NMR Protein Structure Determination in Living E. Coli Cells Using Nonlinear

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© 2010 Nature Publishing Group http://www.nature.com/natureprotocols heteronuclear multidimensional heteronuclear multidimensional protein in living cells for the putative heavy proteinmetal-binding high-resolution 3D structures of proteins in living environments. NOES the nonlinear sampling scheme, the duration of each 3D experiment can be reduced to 2–3 h. dimensions, dimensions, structure calculation and structure refinement. less less than 1 h on a powerful computer system. E. coli for the stable isotope ( R A T Published online 13 May 2010; doi:10.1038/nprot.2010.69 Nishikyo-Ku, Kyoto, Japan. Biomedical and Faculty of Life Sciences, University Glasgow,of Glasgow, UK. 8 Japan. RIKEN, Wako-shi, Saitama, Japan. Goethe University, am Frankfurt Main, Germany. 1 cal macromolecules inside living cells to combined obtain information on been the conformation have and dynamics of biologi spectroscopy NMR of advantages The In-cell NMR assignment, and calculation structure refinement. structure preparation, NMR measurements, resonance assignment, NOESY article, we describe the protocol of our method that includes sample thermophilus approach has been used to characterize the The structure programs. computer and of techniques the NMR existing of range in structures protein investigating for ideally suitable for NMR the task make resolution atomic at data provide to ability its and tions inside cells. The noninvasive character of NMR spectroscopy their func is requiredbasis of to better the understand structural regarding the 3D structures, dynamics and interactions of proteins influences the behavior of proteins. Therefore, cells in macromolecules by crowding extreme the whether tion the cellular environment many processes.biological It is, however, of very difficult to replicate understanding the to contributions valuable very in resulted ture of purified proteins in single crystals or in solution, which have There are many widely used methods for determining the 3D struc I Daniel Nietlispach Tomomi Hanashima Teppei Ikeya E. coli NMR protein structure determination in living require labeling of target proteins with NMR-active stable isotopes exclusively on the basis of information obtained in living cells. cells, the rapid measurement of the three-dimensional (3D) Department of Biochemistry,of Department University Cambridge, of Cambridge, UK. Chemistry,of Department Tokyo Metropolitan University, Hachioji, Tokyo, Japan. NTRO he he cell is a crowded environment in which proteins interact specifically with other proteins, nucleic acids, cofactors and ligands. ecent ecent improvements to nuclear magnetic resonance ( tomic tomic resolution structural explanation of proteins functioning in this environment is a main goal of biochemical research. Y Y peak lists have been prepared, structure calculation with the program 6 Research Group for Structure-Function, Bio-Supramolecular RIKEN, Yokohama, Kanagawa, Japan. cells. D UCT I A HB8 protein TTHA1718 in living ON s s no protein purification is necessary, a sample for in-cell 1 cells using nonlinear sampling , 2 , Atsuko Sasaki 8 12 , Markus Wälchli Frankfurt Institute Frankfurt for Advanced Studies, am Frankfurt Main, Germany. Correspondence should be addressed to Y.I. ([email protected]). 13 1 in vitro , Masaki Mishima C 2 , , . We have recently developed a method 5 15 Department of Life of Science,Department Graduate Bioscience School of and Biotechnology, Tokyo Institute Technology,of Yokohama, Kanagawa, N and and it is an interesting open ques N 1 M , 3 2 4–10 , Daisuke Sakakibara R H) H) labeling and structure ofdetermination proteins overexpressed in spectra of macromolecules in living cells (in-cell E. coli E. . In-cell NMR experiments 3 CREST, Japan Science and Technology Agency, Kawaguchi, Saitama, Japan. 9 , Brian O Smith cells by combining a combining by cells 1 in vivo , E. coli 3 , Masatoshi Yoshimasu cells information N Thermus M 3 9 . In this Bruker BioSpin, Yokohama, Kanagawa, Japan. R ) ) hardware and methodology allow the measurement of high-resolution 1 11 U 10 , N 3 Department of Molecular of Graduate Kyoto Engineering, Engineering, School Department of University, , Yoshiki Shigemitsu nder nder favorable circumstances, this in-cell , Masahiro Shirakawa M - - - - 1 R T

2 he he protocol combines the preparation of the protein in Center for Biomolecular Magnetic Resonance, Institute Biophysicalof Chemistry, spectra by nonlinear sampling of the indirectly acquired T he he protocol has been used to solve the first 3D structure of a TT such as yet, as the intracellular concentration of the introduced target target introduced the of concentration intracellular the as yet, feasible not is cells human cultured inside proteins of structures determine protein structures by in-cell NMR. Determining the 3D to information structural sufficient of acquisition the prevented Until recently, the low sensitivity and short lifetime of samples have inside Protein cells determination structure through resealable pores formed by streptolysin O toxin was reported, in which labeled proteins were introduced into cells structures. Recently, another method for mammalian in-cell NMR intracellular large to binds it because NMR to invisible becomes tag cleaved The released. is protein target the and cell, the in off peptide cell-penetrating a proteintarget with labeled a tagging by cells human in NMR in-cell enables method limited to extraordinarily large cells such as oocytes. An alternative is microinjection by proteins labeled of delivery artificial the and vidual proteins are too low for in-cell NMR spectra to be collected, indi of concentrations the because cells, human cultured inside proteins study to difficult more even is It microinjection. by cells eggs) to applied been has which approach, ond sec The cells. host in systems expression protein intrinsic using studies NMR in-cell bacterial for this condition.achieving The first approach, which has been used H A N 1718 1718 from 14–16 4 M , Nobuhiro Hayashi 13 R , is to use purified proteins and incorporate them into into them incorporate and proteins purified use to is , C and measurements can be obtained within 2–3 d. With C Y ANA 15 7 Biometal Biometal Science Laboratory, RIKEN, Sayo-gun, Hyogo, Japan. and energy refinement can be completed in Thermus Thermus thermophilus N inside host cells. There were two approaches for 1 , 3 3 , Junpei Hamatsu N , 11 M , Peter Güntert R natureprotocols ). ). In this study, we describe a protocol 10 O Division of Molecular of Division and Cellular Biology, nce nce chemical shift assignments and 5 4 , Tsutomu Mikawa Cellular Cellular and Molecular Laboratory,Biology 11–13 ,toproteins prepareis target N M HB8 overexpressed in Escherichia Escherichia coli 1 1 R , 2 ,

