Name: microcrystallography of protein crystals and in cellulo diffraction
Uploaded: 2017-07-21T14:00:00
Description: Watch this Scientific Journal Video about Microcrystallography of Protein Crystals and In Cellulo Diffraction at JoVE.com

저자들은 오스트레일리아 싱크로트론의 MX2 빔라인에서의 지원을 위해 정화 된 미세 결정, Daniel Eriksson 및 Tom Caradoc-Davies의 사진을 제공 한 Chan-Sien Lay과 Monash University의 FlowCore 시설에서 나온 Kathryn Flanagan 및 Andrew Fryga 귀중한 도움.

참고 : 생체 내 결정화는 박테리아, 효모, 식물, 곤충 및 포유 동물 (참고 문헌 21 에서 검토 됨)을 비롯한 많은 유기체에서보고되었습니다. 포유류 세포의 일시적인 형질 감염 및 곤충 세포의 바큘로 바이러스 감염을 사용하여 재조합 단백질의 결정화가 실험실에서도 달성되었다. 다음 프로토콜은 참조 22의 지침에 따라 생성 된 baculovirus polyhedrin promoter하?...

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이 프로토콜은 과거에 간과되었을 작은 결정의 분석을 용이하게하기 위해 미정을 분석하는 두 가지 접근법을 제공합니다. 미세 결정 정제를위한 중요한 단계 제시된 프로토콜은 Sf9 세포에서 발현 된 Bombyx mori CPV1 폴리 헤드린을 모델 시스템으로 사용하여 최적화 하였다. 그러나, 생체 내 미결정은 기계적 저항성에 큰 변화를 보인다. 예를 들면...

생물 고분자의 고해상도 구조 결정을위한 X 선 결정학의 사용은 지난 20 년간 꾸준히 발전해 왔습니다. 전문가가 아닌 연구자에 의한 X 선 결정학 (X-ray crystallography)의 증가 추세는 생명 과학 분야의 많은 분야에서이 접근법의 민주화를 보여줍니다 1 . 역사적으로 ~ 10 μm 이하의 크기를 가진 결정은 구조 결정을 위해 사용하기에 어렵지 않은 경우 도전적인 것으로 간주되었습니다. 싱크로트론 방사선 소스의 전용 미세 초점 빔라인의 가용성 증가와 미세 결정 조작 도구 개발과 같은 기술적 진보로 인해 X 선 미세 결정의 폭 넓은 사용을 방해하는 이러한 장벽이 제거되었습니다. 직렬 X 선 마이크로 결정학 2 , 3 및 마이크로 전자 회절 4 ha의 발전구조 결정을위한 마이크로 및 나노 결정의 사용이 가능할뿐만 아니라 때로는 큰 결정 5 , 6 , 7 의 사용보다 바람직하다는 것을 보여 주었다. 이러한 발전은 제 8 펩티드 곤충 바이러스 9, 10에 의해 생성 된 천연 결정의 연구에 적용 하였다. 이들은 이제 막 단백질 복합체 크고 11 가장 어려운 시스템을 포함하는 생물학적 고분자의 다양한 사용된다. 이 미세 결정의 분석을 용이하게하기 위해 meso , 특히 멤브레인 단백질 12 와 마이크로 유체 칩 13 에서 분석되었습니다. 이러한 신규 microcrystallography 방법론의 가용성에 사용할 수있는 가능성을 제기in vivo 결정학에 대한 대안을 제공하는 구조 생물학 14 , 15 , 16 의 새로운 경로로서의 생체 내 결정화. 불행하게도, 생체 내 결정체가 생성 될 수있을지라도, 세포로부터의 정제 동안 리간드의 분해 또는 손실, 싱크로트론 빔라인에서의 수정 및 시각화의 어려움 및 지루한 X- 선 회절 실험과 같은 몇 가지 장애물이 남아있다. 대안으로 결정은 정제 단계 17 , 18 , 19 없이 셀 내에서 직접 분석되었습니다. 비교 분석은 cellulo 접근 그러한 정제 된 결정과 높은 해상도 (20)의 수율 데이터의 분석을보다 더 효율적이 될 수 있음을 시사한다. 이 프로토콜은단백질 미세 결정학에 새로운 연구원을 돕는 경향이 있었다. 싱크로트론 빔라인에서 X 선 회절 실험을위한 시료 준비 및 조작에 초점을 맞춘 방법론을 제공합니다. 두 가지 옵션이 고전 microcrystallography 또는 cellulo 분석 ( 그림 1 )에 대한 유동 세포 계측법에 의해 정렬 크리스탈 포함 세포에 대한 격리 된 결정을 사용하여 제안하고 있습니다.

