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Polymer Synthesis & Physics Laboratory

Current status of 4C SAXS beamline and its long-term plan for advanced facility:

SAXS Studies on Structures of Biological Macromolecules in Solution at 4C Beamline

Kyeong Sik Jin* & Byoungseok Min Pohang Accelerator Laboratory, Pohang University of Science & Technology, Pohang 790-784, Republic of Korea Email: jinks@postech.ac.kr

Contents

• 1. Introduction of 4C SAXS Beamline at PAL
• 2. SAXS Data Treatment and Analysis Procedures [Skip]
• 3. Research Results of Biomacromolecules in Solution

2016 PLS – II Operation Schedule and Status

Beamline Map and Status

30 operation BLs, 10 ID BLs, 3 ID + 1 Bending SAXS BLs (Material Chemistry)

4C SAXS BL : Small-angle X-ray Scattering

Small-angle X-ray Scattering (SAXS) is a small-angle scattering (SAS) technique where the elastic scattering of X-rays (wavelength 0.1 ~ 0.2 nm) by a sample which has inhomogeneities in the nm-range, is recorded at very low angle (typically 0.1 ~ 10°). This angular range contains information about the shape and size of macromolecules, characteristic distances of partially ordered materials, pore sizes, and other data. SAXS is capable of delivering structural information of macromolecules between 3 and 25 nm, of repeat distances in partially ordered systems of up to 150 nm. U-SAXS (ultra-small angle X- ray scattering) can resolves even larger dimensions. 4C SAXS beamline is dedicated to conventional transmission small-angle X-ray scattering and diffraction studies for interpreting the structure, structural changes, and relationship between structure and function of molecular/nano structured polymer, self-assembled organic/inorganic nanostructure, composite nanomaterials, biological macromolecules (protein, DNA, and RNA) and their complexes in nearly physiological environments.

SAXS is the universal method for nanostructure analysis

• Sensitive – low concentration (down to 0.1 %)
• Noninvasive – no preparation, staining or drying
• In-situ, real-time capabilities
• Extremely versatile (solids, liquids, gas phase)

The method is accurate, non-destructive and usually requires only a minimum of sample preparation. Applications are very broad and include colloids of all types, metals, cement, oil, polymers, plastics, proteins, foods and pharmaceuticals can be found in research as well as in quality control.

Applications

SAXS is used for the determination of nanoscale structure of particle systems

• Synthetic Polymer Nanostructure Studies: Drug Delivery System, Electronic Device
• Biological Nanostructure Studies: Disease, Drug, Molecular Device
• Energy Material: Organic Solar Cell, Battery
• Flexible Display: Organic Thin-film Transistor
• Ultra Low-k Dielectric Material
• Nano-template Fabrication
• Micro-array Fabrication

Resolution

) q ( ) q ( ) q ( S F I  

Form factor F(q)

size, shape, orientation

Structure factor S(q) local order, relative position

Scattering Intensity

0.0 0.5 1.0 1.5 I(q) S(q)

log I (a.u.)

F(q)

1 S(q) 

For BioSAXS,

X-ray Scattering: SAXS vs. WAXS?

WAXS patterns contain data concerning correlations on an intra-molecular, inter- atomic level SAXS patterns contain data concerning correlations on an inter-molecular level: necessary samples where there is macromolecular or aggregate order As synthesis design/control improves, SAXS becomes more relevant than ever before

Incident beam height = 1400 mm

4C SAXS II Beamline Layout

Beam Size [mm] Beam Size [mm] 1.450 1.650 0.700 0.700 23 um 300 um 0.400 1.030

Undulator

Incidence Angle 2.6 mrad 88.4 mm

Source 0 m DCM 18.0 m FM 21.3 m Defining Slit 11.4 m

Horizontal (33.00 urad) Vertical (29.00 urad) Wall Movable mask Beam stopper

Optical Hutch Experimental Hutch Detector 37 m Front End

(including copper screen/tungsten wire monitors) (including graphite filter)