3 approach can provide , | 12 , VOL.5 NO.6VOL.5 Xenopus

& Yutaka Ito 1 7 . cleaved is tag The protocol oocytes (frog (frog oocytes

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© 2010 Nature Publishing Group http://www.nature.com/natureprotocols C 100% D Total volume Metal mixture stock solution FeCl Thiamine stock solution CaCl MgSO NaCl KH Na NH Unlabeled leucine Unlabeled glucose [U- [3- [U- 15 T proteins protonationlarger analysis of has a on large impact the structural labeled uniformly on measured as 3D 1 distance restraints can be collected using a sample selectively with NOE of number sufficient a and assigned, and observed be can nances for the backbone and most side-chain of NMR resonances experiments to be performed. As a result, the expected NMR reso dimensions scheme acquired for indirectly the sampling a nonlinear applying by h 2–3 to durations their shortening and experiment each for tube. This can be overcome, however, by a preparing fresh sample NMR is longer than the time that cells can remain viable in an proteinbyNMRtodata an in-cell determine sufficient structure collect obtained in information basis living of the on exclusively calculated structure protein 3D first the with in severely overlapped cross-peaks. These problems were overcome which the broadened lines observed from in-cell samples can result in cases in (NOEs),even effects Overhauser nuclear from mation obtained information on rely not do that assignment resonance for methods requires novo determination. structure protein 3D for method NMR in-cell an on We focus structures. therefore protein on ing crowd molecular systemof for investigating easiest effect the the centration for NOESY-type experiments. in-cell Bacterial NMR is concentration intracellular mM ~0.7 to up at injected were proteins which into to determine the structures of proteins in proteins achieved so far is at the most ~30 1052 H/ δ a protocol NH methyl-selective labeling atisoleucine residues from [U- One important problem is that the length of time required to required time of length the that is problem important One b 2 2 4 13 HPO 13 13 PO 13 Cl le 3 2 4 C] C] alanine C, C, 3- C] glucose stock solution stock solution NMR protein structure determination in living living in determination structure protein NMR

C-labeled methyl groups of alanine, leucine and valine, as well Cl 2 4 | O wasusedfor the preparation of media for selectiveprotonation atside-chain methyl groups, whereas H stock solution 4 15 VOL.5 NO.6VOL.5 1 4 N-separated NOESY and 3D 2 | 2 2 , and has also been used to provide site-specific probes in

H] H] M9 M9 minimal medium for 19–21 α 1 -ketoisovalerate 4 . This makes it possible for the requisite 3D NMR NMR 3D requisite the for possible it makes .This , thus fulfilling the requirement on protein con protein on requirement the fulfilling thus , | 2010 in vitro in | natureprotocols , and for obtaining distance infor distance obtaining for and , E. coli E. 13 C/ 13 15 C-separated NOESY spectra 13 N N uniform labeling (UL) and selective protonation at side-chain methyl groups. samples. Methyl-selective Methyl-selective samples. E. coli C, 3,3- X. laevis µ M 2 H] 13 cells 1 C/ 100 100 ml α 100 100 200 200 7 40 40 40 0.2 0.2 g 0.1 g 0.1 0.1 g 0.6 g 1.2 g . It may be possible -ketobutyrate isnot included inthistable. 15 — — — — — N UL oocytes or eggs, µ µ 3 µ µ µ . l l l l l E. coli E. E. coli E. -based -based cells cells De De - - - - Ala/Leu/Val the case of rat calmodulin, in which, for reasons unknown, good good unknown, reasons for which, in calmodulin, rat of case the observed However,also we strains. the both from results similar obtain we cases, many In protein. new a with experiments NMR JM109 (a K-12 strain) and BL21 (a B strain), when in-cell starting two least experiments. Weat large-scale try usually the protein expression at scale beforebe should small determined for times incubation the and temperature optimal the induction, isopropyl of tion Table transfer into then M9 minimal medium containing stable isotopes and medium, LB unlabeled in plasmid expression the ing with labeled is To produce a sample of design Experimental (17 calmodulin kDa) in bone resonance connectivities and sequential H back sequential the kDa, identify tocouldwe ~20 proteins up as for feasible be will approach our by determination structure cell time and proteins.apparent mass molecular of We that expect in- protein can be calculated the with program CYANA NMR in-cell Table in summarized are protonation Ala/Leu/Val and methyl selective uniform a in residues Val and Leu Ala, of groups methyl side-chain nated proto selectively with generated are samples more NMR, in-cell by restraints conformational of collection the Forresults). lished were observed when using BL21 (M.Y., N.H., T.M. and Y.I., unpub were observed when using JM109, whereas broadened cross-peaks 1 H- 100 100 ml 100 100 200 200 200 0.01 0.01 g 0.01 g 40 40 40 40 0.1 0.1 g 0.6 g 1.2 g 0.1 g 0.2 g 0.1 g The viscosity inside cells inside viscosity The — — 15 2 O wasusedfor µ µ N heteronuclear single quantum coherence (HSQC) spectra spectra coherence (HSQC) quantum single heteronuclear N µ µ µ 1 l l 1 l l l ) ) in which protein expression is induced (e.g., by the addi . Cells are harvested by gentle centrifugation and placed placed and centrifugation gentle by harvested are Cells . 2 H background. The M9 media for Val/Leu, Ala/ValVal/Leu, for media M9 The background. H 2 3 . On the basis of these data, the 3D structure of the the of structure 3D data,the these of basis .the On 13 C/ 13 15 C and and C N uniform labeling. BecauseTTHA1718 protein hasno isoleucine residues, β - D -1-thiogalactopyranoside). The timing of of timing The -1-thiogalactopyranoside). E. coli 15 E. coli Val/Leu N, we generally first grow cells harbor cells grow first generally we N, 100 100 ml 100 100 200 200 0.01 0.01 g 40 40 40 0.1 0.1 g 0.6 g 1.2 g 0.1 g 0.2 g 0.1 g 2 — — — 5 increases the rotational correlation correlation rotational the increases

cells in which only the target protein µ µ cells µ µ µ l l l l l 3 . N -H Ala/Val 100 100 ml 100 100 200 200 200 0.01 0.01 g 0.01 g 0.01 g 40 40 40 0.1 0.1 g 0.6 g 1.2 g 0.1 g 0.2 g 0.1 g N 2 E. coli E. NOEs for rat 4 — . µ µ µ µ µ l l l l l strains, strains, ( see see - - - - -

