우리는 그라츠 (Graz), 오스트리아 과학 아카데미 오스트리아에서 박사 만프레드 Hecus의 XRS의 Kriechbaum 및 생물 물리학 및 Nanosystems 연구 연구소 감사드립니다. LL 및 XW는 수상 번호 NRC-38-08-950 아래 네리-C 상 번호 DE-FG07-07ID14889, 미국 원자력 규제위원회에서, 에너지 미국학과에 의해 부분적으로 지원되었다. SWAXS기구는 수상 번호 DE-NE0000325 아래, 에너지의 미국학과에 의해 부분적으로 지원됩니다.

1. 샘플의 작성 <ol><li> 샘플 컨테이너에서 샘플의 일부를 제거하는 바늘을 사용합니다. * </li><li> 모세관을 채우기 위해 바늘을 사용 (2.2 mm 최대 직경) 샘플. 모세는 바닥에서 2 3cm 사이에 작성해야합니다. </li><li> 그 끝에 녹는 왁스로 모세관을 닫습니다. </li><li> 나사가 빠지다 시스템에서 진공 샘플 홀더를. </li><li> 융합 끝 (왁스 끝)에 의해 모세관를 타고 샘플 홀더에 해제 - 융합 끝을 삽입합니다. </li><li> 장소에 시스템 및 나사에 다시 홀더를 배치합니다. </li></ol> 액체 샘플이 모세관에 삽입해야합니다 반면, * 고체 시료 (분말 샘플 포함) 직접 샘플 홀더 (NO 모세관 필요)에 배치 할 수 있습니다. 시작 - 업 SWAXS 기계의 2. 소스 냉간 시동 절차 <ol><li> 안전 셔터 버튼을 눌러 셔터를 닫습니다. </li><li> 주 전원 스위치버튼을 녹색을 눌러 &quot;ON&quot;. </li><li> 긴급 &quot;시스템 종료 - 오프 &#39;버튼을 확장 위치에 있는지 확인합니다. </li><li> 녹색 버튼을 눌러에 주 전원을 전환합니다. </li><li> 연동 LED의 빛은 모든 연동 괜찮 니 나타내는 녹색 확인합니다. </li><li> 로드 터치 스크린이 나올 때까지 기다리는 것이 좋습니다. 일단로드, 메뉴의 &quot;R6&quot;를 누르십시오. </li><li> &quot;ON&quot;위치로 대기 모드 키를 설정합니다. </li><li> &quot;ON&quot;위치로 X-레이 키를 설정합니다. 빨간색 X-선 라이트가 점등합니다. </li><li> 대기 수준 (30 KV와 0.4 mA)을 읽는 터치 스크린이 나올 때까지 기다리는 것이 좋습니다. </li><li> &quot;OFF&quot;위치에 대기 안전 키를 설정합니다. 터치 스크린 하단에있는 &quot;시작주기&quot;버튼을 누르십시오. </li><li> 정격 출력 (50 KV와 1mA)를 읽어 터치 스크린이 나올 때까지 기다리는 것이 좋습니다. 다른 전원 수준이 필요하다면, 터치 스크린에 &#39;R4&#39;를 누르고 원하는 설정을 입력하여 구성 화면을 입력합니다. </li></ol> 3. 냉각기 절차 <ol><li> 전원을 켜 swi온도 제어 패널의 &quot;ON&quot;위치로 tch. 제어 빛과 LED 온도 표시가 점등됩니다. </li><li> 냉각기에있는 &quot;ON&quot;위치로 전원 스위치를 켜십시오. </li><li> 온도 디스플레이가 읽 때까지 기다리십시오 &quot;OFF.&quot; 이 대기 모드입니다. </li><li> 누르면 약 4 초 또는 온도 표시 될 때까지 ENTER 버튼 (반환 기호)를 개최는 실제 온도를 나타냅니다. </li><li> 온도를 변경하려면 위 또는 아래 화살표를 누르십시오. 그것이 실제 값으로 돌아갑니다 전에 설정 값은 약 8 초 동안 온도 디스플레이에 표시됩니다. </li><li> 원하는 설정 값이 표시되면 ENTER 버튼을 누릅니다. 이것은 값을 저장합니다. </li><li> 실제 온도가 원하는 설정 값을 도달 할 때까지 기다리십시오. </li></ol> 참고 : 언제든지 오류 &quot;E 01&quot;온도 디스플레이에 표시되면, 탱크 fil에 정화 물을 붓는하여 냉각 시스템 종료 방법, 충전 목욕 단위를 따르십시오저는 다음 다시 냉각기의 시작 절차를 수행합니다. 4. 냉각기의 종료 <ol><li> 약 4 초에 &quot;를 입력&quot;키를 누릅니다. </li><li> &quot;OFF&quot;위치로 전원 스위치를 켜십시오. </li></ol> 5. 진공 켜기 <ol><li> 진공 문이 곳에서 고정되어 있는지 확인합니다. </li><li> &quot;ON&quot;위치로 진공 1, 2 노브를 켜십시오. </li><li> VAP5 진공 게이지 1.5 mbar보다 진공 레벨 이하 읽습니다 때까지 기다리십시오. </li></ol> 6. 