Experimental Components in Hutch

A Brief Introduction about 4C SAXS II Beamline

Beamline Specifications

Beamline 4C SAXS-II

Status Operational Source In-vacuum Undulator 20 (1.4 m short, 20 mm period) Monochromator DCM Si (111) Wavelength 0.06-0.12 nm, currently 0.07 nm Mirror Vertical focusing toroidal, rhodium coated Beam flux 1×1012 ph/sec Beam size 100(V)×300(H) µm2 Resolution 200 nm ~ 0.3 nm Slits Individually motorized blades of tungsten (W) Sample-to-detector distance 5.0/4.0/3.0/2.0/1.0/0.5/0.2 m Detector Rayonix 2D SX 165 Experimental methods Bulk, solution, liquid crystal, film, powder, sol-gel T-SAXS, T-WAXD User groups 50 user groups (Polymer, bio-,

• rganic, inorganic groups)

Research results 40 ~ 50 papers/year Data analysis software ATSAS package, SCATTER, etc. (free download) Staff science SAXS studies for self-assembled nanostructures and biomacromolecules in solution

• Development and construction of EPICS-based software

package for electronic

• ptical

component and experimental equipment control (Self-development)

• Development

and construction

• f

user friendly integrated software package for data measurement and data treatment (Self-development)

• Design and fabrication of a variety of sample cell &

stage systems for bulk/powder/film/solution SAXS and WAXD experiments (Self-development)

• Construction of experimental equipments for sample

storage, preparation, treatment, and basic material property analysis

• Activation of user groups and of joint researches
• Further development of methods and approaches for

high-throughput application measurements, which is available at 4C beamline

• Further

improvement

• f

the performance

• f

4C beamline, including automation of SAXS experiments and data analysis, and time-resolved SAXS setups

• Embarkation on a collaborative research project to

study the structure of a wide range of self-assembled nanostructures and biological macromolecules

• Construction of computer system for molecular dynamic

(MD) simulation that could be combined well with time- resolved SAXS study – Pre-dynamic SAXS studies aimed at the 4th generation XFEL experiment

Future Projects and Goals Previous and Current Status

4C Small angle X-ray scattering (SAXS) Beamline at PAL

Sample Environments:

• Multi (100 holes) Sample Stage for Powder, Bulk, Film (25 ºC)
• Multi (10 lines) Sample Stage for Scan of Film (25 ºC)
• Multi (6 holes) Heating Sample Stage equipped with Eurotherm Controller for Powder,

Bulk, Film (25 ~ 400 ºC)

• Multi (5 holes) Cooling/Heating Sample Stage equipped with Julabo Circulation for Solution

(-15 ~ 200 ºC)

• Multi (5 holes) Heating Sample Stage equipped with Eurotherm Controller for Solution

(25 ~ 400 ºC)

• Single Cooling/Heating Sample Stage for Sol-gel (-15 ~ 400 ºC)

Equipments:

1)Malvern Zetasizer Nano Series DLS, 2)Refrigerated/multipurpose/high speed Centrifuge, 3)Ultarsonic Cleaner, 4)Water Purification System, 5)Cold Storage (4ºC), 6)Ultra-Low Temperature Freezer (-40ºC), 7)Bench Mixer, 8)Hanil Micro-12, 9)Thermo Scientific Heater, 10)METTLER TOLEDO EL204-IC Electronic Balance, 11)Pipettes, 12)S-1700, 13)NanoDrop, 14)GPC-FFF-MALS System

Multi (100 holes) sample stage for powder, bulk, film (25 ºC) Multi (10 lines) sample stage for film (25 ºC) N2 inlet Cooling water inlet Sample cell Heating bar cable Temperature sensor Multi (6 holes) heating sample stage equipped with Eurotherm Controller for powder, bulk, film (25 ~ 400 ºC) Single heating sample stage equipped with Eurotherm Controller for powder, bulk, film (25 ~ 400 ºC)