© 2010 Nature Publishing Group http://www.nature.com/natureprotocols size usage of ‘sample2’ command is as follows: sample2 numbers for chosen points are written in order of measurement. The defines the sampling list (text format), in which AZARA-defined index iterations for 2D MaxEnt processing). The line starting with ‘sample2’ the algorithm should converge in less iterations (typically within 10–20 of iterations that the algorithm will use on one slice of data. Normally, an exclamation mark. The parameter ‘iter’ defines the maximum number with AZARA. A brief explanation for each line is presented, following processing. ( defined index numbers, which should be specified in the sampling lists for omitted (blue circles) data points are represented with their AZARA- pairs for both 48, respectively). Real and imaginary data points are measured in of for measurement in a pseudorandom manner from the regularly spaced grid example of a nonlinear sampling scheme, in which data points are chosen using a nonlinear sampling scheme. Adapted from reference cells. ( Figure 1 • • • • • • • • • • REAGENTS M of grid conventionalfrom the manner spaced pseudorandom a regularly Briefly, about 1/8–1/4 of the points are chosen for measurement in time. experimental reduce to dimensions observed indirectly the 3D NMR experiments, we use a nonlinear sampling scheme in-cell and werethat for used the 2D and 3D NMR spectra of the set marizes information. useful yields it that provided applied, be can chains side amino-acid and backbone polypeptide the of tra. Any of the common 2D and 3D spectra used for the assignment the end-point health of cells. and initial the between comparison a for allows which tests, ony 2D by samples D 10% containing medium M9 with slurry ~60% an as tubes NMR into azara/azara_docs/azara.html). information, please see the AZARA manual pages (http://www.ccpn.ac.uk/ chemical shifts, e.g., using the program TALOS program the using e.g., shifts, chemical distance restraints, backbone torsion angle restraints obtained from assignment tion with the program CYANA used for processing the sampled nonlinearly dimensions. Glucose (Wako, cat. no. 041-00595) l l [U- [3- U- 15 BL21 (DE3) JM109 (DE3) respectively) cat. no. 69660-3) expression systems for TTHA1718 and rat calmodulin, we used pET-11a (Novagen, cat. no. 69436-3) and pET-14b (Novagen, Expression plasmidencoding theproteininterest (for of example, ATER -Leucine (Sigma, cat. no. L8000) -Lysine- The present protocol does not require a specific set of NMR spec The The 3D structures of proteins are obtained by structure calcula t NH 1

13 > , 13 13 t

C-glucose (ProSpect Pharma, cat. no. PT100803)

C] alanine(ISOTEC, cat. no. 489948) 2 C, 3-

4 points (32 and 24 complex points, i.e., time domain data size 64 and < a Cl (Spectra, cat. no. 5300)

) ) Schematic representation of rapid acquisition of 3D NMR spectra d3 d3 time domain data size I t

2 | 1 ALS O. We recommend monitoring the stability of of stability the monitoring recommend We O. , Rapid acquisition of 3D NMR spectra of proteins in living 15 t 2 2 H] N points ( c in in vitro 2 2 E. coli ) ) An example of a part of a script for 2D MaxEnt processing 7 t HCl (ISOTEC, cat. no. 609021) and torsion angle dynamics angle torsion and 1 α E. coli and -ketoisovalerate (CIL, cat. no. CDLM-4418-0) 1 (Novagen, cat. no. 69450) H- t (Promega, cat. no. P9801) structure determinations of TTHA1718. For all 2 evolution times. Chosen (filled blue circles) and Fig. 15 HQ seta floe b paig col plating by followed spectra, HSQC N 1 ). The 2D maximum entropy method

4 < version 3.0 using automated NOE

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restraints. OPALp program the with water shell and energy minimized against the AMBER force field a CYANA in lowestfinal the areembedded with values function target conformers 20 The elements. structure secondary regular in bonds hydrogen for restraints distance as well as input, the to of TTHA1718. T Complex c b a a 3D 3D CBCANH 3D CBCA(CO)NH 3D HN(CA)CO 3D HNCO 3D HNCA 3D HN(CO)CA 2D assignment For backbone points (Acquisition dim.) end_maxent phase0 npts128 complex dim2 phase0 npts256 dim1 sample26448sampling.lst logmaxent.log rate0.3 maxent2_com 2 t2 24th evolution time t b 2n complex 2 iter30

1st (1st indirect dim. ! ! noise 3500 (2nd indirect dim.) le d t 1 r r H- i i

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2951 3015 r 19 13 71 r 8 7 4th 2 9 natureprotocols 1 5 1 1 H- H- 3016 2952 72 i 00 36 t 1 31,32 t t evolutiontime 13 13 2 2 3017 2953 20 13 73 r 1 9 C C HMQC C HSQC 5th 2 1 1 7 in the presence of the experimental experimental the of presence the in 3018 2954 74

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1 2 2D MaxEnt 2D MaxEnt 20 14 3021 2957 77 r 1 3 DFT or 7th l 2 5 1 1 3022 2958 78 i 06 42 4

(ALV-selective samples) HMQC-NOESY-HMQC 3D NOESY-HSQC 3D NOESY-HSQC 3D measurement For distancerestraint | VOL.5 NO.6VOL.5 F2 13 13 15 3069 3005 25 12 18 F1 61 C/ C-separated N-separated 31st r 3 5 9 3006 3070 25 12 19 62 13 F3 i protocol 4 6 0 C-separated 3007 m m 25 12 3071 19 63 32nd r 5 7 1 3008 25 12 3072 19 6 | i 4 6 8 2010 2 Real/imag.

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© 2010 Nature Publishing Group http://www.nature.com/natureprotocols ! 6| 5| 4| ● reaches 0.5–0.6. 3| ● 2| 1| Preparation of proteins insideliving PROCEDURE • • • • EQUIPMENT • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • 1054 7| Metal mixture stock solution (4mMZnSO FeCl CaCl MgSO LB medium (10 g Mark 12Unstained Standard (Invitrogen, cat. no. 354377) NuPAGE Buffer LDSSample (4×; Invitrogen, cat. no. NP0007) NuPAGE MESSDSRunning Buffer (20×; Invitrogen, cat. no. NP0002) NuPAGE 12%Bis-TrisGel (Invitrogen, cat. no. NP0349BOX) Simply Blue Safe Stain(Invitrogen, cat. no. LC6065) Benzamidine, hydrochloride (Calbiochem, cat. no. 199001) Bug Buster Protein Reagent Extraction (Novagen, cat. no. 70584-3) Ampicillin sodium(Wako, cat. no. 018-10372) D Isopropyl thio- hydrochlorideThiamine (Wako, cat. no. 201-00852) H MnSO CuSO ZnSO FeCl CaCl MgSO KH Na NaCl (Wako, cat. no. 191-01665) Bacto Agar (BD, cat. no. 214010) Bacto Yeast (BD, Extract cat. no. 212750) (BD,Bacto tryptone cat. no. 211705) NH protocol nmr.ch/doku.php) biol.ethz.ch/groups/wuthrich_group/software) NMRView (OneMoon Scientific Inc.) cam.ac.uk/nmr/ccmr/public/ANSIG/ansig.html) Software foranalysis, interactive spectra e.g., ANSIG 3.3(http://www.bio. mum entropy method Software for processing the2Dmaxi nonlinearlyNMRdatawith sampled ture calculations(multiple processors recommended) Linux computer system for andstruc dataprocessing, spectra analysis of a cryoprobe recommended) thatishighly NMR spectrometer ( (see M9 minimalmedium for selectively protonated side-chain methyl groups M9 minimalmedium for uniformly stockThiamine solution 0.3Mthiaminehydrochloride 4.7 mMH