<table border="0" cellpadding="0" cellspacing="0" width="629">
	<colgroup>
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		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sf9 cells</td>
			<td style="width:127px;">Life Technologies</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
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			<td height="21" style="height:21px;width:216px;">SF900-SFM insect medium</td>
			<td style="width:127px;">Life Technologies</td>
			<td style="width:119px;"></td>
			<td></td>
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		<tr height="42">
			<td height="42" style="height:42px;width:216px;">1L cell culture flask</td>
			<td style="width:127px;">Thermofisher Scientific</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Shaking incubator for insect cell culture</td>
			<td style="width:127px;">Eppendorf</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">50mL conical tubes</td>
			<td style="width:127px;">Falcon</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Centrifuge with swing buckets for 50mL tubes</td>
			<td style="width:127px;">Eppendorf</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Sonicator equiped with a 19mm probe</td>
			<td>MSE Soniprep 150&#160;</td>
			<td style="width:119px;"></td>
			<td></td>
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		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Glass slides</td>
			<td style="width:127px;">Hampton Research</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Hemacytometer</td>
			<td style="width:127px;">Sigma-Aldrich</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Propidium iodide</td>
			<td style="width:127px;">Thermofisher Scientific</td>
			<td style="width:119px;"></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">BD Influx cell sorter&#160;</td>
			<td style="width:127px;">BD Biosciences</td>
			<td style="width:119px;"></td>
			<td></td>
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		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Hampton Heavy atom screens</td>
			<td style="width:127px;">Hampton Research</td>
			<td style="width:119px;"></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Microcentrifuge</td>
			<td style="width:127px;">Eppendorf</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:216px;">Micromesh</td>
			<td style="width:127px;">Mitigen</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">700/25 meshes offer a larger surface. Indexed meshes can be purchased for systematic studies.</td>
		</tr>
		<tr height="147">
			<td height="147" style="height:147px;width:216px;">Paper wick</td>
			<td style="width:127px;">Mitigen</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">The size of the paper wick can be varied for optimal flow. This will largely depend on the nature of the crystals and cryoprotectant used.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ethylene glycol</td>
			<td style="width:127px;">Sigma-Aldrich</td>
			<td style="width:119px;"></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Trypan blue</td>
			<td style="width:127px;">Life Technologies</td>
			<td style="width:119px;"></td>
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		<tr height="231">
			<td height="231" style="height:231px;width:216px;">MX2 microfocus beamline</td>
			<td style="width:127px;">Australian Synchrotron</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">A list of available microfocus beamlines can be found in Boudes et al. (2014) Reflections on the Many Facets of Protein 
			Microcrystallography. Australian Journal of Chemistry 67 (12), 1793&#8211;1806, 
			doi:10.1071/CH14455.</td>
		</tr>
	</tbody>
</table>

