【For INTERNATIONAL COLLABORATIVE RESEARCH 2017】

DATE：Day 20 Month 5 Year 2018

SUMMARY of2017 RESEARCH RESULTS REPORTFor International Collaborative Research with IPR, Osaka University
Research Title

Structural and functional research on the survival-essential factors frombacterial pathogens for the development of novel antibiotics which induce
suicide effect (phase II)

ApplicantName

Bong-Jin Lee

Affiliation

Seoul National University

Present Title

Professor

Research Collaborator (Host PI)

Professor Atsushi Nakagawa

Summary
We tried to collect all data over ~3.5 Å after calculating the appropriate distance, the measurement time, the wavelength,and choice of a start degree for efficient data collection but had many difficulties to get good results.
For low-diffraction cases or bad quality of data, we tried flash-annealing or dehydration method to improve resolution
or quality. Actually, a kind of quick solutions to improve quality of data show a little improvement. Therefore, werecalculated the data using various tools, reinterpreted them, and extracted meaningful information from low-resolution
data.
For a set being capable of molecular replacement, we tried to solve a structure using known a structural template by
Phenix or CCP4i program suites. For sets which have no structural homologs based upon sequences, we tried single(or multi-) wavelength anomalous dispersion method to solve protein structures. For efficient performance, we preparedand mounted SeMet-derivatized crystals at first and used heavy atoms-derivatized crystals which were treated withplatinum or mercury compounds etc. In addition, we tried to solve phasing problems using non-covalently bound atoms
including sodium bromide. For efficient management of beam time, we screened optimum conditions of concentrationand soaking time of a specific heavy atom in prior to works in the assigned beam time. For a weak phasing set, we tried
to calculate data using multiple isomorphous replacement method. Data were processed using HKL2000. Structural
determination was tried using various programs including CCP4, CNS, and Phenix with either manual or automatedmethod.

Experimental Results
The Whi protein from M. tuberculosis- Not diffracted. Crystals were improved but showed low diffraction.
SAV2069-DNA complex- Diffracted poorly to ~10 A.
- Tried to improve crystal samples by addition of various additives and detergents.
- Low improvement were observed.
- Quality improvement is still in progress.

【For INTERNATIONAL COLLABORATIVE RESEARCH 2017】

SP1143-1144 complex- Native data sets were collected at 3 - 4 A (five sets were collected and processed.)- SeMet data sets were collected at 3 - 4 A.
- Obtaining the other crystallization conditions is in progress.
Rv0239-0240 complex- Native crystals of the protein complex were diffracted to ~ 3.0 A.
- Three data sets were collected.
- Improvement of the crystal samples is in progress.
Rv1494-Rv1495 complex with DNA- Three DNA constructs were soaked with the protein complex.
- No electron density maps of DNA were observed.
- Truncated constructs of DNA were not better than previous samples.
Rv0581-Rv0582 complex with DNA- Two DNA constructs were soaked with the protein complex.
- No electron density maps of DNA were observed.
- Construct optimization is in progress.
- Truncated constructs of DNA were not better than previous samples.
Rv3183- Sodium bromide soaking was performed (100 and 300 mM, 10/30/60 mins)- Three data sets were collected.
- Phasing was not successful.
- EMTS were soaked in various conditions: phasing was not successful.
SP1143-SP1144 complex- 1 set of SeMet data were collected to ~3.6: phasing problem was not solved.
- Improvement of crystals are in schedule.
Rv0239-Rv0240- The other 10 conditions were tested.
- Native crystals were diffracted to ~ 4.0 Å.
- Crystals from the other conditions were diffracted to 4.0 ~ 8.0 Å.
- Optimization is in progress.

*Deadline: May 18, 2018
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