CAUT

2 3 TIMING TIMING O (Spectra, cat. no. 5150) BO 2 HPO 2 4

PO Cl (Wako, cat. no. 017-02995) Table 1 3 3 Remove the supernatant by pipetting, and resuspend the cellsin100mlof stable isotope-labeledM9media. Decant the supernatant. Centrifuge the pelletagain at~800 Centrifuge the culture at ~800 Subculture Grow Transform Incubate the cellsat37°Cwithout shaking for 1h. 2 2 | stock solution 5mMFeCl ·6H stock solution 50mMCaCl (Wako, cat. no. 039-00475) VOL.5 NO.6VOL.5 4 3 4 4 4 ·7H 4 (Wako, cat. no. 021-02195) ·5H stock solution 1MMgSO (Wako, cat. no. 137-12335) ·5H 4 I (Wako, cat. no. 169-04245) 4 ON 2 (Wako, cat. no. 197-02865) O (Wako, cat. no. 091-00872) 2 3 2 O (Wako, cat. no. 264-00402) 2 BO O (Wako, cat. no. 039-04412) E. coli 12–14h ~10h O (Wako, cat. no. 130-13182) ) The samples selectively labeled at side-chain methyl groups are expressed in 100% D . 3 β , 0.7mMCuSO -

D l E. coli

− | E. coli

-thiogalactoside (Wako,-thiogalactoside cat. no. 099-05013) 1 cellsin2mlLBmedia at37°Cwithshaking toahigh OD 2010 Bacto tryptone, 5 g

1 H frequency 500 MHz or higher and equipped with H frequency with andequipped 500MHzor higher 26,33 cellswiththe overexpression plasmid. (2DMaxEnt), e.g., AZARA 2.7(W. Boucher). | cells(100 natureprotocols 4 ·5H 3 ·6H 4 2 2 O) 2 13 O C/ 37,38

− 15 µ

1 , Sparky, XEASY(http://www.mol. N-labeled samples (see samples(see N-labeled Bacto Yeast Extract, 10 g l) in100mlunlabeledM9media, and incubate the culture at37°Cuntil the OD 4 ·7H g for 20minatroom temperature (25°C). 3 2 9 O, 1mMMnSO andCARA (http://cara. E. coli 34,35 , CcpNmr Analysis cells 4 ·5H Table 1

−

1 2 NaCl) O, 3 6 ,

- ) -

• • NMR spectra after conversion. format NMR spectra bi.a.u-tokyo.ac.jp/~takeshi/ansig4opengl/) or CcpNmr Analysis software laboratory, version either anOpenGL ANSIG 3.3software of foris needed theresonance obtaining from assignment thespectra. In our nonlinearly data. sampled An analysis interactive program NMRspectrum other processing procedures, such asmultidimensional decomposition processing.tive of Note software for this type that, to inaddition MaxEnt, a a simple computer program was used to generate VC lists for nonlinear for functions constant-time evolution dimensions. In our group, other decaying) for functions conventional evolution dimensions; constant for each dimension according time to evolution:of the exponential (or type mddnmr/), in which sampling points are selected using a function weighting and NUSSAMPLER med.harvard.edu/) to generate VC lists for nonlinear sampling: e.g., COAST ac.jp/osbc/GROUP/ITO/ito_nls_utility.html. There are availableprograms are available from the corresponding author at http://www.comp.tmu. defined by2–4 linesin (digits) variable (VC) counter lists. Pulsesequences to be acquired from the conventional regularly spaced of grid to the procedure reported by Rovnyak points according to sampling schedule lists as opposed to conventional NMR pulse sequences 500 MHz are recommended.highly head and field a strength corresponding magnetic to a probe cryogenic a experiments, NMR in-cell of factors limiting the of one is resonance ( spectrometer NMR EQUIPMENT SETUP Inc.) were used. Alternative such programs asNMRView (OneMoon Scientific and multidimensional Fourier transform Rowland NMRToolkit (http://webmac.rowland.org/rnmrtk/) generated from thecorresponding VC listsfor theNMRmeasurement. The sampling listsfor dataprocessing the with AZARA 2.7software shouldbe for used can be processing 2DMaxEnt nonlinearlydatawith sampled efficiently run can be on Linux cluster systems.The AZARA 2.7software CYANA Computer andsoftware setup ito_nls_utility.html. sampling, which is also at http://www.comp.tmu.ac.jp/osbc/GROUP/ITO/ spectrometer spectrometer (Bruker Avance 600), pulse sequences were accordingmodified point every on sampling of a regularly spaced grid. For our NMR g restraints on the basis of thechemical shifts onrestraints thebasisof TALOS calculation structure CYANA 3.0software package for automated and NOESYassignment for 5minatroom temperature. 37,38 , Sparky, XEASY 2 2 4 9 and restrained energy refinements with the program OPALp refinementswith the program energy andrestrained or TALOS 1 H/ 600 13 of ~2.0. C/ 15 N) experiments with pulsed field gradients. As sensitivity gradients. field Aspulsed sensitivity with N) experiments

+ h NR pcrmtr ut e qipd o triple- for equipped be must spectrometer NMR The 2

4 4 0 These These should be prepared so as to control sampling software backbone torsion for deriving angle 3 9 and CARA can also handle andCARA canalsohandle AZARA-processed