Sample Environments

Quartz-based solution cell Pipette + Tip Quartz capillary heating bar cable Sample cell Julabo connection part Single cooling/heating sample stage for sol-gel (-15 ~ 400 ºC) Single cooling/heating sample stage equipped with Julabo circulation for solution (-15 ~ 200 ºC) Multi (20 holes) cooling/heating sample stage for sol-gel (-15 ~ 200 ºC)

Sample Environments

Dual Detector System for Rayonix 165 and Pilatus 1M

GPC-FFF-MALS System

Pump Sample injector GPC column FFF channel UV/Vis MALS (18 channel) FFF RI Fraction collector

World-class Beamline

[FFF]

1) Injection flow 2) Cross flow 3) Channel flow

Before After

GPC-FFF-MALS System

◈ Test Result of BSA Protein Standard (MW = 66.450kDa, C= 2mg/ml, Injection volume = 15 ㎕, Experiment time = 13 min)

EPICS IOC-based Control for Main Optics STEPⅠ

Python-based Data Treatment Software

Automatic/selective background - transmission corrected 2D data acquisition - 1D radial averaged data acquisition – save, batch processing program buildup

STEP Ⅱ

(1) (2) (3) (4) (6) (5)

◈ Bio SAXS P12 beamline at the Petra-Ⅲ storage ring (Leader: Dr. Dmitri Svergun)

Automatic sample changer Purification system Dynamic and movable detector

A World-wide Leading Beamline

Medium- and Long-term Plan for 4C SAXS BL

Final Goal: High-throughput SAXS environmental instruments construction

• GPC-FFF-MALS System – Test and user service (2015 ~ 2016)
• PBPM orbit feedback system in FE (2016)
• Dual detector system of Rayonix 165 CCD (2009) and Pilatus 1M (2012, 9C BL) (2016)
• Upgrade of multi measure-data treatment-save related batch processing program (2016 ~ 2017)
• Inline construction of FPLC (Fast Protein Liquid Chromatography) system (2016 ~ 2017)

to purify mixtures of proteins (as requested by user groups)

• Rapid SDD exchanging system (Detector wagon in the vacuum chamber)
• Robotic sample exchanger for rapid and reliable high-throughput
• Feedback system of FE-PTL-End station
• K-B mirror
• Micro-DSC, mixer, micro-channel, UV, electric/magnetic field, pressure, etc.

Contents

• 1. Introduction of 4C SAXS Beamline at PAL
• 2. Research Results of Biomacromolecules in Solution

Experimental Procedure of Bio-SAXS

Synchrotron X-ray Beam Sample 2θ CCD Camera qz qx qy z x y Beamstop )

IMolecules + Buffer solution (q) IBuffer solution (q) IMolecules (q)

• 1. Sample measurement
• 2. Background measurement
• 4. Corrected 2D SAXS image
• 5. Radial averaged 1D SAXS curve
• 3. Background correction

▪ Experimental procedures 1 2 3 4 5

0.0 0.1 0.2 0.3 0.4 0.5

q (nm-1)

6 5 4 3 2 1

log I (a.u.)

Solid sphere Hollow sphere Dumbbell Flat disk Long rod D 2 4 6 8 10

r (nm) p(r) (a.u.)

Data Treatment and 3D Structural Model

(1) Radial Averaged 1D SAXS Curve (2) Indirect Fourier Transform Method → Pair Distance Distribution Function (PDDF) (3) Ab initio Shape Determination Method → Reconstructed 3D Structural Model

(1) (2) (3)

2D SAXS Pattern

STEP 2. 3D Modeling using Simulated Annealing STEP 1. Data Treatment and p(r) Function

1D SAXS Curve p(r) Function Initial 3D Model Final 3D Model Intermediate 3D Model