감지기 시스템 설치 <ol><li> 가스 압력을 설정하려면 확인 감소 밸브의 주요 압력 게이지는 최소 10 줄입니다. </li><li> 메인 밸브를 엽니 다. </li><li> 압력이 운영 압력 게이지 8 줄을 도달 할 때까지 천천히 작동 압력 밸브를 엽니 다. </li><li> 두 번째 메인 밸브를 엽니 다 </li><li> 가스의 흐름을 조정하려면 가스 제어 패널의 수직 위치에 노브를 돌려 메인 압력 밸브를 엽니 다. </li><li> 흐름을 조정니들 밸브와 속도 게이지가 약 8 줄을 읽습니다 때까지. </li><li> &quot;ON&quot;위치로 주 전원 스위치를 켜십시오. LED 빛은 점등합니다. </li><li> 높은 전압을 조정하려면 &quot;ON&quot;위치로 스위치를 설정하여 높은 전압을 활성화합니다. LED 빛은 점등합니다. </li><li> 약 3.5kV이 게이지에 도달 할 때까지 천천히 전압 제어 노브를 켜십시오. 4kV를 초과하지 마십시오. </li></ol> 7. 구경 측정 <ol><li> 위의 실험 단계는 알려진 봉우리의 샘플로 수행됩니다. </li><li> ASA 3.3은 5 봉우리와 강도 대 채널의 그래프를 얻을하는 데 사용됩니다. </li><li> 메뉴로 이동 → 옵션 → 채널과 해당 농도를 입력합니다. </li><li> 첫번째 &quot;TPF &#39;탭을 ASA3로 이동합니다. </li><li> 필터 변경, 상단 1mm 니켈을 (빔 필터) 떠났다. </li><li> → 위치로 이동 검출기는 정말이에요 있는지 확인해야 진공은 아래 5 mbar인지 확인 위치 32000을 (32000에 이르는 28,000, 28,000 주위에 중요한 지역) 설정1,000 - 10,000 사이 땡. </li><li> 초 당 약 52 카운트에 대한 10 초에 실행합니다. </li><li> 에너지 창을 (또는하지 필터 케이스 포함 만 샘플로 에너지 창을 측정) 변경합니다. 10 초에 대한 최종 실행을 설정합니다. </li><li> 주 빔 (예를 들어 채널 233)의 위치를​​ 찾습니다. </li><li> 다른 실행를 들어, 2mm 텅스텐 (W, 빔 스토퍼)로 다시 필터링하고 30,000부터 시작을 변경할 수 있습니다. </li><li> 샘플 다른 모든 봉우리의 위치를​​ 찾습니다. </li><li> SAXS을 보정 ImageJ - 매크로 소프트웨어를 사용하여 - (그림 1) 강도 대 채널에서 강도 대 Q (또는 D 간격)로 전환하는. </li><li> WAXS을 보정 ImageJ - 매크로 소프트웨어를 사용 - 강도 대 채널에서 강도 대 Q (또는 D 간격)로 전환하기 (그림 2). </li></ol> 8. 소프트웨어 절차 <ol><li> 라이브 데이터를 수집하기 위해, 소프트웨어에게 ASA 3.3 사용 : 라이브 데이터 → TPF 탭 → 파일을 저장합니다. </li><li> EasySWAX에 대한S는 장치 설정 탭을 클릭합니다. </li><li> &quot;A&quot;, 람다, 및 D 값뿐만 아니라 무게 중심을 입력합니다. </li><li> 포인트 시준을 선택합니다. </li><li> 구상 입자 유형을 선택합니다 (이것은 평면 모양의 입자의로드로 알려져 있지 않는 한). </li><li> Guinier - 플롯 탭을 클릭합니다. I 대 Q이 그래프가 표시됩니다. </li><li> 약 선형 기울기의 한 부분을 둘러싸고있는 수직 라인을 끕니다. </li><li> 화면의 하단에서 R 값이 표시됩니다. 이 옆에 유효성 검사 요구 사항이 있습니다. </li><li> 유효성 검사 값이 1과 2 사이에 때까지 서로 가깝게 수직 라인을 드래그하고 있습니다. 이 주관적입니다. 획득 R 값, 단면 2 차 반경, 추정치입니다. </li></ol> 9. 소스 종료 절차 <ol><li> 안전을 확인하고 빠른 셔터은 실행 종료와 함께 해제된다. </li><li> &quot;ON&quot;위치로 대기 모드 키를 설정합니다. </li><li> 대기 수준을 읽을 터치 스크린 기다립니다. </li><li> t에 보도 R5ouchscreen. </li><li> 0 A.에 전류를 감소 </li><li> 0 A.에 전압을 감소 </li><li> &quot;OFF&quot;위치에 X-레이 키를 설정합니다. </li><li> 빨간색 해제 버튼을 눌러 주 전원 &quot;OFF&quot;를 전환합니다. </li></ol>

