Oral Presentation 4

日時： 2020 年 10 月 29 日 17:30-19:00
チャンネル： １

口頭発表４『構造生命科学』
Structural Bioscience
座長： 上村 みどり （帝人ファーマ株式会社）
Midori Kamimura (Teijin Pharma Limited)
O1-05"Protocol for Assessing the Optimal Pixel Size to Improve the Accuracy ofSingle-Particle Cryo-Electron Microscopy Maps"
Sandhya Tiwari(RIKEN Center for Computational Science)
O1-06"High-resolution neutron crystallography visualizes an OH-bound resting state of acopper-containing nitrite reductase"
Yohta Fukuda(Graduate School of Pharmaceutical Science, Osaka University)
O1-07"Innovative Small-Angle Scattering for Structural Analysis of Biomacromolecule inSolution"
Ken Morishima(Institute for Integrated Radiation and Nuclear Science, Kyoto University)

O1-05

Protocol for Assessing the Optimal Pixel Size toImprove the Accuracy of Single-Particle Cryo-Electron
Microscopy Maps
Sandhya P Tiwari 1

sandhya.tiwari@riken.jp

Florence Tama 1,2,3

florence.tama@nagoya-u.jp

Osamu Miyashita 1

osamu.miyashita@riken.jp

123

RIKEN Center for Computational Science, 6-7-1, Minatojima-minami-machi, Chuo-ku, Kobe,Hyogo, 650-0047, Japan
Graduate School of Science, Department of Physics, Nagoya University, Nagoya, Aichi
464-8601, Japan
Institute of Transformative Bio- Molecules (WPI-ITbM), Nagoya University, Nagoya, Aichi
464-8601, Japan

Keywords: Cryo-EM, Protein structure modelingWith the advances of hardware and software technologies, detailed structural models ofbiomolecules can now be obtained from cryo-EM experiments. We have been developing
algorithms and software to assist such modeling [1]. The three-dimensional volume obtained fromthe cryo-EM is given as data on a three-dimensional lattice. Here, the parameter that specifies theactual size of each lattice determines the size of the whole molecule and thus affects the structure
modeling results. However, it is known that there can be an error of a few % in the estimation ofthis parameter. Therefore, in order to improve the accuracy of structural modeling, we havedeveloped an algorithm that can estimate an accurate value for this lattice size parameter [2].
In this algorithm, many EM maps with different grid sizes are prepared and a large number ofmodels are created by performing structure refinement against these maps. Then, the correct latticesize is estimated by evaluating the "quality" of each of these structures. Here, evaluation of the"quality" of the obtained structures needs to be performed independent of the fitting procedure.
The algorithm was tested using the maps created by simulation for several model systems, andvarious "quality" evaluation approaches were examined. First, we showed that the general
correlation coefficient, which measures the degree of agreement between the structure and the map
as corresponding electron densities, is not sufficient to detect correct grid size. It was also foundthat the Molprobity index, which is commonly used to evaluate X-ray crystal structure models, isinsufficient because there is no physicochemical aspect in the evaluation of interatomicinteractions. Finally, we found that GOAP, which is an index used for protein structure prediction,is effective in evaluating the local structure and interatomic distance in proteins, which is the"quality" of the structure necessary to identify correct grid size. We applied the developed
algorithm to experimental data and demonstrated the usability of the algorithm. The details ofcomputational protocols and tools will be discussed.
[1] Miyashita, O., Kobayashi, C., Mori, T., Sugita, Y., Tama, F., Flexible fitting to cryo-EM density
map using ensemble molecular dynamics simulations. J Comput Chem. 2017;38:1447-1461[2] Tiwari, S.P., Chhabra, S., Tama, F., Miyashita, O., Computational Protocol for Assessing theOptimal Pixel Size to Improve the Accuracy of Single-Particle Cryo-Electron Microscopy Maps,
J Chem Inf Model. 2020;60:2570-2580

O1-06High-resolution neutron crystallography visualizes anOH-bound resting state of a copper-containing nitrite
reductase
Yohta Fukuda 1Yu Hirano 2

y_fukuda@phs.osaka-u.ac.jp

hirao.yu@qst.go.jp

Katsuhiro Kusaka 3Tsuyoshi Inoue 1

Taro Tamada 2

katsuhiro.kusaka.1129@vc.ibaraki.ac.jp

t_inoue@phs.osaka-u.ac.jp

tamada.taro@qst.go.jp

12

3

Graduate School of Pharmaceutical Science, Osaka University, Suita, Osaka, 565-0871, Japan
Institute for Quantum Life Science, National Institutes for Quantum and Radiological Scienceand Technology, Tokai, Ibaraki, 319-1106, Japan
Frontier Research Center for Applied Atomic Sciences, Ibaraki University, Tokai, Ibaraki,319-1106, Japan