Solid sphere Hollow sphere Dumbbell Flat disk Long rod

Detailed Experimental Procedures of Bio-SAXS

• Determination of optimum temperature, pH, and buffer conditions
• Analysis of purity and homogeneity of solution sample using basic biological/chemical analysis
• Determination of direct main beam center from standard sample and CCD detector
• Elimination of parastic scattering by means of 4-axis slit components
• Determination of sample-to-detector distance (SDD) : optimum scattering vector q range
• Determination of solution sample concentration : elimination of inter-particle effect
• Determination of X-ray exposure time : elimination of X-ray damage effect
• Background correction of air/cell window/solvent scattering using scintillation counter detector
• Corrected 2D SAXS image → 1D SAXS curve conversion
• Determination of reliable q range from standard and water samples
• Estimation of radius of gyration Rg from Guinier plot
• Basic structural analysis from Kratky plot
• Calculation of pair distance distribution function (PDDF) from Indirect Fourier Transform method
• Acquisition of 3D reconstructed model using ab-initio shape determination method
• Investigation of overall and internal structures from structural comparison
• 1. Check Points before SAXS Measurement
• 2. Data Treatment and Analysis after SAXS Measurement

among solution and crystal and theoretical calculated models

Schematic for SAXS data collection, evaluation, analysis, modeling, and interpretation

Data extrapolation, merging, analysis, modeling and interpretation

Proton-sensitive i-motif DNA (telomer) ?

Sequence : H2NC6H12-5’-CCC TAA CCC TAA CCC TAA CCC-3’-C6H12(CH2OH)NH2 H+ OH-

= C60 (1) (2)

Fu Fully lly Closed [contractile ile] ] State : stable le 완전히 닫힌 (수축) ) 상태 Less ss Ope pen [rela laxed] d] State : unstable le 덜 열린 (이완) ) 상태 Less ss Closed [contractile ile] State : unstable le 덜 닫힌 (수축) ) 상태

H+: dissociation OH- : association

Fu Fully lly Open [rela laxed] ] State : stable le 완전히 열린 (이완) ) 상태

Compl plementary ary DNA NA

(3)

Ful ullere rene

Result of Research

Representative models used to simulate the structure of i-motif DNA in solution at various pH values.

1.1. Theoretical Model of i-motif DNA

Fu Fully lly Closed [contractile ile] State 닫힌 (수축) ) 상태 Fu Fully lly Open [rela laxed] ] State 열린 (이완) ) 상태

Strong acidic induced fragmentation

1.2. SAXS Curves of i-motif DNA

▪ Color lines - Theoretical SAXS curves ▪ Symbols - Experimental SAXS curves ▪ Black solid lines - SAXS curves from the p(r) fit

(a) (b) (c)

pH 6.15

1nm

(b)

pH 5.33

1nm

5’ end 3’ end C3 C15

(a)

ⅹ

Thymine (T) Adenine (A) Cytosine (C)

pH 8.03

1nm

(c)

VV

pH 11.2

1nm

(d)

Structural models of the i-motif DNA in solutions of various pH conditions. The reconstructed models were

• btained without imposing symmetry restrictions by the program DAMMIN, respectively.
• J. Phys. Chem. B 2009, 113, 1852-1856.

☞ Improvement o

• f L

Less s Closed [contractil ile] State ? ?

1.3. Structural Model of i-motif DNA

H+ OH-

= C60

(1) Fullerene binding-induced transformation (2) pH-induced conformational switching

(1) (2)

Representative models of non-functionalized DNA and fullerene DNA hybrid (FDH) nanomachine and the working switching cycle by protons.

Close Open Proton-sensitive i-motif DNA

☞ Hy Hydrophobic ic int nteractio ion (소수성 상호작용)

2.1. Theoretical Model of C60/i-motif DNA Hybrid

Fu Fully lly Closed [contractile ile] ] State : stable le 완전히 닫힌 (수축) ) 상태 Less ss Open [rela laxed] State : unstable le 덜 열린 (이완) ) 상태 Less ss Closed [contractile ile] State : unstable le 덜 닫힌 (수축) ) 상태

Stabilizing by C60 fullerene : C60 proton-sensitive DNA fullerene DNA hybrid nanomachine

The distance distribution p(r) functions for non-functionalized DNA and FDH in solution at pH ≈ 5 conditions, based on an analysis of the experimental SAXS data.