<ol>
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관심 없음 충돌이 선언 없습니다.

SWAXS 시스템의 비교 절차는 실험 분석에서 결정되는 수많은 변수를 할 수 있습니다. 분석 달성하는 매개 변수 샘플 및 실험 설정에 따라 다양한 용도로 사용할 수 있습니다. WAXS는 원자 및 마이크로 규모의 구조 (예 : 분자 격자, 단위 셀 치수 대칭)에 초점을 맞추고 반면 SAXS는 나노 규모의 크기와 물체의 모양에 대한 정보를 제공합니다. 좀 더 구체적으로 말하자면, 희석 솔루션의 입자를 들?...

<table border="1"><tbody><tr><td> 제품의 이름 </td><td> 회사 </td></tr><tr><td> System3 소형 및 와이드 앵글 X-레이 산란 (SWAXS) 카메라 </td><td> Hecus XRS와 이븐, 그라츠, 오스트리아 </td></tr><tr><td> GNOM </td><td> D. Svergun EMBL - 함부르크 (Hamburg)의 ATSAS 2.3 패키지 </td></tr></tbody></table>

small and wide angle x-ray scattering studies of biological macromolecules in solution

본 논문에서는 고분자의 소형 및 와이드 앵글 X-선 산란 (SWAXS) 분석 실험을 통해 입증된다. 넓은 각도 (일반적으로&gt; 5 °) - SWAXS는 X-선을 탄 성적으로 작은 각도 (5 ° 일반적으로 0.1)에서 나노 미터 범위의 inhomogeneous 샘플에 의해 분산되어 기술입니다. 이 기술은 모양, 크기, 고분자, 부분적으로 주문 자료, 기공 크기 및 표면 - 투 - 볼륨 비율의 특성 거리의 분포에 대한 정보를 제공합니다. 와이드 앵글 X-선 산란 (WAXS)가에 따라 0.33 나노 미터와 0.49 나노 미터 사이의 샘플도 작은 브래그 간격을 해결할 수 있습니다 반면, 작은 각도 X-선 산란 (SAXS)는, 1, 200 nm의 사이에 고분자의 구조 정보를 제공 할 수 있습니다 특정 시스템 설정 및 검출기. 간격은 브래그의 법칙에서 결정하고 파장과 사건 각도에 따라 달라입니다. SWAXS 실험에서, 자료는 고체 수 있습니다또는 액체와 조합에서 동일한 또는 다른 재료의 고체, 액체 또는 기체 도메인 (소위 입자)를 포함 할 수 있습니다. SWAXS 응용 프로그램은 매우 광범위하고 모든 유형의 colloids은 다음과 같습니다 금속, 복합 재료, 시멘트, 석유, 폴리머, 플라스틱, 단백질, 식품 및 제약합니다. 고체 시료를 들어, 두께는 약 5mm로 제한됩니다. 실험실 기반의 SWAXS 장비의 사용이 논문에 자세히 설명되어 있습니다. SWAXS 시스템에 사용할 수있는 소프트웨어 (예를 들어, D. Svergun EMBL - 함부르크 (Hamburg)와 EasySWAXS 소프트웨어를 사용하여 GNOM - ATSAS 2.3 패키지)를 통해 실험은 주어진 샘플에 대한 관심의 특정 매개 변수를 결정하기 위해 실시 할 수 있습니다. 생물 macromolecule 실험 중 하나 예 선택하고 다양한 방법으로 준비 할 수있는 물을 기반 수성 버퍼 2 중량 %의 라이소자임의 분석이다. 샘플의 준비는 샘플 섹션의 준비에 아래의 지침을 따릅니다. SWAXS 실험을 통해,라이소자임의 중요한 구조 매개 변수는 단면 2 차 반경을 예를 들어, 분석 할 수 있습니다.

