# Presentation: 076802

## Structural Studies on Single Particles and Biomolecules 

**Janos Hajdu, ***Uppsala University*
**Gyula Faigel, ***Institute for Solid State Physics and Optics, Budapest*
**Keith Hodgson, ***Stanford Synchrotron Radiation Laboratory*
**Chris Jacobsen, ***State University of New York at Stony Brook*** **
**Janos Kirz, ***State University of New York at Stony Brook*** **
**Gerd Materlik, ***HASYLAB, DESY, Hamburg*
**John Miao, ***Stanford Synchrotron Radiation Laboratory*
**Richard Neutze, ***Uppsala University*
**Carol V. Robinson, ***New Chemistry Laboratory, Oxford University*
**David Sayre, ***State University of New York at Stony Brook*
**Abraham Szöke, ***Lawrence Livermore National Laboratory*
**Edgar Weckert, ***HASYLAB, DESY, Hamburg*
**David van der Spoel, ***Uppsala University*

## Mycoplasmas

**MYCOPLASMAS**
**The smallest creatures capable of self-replication**

**                      ****1 DNA (genome size: 600 - 1,300 kbp)**
**                 ****400  ribosomes**
**            ****10,000  RNA molecules**
**            ****50,000  protein molecules**
**   ****400,000,000  water/solute molecules**

**~300 nm Ø (cell membrane: 8 nm)**
**Solvent content: 60-70%**

## Structural Biology: The Damage Problem

**Conventional methods cannot achieve atomic resolution on non- repetitive (or non-reproducible) structures **
**The limit to damage tolerance is about 200  X-ray  photons/Å****2****  **
**   ****in crystals (conventional experiments)**
**The conventional damage barrier can be stretched by very **
**   ****fast imaging**

***Structural Biology: The Damage Problem***

**(Neutze, R., Wouts, R., van der Spoel, D., Weckert, E. Hajdu, J. (2000) *****Nature***** 406, 752-757)**

## Scattering by a Crystal and by a Single Molecule

**Planar section, simulated image**

**SINGLE MACROMOLECULE**

**PROTEIN CRYSTAL**

**Max. resolution does not depend on sample quality**

**Max. resolution is a function of crystal quality**

***Scattering by a Crystal and by a Single Molecule***

## Scattering and Damage by X-rays (biological samples: C,N,O,H,S)

**(1) Photoelectric effect**** (~90%) followed by ****Auger**** emission, shake-up**
**           ****excitations, and interactions between decay channels**
**(2) Elastic scattering (~7-10%)**
**(3) Inelastic scattering**** (~3%)**

- The ***LCLS*** Beam Interacts with the Matter Through
- Scattering and Absorption:

***LCLS***** -  *****a “never seen regime”, for which only predictions and simulations exist***

## LCLS

***Coulomb Explosion of Lysozyme (50 fs) ***

***LCLS***

**Radiation damage interferes with atomic positions and the atomic scattering factors**

## Modelling Sample Dynamics

**Bond break**   **through Morse potential**
**Ionisation**  **primary and secondary effects  **
**Ionisation dynamics**   **calculate changes in the elastic, inelastic and photoelectric cross-sections for each atom during exposure**

***Modelling Sample Dynamics***

***XMD****** interfaced with ******GROMACS****** ******(van der Spoel et al.)***

**Inventory**  **kept on all electrons in the sample**

- (Neutze, R., Wouts, R., van der Spoel, D., Weckert, E. Hajdu, J. (2000) *Nature* 406, 752-757)

## Ionisation and Coulomb Explosion of a Protein Molecule (Lysozyme) in Intense X-ray Pulses

- Ionisation events

- Energy

***Ionisation and Coulomb Explosion of a Protein Molecule****** (Lysozyme) in Intense X-ray Pulses***

**3x10****12**** photons/100 nm diameter spot (3.8x10****6**** photons/Å****2****, 12 keV)**

## Landscape of Damage Tolerance

**Photons/pulse/100 nm spot**

***Landscape of Damage Tolerance***

- Ionisation and subsequent sample explosion causes  diffraction intensities to change

- Agreement factor:

**Time (fs)**

## Sample Size and Scattering

- RUBISCO  562,000 Da

- HRV ~3,000,000 Da

- LYSOZYME  19,806 Da

**Structure of content unknown**

## Single virus particles look very promising

- .