Structure calculations with the program theprogram calculationswith Structure et et al. 4 2 1 (http://groups.google.com/group/ 43,44 O. 2 1 , have for used been processing , in which each indirect point 1 H H frequency at least of 2 1 (http://gwagner. 34,35 t 3 1 3 , isanalterna t (http://www. 600 2

points is

31,32 6

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© 2010 Nature Publishing Group http://www.nature.com/natureprotocols the condition of the sample. Combine these 3D data to generate a new data set with improved signal-to-noise ratio up to the measurement of each 3D experiment several (3–4) times interleaved with a short 2D  cells under measurement conditions. ● sample for each3Dexperiment. selected inapseudorandom manner from the conventional regularly spaced grid of time. Although optimal reduction should beconsidered ineachcase, approximately 1/4–1/8of the data points were typically experiments, anonlinear sampling scheme 16| 15| 14| unlabeled M9media (10%D 13| N ? be checked experimentally ( known concentration. The localization of overexpressed proteins can be predicted from its amino-acid sequence, and can also comparing the density of the Coomassie-stained bands in SDS–PAGE with those of proteins with similar molecular size and tion from extracellular proteins is negligible. The concentration of the expressed protein in  and viability of the sampleshould bechecked ( 12| (140–160 11| 10| ● 9| concentration of 0.5mM). 8| point that the 2D spectra show significant changes. In the case of TTHA1718, two 3D data sets were combined.

M Box 1 EXPERIMENTS structure and physical properties. 4. Optionally, prepare purifiedprotein for (http://www.cbs.dtu.dk/services/SignalP/). dicted from itsamino-acid sequence byPSORTbversion 2.0(refs. 47,48)(http://www.psort.org/psortb/) and SignalP 3.0(refs. 49,50) Thorstenson which are fractionated from target protein-expressing 3. Analyze the localization of overexpressed proteins bymeasuring 2D new samplesbefore designing the measurement schedule of in-cellNMRspectra. which time point viability was85±11%,then decreased withgradually increasing pace. We recommend plotting the viability curvefor 37 °Cand count the colonies. We experienced that,under ourmeasurement condition, viability decreased slowlyfor atleast6h, (~10 2. The viability of cellsduring NMRexperiments canbeinvestigated byaplating colony test.Spread asmall volume of the NMRsample microfuge tubeand centrifuge until allbacteria are collectedinapellet.Store the pelletand supernatant separately for SDS–PAGE. from the bacteria becauseof celllysis, aftereachNMRmeasurement, transfer asmall amount (~10 1. To ensure thatthe observedNMRspectrumrepresents intracellular protein and thatthe signals are not causedbyproteins released THE LOCALIZATION F

TROUBLESHOOTING

TIMING TIMING CR CR R Collect3DNMRspectra required for chemical shiftassignments and structure calculation (see Check the sample’scondition bymeasuring 1D Insert the in-cellNMRsampleinto the magnet, and tune the probe head. Before the measurement of the in-cellNMRsample, shimthe magnetic fieldwithaseparate NMRsamplecontaining Add D Remove the supernatant byaspiration, and resuspend the cellsbyadding small amounts of unlabeled M9media Harvest the cellsbycentrifugation at~400 measurements and data processing Continue protein expression withshaking atoptimal temperature. Induce the production of the target protein (for example, byadding isopropyl thio- I I T T µ I I l) taken before and afterthe experiments onLBplatescontaining the appropriate antibiotic. Incubate the platesovernight at CAL CAL µ The duration of the NMR experiments for anin-cellNMRsamplemust besetconsidering the lifetime of the ~3h 2 O (10%of the final samplevolume) tothe bacterial slurry, and transfer the sampletoanNMRtube. The condition l) and carefully pipetting the solution upand down until the entire cellpellethasbeensuspended. |

STEP STEP CHECKINGTHESAMPLECONDITIN,VIABILITYFCELLS,AND et al. With the nonlinear sampling scheme, the duration of each 3D experiment is reduced to 2–3 h. Repeat the Ensure that the proteins providing NMR spectra are indeed inside the living cells, and that the contribu 4 6 . Ensure spheroplast formation bylight microscopy. The localization of the protein in Box 1 2 O), which isprepared with the same samplelength asfor the in-cellNMRsample.

). VEREXPRESSED PRO in vitro 19–21 NMRexperiments tocompare the effectsof in-celland should beusedfor the indirectly observeddimensions toreduce measurement Box 1 g for 30minatroom temperature. 15 1 N-labeled cellsbylysozyme-EDTA treatment using the conditions described by H-NMR spectra and 1D(or2D) ). 1 H- 15 TEINS DURINGIN-CELLNMR N HSQC spectra of spheroplasts and periplasmic extracts,

1 t H- 1 1 , H- t 15 natureprotocols 2 points inourcase. Prepare afresh N HSQC spectra. 15 β N N HSQC experiment used to monitor - D µ E. E. coli -thiogalactoside toafinal l) from the NMRsampletoa in vitro E. coli cells can be estimated by T able cellscanalsobepre conditions onthe

| VOL.5 NO.6VOL.5 2 ). Forall3DNMR protocol

| 2010

E. coli | -

- 1055

© 2010 Nature Publishing Group http://www.nature.com/natureprotocols the existence of hydrogen bonds isstrongly supportedbymedium-range orinterstrand NOEs. 23| 22| Steps 22 and 23. ! spectra. 21| likely to correlate resonances of the target protein. ( ! HMQC spectra measured onmethyl-selectively protonated samplesare useful. sample. Forthe analysis of NOEs involving methyl protons, 3D 20| amino-acid classification of methyl assignment. selectively methyl-protonated samples. Intraresidue and sequential NOEs involving methyl protons are alsousedfor the 19| NOE assignment in the subsequent structure calculation.  18| S ? 17| 1056 Fig. Fig. protocol