선전, 입자의 크기와 모양, 솔루션 구조 요소, 특정 내부 표면 및 기공 크기, 격자 유형 및 크기, 그리고 전자 밀도 1의 반경 : SAXS와 WAXS 교수는 다음 매개 변수를 통해 샘플의 구조 정보를 제공 할 수 있습니다. SAXS 및 WAXS 또한 단백질 역학 (2)의 연구에 적용 할 수 있습니다. SWAXS 실험의 구조 정보는 실험적으로 발견 스펙트럼과 시스템의 계산 결과를 비교하여 ...

Watch this Scientific Journal Video about Small and Wide Angle X-Ray Scattering Studies of Biological Macromolecules in Solution at JoVE.com

Small and Wide Angle X-Ray Scattering Studies of Biological Macromolecules in Solution

작고 다양한 각도 X-선 산란 (SWAXS) 절차의 데모는 생물 고분자의 연구 수단이되고있다. 특정 각도 방법과 준비 계측 및 절차의 사용을 통해, SWAXS의 실험 데이터는 고분자의 원자 및 나노 규모 특성을 표시합니다.

솔루션의 생물 고분자의 작은 및 와이드 앵글 X-레이 산란 연구

In this paper, Small and Wide Angle X-ray Scattering (SWAXS) analysis of macromolecules is demonstrated through experimentation. SWAXS is a technique ...

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18.1K 조회수.  Rensselaer Polytechnic Institute. 작고 다양한 각도 X-선 산란 (SWAXS) 절차의 데모는 생물 고분자의 연구 수단이되고있다. 특정 각도 방법과 준비 계측 및 절차의 사용을 통해, SWAXS의 실험 데이터는 고분자의 원자 및 나노 규모 특성을 표시합니다.

비디오: Small and Wide Angle X-Ray Scattering Studies of Biological Macromolecules in Solution

Online Size-exclusion and Ion-exchange Chromatography on a SAXS Beamline

Biological small angle X-ray scattering (BioSAXS) is a powerful technique in molecular and structural biology used to determine solution structure, particle size and shape, and surface-to-volume ratio of macromolecules. The technique is applicable to a very wide variety of solution conditions spanning a broad range of concentrations, pH values, ionic strengths, temperatures, additives, etc., but the sample is required to be monodisperse. This caveat led to the implementation of liquid chromatography systems on SAXS beamlines. Here, we describe the upstream integration of size-exclusion (SEC) and ion-exchange chromatography (IEC) on a beamline, different methods for optimal background subtraction, and data reduction. As an example, we describe how we use SEC- and IEC-SAXS on a fragment of the essential vaccinia virus protein D5, consisting of a D5N helicase domain. We determine its overall shape and molecular weight, showing the hexameric structure of the protein.

The determination of the solution structure of a protein by small angle X-ray scattering (SAXS) requires monodisperse samples. Here, we present two possibilities to ensure minimal delays between sample preparation and data acquisition: online size-exclusion chromatography (SEC) and online ion-exchange chromatography (IEC).

SAXS 빔라인에 온라인 크기 배제 및 이온 교환 크로마토 그래피

Biological small angle X-ray scattering (BioSAXS) is a powerful technique in molecular and structural biology used to determine solution structure, ...