- 0 Å

- <2

- .

- 0 Å

- 2

- .

- 4 Å

**Single virus particles look very promising**

***Calculated Limits of Resolution with R******electronic****** = 15 %***

## First Experiments

**Single viral particles - structure of the viral genome**
**Nanoclusters**
**Structural kinetics on nanometer-sized samples**
**Nanocrystals**
**Two dimensional crystalline arrays**
**X-ray diffraction tomography of whole cells**
**X-ray scattering from intact cells**

## Sample Handling 

**1.**** ****Spraying Techniques**

**2. Sample embedded in vitreous ice**

**Native proteins,**
**Viruses,**
**Nanoclusters,**
**Nanocrystals, **
**Cell organelles,**
**Intact cells**

**Sample selection and injection**

**Nanodroplets, Cryogenic Temperatures, High Vacuum**

**Intact cells, cell organelles**

**Goniostat, Cryogenic Temperatures, High Vacuum**

## Prototype High Mass Q-TOF Mass Spectrometer

**COLLECTOR PROBE**

**REFLECTRON**

**TIME-OF-FLIGHT **
**ANALYSER**

**LOW FREQUENCY QUADRUPOLE MASS FILTER**

**SAMPLE INJECTOR**

**EXTRACTION LENS**

**RF HEXAPOLE**

**RF HEXAPOLE**

**PUSHER**

**DETECTOR**

**SAMPLE GRID**

- FIRST MASS MEASUREMENT:
- MS2 virus: 2,484,700 Da

**(Tito et al. (2000) *****J. Am. Chem. Soc.***** **
**122, 3550-3551)**

## Interaction Chamber and Detector Arrangement

- To MASS/CHARGE ANALYSIS
- and ELECTRON MICROSCOPY

- DIAGNOSTICS
- and LASER PORT

- X-RAY
- PULSE

- SAMPLE INJECTION

- DETECTOR 1

- DETECTOR 2

- INTELLIGENT
- BACK-STOP

***Interaction Chamber and Detector Arrangement***

## A Planar Section across the Molecular Transform of TBSV

**2 Å resolution**

***A Planar Section across the Molecular Transform of TBSV***

## A Planar Section across the Molecular Transform of TBSV

***A Planar Section across the Molecular Transform of TBSV***

**8 Å resolution**

## The Phase Problem

**Tools of classical crystallography (these should work with reproducible structures)**
**Holography**

***The Phase Problem***

## How is Nucleic Acid Organised inside a Virus?

***How is Nucleic Acid Organised inside a Virus?***

- Courtesy of D. I. Stuart, Oxford

- Experiment:

**Artist’s view:**

- Gouet et al. *Cell * **97**, 481-490 (1999)

**Nucleic acid is released at the right time in the right order**

## Structural Kinetics on Nanocrystals and Nanoclusters

**Most biochemical processes involve diffusion of reactants**

- Decorated nanoclusters may help to overcome this limitation

- Implications for Functional Genomics

**Kinetic studies in crystals suffer from “the mixing problem”**

***Structural Kinetics on Nanocrystals and Nanoclusters***

## Mycoplasmas

**MYCOPLASMAS**
**The smallest creatures capable of self-replication**

**                      ****1 DNA (genome size: 600 - 1,300 kbp)**
**                 ****400  ribosomes**
**            ****10,000  RNA molecules**
**            ****50,000  protein molecules**
**   ****400,000,000  water/solute molecules**

**~300 nm Ø (cell membrane: 8 nm)**
**Solvent content: 60-70%**