Keywords: Neutron crystallography, Structural Biology, Quantum chemistry
Copper-containing nitrite reductases (CuNIRs) transform nitrite to gaseous nitric oxide, which is a
key process in the global nitrogen cycle. The catalytic mechanism has been intensively studied toultimately achieve rational control of this important geobiochemical reaction. However,accumulated structural biology data show discrepancies with spectroscopic and computationalstudies, and hence the reaction mechanism is still controversial. In particular, the details of theproton transfer involved in it are largely unknown. This situation has arisen from that even atomic
resolution X-ray crystallography fails to determine positions of hydrogen atoms and protons,
which play essential roles at the catalytic site of CuNIRs. We determined the 1.50 Å resolution
neutron structure of a CuNIR from Geobacillus thermodenitrificans (trimer molecular mass of ~106 kDa) in its resting state at low pH [1]. Our neutron structure reveals protonation states ofcatalytic residues (deprotonated aspartate and protonated histidine), providing insights into thecatalytic mechanism. We found that a hydroxide ion, not a water molecule, can exist as a ligand tothe catalytic Cu atom in the resting state even at low pH. This OH-bound Cu site is unexpected
given previous X-ray structures, but consistent with a reaction intermediate suggested bycomputational chemistry. Furthermore, the hydrogen-deuterium exchange ratio in our neutron
structure suggests that the intramolecular electron transfer pathway has a hydrogen-bond jump,which is proposed by quantum chemistry. Our study can seamlessly link the structural biology to
the computational chemistry of CuNIRs, boosting our understanding of the enzymes at the atomicand electronic levels.
[1] Fukuda, Y., Hirano, Y., Kusaka, K., Inoue, T., and Tamada, T., High-resolution neutron
crystallography visualizes an OH-bound resting state of a copper-containing nitrite reductase,Proc. Natl. Acad. Sci. USA , 117:4071-4077, 2020.

O1-07
Innovative Small-Angle Scattering for Structural Analysis
of Biomacromolecule in Solution
Ken Morishima 1

Rintaro Inoue 1

morishima@rri.kyoto-u.ac.jp

rintaro@rri.kyoto-u.ac.jp

Tatsuo Nakagawa 2

tatsuo@unisoku.co.jp

Masahiro Shimizu 1

shimizu.masahiro.3n@kyoto-u.ac.jp

Nobuhiro Sato 1

sato-n@rri.kyoto-u.ac.jp

Yousuke Miyamoto 1

miyamoto.yousuke.78w@st.kyoto-u.ac.jp

Aya Okuda 1

okuda.aya.5r@kyoto-u.ac.jp

Reiko Urade 1

urade.reiko.8w@kyoto-u.ac.jp

Masaaki Sugiyama 1

sugiyama@rri.kyoto-u.ac.jp
12Institute for Integrated Radiation and Nuclear Science, Kyoto University, 2-1010 Asashironishi,Kumatori, Sennan, Osaka, 590-0494, Japan.
Unisoku Co. Ltd., 3-4-2 Kasugano, Hirakata, Osaka, 573-0131, Japan.

Keywords: Small-Angle X-ray Scattering (SAXS), Laboratory-based Size exclusionchromatography SAXS System (La-SSS), Analytical UltraCentrifugation Small-Angle Scattering(AUC-SAS)
Small-Angle Scattering (SAS) offers overwhelming opportunities for structural analysis onvarious biomacromolecules in solution close to physiological condition. To reveal structure of asingle biomacromolecule in multi-component solution, it is indispensable to selectively acquire
scattering profile of the concerned biomacromolecule. In particular, contamination of undesirable
aggregates could hinder the scattering profile and then lead to failure of the structural analysis.
Therefore, we developed two methods for realizing the selective observation of multi-component
systems. In addition, the developed methods are utilized with laboratory-based instruments,
meaning that the problem on the limitation of the beam time of the synchrotron-based Small-Angle
X-ray Scattering (SAXS) could be also solved.
The first development is Laboratory-based Size exclusion chromatography SAXS System(La-SSS) by utilizing a state-of-the-art laboratory-based SAXS instrument and optimization ofvarious parameters [1] . The components with the different sizes in solution are separated with size
exclusion chromatography just prior to SAXS measurements and then are directly injected into
SAXS instrument. La-SSS makes the selective observation of the concerned component not only insolutions including aggregates but also in general multi-component solutions. The second
development is a combination analysis method with Analytical UltraCentrifugation and SAS(AUC-SAS) [2] . The scattering profile of the concerned component is extracted from that ofmulti-component solution by utilizing the weight fraction of each component obtained with AUC.
In the aggregation-removal, La-SSS works even with the sample including large amount ofaggregates, while AUC-SAS is applicable only to that with relatively small amount of aggregates(ca. < 15 % of weight fraction). On the other hand, AUC-SAS requires the smaller amount ofsample (~ 0.2 mg of biomacromolecule for AUC-SAS, and > 2 mg for La-SSS in a typical case)and enables to observe a weakly-bound complex which dissociates in a SEC-column. Consequently,
complementary use of La-SSS and AUC-SAS makes it possible to analyze variousmulti-component biomacromolecular solutions. In the presentation, we will demonstrate some
applications of La-SSS and AUC-SAS to aggregation-prone systems and general multi-component
systems such as solutions under the association-dissociation equilibrium.
[1] R. Inoue, T. Nakagawa, K. Morishima, N. Sato, A. Okuda, R. Urade, R. Yogo, S. Yanaka, M.
Yagi-Utsumi, K. Kato, K. Omoto, K. Ito, M. Sugiyama, Scientific Reports, 9:12610, 2019.
[2] K. Morishima, A. Okuda, R. Inoue, N. Sato, Y. Miyamoto, R. Urade, M. Yagi-Utsumi, K. Kato,R. Hirano, T. Kujirai, H. Kurumizaka, M. Sugiyama, Communications Biology, 3:294, 2020.