연구

생화학

Combining X-Ray Crystallography with Small Angle X-Ray Scattering to Model Unstructured Regions of Nsa1 from S. Cerevisiae

Determination of the full-length structure of ribosome assembly factor Nsa1 from Saccharomyces cerevisiae (S. cerevisiae) is challenging because of the disordered and protease labile C-terminus of the protein. This manuscript describes the methods to purify recombinant Nsa1 from S. cerevisiae for structural analysis by both X-ray crystallography and SAXS. X-ray crystallography was utilized to solve the structure of the well-ordered N-terminal WD40 domain of Nsa1, and then SAXS was used to resolve the structure of the C-terminus of Nsa1 in solution. Solution scattering data was collected from full-length Nsa1 in solution. The theoretical scattering amplitudes were calculated from the high-resolution crystal structure of the WD40 domain, and then a combination of rigid body and ab initio modeling revealed the C-terminus of Nsa1. Through this hybrid approach the quaternary structure of the entire protein was reconstructed. The methods presented here should be generally applicable for the hybrid structural determination of other proteins composed of a mix of structured and unstructured domains.

Combining X-Ray Crystallography with Small Angle X-Ray Scattering to Model Unstructured Regions of Nsa1 from S. Cerevisiae

This method describes the cloning, expression, and purification of recombinant Nsa1 for structural determination by X-ray crystallography and small-angle X-ray scattering (SAXS), and is applicable for the hybrid structural analysis of other proteins containing both ordered and disordered domains.

S. Cerevisiae 에서 Nsa1의 구조화 되지 않은 영역을 모델링 하는 데 작은 각 x 선 산란 된 x-선 결정학을 결합

Determination of the full-length structure of ribosome assembly factor Nsa1 from Saccharomyces cerevisiae (S. cerevisiae) is challenging because of the ...

Contrast-Matching Detergent in Small-Angle Neutron Scattering Experiments for Membrane Protein Structural Analysis and Ab Initio Modeling

The biological small-angle neutron scattering instrument at the High-Flux Isotope Reactor of Oak Ridge National Laboratory is dedicated to the investigation of biological materials, biofuel processing, and bio-inspired materials covering nanometer to micrometer length scales. The methods presented here for investigating physical properties (i.e., size and shape) of membrane proteins (here, MmIAP, an intramembrane aspartyl protease from Methanoculleus marisnigri) in solutions of micelle-forming detergents are well-suited for this small-angle neutron scattering instrument, among others. Other biophysical characterization techniques are hindered by their inability to address the detergent contributions in a protein-detergent complex structure. Additionally, access to the Bio-Deuteration Lab provides unique capabilities for preparing large-scale cultivations and expressing deuterium-labeled proteins for enhanced scattering signal from the protein. While this technique does not provide structural details at high-resolution, the structural knowledge gap for membrane proteins contains many addressable areas of research without requiring near-atomic resolution. For example, these areas include determination of oligomeric states, complex formation, conformational changes during perturbation, and folding/unfolding events. These investigations can be readily accomplished through applications of this method.

Contrast-Matching Detergent in Small-Angle Neutron Scattering Experiments for Membrane Protein Structural Analysis and Ab Initio Modeling

This protocol demonstrates how to obtain a low-resolution ab initio model and structural details of a detergent-solubilized membrane protein in solution using small-angle neutron scattering with contrast-matching of the detergent.

막 단백질 구조 분석 및 Ab 론 적 모델링 작은 각 중성자 산란 실험에서 일치 하는 대비 세제

The biological small-angle neutron scattering instrument at the High-Flux Isotope Reactor of Oak Ridge National Laboratory is dedicated to the ...

Structural Studies of Macromolecules in Solution using Small Angle X-Ray Scattering

Protein-protein interactions involving proteins with multiple globular domains present technical challenges for determining how such complexes form and how the domains are oriented/positioned. Here, a protocol with the potential for elucidating which specific domains mediate interactions in multicomponent system through ab initio modeling is described. A method for calculating solution structures of macromolecules and their assemblies is provided that involves integrating data from small angle X-ray scattering (SAXS), chromatography, and atomic resolution structures together in a hybrid approach. A specific example is that of the complex of full-length nidogen-1, which assembles extracellular matrix proteins and forms an extended, curved nanostructure. One of its globular domains attaches to laminin &#947;-1, which structures the basement membrane. This provides a basis for determining accurate structures of flexible multidomain protein complexes and is enabled by synchrotron sources coupled with automation robotics and size exclusion chromatography systems. This combination allows rapid analysis in which multiple oligomeric states are separated just prior to SAXS data collection. The analysis yields information on the radius of gyration, particle dimension, molecular shape and interdomain pairing. The protocol for generating 3D models of complexes by fitting high-resolution structures of the component proteins is also given.

