystal volume to the asymmetric unit volume, from lowest to highest. Those with the lowest ratios can be considered the most challenging. Highlighted in red is the study of HIV-1 protease with the antiretroviral drug amprenavir bound (Weber et al., 2013 ▸) that has the lowest ratio of the crystal volume to the asymmetric unit volume. Highlighted in orange is the study of inorganic pyrophosphatase from Thermococcus thioreducens (I-PPase; Hughes et al., 2012 ▸) that currently is the largest unit cell and asymmetric unit volume to be studied. Highlighted in blue is a study of rubredoxin from Pyrococcus furiosus (RdPf; Munshi et al., 2012 ▸) that is the fastest data collection to date at 14 h. Highlighted in green is another study of RdPf (Cuypers et al., 2013 ▸) which is currently the highest resolution study at 1.05 Å. Highlighted in purple are neutron cryo-crystallography studies performed at 100 K for cytochrome c peroxidase (MW ∼34 kDa) (Casadei et al., 2014 ▸) and β-lactamase (MW ∼28 kDa) (Coates et al., 2014 ▸), and highlighted in yellow are the studies of Cu nitrite reductase (MW ∼37 kDa) from Achromobacter cycloclastes (AcNiR) and cytochrome c′ (MW ∼14 kDa) from Alcaligenes xylosoxidans (AxCytCp) presented here.

Mentions: The application of neutron crystallography for the location of H atoms in macromolecules has historically been restricted to the study of small unit-cell systems (cell edges <30 Å) for which large crystals of several mm3 could be grown. Even then, long data collection times of several months were required due to the lack of optimized instrumentation (Schoenborn, 2010 ▸). This is no longer the case, with neutron crystallography expanding beyond its traditional boundaries, to address larger and more complex problems, with smaller samples and shorter data collection times. The origin of this transformation can be found in a number of advances including the development of quasi-Laue (Blakeley et al., 2010 ▸; Meilleur et al., 2013 ▸) and monochromatic (Kurihara et al., 2004 ▸; http://www.mlz-garching.de/biodiff) diffractometers with cylindrical image-plate detectors at nuclear reactor neutron sources, time-of-flight (TOF) Laue diffractometers at spallation neutron sources (Langan et al., 2004 ▸; Coates et al., 2010 ▸; Kusaka et al., 2013 ▸), centralized facilities for sample deuteration and new computational tools for structural refinement (Afonine et al., 2010 ▸; Gruene et al., 2014 ▸). Of the 83 neutron structures in the PDB, more than half (49/83) were deposited since 2010 (Fig. 1 ▸). Many of these illustrate the complementarity of combining X-ray and neutron crystallography in order to locate important H-atom positions to improve our understanding of macromolecular structure and function.


Sub-atomic resolution X-ray crystallography and neutron crystallography: promise, challenges and potential.

Blakeley MP, Hasnain SS, Antonyuk SV - IUCrJ (2015)

Neutron and joint X-ray/neutron structures of macromolecules deposited in the PDB since 2010, including data collection details and crystallographic parameters for each. The structures are ordered in terms of the ratio of the crystal volume to the asymmetric unit volume, from lowest to highest. Those with the lowest ratios can be considered the most challenging. Highlighted in red is the study of HIV-1 protease with the antiretroviral drug amprenavir bound (Weber et al., 2013 ▸) that has the lowest ratio of the crystal volume to the asymmetric unit volume. Highlighted in orange is the study of inorganic pyrophosphatase from Thermococcus thioreducens (I-PPase; Hughes et al., 2012 ▸) that currently is the largest unit cell and asymmetric unit volume to be studied. Highlighted in blue is a study of rubredoxin from Pyrococcus furiosus (RdPf; Munshi et al., 2012 ▸) that is the fastest data collection to date at 14 h. Highlighted in green is another study of RdPf (Cuypers et al., 2013 ▸) which is currently the highest resolution study at 1.05 Å. Highlighted in purple are neutron cryo-crystallography studies performed at 100 K for cytochrome c peroxidase (MW ∼34 kDa) (Casadei et al., 2014 ▸) and β-lactamase (MW ∼28 kDa) (Coates et al., 2014 ▸), and highlighted in yellow are the studies of Cu nitrite reductase (MW ∼37 kDa) from Achromobacter cycloclastes (AcNiR) and cytochrome c′ (MW ∼14 kDa) from Alcaligenes xylosoxidans (AxCytCp) presented here.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4491318&req=5