pectra analysis and structure determination

Box 2 it isrunonthe entire data filetoensure thatconvergence occurs for the chosen parameters. 6. Perform processing of the data withthe scriptfile. It is recommended thatthe MaxEnt algorithm betestedonaslice of data before convergence of the calculations (typically within10–20iterations). noise levelhastobeestimated from spectra withsimilaracquisition parameters, and modified bytrial and error with monitoring of the However, itisimpossibletousethisequation for nonlinearly sampleddata, asFFT-processed spectra cannot beproduced. Instead, the Azara isthe noise level, which isusually estimated byusing the equation: above) and the 2DMaxEnt partfor the d2and d3dimensions ( 5. Generate ascriptfileconsisting of the FFTpart for the dimension d1(including phasing withthe phaseparameters determined 4. Determine the 0thand 1storder phasecorrection parameters for the dimension d1using the phaseutilityof ‘plot2’. omitted. Process the data withthe script. 3. Generate ascriptfile for the FFTprocessing of the acquisition dimension (d1),inwhich commands for phasecorrection should be number of collectedFIDs. number of real points for indirect dimensions (d2and d3dimensions), for which nonlinear sampling isapplied, isequal tothe total 2. Generate aparameter filethatisusedto describe the dimensions, referencing, etc., of the data set.Ensure thatthe product of the defined inthe sampling listfileisconsistent with the number ofcollectedFIDs. ement of the hypercomplex points sampledinthe conventional sampling scheme (see been sampledinthe inputdata setrelative tothe usual uniformly sampledset.Eachinteger in the listidentifies the location of anel made availableatthe URL,http://www.comp.tmu.ac.jp/osbc/GROUP/ITO/ito_nls_utility.html. The listdescribes the points thathave 1. Prepare asampling listfile(text format) for 2D MaxEnt processing. Examples of allfiles and scripts describedinthissection are MAXIMUM ENTROPYMETHDIMPLEMENTEDINTHEAZARASFTWARE 7. Check the calculatedspectra with ‘plot2’. CAUT CAUT nois TROUBLESHOOTING

CR Prepare distance restraints for hydrogen bonds for those positions inthe regular secondary structure regions inwhich Prepare backbone torsion angle restraints onthe basisof chemical shiftswiththe programs TALOS Prepare NOESY peak lists containing the chemical shifts and volumes (or intensities) of the cross-peaks in the NOESY Analyze 3D Perform side-chain methyl resonance assignments byanalyzing 3D(H)CC(CO)NHand H(CCCO)NHspectra measured on Perform backbone and side-chain resonance assignments byanalyzing the 3DNMRspectra listedin Process all3Dspectra using the 2Dmaximum entropy method (

| I 2 VOL.5 NO.6VOL.5 = e T ) I I I 2 ON ON CAL 3 . . NOE cross-peaks in the 3D Use consistent chemical shift referencing in NOESY spectra and for the chemical shifts determined in In contrast to | (numbe

1 STEP PROCESSINGFNNLINEARLY SAMPLED3DNMR DATA WITHTHE2D H- 13 | 15 2010 C HMQCspectra of in-cellNMRsampleswithdifferent methyl-selective labeling patterns are usedfor N-separated NOESY-HSQC and 3D Backbone and side-chain resonances should be assigned as completely as possible to assist the automated o r standard | c f natureprotocols omplex 15 N-labeling, N-labeling, uniform deviatio point 1 H- sf no 13 C-separated C-separated NOESY should be analyzed carefully and selected only if they are highly 13 or d f C correlation cross-peaks. d2 at × v a number alue 13 C-labeling C-labeling gives rise to a considerable number of ‘background’ cross-peaks 13 sf C-separated NOESY-HSQC spectra measured onthe uniformly labeled or of nois comp Fig. 1 er egions lex 13 c C-separated NOESY-HSQC and 3D ). The most important parameter for MaxEnt processing with point Box 2 sf or ). d3) Fig. 1 b ). Ensure thatthe totalnumber of points 13 C/ 13 C-separated HMQC-NOE- 2 9 orTALOS T able

2 . + 4 0 .

- © 2010 Nature Publishing Group http://www.nature.com/natureprotocols several hours depending on the performance of the computer, the size of the protein, the quantity of NMR data and the Step P 17 12 T conformer Number of torsion angle dynamics steps per Number of conformers analyzed Number of conformers calculated Median distance for automated NOE calibration Tolerance for NOE assignment and structure calculation cycles T necessary, manually assigned NOE peaks can also be included in the CYANA calculation. RMSD of less than 3 Å for the backbone atoms, excluding flexible regions, in the first cycle of the structure calculation ! number of structures computed in CYANA.  ● calculation bysimulated annealing using torsion angle dynamics 24| TTHA1718 (blue). Adapted from reference TTHA1718 (black) and ( aliased cross-peaks are represented in red. For harvested cells after 6-h NMR measurement. The of the in-cell sample after 6 h measurement. ( ( sample immediately after sample preparation. 1 HSQC spectrum of purified TTHA1718. ( TTHA1718 in living Figure 2 H- e c a a

arameter ) The ) Overlay of the CAUT

b b 15 TIMING PAUSE a 1 le le N HSQC spectrum of a TTHA1718 in-cell NMR Use the program CYANA H- – d 4 3 15 , negative contours originating from 1 H-

N HSQC spectrum of the lysate of the I Problem MaxEnt processing No, or very slow convergence in 2D cells during NMR experiments Leakage of the target protein from target protein in in-cell NMR spectra No, or very weak cross-peaks from the | ON | | 2D 15

Troubleshooting table. Parameters for in-cell structure calculations with CYANA. N HSQC spectrum of the supernatant PO 0.2–5h,depending oncomputerperformance and protein size. The reliability of the structure obtained by automated NOE assignment with CYANA should be ascertained by an 1 H- 1 I H/ NT 15 1 N or H- 13 E. coli CYANA calculations can usually be accomplished in C/ 13 E. coli C HSQC spectra of purified 15 1 H- N N chemical shift matching cells. ( 13 C HSQC spectra of cells expressing a ) The 2 4 torunmultiple (typically seven)iterative cyclesof automated NOE assignment b 1 ) The H- 3.

15 N

d )

P be be one of the possible reasons actions inside tions due to nonspecific inter Unknown. Motional restric rate is too low Noise level is too low or Unknown ossible reason 15N (p.p.m.) 15N (p.p.m.) T 128 126 124 122 120 118 116 114 128 126 124 122 120 118 116 114 ypical value 0.3 0.3 p.p.m. 0.03/0.3/ 10,000 d c a 4.2 4.2 Å 10 10 100 20 7 1 1 8 9 8 9 H (p.p.m.) H (p.p.m.) E. E. coli

6 7 6 7 cells can Troubleshooting advice canbefound in ? AMBER force field 26| ? program. and the final 3Dstructure witha molecular graphics table of the structure calculation provided byCYANA the extent of NOESY cross-peak assignments, the summary 25| 2 8

( b TROUBLESHOOTING

TROUBLESHOOTING 10 10 T - Perform energy refinement of the structure against the Analyze the results of the CYANA calculation. Check

< able

1 1 h of unattended computation time, but may take 1 1 8 9 8 9 - H (p.p.m.) H (p.p.m.) expression, e.g., host but it is worth altering the conditions for protein Usually drastic improvement cannot be guaranteed, tions) indicated in the output log files or increase rate the convergence of the calculations (within 10–20 itera Increase the value of ‘noise’ parameter in the script. Check capsules stabilizes Li NMR measurement by reducing the temperature for protein expression and As above. In one case, leakage was largely prevented Solution optimal temperature and incubation times 3 ) et et al. 6 7 6 7 4 5 .