Here, we present how Small Angle X-Ray Scattering (SAXS) can be utilized to obtain information on low-resolution envelopes representing the macromolecular structures. When used in conjunction with high-resolution structural techniques such as X-Ray Crystallography and Nuclear Magnetic Resonance, SAXS can provide detailed insights into multidomain proteins and macromolecular complexes in-solution.

작은 각 엑스레이 뿌리기를 사용 하 여 솔루션에서 고분자의 구조 연구

Protein-protein interactions involving proteins with multiple globular domains present technical challenges for determining how such complexes form and ...

An All-in-one Sample Holder for Macromolecular X-ray Crystallography with Minimal Background Scattering

Macromolecular X-ray crystallography (MX) is the most prominent method to obtain high-resolution three-dimensional knowledge of biological macromolecules. A prerequisite for the method is that highly ordered crystalline specimen need to be grown from the macromolecule to be studied, which then need to be prepared for the diffraction experiment. This preparation procedure typically involves removal of the crystal from the solution, in which it was grown, soaking of the crystal in ligand solution or cryo-protectant solution and then immobilizing the crystal on a mount suitable for the experiment. A serious problem for this procedure is that macromolecular crystals are often mechanically unstable and rather fragile. Consequently, the handling of such fragile crystals can easily become a bottleneck in a structure determination attempt. Any mechanical force applied to such delicate crystals may disturb the regular packing of the molecules and may lead to a loss of diffraction power of the crystals. Here, we present a novel all-in-one sample holder, which has been developed in order to minimize the handling steps of crystals and hence to maximize the success rate of the structure determination experiment. The sample holder supports the setup of crystal drops by replacing the commonly used microscope cover slips. Further, it allows in-place crystal manipulation such as ligand soaking, cryo-protection and complex formation without any opening of the crystallization cavity and without crystal handling. Finally, the sample holder has been designed in order to enable the collection of in situ X-ray diffraction data at both, ambient and cryogenic temperature. By using this sample holder, the chances to damage the crystal on its way from crystallization to diffraction data collection are considerably reduced since direct crystal handling is no longer required.

A novel sample holder for macromolecular X-ray crystallography along with a suitable handling protocol is presented. The system allows crystal growth, crystal soaking and in situ diffraction data collection at both, ambient and cryogenic temperature without the need of any crystal manipulation or mounting.

최소한의 배경 산란을 갖춘 거대 분자 X선 결정학을 위한 올인원 샘플 홀더

Macromolecular X-ray crystallography (MX) is the most prominent method to obtain high-resolution three-dimensional knowledge of biological macromolecules. ...

Analysis of SEC-SAXS data via EFA deconvolution and Scatter

BioSAXS is a popular technique used in molecular and structural biology to determine the solution structure, particle size and shape, surface-to-volume ratio and conformational changes of macromolecules and macromolecular complexes. A high quality SAXS dataset for structural modeling must be from monodisperse, homogeneous samples and this is often only reached by a combination of inline chromatography and immediate SAXS measurement. Most commonly, size-exclusion chromatography is used to separate samples and exclude contaminants and aggregations from the particle of interest allowing SAXS measurements to be made from a well-resolved chromatographic peak of a single protein species. Still, in some cases, even inline purification is not a guarantee of monodisperse samples, either because multiple components are too close to each other in size or changes in shape induced through binding alter perceived elution time. In these cases, it may be possible to deconvolute the SAXS data of a mixture to obtain the idealized SAXS curves of individual components. Here, we show how this is achieved and the practical analysis of SEC-SAXS data is performed on ideal and difficult samples. Specifically, we show the SEC-SAXS analysis of the vaccinia E9 DNA polymerase exonuclease minus mutant.

