One-stop service for protein structure analysis to accelerate R&D

C-Platform
Comprehensive X-ray crystallography (XRD) services for improving drug candidate
compounds and understand protein functions.

Services based on experience with space experiments
X-ray crystallography (XRD: X-Ray Diffraction Structure Analysis) of proteincompound complexes can confirm the electron density map derived from thebound compounds. It has been reported that high-quality crystals provide amore detailed electron density map, which enables us to accurately determine
the coordinates of not only the atoms that make up the bound compounds andprotein molecules, but also the coordinates of the binding water, thereby
enabling us to understand the important interaction sites.
Based on the experience obtained through technical support for JAXA spaceexperiments, Confocal Science has developed crystallization devices for
growing high-quality crystals, and has accumulated know-how in this area.
Technical Information:
The technologies provided by C-Platform are introduced in the following review.
Y. Hashizume, K. Inaka, N. Furubayashi, M. Kamo, S. Takahashi and H. Tanaka,
Methods for Obtaining Better Diffractive Protein Crystals: From Sample
Evaluation
to Space Crystallization, Crystals 2020, 10(2), 78;
doi:10.3390/cryst10020078◆Services are customized for the user’s demand
C-Platform can undertake the entire process as a single contract, or canoptimize the processes. As a whole, more rational services with shorter periods
and lower costs that match the budget can be provided.
Some of the processes provided by C-Platform have a strong R&D element, and
it is sometimes difficult to guarantee the final result from the beginning. For thisreason, a contract with milestones for each process is provided. Roughestimate without detailed information on the target protein can be provided.

Confocal Science Inc.
Musashino bld. Fukasawa 5-14-15, Setagaya-ku
Tokyo, 158-0081 Japan
TEL: +81-3-5809-1561 FAX: +81-3-6411-6261
E-MAIL：info@confsci.co.jp
Web: http://www.confsci.co.jp/(Ver. 2406)

＜Service and Process Overview＞

1. Expression
E. coli, insect cells, or mammalian cells are available as hosts. DNA sequence is optimized for each system. The culture

conditions are examined. Then, mass expression and purification of protein samples are performed.

2. Purification
A purification is applied to the expressed protein to obtain a stable and highly homogeneous protein sample. The quality of it isevaluated by the own methods and experience based on the consistency between the results of SDS/Native-PAGE
electrophoresis, HPLC, DLS, etc. and the physical properties predicted from the amino acid composition of the protein sample. Atag cleavage is applied, if necessary. In the purification process, it is not necessary to disclose the details of the protein. If it isdifficult to disclose the amino acid sequence, only the physicochemical properties of the protein calculated by C-Profile (soldseparately) is required.

3. Search & optimize crystallization condition
For protein samples for which crystallization conditions have already been reported, those crystallization conditions and methods
are used as a starting point. For unknown samples, searching and optimization of the crystallization condition and method will bebased on our own experience. Confocal Science has experienced more than 500 protein samples using various crystallization
methods (batch method, VD method, CD method, dialysis method, etc.). In addition, the crystallization condition will finally beoptimized for the good cryoprotection.

4. Co-crystallization
Based on the optimized crystallization conditions, co-crystallization of the prepared protein with the given ligand compound will beapplied. Sometimes crystallization conditions may vary greatly when adding the ligand, but based on the extensive experience,some countermeasure will be applied including the seeding and the post soaking. In the co-crystallization, it is not necessary todisclose any details other than information on the molecular weight and solubility of the compound.
Once an experimental protocol has been established, it is possible to order at any time to prepare complex crystals of the newcompound with the prepared samples. Optionally, it is possible to deliver samples, optimized crystallization reagents, andcrystallization device, and to allow customer to attempt co-crystallization at the customer’s site without sending the compound.

5. Cryo-cooling the crystal and X-ray diffraction data collection
To obtain X-ray diffraction data, crystals must be frozen with cryoprotection. After optimizing the cryoprotection conditions, weperform the cryo-cooling, preparing about 5 frozen crystals per compound. Diffraction data will be obtained at Diamond Light
Source in the UK. Beamtime is set aside according to crystal preparation, and data set is acquired with the simultaneous
evaluation. Confocal Science also handles all the procedures related to the transportation of the crystals beyond the boarder.

6. Scaling
The obtained diffraction data sets are used for indexing, integration, and scaling. Diffraction data sets from multiple crystals areevaluated for subsequent analysis.

7. Phasing and structure determination
Based on the coordinate data in PDB, the phasing is applied to determine the structure by molecular replacement method. In thisprocess, the customer is expected to disclose information related to the structure of the protein sample. If the molecularreplacement method is not applicable, it is necessary to consult with the customer to solve the situation. Once the phase andstructure have been determined, in most cases the electron density derived from the compound can be confirmed.

8. Refinement
Based on the determined coordinates, the refinement of the structural coordinate will be performed. If the structure of thecompound is disclosed, the refinement including the atomic coordinates of the compound will be performed. Confocal Science hasabundant experience in structural analysis, including ultra-high resolution structural analysis that exceeds 1.0 Å.

9. In space crystallization (optional)
Confocal Science offers high-quality protein crystallization in microgravity using “Kirara” space experiment of the Japan Manned
Space Systems Corporation (JAMSS). (https://www.jamss.co.jp/kirara/index.html). Kirara is highly confidential, and it has beencommissioned to create high-quality crystals for pharmaceutical companies in Japan and overseas.
Confocal Science has contributed to JAXA and NASA space experiments with more than 25 years and has world-leading knowhow in space experiments.

※Some of these processes will be outsourced as necessary.