5 1 reported that encapsulation in alginate micro 3 0 . 13C (p.p.m.) 45 40 35 30 25 20 15 natureprotocols 8 e

E. E. coli 7 E. E. coli cells and prevents leakage 6 strains, timing of induction, 5 1

H (p.p.m.) | VOL.5 NO.6VOL.5 T able 4 2 7 protocol and structure 4 3 . | 2010 (Continued) 2

3 . . If 1 |

1057 - - © 2010 Nature Publishing Group http://www.nature.com/natureprotocols binding protein of 66amino acids thatwasoverexpressed in Step tive heavy metal-binding loop, inter loop, metal-binding heavy tive (blue). Incontrast to methyl-protonated in-cell NMR NMR in-cell methyl-protonated selectively in obtained groups methyl involving NOEs from derived restraints distance without calculated when Å, 5.46 of RMSD backbone a to drastically dropped structures of convergence the as structures, 3D of determination cise pre the for indispensable were NOEs methyl–methyl Long-range region. the of conformation the affect may cytosol the in ions metal with actions puta the Particularly, in cytosol. the in crowding molecular and viscosity of effects the to due be may gions,which re loop dynamic more the in found were differences structural Slight and in-cell the between RMSD backbone ( sample purified a from independently determined was that structure the to similar is and coordinates, mean the to Å 0.96 of RMSD backbone a with converged well is structure resulting The hydrogenbonds. for restraints and restraints angle torsion backbone restraints, distance selectively were residues Val and Leu Ala, of groups methyl which in sample, NMR 1 in-cell TTHA1718 a on measured living in TTHA1718 labeled formly ( spectrum NOESY-HSQC living in TTHA1718 for measured assignment resonance side-chain and backbone the for spectra NMR triple-resonance 3D from p.p.m.) (120.06 showed much sharpercross-peaks ( appeared onremoval of bacteria bygentle centrifugation ( inside the living cells, and thatthe contribution from extracellular proteins isnegligible. Most samples are stablefor atleast6h.It iscrucial for in-cellNMRtoensure thatthe proteins providing NMRspectra are indeed sample preparation ( broader line shapefor both The protocol wasapplied toamodel system,the ANTICPATED RESULTS 25 T 1058 ( samples H/ a protocol Figure 5 Figure Figure Figure The b le 13 in vitro

| C labeled. C VOL.5 NO.6VOL.5 4 1 H- No convergence in structure calculation lating Excessively rapid convergence or oscil Problem |

15 3 Troubleshooting table (continued). Fig. 5 Fig. 4 structures is 1.16 Å ( N HSQC spectra of TTHA1718 a shows shows shows slices from 3D NOESY-type experiments for TTHA1718 in living living in TTHA1718 for experiments NOESY-type 3D from slices shows shows the 3D structures of TTHA1718 in in TTHA1718 of structures 3D the shows ω value in 2D MaxEnt processing d | 2010 ). 13 C(F1)- Fig. 2 | 15

natureprotocols N-labeling, uniform E. coli E. Fig. 5 Fig. Fig. 4 Fig. 1 b H in vitro in 1 ) and after6hinanNMRtube at37°C(data not shown) shows thatTTHA1718in-cellNMR N H and (F3) or or (F3) cells. Fig. 5 b E. coli E. a ). The ).

) and the 3D 3D the and )

- Fig. 2 15 c

- N dimensions. The virtual identity of the ). -

1 E. coli E.

H(F1)-

in vitro - d ). correlations are assigned in red. Intraresidue correlations are indicated by blue boxes and annotated. chain resonance assignments of TTHA1718 in living HBHA(CBCACO)NH, H(CCCO)NH and (H)CC(CO)NH spectra (black) used for backbone and side- shift of Lys30 (120.06 p.p.m.) from 3D HNCA, HN(CO)CA, CBCANH, CBCA(CO)NH, HN(CA)CO, HNCO, Figure 3 13 cells. The 3D 3D The cells. P NOE distance restraints Too few, or inconsistent rate is too high Noise level is too high or 1 Figure 2 C-labeling gaverisetoaconsiderable number of ‘background’ cross-peaks H 13C (p.p.m.) ossible reason N and in-cellare presented in 13 45 65 60 55 50 (F3) slices corresponding to the amide amide the to corresponding slices (F3) C-separated NOESY-HSQC spectrum ( spectrum NOESY-HSQC C-separated T. thermophilus

C | 7.5

1 α 13

H (p.p.m.) HNCA C(F1)- e E. coli E. shows E29 C 7.5 α

1 HN(CO)C H Fig. 2 13 N cells calculated using the program CYANA program the using calculated cells (F3) (F3) or C/ E. coli

13 A 1 H- C (p.p.m.) 13 70 60 50 40 30 20 10 HB8TTHA1718gene product, aputativeheavy metal- C-separated HMQC–NOE–HMQC spectrum ( spectrum HMQC–NOE–HMQC C-separated c 13 ), whereas the spectrumof the lysateof the harvestedcells

1 C HSQC spectra of TTHA1718 H(F1)- toaconcentration of 3

7.5 C 1 CBCANH H (p.p.m.) β 1 H E29 7.5 C E29 spectral dimensions in the peak list headers chemical shift referencing, and the specification of the Check chemical shift assignment, NOESY peak picking, Decrease the value of the ‘noise’ or ‘rate’ parameter Solution C N

β CBCA(CO)N (F3) (F3) cross-sections corresponding to the amide α Figure 2a, 1 13C (p.p.m.) H- 182 180 174 172 170 178 176 H 15 N HSQC spectra recorded immediately after 7.5 1 C E. E. coli

H (p.p.m.) HN(CA)CO ′ b E. coli E. Fig. 4 Fig. 15 . The in-cellspectrumshows amuch N chemical shift of Lys30 Lys30 of shift chemical N cells. The cross-peaks due to sequential 7.5 E29

HNCO C − ′ 4 mM(ref. cells. The 3D 3D The cells. b 1 ) were measured on uni on measured were ) in vitro 1H (p.p.m.) H- 6 5 4 3 2 1 15 N HSQC cross-peaks dis 7.5 E29 1

E29

H H (p.p.m.)