SEC-BioSAXS measurements of biological macromolecules are a standard approach for determining solution structure of macromolecules and their complexes. Here, we analyze SEC-BioSAXS data from two types of commonly encountered SEC traces&#8212;chromatograms with fully resolved and partially resolved peaks. We demonstrate the analysis and deconvolution using scatter and BioXTAS RAW.

EFA 감소 및 분산을 통한 SEC-SAXS 데이터 분석

BioSAXS is a popular technique used in molecular and structural biology to determine the solution structure, particle size and shape, surface-to-volume ...

Study of Protein Dynamics via Neutron Spin Echo Spectroscopy

Most human body proteins' activity and functionality are related to configurational changes of entire subdomains within the protein crystal structure. The crystal structures build the basis for any calculation that describes the structure or dynamics of a protein, most of the time with strong geometrical restrictions. However, these restrictions from the crystal structure are not present in the solution. The structure of the proteins in the solution may differ from the crystal due to rearrangements of loops or subdomains on the pico to nanosecond time scale (i.e., the internal protein dynamics time regime). The present work describes how slow motions on timescales of several tens of nanoseconds can be accessed using neutron scattering. In particular, the dynamical characterization of two major human proteins, an intrinsically disordered protein that lacks a well-defined secondary structure and a classical antibody protein, is addressed by neutron spin echo spectroscopy (NSE) combined with a wide range of laboratory characterization methods. Further insights into protein domain dynamics were achieved using mathematical modeling to describe the experimental neutron data and determine the crossover between combined diffusive and internal protein motions. The extraction of the internal dynamic contribution to the intermediate scattering function obtained from NSE, including the timescale of the various movements, allows further vision into the mechanical properties of single proteins and the softness of proteins in their nearly natural environment in the crowded protein solution.

Study of Protein Dynamics via Neutron Spin Echo Spectroscopy

The present protocol describes methods for investigating the structure and dynamics of two model proteins that have an important role in human health. The technique combines bench-top biophysical characterization with neutron spin echo spectroscopy to access the dynamics at time and length scales relevant for protein interdomain motions.

중성자 스핀 에코 분광법을 통한 단백질 역학 연구

Most human body proteins' activity and functionality are related to configurational changes of entire subdomains within the protein crystal structure. The ...

공학

Crystallization of Proteins on Chip by Microdialysis for In Situ X-ray Diffraction Studies

This protocol describes the manufacturing of reproducible and inexpensive microfluidic devices covering the whole pipeline for crystallizing proteins on-chip with the dialysis method and allowing in situ single-crystal or serial crystallography experiments at room temperature. The protocol details the fabrication process of the microchips, the manipulation of the on-chip crystallization experiments and the treatment of the in situ collected X-ray diffraction data for the structural elucidation of the protein sample. The main feature of this microfabrication procedure lies on the integration of a commercially available, semipermeable regenerated cellulose dialysis membrane in between two layers of the chip. The molecular weight cut-off of the embedded membrane varies depending on the molecular weight of the macromolecule and the precipitants. The device exploits the advantages of microfluidic technology, such as the use of minute volumes of samples (&#60;1 &#181;L) and fine tuning over transport phenomena. The chip coupled them with the dialysis method, providing precise and reversible control over the crystallization process and can be used for investigating phase diagrams of proteins at the microliter scale. The device is patterned using a photocurable thiolene-based resin with soft imprint lithography on an optically transparent polymeric substrate. Moreover, the background scattering of the materials composing the microchips and generating background noise was evaluated rendering the chip compatible for in situ X-ray diffraction experiments. Once protein crystals are grown on-chip up to an adequate size and population uniformity, the microchips can be directly mounted in front of the X-ray beam with the aid of a 3D printed holder. This approach addresses the challenges rising from the use of cryoprotectants and manual harvesting in conventional protein crystallography experiments through an easy and inexpensive manner. Complete X-ray diffraction data sets from multiple, isomorphous lysozyme crystals grown on-chip were collected at room temperature for structure determination.

Crystallization of Proteins on Chip by Microdialysis for In Situ X-ray Diffraction Studies

This paper details the fabrication protocol of microfluidic chips developed for on-chip protein crystallization with the dialysis method and in situ X-ray diffraction experiments. The microfabrication process makes it possible to integrate a semipermeable regenerated cellulose dialysis membrane with any molecular weight cut-off, between two layers of the chip.