fig1: Neutron and joint X-ray/neutron structures of macromolecules deposited in the PDB since 2010, including data collection details and crystallographic parameters for each. The structures are ordered in terms of the ratio of the crystal volume to the asymmetric unit volume, from lowest to highest. Those with the lowest ratios can be considered the most challenging. Highlighted in red is the study of HIV-1 protease with the antiretroviral drug amprenavir bound (Weber et al., 2013 ▸) that has the lowest ratio of the crystal volume to the asymmetric unit volume. Highlighted in orange is the study of inorganic pyrophosphatase from Thermococcus thioreducens (I-PPase; Hughes et al., 2012 ▸) that currently is the largest unit cell and asymmetric unit volume to be studied. Highlighted in blue is a study of rubredoxin from Pyrococcus furiosus (RdPf; Munshi et al., 2012 ▸) that is the fastest data collection to date at 14 h. Highlighted in green is another study of RdPf (Cuypers et al., 2013 ▸) which is currently the highest resolution study at 1.05 Å. Highlighted in purple are neutron cryo-crystallography studies performed at 100 K for cytochrome c peroxidase (MW ∼34 kDa) (Casadei et al., 2014 ▸) and β-lactamase (MW ∼28 kDa) (Coates et al., 2014 ▸), and highlighted in yellow are the studies of Cu nitrite reductase (MW ∼37 kDa) from Achromobacter cycloclastes (AcNiR) and cytochrome c′ (MW ∼14 kDa) from Alcaligenes xylosoxidans (AxCytCp) presented here.
Mentions: The application of neutron crystallography for the location of H atoms in macromolecules has historically been restricted to the study of small unit-cell systems (cell edges <30 Å) for which large crystals of several mm3 could be grown. Even then, long data collection times of several months were required due to the lack of optimized instrumentation (Schoenborn, 2010 ▸). This is no longer the case, with neutron crystallography expanding beyond its traditional boundaries, to address larger and more complex problems, with smaller samples and shorter data collection times. The origin of this transformation can be found in a number of advances including the development of quasi-Laue (Blakeley et al., 2010 ▸; Meilleur et al., 2013 ▸) and monochromatic (Kurihara et al., 2004 ▸; http://www.mlz-garching.de/biodiff) diffractometers with cylindrical image-plate detectors at nuclear reactor neutron sources, time-of-flight (TOF) Laue diffractometers at spallation neutron sources (Langan et al., 2004 ▸; Coates et al., 2010 ▸; Kusaka et al., 2013 ▸), centralized facilities for sample deuteration and new computational tools for structural refinement (Afonine et al., 2010 ▸; Gruene et al., 2014 ▸). Of the 83 neutron structures in the PDB, more than half (49/83) were deposited since 2010 (Fig. 1 ▸). Many of these illustrate the complementarity of combining X-ray and neutron crystallography in order to locate important H-atom positions to improve our understanding of macromolecular structure and function.

Bottom Line: Although the development of neutron macromolecular crystallography over the years has been far less pronounced, and its application much less widespread, the availability of new and improved instrumentation, combined with dedicated deuteration facilities, are beginning to transform the field.Sub-mm(3) crystals are now regularly being used for data collection, structures have been determined to atomic resolution for a few small proteins, and much larger unit-cell systems (cell edges >100 Å) are being successfully studied.New results from two metalloproteins, copper nitrite reductase and cytochrome c', are also included, which illustrate the type of information that can be obtained from sub-atomic-resolution (∼0.8 Å) X-ray structures, while also highlighting the need for complementary neutron studies that can provide details of H atoms not provided by X-ray crystallography.

View Article: PubMed Central - HTML - PubMed

Affiliation: Large-Scale Structures Group, Institut Laue-Langevin , 71 Avenue des Martyrs, Grenoble 38000, France.

ABSTRACT
The International Year of Crystallography saw the number of macromolecular structures deposited in the Protein Data Bank cross the 100000 mark, with more than 90000 of these provided by X-ray crystallography. The number of X-ray structures determined to sub-atomic resolution (i.e. ≤1 Å) has passed 600 and this is likely to continue to grow rapidly with diffraction-limited synchrotron radiation sources such as MAX-IV (Sweden) and Sirius (Brazil) under construction. A dozen X-ray structures have been deposited to ultra-high resolution (i.e. ≤0.7 Å), for which precise electron density can be exploited to obtain charge density and provide information on the bonding character of catalytic or electron transfer sites. Although the development of neutron macromolecular crystallography over the years has been far less pronounced, and its application much less widespread, the availability of new and improved instrumentation, combined with dedicated deuteration facilities, are beginning to transform the field. Of the 83 macromolecular structures deposited with neutron diffraction data, more than half (49/83, 59%) were released since 2010. Sub-mm(3) crystals are now regularly being used for data collection, structures have been determined to atomic resolution for a few small proteins, and much larger unit-cell systems (cell edges >100 Å) are being successfully studied. While some details relating to H-atom positions are tractable with X-ray crystallography at sub-atomic resolution, the mobility of certain H atoms precludes them from being located. In addition, highly polarized H atoms and protons (H(+)) remain invisible with X-rays. Moreover, the majority of X-ray structures are determined from cryo-cooled crystals at 100 K, and, although radiation damage can be strongly controlled, especially since the advent of shutterless fast detectors, and by using limited doses and crystal translation at micro-focus beams, radiation damage can still take place. Neutron crystallography therefore remains the only approach where diffraction data can be collected at room temperature without radiation damage issues and the only approach to locate mobile or highly polarized H atoms and protons. Here a review of the current status of sub-atomic X-ray and neutron macromolecular crystallography is given and future prospects for combined approaches are outlined. New results from two metalloproteins, copper nitrite reductase and cytochrome c', are also included, which illustrate the type of information that can be obtained from sub-atomic-resolution (∼0.8 Å) X-ray structures, while also highlighting the need for complementary neutron studies that can provide details of H atoms not provided by X-ray crystallography.

No MeSH data available.


Related in: MedlinePlus