• Adv. Mater. 2009, 21, 1907-1910.

2.2. Structural Model of C60/i-motif DNA Hybrid

☞ Improvement of Less ss open [rela laxed] State ?

산성: 분해 H+: dissociation 염기성: 결합 OH- : association

Representative models of i-motif DNA with a carboxyl functionalized C60 and working cycle of the pH-induced conformational switching between the duplex (open state) and the i-motif (closed state) structure in the presence of cDNA.

Fu Fully lly Closed [contractile ile] ] State : stable le 완전히 닫힌 (수축) ) 상태 Fu Fully lly Open [rela laxed] ] State : stable le 완전히 열린 (이완) ) 상태 Comple lementary DNA 상보 DNA

3.1. Theoretical Model of Duplex C60/i-motif DNA Hybrid

Structural models of duplex conformation of (a, b) i-motif DNA system and (c, d) C60/i-motif DNA hybrid system at different pH values. The reconstructed models were obtained without imposing symmetry restrictions by the program DAMMIN, respectively.

• J. Phys. Chem. B 2010, 114, 4783-4788.

3.2. Structural Model of Duplex C60/i-motif DNA Hybrid

C60 : fullerene Complementary DNA Sample (pH 5.0) Tm (oC) ΔG a (kJ/mol) Power stroke b (%) Closing force c (pN)

i-motif DNA 50.2

• 11.1

80 2.8 C60/i-motif DNA hybrid 65.6

• 26.3

86 8.5

a Van’t Hoff plot b Contraction Strain = Lmax-Lmin/ Lmax c Fclose= Wclose(=ΔG) / ΔL

Sample (pH 8.0) Transition pH Tm (oC) ΔG a (kJ/mol)

Duplex of i-motif DNA system 4.5 51.0

• 18.9

Duplex of C60/i-motif DNA hybrid system 5.5 57.8

• 20.1
• 1. Structural aspect (구조적 측면)
• 2. Thermodynamical/mechanical aspect [열역학적/기기학적 측면]

cf) Kinesin (2pN), Myosin (3-4 pN)

Step ep 1 Less ess Cl Close

• sed [con
• ntr

tractile tile] st state Step ep 3 Full lly op

• pen

en [rel elaxed] State te Step ep 2 Full lly Cl Clos

• sed [contractile

tile] st state

Conclusions

• 1. Digestion-Related Pepsin Protein

Structural models of porcine pepsin at different pH values. The reconstructed models were obtained without imposing symmetry restrictions by the program GASBOR, respectively.

Structural Model in Solution

Jin, K. S.; Yoon, J.; Heo, K.; Jin, S.; Kim, J.; Kim, S; Ree M, J. Phys. Chem. B 2008, 112, 15821-15827.

Structural Model in Solution

• 2. Estrogen-Related Receptor (ERR) Protein

(i)

Collaboration : Dr. Eunice EunKyeong Kim (KIST)

Jin, K. S.; Park, J. K.; Yoon, J.; Rho, Y.; kim, J. H.; Kim, E.; Ree M, J. Phys. Chem. B 2008, 112, 9603-9612.

• 3. Stress-Related RseA/RseB Protein

Collaboration : Prof. Kyeong Kyu Kim (Sungkyunkwan Univ.)

(1) Kim, D. Y.; Jin, K. S.; Kwon, E.; Ree, M.; Kim, K. K. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 8779-8784. (2) Jin, K. S.; Kim, D. Y.; Rho, Y.; Le, V. L.; Kwon, E.; Kim, K. K.; Ree. M. J. Synchrotron Rad. 2008, 15, 219-222.