시투 X선 회절 연구를 위한 미세 투약에 의한 칩에 단백질의 결정화

This protocol describes the manufacturing of reproducible and inexpensive microfluidic devices covering the whole pipeline for crystallizing proteins ...

화학

Studying Soft-matter and Biological Systems over a Wide Length-scale from Nanometer and Micrometer Sizes at the Small-angle Neutron Diffractometer KWS-2

The KWS-2 SANS diffractometer is dedicated to the investigation of soft matter and biophysical systems covering a wide length scale, from nm to &#181;m. The instrument is optimized for the exploration of the wide momentum transfer Q range between 1x10-4 and 0.5 &#197;-1 by combining classical pinhole, focusing (with lenses), and time-of-flight (with chopper) methods, while simultaneously providing high-neutron intensities with an adjustable resolution. Because of its ability to adjust the intensity and the resolution within wide limits during the experiment, combined with the possibility to equip specific sample environments and ancillary devices, the KWS-2 shows a high versatility in addressing the broad range of structural and morphological studies in the field. Equilibrium structures can be studied in static measurements, while dynamic and kinetic processes can be investigated over time scales between minutes to tens of milliseconds with time-resolved approaches. Typical systems that are investigated with the KWS-2 cover the range from complex, hierarchical systems that exhibit multiple structural levels (e.g., gels, networks, or macro-aggregates) to small and poorly-scattering systems (e.g., single polymers or proteins in solution). The recent upgrade of the detection system, which enables the detection of count rates in the MHz range, opens new opportunities to study even very small biological morphologies in buffer solution with weak scattering signals close to the buffer scattering level at high Q.
In this paper, we provide a protocol to investigate samples with characteristic size levels spanning a wide length scale and exhibiting ordering in the mesoscale structure using KWS-2. We present in detail how to use the multiple working modes that are offered by the instrument and the level of performance that is achieved.

Here, we present a protocol to investigate soft matter and biophysical systems over a wide mesoscopic length scale, from nm to &#181;m that involves the use of the KWS-2 SANS diffractometer at high intensities and an adjustable resolution.

작은 각도 중성자 회절 계 KWS-2에서 나노 미터와 마이크로 미터 크기에서 넓은 길이 규모 이상 소프트 물질과 생물학적 시스템을 공부

The KWS-2 SANS diffractometer is dedicated to the investigation of soft matter and biophysical systems covering a wide length scale, from nm to &#181;m. ...

생체공학

High-Resolution Neutron Spectroscopy to Study Picosecond-Nanosecond Dynamics of Proteins and Hydration Water

Neutron scattering offers the possibility to probe the dynamics within samples for a wide range of energies in a nondestructive manner and without labeling other than deuterium. In particular, neutron backscattering spectroscopy records the scattering signals at multiple scattering angles simultaneously and is well suited to study the dynamics of biological systems on the ps-ns timescale. By employing D2O-and possibly deuterated buffer components-the method allows monitoring of both center-of-mass diffusion and backbone and side-chain motions (internal dynamics) of proteins in liquid state.
Additionally, hydration water dynamics can be studied by employing powders of perdeuterated proteins hydrated with H2O. This paper presents the workflow employed on the instrument IN16B at the Institut Laue-Langevin (ILL) to investigate protein and hydration water dynamics. The preparation of solution samples and hydrated protein powder samples using vapor exchange is explained. The data analysis procedure for both protein and hydration water dynamics is described for different types of datasets (quasielastic spectra or fixed-window scans) that can be obtained on a neutron backscattering spectrometer.
The method is illustrated with two studies involving amyloid proteins. The aggregation of lysozyme into &#181;m sized spherical aggregates-denoted particulates-is shown to occur in a one-step process on the space and time range probed on IN16B, while the internal dynamics remains unchanged. Further, the dynamics of hydration water of tau was studied on hydrated powders of perdeuterated protein. It is shown that translational motions of water are activated upon the formation of amyloid fibers. Finally, critical steps in the protocol are discussed as to how neutron scattering is positioned regarding the study of dynamics with respect to other experimental biophysical methods.

Neutron backscattering spectroscopy offers a nondestructive and label-free access to the ps-ns dynamics of proteins and their hydration water. The workflow is presented with two studies on amyloid proteins: on the time-resolved dynamics of lysozyme during aggregation and on the hydration water dynamics of tau upon fiber formation.