microcrystallography of protein crystals and in cellulo diffraction

많은 싱크로트론 설비에서 고품질의 미세 초점 빔라인이 출현함에 따라 도전을 대표하는 가장 큰 차원에서 10μm보다 작은 결정의 일상적인 분석이 가능 해졌다. 우리 는 생체 내에서 성장한 결정에 특별한 초점을 맞춘 X 선 결정학에 의한 단백질 미결정의 구조 결정을위한 두 가지 대안 적 작업 흐름을 제시합니다. 미세 결정은 초음파 처리에 의해 세포로부터 추출되고 차등 원심 분리에 의해 정제되거나 결정 함유 세포의 유동 세포 계측법에 의한 세포 분류 후에 셀룰로오스로 분석된다. 선택적으로, 정제 된 결정 또는 결정 - 함유 세포를 실험적 단계적 조정을 위해 중질 원자 용액에 침지시킨다. 이 샘플들은 액체 질소에서의 마이크로 메쉬지지 및 플래시 냉각에 대한 적용에 의해 유사한 방식으로 회절 실험을 위해 준비됩니다. 격리 된 미세 결정과 수정 결정의 연속 회절 실험을 간략하게 기술하고 비교한다.microfocus 싱크로트론 빔라인을 사용하여 셀을 포함하는 셀을 조정하여 위상 조정, 모델 구축 및 미세 조정에 적합한 데이터 세트를 생성합니다. 이러한 워크 플로우는 곤충 세포에 재조합 배큘로 바이러스 (baculovirus)가 감염되어 생성 된 Bombyx mori cypovirus 1 (BmCPV1) 폴리 헤드린의 결정체를 예로들 수 있습니다. 이 사례 연구 에서 셀룰로오스 분석은 정제 된 결정의 분석보다 효율적이며 표현에서부터 정제까지 ~ 8 일 내에 구조를 산출합니다.

생체 내 미세 결정을 이용한 구조 결정을위한 두 가지 대체 방법의 개요가 제시됩니다 ( 그림 1 ). Polyhedra는 초음파 처리 및 원심 분리로 쉽게 정제 할 수 있습니다. 밀도 때문에, 그들은 pipetting ( 그림 3a 와 3b )에 의해 제거 할 수있는 파편 층 밑에 튜브의 바닥에 레이어를 형성합니다. 그런 다음 샘플을 여러 ?...

Watch this Scientific Journal Video about Microcrystallography of Protein Crystals and In Cellulo Diffraction at JoVE.com

Microcrystallography of Protein Crystals and In Cellulo Diffraction

단백질 미세 결정을 사용하는 X 선 결정학을위한 프로토콜이 제시됩니다. 정제 후 또는 cellulo에서 미세 -grown 생체 분석 두 예가 비교된다.

단백질 결정의 미세 결정학 및<em&gt; 셀룰로에서</em&gt; 회절

The advent of high-quality microfocus beamlines at many synchrotron facilities has permitted the routine analysis of crystals smaller than 10 &#181;m in ...

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Biochemistry

Microcrystallography of Protein Crystals and In Cellulo Diffraction

Growing Protein Crystals with Distinct Dimensions Using Automated Crystallization Coupled with In Situ Dynamic Light Scattering

Growing Protein Crystals with Distinct Dimensions Using Automated Crystallization Coupled with In Situ Dynamic Light Scattering

사용 하 여 다른 크기와 성장 단백질 결정 자동화 현장에서 동적 산란과 함께 결정 화

The automated crystallization device is a patented technique1 especially developed for monitoring protein crystallization experiments with the aim to ...

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Radioactive pulse-chase labeling is a powerful tool for studying the conformational maturation, the transport to their functional cellular location, and ...

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High-brilliance X-ray beams coupled with automation have&#160;led to the use of synchrotron-based macromolecular X-ray crystallography (MX) beamlines for ...

Application of Biolayer Interferometry (BLI) for Studying Protein-Protein Interactions in Transcription

Application of Biolayer Interferometry BLI for Studying Protein-Protein Interactions in Transcription

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A transcription factor&#160;(TF) is a protein that regulates gene expression by interacting with the RNA polymerase, another TF, and/or template DNA. GrgA ...

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Protein dimerization events that occur only in the presence of a small-molecule ligand enable the development of small-molecule biosensors for the ...

Derivatization of Protein Crystals with I3C using Random Microseed Matrix Screening

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Protein structure elucidation using X-ray crystallography requires both high quality diffracting crystals and computational solution of the diffraction ...

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Here, a protocol is presented to facilitate the creation of large volumes (&#62; 100 &#181;L) of micro-crystalline slurries suitable for serial ...

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