(a) Crystal structure of Escherichia coli RseB at a resolution of 0.24 nm The solution models of RseB (b) and RseA121–216/RseB complex (c) restored from the SAXS data at a resolution of 1.25 nm. The ribbon diagram of the RseB is overlapped onto the solution model of RseB for the comparison of overall shape and dimension

Structural Model in Solution

SAXS Studies of a Wild-Type and Ketosteroid Isomerase and Its Single Mutant

• H. J. Cha, D. S. Jang, K. S. Jin*, H. J. Lee, B. H. Hong, E.-S. Kim, J. Kim, H. C. Lee, K. Y. Choi*, and M. Ree*,

Science of advanced Materials 6, 2325 (2014)

Research Background

4.1. Ketosteroid Isomerase (KSI) Protein

Porod Plots p(r) function SAXS curves Guinier Plots

4.2. Ketosteroid Isomerase (KSI) Protein

4.3. Ketosteroid Isomerase (KSI) Protein

Structural Model in Solution

Volume of active-site cavity: WT-KSI (899Å3) Mutant KSI(1116Å3)

<

Yaoyao Fu, Youngran Kim, Kyeong Sik Jin, Hyun Sook Kim, Jong Hyun Kim, DongMing Wang, Minyoung Park, Chang Hwa Jo, Nam Hoon Kwon, Doyeun Kim, Myung Hee Kim, Young Ho Jeon, Kwang Yeon Hwang, Sunghoon Kim, Yunje Cho Proceedings of the National Academy of Sciences of the United States of America 2014, 111, 15084-15089.

[ Hexameric structure of the RQA subcomplex ] “Sturcture of the ArgRS-GlnRS-AIMP1 complex and its implications for mammalian translation”

5.1. Mammalian Translation-related Protein

5.2. Mammalian Translation-related Protein

Structural Model in Solution

“Molecular basis for SMC rod formation and its dissolution upon DNA binding”

Young-Min Soh, Frank Bürmann, Ho-Chul Shin, Takashi Oda, Kyeong Sik Jin, Christopher P. Toseland, Cheolhee Kim, Han-Sol Lee, Su Jin Kim, Min-Seok Kong, Marie-Laure Durand-Diebold, Yeon-Gil Kim, Ho Min Kim, Nam-Ki Lee, Mamoru Sato, Byung-Ha Oh*, Molecular Cell 2015, 57, 290-303.

• 6. Chromosom organization-related SMC Protein

SMC (Structural Maintenance of Chromosome)

Collaboration : Prof. Kyeung-Jin Kim (Kyungpook National Univ.) Jae-Woo Ahn†, Kyeong Sik Jin†, Hyeoncheol Francis Son, Jeong Ho Chang, and Kyung-Jin Kim* Scientific Reports 2015, 5, 14251

• 7. mRNA Splicesome-related Protein Complex

Collaboration : Prof. Eun Chul Cho (Amorepacific & Hanyang Univ.) Do-Hoon Kim, Sora Lim, Jongwon Shim, Ji Eun Song, Jong Soo Chang, Kyeong Sik Jin*and Eun Chul Cho* ACS Applied Materials & Interfaces 2015, 7, 20438-20446

• 8. Liquid Crystalline Lipid Nanoparticles

Sample NCPMs CP CPMs Mo Model sphere sphere Typ ype homogeneous core e and and shell ll homogeneous core e and and shell ll

aRc (nm)

3. 3.22 220

b(4.

4.317) 1. 1.46 460

b(1.

1.914)

cσR

0. 0.32 326

• 0.

0.32 322

• dRc, max (nm)

2. 2.88 888

e(3.

3.944) 1. 1.22 228

e(1.

1.732)

fRm (nm)

11 11.9 .90

g(15

15.69) 13 13.7 .70

g(17

17.96)

hRm, max (nm)

10 10.6 .61

i(14

14.48) 12 12.2 .21

i(16

16.40)

jRho

0. 0.10 106

• 0.

0.01 019

• Collaboration : Dr. Hyun-Chul Kim (DGIST)

Hyun-Chul Kim*, Kyeong Sik Jin*, Se Guen Lee, Eunjoo Kim, Sung Jun Lee, Sang Won Jeong, Seung Woo Lee, and Kwang-Woo Kim. Journal of Nanoscience and Nanotechnology 2016, 16, 6432-6439.

• 9. Cross-linked Polymer Micelles

Thank you for your attention