H HBHA(CBCACO)NH α (black)and in-cell β 2 4 3 on the basis of NOE of basis the on ). 7.5 E29

H H(CCCO)NH γ 15 Fig. 4 Fig. N-separated N-separated 13C (p.p.m.) 15 80 70 60 50 40 30 20 10 N N chemical 1 H (p.p.m.) c

) was was ) 7.5 E29 C

E29 (H)CC(CO)NH C α 2 γ 3 . - - © 2010 Nature Publishing Group http://www.nature.com/natureprotocols Published online at http://www.natureprotocols.com/. interests. CO development for rapid NMR data acquisition and MaxEnt processing. and resonance assignment. Y.S., M.M., D.N. and M.W. contributed the protocol protocols, including sample preparation and characterization, data acquisition and refinement. A.S., D.S., J.H., T.H., M.Y., N.H. and T.M. developed the and wrote the article. T.I. developed the protocol for structure calculation AUT Process’, and by the Volkswagen Foundation. Systems—Development of Advanced Methods for Exploring Elementary of Biological Systems’ and ‘Molecular Science for Supra Functional Technology on ‘Molecular Soft Interactions Regulating Membrane Interface Areas from the Japanese Ministry of Education, Sports, Culture, Science, and Ensemble Program of RIKEN, Grants-in-Aid for Scientific Research of Priority program of the Japan Science and Technology Agency (JST), the Molecular plasmid encoding TTHA1718. This work was supported in part by the CREST A methyl groups obtained in methyl-selectively protonated in-cell NMR samples. Adapted from reference a ensembles is shown with the same color code in (N, C in vitro TTHA1718 structures in living purified TTHA1718 ( cells, showing backbone (N, C 20 final structures of TTHA1718 in living in living Figure 5 that are otherwise unstable and difficulttopurify. protein modification, the conformations of proteins thatare intrinsically disordered provides toolsfor investigating inliving cellsthe effectsof molecular crowding inthe cytosol,protein stabilityand covalent manner inwhich protein conformations change inresponse tobiological events inliving environments. The approach com/reprintsandpermissions/. http://npg.nature. at online available is information permissions and Reprints b and ckno a ) ) A superposition of the 20 final structures of M In conclusion, in-cellprotein structure determination byNMRopens new avenues for studying inatomic resolution, the 1

H H (p.p.m.) PET α OR 9 8 7 6 5 4 3 2 1 , , C b w . . The best-fit superposition of backbone . . ( I H H V31 H

L41H V31 ′ N le CONTR

α E32 β E. E. coli ) ) atoms of the two conformational γ V42 | d G G FI 8. NMR solution structure of TTHA1718 H dgm H ) ) A superposition of the 20 final structures of TTHA1718 in living 5 γ H N α α NANC H H E32 cells. ( IB ents V33 γ β 1 UT H (p.p.m.) H α 8. in in vitro I I

AL 5 We thank Professor Seiki Kuramitsu for providing the ONS H H G38H A40 V33H V33H A40 V33H S34 β a α I 8. ) ) A superposition of the NTERESTS 5 B.O.S., M.S., P.G. and Y.I. designed the research H H α α α β β γ . . ( H H H H G38H G38H S34 K37 K37 c V33H E39 β β α γ ) ) A comparison of ' α E. E. coli , , C H 7. H H S34 N N β α 0 α γ ′

) ) atoms. H The authors declare no competing financial b β cells and 1H (p.p.m.) 9 8 7 6 5 4 3 2 1 0. 5 L22 E. E. coli

H H H δ H H H α N β γ δ ′ β ′

V33 1 0. H (p.p.m.) H γ H 5 H

α N β 5. H E39 5 β

a C H H α N γ N Y60

H H ε 6. N β V18

′ 5

13 H γ C (p.p.m.) c 26 24 22 20 18 γ′ V18 V6 24 E. E. coli γ γ C b 10. 4. 9. 8. 7. 5. 1. 3. 6. 2. 24 L35 V15 cells calculated without distance restraints derived from NOEs involving 13 N 23 C (p.p.m.) γ′ Selenko, P. & Wagner, G. Looking into live cells with in-cell NMR in-cell with cells live into Looking Wagner,G. P.& Selenko, Serber, Z. & Dotsch, V. In-cell NMR spectroscopy.NMR V.In-cell Dotsch, & Z. Serber, spectroscopy. Sp. Res. Mag. Nucl. Prog. spectroscopy.NMR V.In-cell Dötsch, & F.Löhr, R., Hänsel, S., Reckel, (2006). (STINT-NMR).interactions protein-protein for NMR In-cell A. Shekhtman, & D. Cowburn, K., Dutta, D.S., Burz, (2006). 91–93 (STINT-NMR). spectroscopy NMR in-cell usinginteractions structural Mapping A. Shekhtman, & D. Cowburn, K., Dutta, D.S., Burz, (2006). spectroscopy.NMR in-cell by cells injected Enzymol. Method. spectroscopy.NMR V.In-cell Dötsch, & F. Durst, L., Corsini, Z., Serber, (2001). 14317–14323 in-cell NMR spectroscopy.NMR in-cell conditions. Sci. Biochem. Ellis, R.J. Macromolecular crowding: obvious but underappreciated.but obvious crowding:Macromolecular R.J. Ellis, Sakakibara, D. Sakakibara, Serber, Z. Serber, Pielak, G.J. Pielak, δ δ′ 22 V28 A46 V28 γ 21 β γ′ et al. et A50 Biochemistry 19 et al. et V25 V25

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Protein nuclear magnetic resonance under physiologicalunder resonance magnetic nuclear Protein 394 3. C c Protein structure determination in living cells by cells living in determinationstructure Protein in vitro , 17–41 (2005). 17–41 , N are indicated as in in as are indicated and NOEs intraresidue The inter- spectrum. the 3D from extracted groups methyl representative the to corresponding in as and are NOEs intraresidue The indicated inter- the 3D from extracted nuclei carbon representative the to corresponding ( and annotated. boxes blue by Intraresidual are NOEs indicated red.in are NOEs assigned interresidual to due The cross-peaks spectrum. NOESY-HSQC the from 3D extracted groups amide backbone of selected shifts chemical cross-sections corresponding to the to corresponding cross-sections living in TTHA1718 in restraints distance effect-derived 4 Figure natureprotocols

51 Nature , 226–234 (2009). 226–234 ,

, 91–101 (2007). 91–101 , 158 a and the 3Dstructures of proteins . . ( 13 13

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protocol 1 15

1 , 146–152 , N-separated N-separated , 2701–2709 , 1 Nat. Methods Nat. H | N 2010 (F3) (F3) 15

40 N

Trends |

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